functional analysis of dehydration responsive genes in the ... - monash … · 2017. 3. 2. ·...

MONASH UNIVERSITY

Functional analysis of dehydration responsive genes in the

resurrection grass Sporobolus stapfianus

Cara Griffiths

Bachelor of Science (Honours)

For the award of Doctor of Philosophy

School of Biological Sciences

Monash University

2015

Copyright notice

Notice 1 Under the Copyright Act 1968, this thesis must be used only under the normal conditions of scholarly fair dealing. In particular no results or conclusions should be extracted from it, nor should it be copied or closely paraphrased in whole or in part without the written consent of the author. Proper written acknowledgement should be made for any assistance obtained from this thesis. Notice 2 I certify that I have made all reasonable efforts to secure copyright permissions for third-party content included in this thesis and have not knowingly added copyright content to my work without the owner's permission.

Declaration

I hereby declare that the content of this thesis is original, and has not been used for the

conferral of other awards from other institutions. All sources that have been used throughout

the text which are not my own work have been acknowledged.

Cara Griffiths

July 2015

Acknowledgements

Firstly, I would like to thank Monash University for awarding me the Dean’s Postgraduate

Research Scholarship, allowing me to pursue my PhD. My sincerest thanks belongs with my

supervisors Dr. Alan Neale, Dr. Cecilia Blomstedt and Prof. John Hamill for their support,

guidance and wisdom, for which I am deeply and truly grateful. I would also like to extend

thanks to Dr. Don Gaff for his valuable discussions on all things desiccation tolerance, and

Assoc. Prof. Ros Gleadow for her support throughout my time at Monash University. I also

thank Prof. Ian Godwin and Guoquan Liu at the University of Queensland, for their help with

the Sorghum transformations, as well as the Australian Society of Plant Scientists for funding

my visit there. I value the lasting friendships I have made in the ADN, JDH and RMG labs,

particularly Heidi, Sam, Viviana, Aaron, Nicole, Natalie, Suzy and Huai-Yian, which

provided support, laughter and conversations about science and life. Lastly, I would like to

thank my parents, Sharon and Glenn Griffiths, for their constant support throughout my

completion.

Publications

Blomstedt, CK, Griffiths, CA, Fredericks, DF, Hamill, JD, Gaff, DF & Neale, AD (2010)

The resurrection plant Sporobolus stapfianus: An unlikely model for engineering enhanced

plant biomass? Plant Growth Regulation, 62(3):217-232.

Islam, S, Griffiths, CA, Blomstedt, CK, Le, TN, Gaff, DF, Hamill, JD & Neale, AD (2013),

Increased biomass, seed yield and stress tolerance is conferred in Arabidopsis by a novel

glucosyltransferase from the resurrection grass Sporobolus stapfianus. PLoS ONE, 8(11):

e80035.

Griffiths CA, Gaff DF and Neale AD (2014) Drying without senescence in resurrection

plants. Front. Plant Sci. 5:36.

Nomenclature

ABA abscisic acid

AD activation domain

ATP adenosine triphosphate

BD binding domain

bp base pair

dNTPs deoxynucleotides

DMSO dimethylsulfoxide

EDTA ethylenediamine tetracetic acid

GFP green fluorescent protein

GR24 (3aR*,8bS*,E)-3-(((R*)-4-methyl-5-oxo-2,5-dihydrofuran-2-

yloxy)methylene)-3,3a,4,8b-tetrahydro-2H-indeno[1,2-b]furan-2-one

GUS uidA (β-glucuronidase) reporter gene

kb kilobase

kDa kilodaltons

LD long day

MeJa methyl jasmonate

OD optical density

ORF open reading frame

PCR polymerase chain reaction

PEP phosphoenolpyruvic acid

RT-PCR reverse transcription polymerase chain reaction

RWC relative water content

SD short day

SDS sodium dodecyl sulfate

SDS-PAGE SDS polyacrylamide gel electrophoresis

Tris tris(hydroxymethyl)aminoethane

UDP -glucose uridine-5'-diphosphoglucose

Figures and tables

1.0 Figures

Figure 1.1: Cellular pathways leading to dehydration induced gene expression

Figure 1.2: Summary of ABA signalling factors when ABA is absent (left) and when ABA is

present (right)

Figure 1.3: Northern analysis of S. stapfianus desiccation tolerance genes

Figure 3.1: Genome walking adaptor

Figure 3.2: pDONR/Zeo Gateway® Donor vectors containing SDG4i used to create GFP fusion

destination vectors

Figure 3.3: pMDC43 (N-terminal) and pMDC83 (C-terminal) GFP fusion vectors containing the

SDG4i insert

Figure 3.4: SDG3i sequence alignment from genome walking experiment

Figure 3.5: SDG3i gene structure in S. stapfianus

Figure 3.6: Southern analysis of SDG3i in S. stapfianus

Figure 3.7: Amino acid motifs in SDG3i

Figure 3.8: Amino acid sequence alignment of predicted full SDG3i sequence to known

proteins in the NCBI database


Figure 3.10: Amino acid sequence alignment of SDG4i sequence to known proteins in the

NCBI database



Figure 3.13: SDG4i intracellular localisation

Figure 3.14: Transcript accumulation of SDG3i, SDG4i and SDG8i during the dehydration and

rehydration of S. stapfianus

Figure 3.15: Transcript accumulation after exogenous hormone application in S. stapfianus

leaves

Figure 3.16: Relative expression levels of SDG3i, SDG4i and SDG8i transcripts in hormone

treated tissues

Figure 4.1: Calli grown from differing varieties of Sorghum seed

Figure 4.2: Gus staining of S. bicolor calli

Figure 4.3: Regeneration of S. bicolor calli

Figure 4.4: Transcript levels in individual A. thaliana 35S:SDG4i transgenic lines

Figure 4.5: Western blot of A. thaliana 35S:SDG4i transgenic lines

Figure 4.6: Amino acid sequence alignment of S. stapfianus SDG4i and A. thaliana AT3G43850

Figure 4.7: Predicted secondary structure of SDG4i and T-AT3G43850

Figure 4.8: Germination time for A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines under LD conditions

Figure 4.9: Rosette leaf number of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.10: Leaf length of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines under LD conditions

Figure 4.11: Petiole length in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog


Figure 4.12: Appearance of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines after the completion of flowering under LD conditions

Figure 4.13: Plant height in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog


Figure 4.14: Branch architecture of A. thaliana

Figure 4.15: Inflorescence development and growth in A. thaliana wild-type Columbia,

35S:SDG4i and SDG4i homolog knockout lines under LD conditions

Figure 4.16: Floral development in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.17: Silique morphology in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.18: Seed yield and vitality in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.19: Seed weight per 1000 seeds in A. thaliana wild type Columbia, 35S:SDG4i and

SDG4i homolog knockout lines

Figure 4.20: Senescence in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines

Figure 4.21: Days taken for A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines to present two fully expanded cotyledons under SD conditions

Figure 4.22: Rosette leaf number of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines under SD conditions

Figure 4.23: Leaf size of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines under SD conditions

Figure 4.24: Petiole length in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog


Figure 4.25: Appearance of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines after the completion of flowering in SD conditions

Figure 4.26: Bolt development in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.27: Plant height in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog


Figure 4.28: Floral development in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines in SD conditions

Figure 4.29: Silique morphology in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.30: Seed yield and vitality in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Figure 4.31: Seed weight per 1000 seeds in A. thaliana wild type Columbia, 35S:SDG4i and

SDG4i homolog knockout lines in SD conditions

Figure 4.32: Senescence in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i homolog

knockout lines in SD conditions

Figure 5.1: Germination percentage of each A. thaliana line used throughout abiotic stress

assays

Figure 5.2: Typical growth of A. thaliana lines used throughout abiotic stress assays

Figure 5.3: Primary root length, lateral root number and leaf number of A. thaliana lines used

throughout abiotic stress assays

Figure 5.4: Germination of A. thaliana lines on media containing mannitol

Figure 5.5: Primary root growth of A. thaliana lines on media containing mannitol

Figure 5.6: Primary root length, lateral root number and leaf number of A. thaliana lines

subjected to growth under 300mM mannitol

Figure 5.7: Germination percentage of A. thaliana lines on NaCl

Figure 5.8: Root length of A. thaliana lines across varying concentrations of NaCl

Figure 5.9: Root length, lateral root number and leaf number of A. thaliana lines subjected to

growth under 125mM NaCl

Figure 5.10: Germination percentage of A. thaliana lines on ABA

Figure 5.11: Root length of A. thaliana lines across varying concentrations of ABA

Figure 5.12: Root length, lateral root number and leaf number of A. thaliana lines subjected to

growth under 4µM ABA

Figure 5.13: Hypocotyl and root length of A. thaliana lines following germination after the seed

was heat shocked at 42ºC

Figure 5.14: Percentage survival of A. thaliana lines in vegetative heat treatment

Figure 5.15: Progression of germination of A. thaliana lines at 4ºC

Figure 5.16: Percentage survival of A. thaliana lines after vegetative freezing treatment at -8ºC

Figure 5.17: Total chlorophyll content of A. thaliana lines prior to senescence induction

Figure 5.18: Chlorophyll content of A. thaliana lines during senescence

Figure 5.19: Photosystem II activity of A. thaliana lines during senescence

Figure 6.1: Amplified inserts from the desiccated S. stapfianus cDNA-GAL4AD library in E. coli

Figure 6.2: Amplified inserts from the desiccated S. stapfianus cDNA-GAL4AD library after

transformation into S. cerevisiae Y187

Figure 6.3: Yeast two-hybrid bait construct

Figure 6.4: Yeast diploids growing on SD/-Trp/-Leu/-His/-Ade with 5mM 3-AT

Figure 6.5: Yeast colony PCR of diploids showing a positive interaction on SD/-Trp/-Leu/-His/-

Ade with 5mM 3-AT plates

Figure 6.6: BIFC assay vectors

Figure 6.7: BIFC visualisation of SDG4i interactions with ADF, BIRC and ERD15.

Figure 6.8: Six-frame translation of S. stapfianus ERD15-like

Figure 6.9: Amino acid sequence alignment of S. stapfianus ERD15-like with homologs in the

NCBI database

Figure 6.10: Relative expression levels of SDG4i and ERD15-like transcripts in dehydrating S.

stapfianus leaf tissue

Figure 6.11: Germination of A. thaliana lines under endoplasmic reticulum stress

Figure 6.12: Schematic diagram of potential effect of SDG4i on ERD15 in biological processes

Figure 7.1: Coupled enzyme assay between pyruvate kinase and lactate dehydrogenase

activity

Figure 7.2: S. stapfianus leaf regrowth

Figure 7.3: MagnICON pICH11599 provector containing the SDG8i insert

Figure 7.4: N. benthamiana infiltrated tissue

Figure 7.5: N. benthamiana total protein extracts

Figure 7.6: SDG8i activity in vitro

Figure 7.7: Lineweaver-Burke plot of SDG8i activity

Figure 7.8: SDG8i expression after exogenous auxin treatment.

Figure 7.9: MAX4 expression in dehydrating S. stapfianus leaf tissue

2.0 Tables

Table 1.1: Summary of changes occurring during dehydration in the desiccation tolerant

S.stapfianus

Table 3.1: Primers used for GFP fusion vector generation

Table 4.1: Percentage of predicted secondary structure types and level of disorder within of

SDG4i and T-AT3G43850

Table 6.1: Primers used for BIFC vector construction

Table 6.2: A. tumerfaciens infiltration mixtures

Table 6.3: Primers used for S. stapfianus gene expression study

Table 6.4: Results from a BLASTn analysis of library inserts interacting with SDG4i in the

yeast two-hybrid assay at the nucleotide level

Table 7.1: Primers used for S. stapfianus gene expression study

Summary

Crop plant productivity in Australia and worldwide is facing increasing

environmental constraints brought upon by drought, cold and salinity. The ability of a

plant to survive drought, as well as other abiotic stressors is a trait of particular

interest in crop improvement studies. Current research focuses of the use of genes

from drought-sensitive plants as a means to increase drought tolerance through

genetic modification, however the use of genes from plants that are extremely drought

tolerant is a growing area of interest in genetic modification studies. Resurrection

plants, such as Sporobolus stapfianus, utilised in this study, are anhydrobiotic

organisms that have molecular mechanisms that allow them to dehydrate and go into a

state of suspended animation for several months during a period of drought. These

plants then restore metabolism 24 hours after a rainfall event.

This study presents evidence of molecular mechanisms associated with genes that are

transcribed during the dehydration of S. stapfianus. Genes of interest in this process

had been isolated in previous work, and a selection of these genes, namely, SDG3i,

SDG4i and SDG8i have been chosen for further work in this study. This study

provided further support for the transcription of these genes during the dehydration

phase of S. stapfianus, and interestingly also in the rehydration phase. Bioinformatic

analysis suggests that SDG3i encodes a protein containing at TIM17-like domain, and

may be localized to either the mitochondrial inner membrane or peroxisome (Chapter

3). Intriguingly, the protein product of SDG4i was predicted to be involved in

chromosome remodeling through its potential for interaction with histone

deacetyalase 19 (Chapter 3). Developmental phenotype analysis of transgenic

Arabidopsis thaliana (Columbia) over-expressing SDG4i showed that these plants

produced had changes in petiole size, shoot branching and height, as well as delayed

senescence, increased seed yield, however a reduced seed longevity in long and short

day conditions (Chapter 4). Furthermore, the abiotic stress tolerance, particularly to

osmotic, salt, cold and endoplasmic reticulum stress was increased (Chapter 5). With

these traits of transgenic plants observed, and the potential of SDG4i to be involved in

a protein-protein interaction, a yeast 2-hybrid screen was performed to ascertain if

these changes could be a result of an interaction between SDG4i and a protein

product. It was shown that SDG4i interacts with an early response to dehydration 15-

like protein (ERD15). This ERD15 protein acts to up-regulate asparagine-rich

proteins (NRPs) to facilitate cell death during dehydration and endoplasmic reticulum

stress. The role of SDG4i in S. stapfianus may potentially be to prevent cell death

during dehydration, allowing for the induction and/or facilitation of the complete

desiccation of S. stapfianus.

In addition to revealing protein-protein interactions in desiccation tolerance-

associated genes in S. stapfianus, this study also uncovered the first known UDP-

glycosyltransferase (SDG8i) (chapter 3) to glycosylate a strigolactone-like compound.

This was completed using an in vitro coupled enzyme assay with a variety of plant

hormones, where SDG8i has the highest affinity for the synthetic strigolactone GR24

(Chapter 7). This supports work completed in prior to this study that A. thaliana over-

expressing SDG8i produce plants that have an increased branching phenotype that is

generally associated with strigolactone mutant plants.

This study provides novel evidence that S. stapfianus transcribes genes that may be

affecting hormone homeostasis as well as drought-related senescence processes to

allow the plant successfully negotiate a dehydration/rehydration cycle.

Table of Contents 1.0 Introduction....................................................................................................... 1

1.1 Drought stress signalling ............................................................................. 2

1.1.2 Drought responsive element (DRE) and ABA-response element (ABRE)…………………………………………………………………………....4

1.2 The involvement of ABA in abiotic stress responses.................................. 6

1.3 ABA biosynthesis and signalling ................................................................ 7

1.4 ABA in vegetative development................................................................ 10

1.5 ABA in seed development ......................................................................... 11

1.6 ABA in germination .................................................................................. 12

1.7 Regulation of plant growth by strigolactone ............................................. 13

1.8 Senescence................................................................................................. 15

1.8.1 Hormonal involvement in senescence processes ................................... 16

1.8.2 Drought induced senescence.................................................................. 17

1.8.3 Role of endoplasmic reticulum stress during senescence processes ..... 19

1.9 Anhydrobiotic organisms .......................................................................... 20

1.9.1 Resurrection plants ................................................................................ 21

1.10 Molecular response to water deficit in plants ............................................ 22

1.11.1 Late Embryogenesis Abundant (LEA) proteins...................................... 24

1.11.2 Dehydrins and rehydrins........................................................................ 25

1.12 Physiological changes during dehydration and rehydration in the resurrection plant S. stapfianus .................................................................................................... 26

1.12.1 Sugar content ......................................................................................... 28

1.12.2 Hormonal induction of desiccation tolerance........................................ 29

1.12.3 Cell structure during dehydration ......................................................... 29

1.12.4 Antioxidant systems and photosynthesis ................................................ 30

i

1.12.5 Changes in gene expression during dehydration in S. stapfianus ......... 31

1.13 Aims of the current study ........................................................................... 33

2.0 General materials and methods ..................................................................... 35

2.1 General Solutions and Reagents ..................................................................... 35

2.2 Bacterial methods ...................................................................................... 36

2.2.1 Solutions and media ............................................................................... 36

2.2.2 Bacterial transformation...........................................................................37

2.2.3 Preparation of E. coli and A. tumerfaciens electrocompetent cells ....... 37

2.2.4 Colony PCR ........................................................................................... 38

2.2.5 Small scale isolation of plasmid DNA ................................................... 38

2.2.6 Medium scale isolation of plasmid DNA ............................................... 38

2.3 Plant materials and methods ...................................................................... 39

2.3.1 Solutions and media ............................................................................... 39

2.3.2 Plant material ........................................................................................ 39

2.3.2.1 Sporobolus stapfianus ........................................................................ 39

2.3.2.2 Arabidopsis thaliana ........................................................................... 40

2.3.3 Agrobacterium-meadiated transformation of Arabidopsis thaliana....... 40

2.4 Nucleic Acid methods ............................................................................... 41

2.4.1 Genomic DNA isolation ............................................................................41

2.4.2 Agarose gel electrophoresis......................................................................41

2.4.3 Sequencing and bioinformatic analysis ....................................................42

2.4.4 Isolation of RNA........................................................................................42

2.4.5 Denaturing agarose gel electrophoresis...................................................43

2.4.6 cDNA synthesis ...................................................................................... 43

2.4.7 Restriction digestion of DNA ................................................................. 43

2.4.8 Ligation of DNA ..................................................................................... 43

ii

3

2.4.9 Purification of DNA fragment from agarose gel ................................... 44

2.5 Nucleic acid hybridisation methods .......................................................... 44

2.5.1 Transfer of nucleic acids from agarose gels to nylon membranes ........ 44

2.5.2 Radiolabelling of probe ......................................................................... 45

2.5.3 Northern blot hybridisation ................................................................... 45

2.5.4 Southern blot hybridisation.......................................................................45

2.5.5 Phosphoimaging .................................................................................... .45

2.6 Protein methods ............................................................................................46

2.6.1 Isolation of Protein from plant tissue .................................................... 46

2.6.2 SDS-PAGE ................................................................................................46

2.6.3 Transfer of proteins to nitrocellulose membranes ................................. 46

2.6.4 Western blotting ..................................................................................... 47 3.0 Dehydration and hormonal responses of genes that are transcribed during the desiccation of Sporobolus stapfianus .................................................................. 48

3.1 Introduction ............................................................................................... 48

3.2 Experimental methods ..................................................................................49

3.2.1 Genome walking of SDG3i .................................................................... 49

3.2.2 Confirmation of SDG3i coding region by RT-PCR.................................50

3.2.3 Dehydration/rehydration of S. stapfianus .............................................. 50

3.2.4 Exogenous application of hormones to S.stapfianus ............................. 51

3.2.5 Construction of SDG4i GFP fusion vectors .......................................... 51

3.2.6 Intracellular localisation of SDG4i in Allium cepa (onion) epidermal peels ……………………………………………………………………….…54

3.3 Results ...................................................................................................... 55

3.3.1 Sequence analysis of SDG3i ....................................................................55

3.3.2 Sequence analysis of SDG4i..........................................................................64

3.3.3 Intracellular localisation of SDG4i ..........................................................69

4

3.3.4 Transcript accumulation of dehydration-related genes during dehydration and rehydration .................................................................................70

3.3.5 Transcript accumulation of dehydration related genes after exogenous hormone application ...............................................................................................72

3.4 Discussion.........................................................................................................75

3.4.1 SDG3i, SDG4i and SDG8i transcript accumulation during dehydration and rehydration ....................................................................................................75

3.4.2: SDG3i has strong homology to plant pore proteins and expression is induced by exogenous cytokinin application..........................................................76

3.4.3 SDG4i has strong homology to bnKCP1 and expression is induced by exogenous ABA application ................................................................................. 78

3.4.4 SDG8i expression is induced by exogenous application of abscisic acid and methyl jasmonate ........................................................................................... 80

4.0 Effect of over-expression of SDG4i on the developmental phenotype of Arabidopsis thaliana .................................................................................................... 82

4.1 Introduction ............................................................................................... 82

4.2 Experimental methods ................................................................................ 83

4.2.1 Callus induction and tissue regeneration in S. bicolor........................... 83

4.2.2 Agrobacterium mediated transformation of S. bicolor calli .................. 83

4.2.3 Growth conditions of A. thaliana and developmental phenotype analysis

……………………………………………………………………….…84

4.3 Results ....................................................................................................... 85

4.3.1 Callus induction of Sorghum bicolor ..................................................... 85

4.3.2 Transformation of callus with GUS reporter gene...................................86

4.3.3 Regeneration of callus into S. bicolor plantlets .......................................88

4.3.4 Expression of SDG4i in transgenic A. thaliana under the 35S promoter ………………………………………………………………………….80

4.3.5 Identification of an A. thaliana T-DNA insertion line homologous to SDG4i …………………………………………………………………….……91

5

4.3.5 Developmental phenotype of 35S:SDG4i A. thaliana in long day conditions................................................................................................................94

4.3.6 Developmental phenotype of 35S:SDG4i A. thaliana in short day conditions..............................................................................................................106

4.4 Discussion..................................................................................................118

4.4.1 Transformation of Sorghum species using an Agrobacterium based method ………………………………………………………………………..118

4.4.2 General health of Arabidopsis SDG4i transgenic lines ......................119

4.4.3 35S:SDG4i lines have a reduced leaf length and petiole size.............119

4.4.4 35S:SDG4i lines have an increased primary rosette branching in short day conditions, and reduced secondary rosette branching in long day conditions

…………………………………………………………………..……122

4.4.5 35S:SDG4i lines have a reduced plant height in long day conditions and an increased plant height in short day conditions............................................... 125

4.4.6 35S:SDG4i lines appear to be affected by photoperiod....................... 126

4.4.7 35S:SDG4i lines have an increased seed yield and delayed senescence

………………………………………………………………………..128

4.5 Conclusion .............................................................................................. 130 5.0 Effect of over-expression of SDG4i on abiotic stress tolerance in A. thaliana...................................................................................................................... 131

5.1 Introduction ............................................................................................. 131

5.2 Experimental methods ............................................................................... 132

5.2.1 General stress assay growth conditions ................................................ 132

5.2.2 Osmotic stress assay ............................................................................. 133

5.2.3 Salt stress assay..................................................................................... 133

5.2.4 ABA stress assay................................................................................... 133

5.2.5 Heat stress assay .................................................................................. 133

5.2.6 Cold germination assay......................................................................... 134

6

5.2.7 Freezing tolerance assay ...................................................................... 134

5.2.8 Senescence assay ................................................................................. 134

5.3 Results ..................................................................................................... 136

5.3.1 Germination, root length, lateral root and leaf number of 35S:SDG4i A. thaliana under standard conditions..................................................................... 136

5.3.2 Osmotic stress assay ............................................................................ 138

5.3.3 Salt stress assay.................................................................................... 141

5.3.4 ABA stress assay.................................................................................. 144

5.3.5 Heat stress assay .................................................................................. 147

5.3.6 Cold germination assay........................................................................ 149

5.3.7 Vegetative freezing tolerance assay ..................................................... 150

5.3.8 Senescence assay ................................................................................. 151

5.4 Discussion................................................................................................ 155

5.4.1 35S:SDG4i A. thaliana lines display increased tolerance to osmotic stress and salinity................................................................................................ 155

5.4.2 35S:SDG4i A. thaliana lines display increased tolerance to exogenous ABA application ................................................................................................. 156

5.4.3 35S:SDG4i A. thaliana lines show altered thermotolerance ............... 158

5.4.4 35S:SDG4i A. thaliana lines show delayed senescence in the presence of ABA 159

5.5 Conclusion .................................................................................................... 161

6.0 Interaction of SDG4i with ERD15-like protein using a yeast two-hybrid screen162

6.1 Introduction ............................................................................................. 162


6.2.1 Construction of cDNA-GAL4AD library ............................................. 163

6.2.2 Construction of SDG4i-GAL4BD bait ................................................ 164

6.2.3 Bait autoactivation and toxicity test...................................................... 165

vii

6.2.4 Screening of cDNA-GAL4AD library using yeast mating.................. 165

6.2.5 Selection of positive diploids............................................................... 166

6.2.6 Plasmid rescue from positive diploids .................................................. 166

6.2.7 Bimolecular fluorescence complementation assay (BIFC) vector construction ....................................................................................................... 167

6.2.8 N. bethamiana infiltration, co-incubation and visualisation of positive interactions.......................................................................................................... 169

6.2.9 Semi-quantitative RT-PCR of SDG4i and ERD15 in dehydrating S. stapfianus tissue.................................................................................................. 170

6.2.10 Endoplasmic reticulum stress germination assay ................................ 171

6.3 Results ..................................................................................................... 172

6.3.1 Construction of cDNA-GAL4AD library ............................................. 172

6.3.2 Construction of Yeast two-hybrid bait construct ................................. 175

6.3.3 Bait toxicity, auto-activation, and screening of the cDNA-GAL4AD library 176

6.3.4 Construction of BIFC vectors ............................................................... 179

6.3.5 Transient expression of BIFC vectors in N. benthamiana ................... 181

6.3.6 Sequence analysis of ERD15-like......................................................... 183

6.3.7 Expression levels of ERD15 and SDG4i transcripts during dehydration

187

6.3.8 Endoplasmic reticulum stress germination assay ................................ 188

6.4 Discussion................................................................................................ 189

6.4.1 Attenuation of drought related senescence by SDG4i ......................... 189

6.4.2 ERD15, SDG4i and the modification of abiotic stress and ABA responses............................................................................................................. 193

6.4 Conclusion ............................................................................................... 195

7.0 Glycosyltransferase activity of SDG8i ........................................................ 196

7.1 Introduction ............................................................................................. 196

viii


7.2.1 S. stapfianus leaf regrowth assay ......................................................... 197

7.2.2 ICON vector construction .................................................................... 197

7.2.3 Transient SDG8i expression in N. benthamiana leaves....................... 198

7.2.4 SDG8i coupled enzyme assay.............................................................. 199

7.2.5 SDG8i enzyme kinetics........................................................................ 200

7.2.6 SDG8i and MAX4 expression in S. stapfianus leaf tissue .................... 201

7.3 Results ..................................................................................................... 202

7.3.3 S. stapfianus leaf regrowth assay ......................................................... 202

7.3.1 ICON vector construction .................................................................... 203

7.3.2 Transient SDG8i expression in N. benthamiana leaves....................... 204

7.3.4 SDG8i coupled enzyme assay and kinetics ......................................... 207

7.3.5 SDG8i and MAX4 expression in S. stapfianus leaf tissue .................... 209

7.4 Discussion................................................................................................. 211

7.4.1 SDG8i glycosylates GR-24 in vitro ...................................................... 211

7.4.2 S. stapfianus has an increased leaf growth rate 24 hours post rehydration ………………………………………………………………………...214

7.5 Conclusion ............................................................................................... 215

8.0 General discussion ........................................................................................ 216

8.1 Desiccation tolerance genes for improving biomass production ............. 216

8.2 The effect on hormone signalling pathways by SDG4i overexpression.. 218

8.3 Conclusion and future perspectives ......................................................... 224

9.0 References…………………………………………………………...…………229

Appendix I……………………………………………………………………………I

Appendix II……………………………………………………………….………….V

Appendix III………………………………………………………………………...XI

1

1.0 Introduction

Research into the extreme drought tolerance that is displayed in resurrection plants

involves the exploration of physiological and molecular mechanisms that allows these

plants to withstand extreme water loss. Examples of resurrection plants include

Sporobolus stapfianus, Craterostigma plantagineum and Tortula ruralis. The

resurrection grass S. stapfianus, the focus of this thesis, is a member of the

Sporobolus genus that contains 160 species. Sporobolus spp. are found in tropical and

sub-tropical areas (Watson 1992). S. stapfianus a desiccation-tolerant representative

of the genus is found in the Transvaal region of South Africa and is both drought and

salt tolerant (Gaff & Ellis 1974). S. stapfianus has the ability to reduce growth and

metabolism during drought whilst maintaining viable tissue. This ability to survive

without water is known as anabiosis. S. stapfianus can remain in anabiosis for an

extended period of time, and even if the cellular water content of the plant is reduced

by greater than 95%, the plant can resume full metabolic activity within 24 hours of

watering without significant damage to the overall viability of the plant (Gaff &

Loveys 1993). Drought causes significant crop losses in many regions of the world.

The study of resurrection plants may provide information that could be utilised to

improve drought tolerance in important food crops. The following literature review

will focus on abiotic stress signal perception and signalling, the roles of hormones

during abiotic stress, importance of senescence processes during abiotic stress and

molecular mechanisms of desiccation tolerance in S. stapfianus.

2

1.1 Drought stress signalling

The recognition of cellular stress in response to water deficit is the first step in the

chain of events that initiates the expression of drought tolerance genes. The initial

effects of water deficit include loss of turgor and changes in cell volume or membrane

area (reviewed in Chaves et al., 2003). Intracellular calcium acts as a secondary

messenger in the transduction of the extracellular signal to cellular targets (Harmon

1997). In many plants, cold and drought stress elevate levels of ABA which activates

the transcription of calcium dependent protein kinases (CDPK) which can activate

signalling pathways (Ludwig et al. 2004). Phospholipase D content has also been

shown to increase during dehydration stress in the resurrection plant C. plantagineum

which suggests that phospholipase D is an important factor in early response to

dehydration stress in resurrection plants (Frank et al. 2000). In the non-resurrection

plant A. thaliana, phospholipase D has been implicated in phosphatidic acid

accumulation during dehydration stress. These results indicate that phosphopliase D is

involved in the dehydration stress signal transduction pathway (Katagiri et al., 2001).

The root system in plants is proposed to be the primary sensor of dehydration stress,

however, few stress sensors have been identified in plants. In Arabidopsis thaliana

the ATHK1 (Arabidopsis thaliana histidine kinase 1) encodes a histidine kinase

primarily in the root tissues. ATHK1 acts as an osmosensor during cold and high salt

stress (Urao et al., 1999). In addition, RPK1 (receptor-like protein kinase 1) encodes

a receptor-like kinase, which is induced by dehydration, salt and cold stress and is

proposed to be an environmental stress transducer in A. thaliana. RPK1 is expressed

in roots, leaves, stems and flowers (Hong et al. 1997).

Other components involved in signal transduction pathways associated with the

dehydration response include phospholipase C, CDPKs, mitogen activated protein

(MAP) kinase, ribosomal S6 kinase and MAP kinase kinase (Bray 1997). These

proteins are involved in MAP kinase (MAPK) pathways (Wrzaczek & Hirt 2001).

The MAPK pathway generates a phosphorylation cascade which translocates MAPKs

into the nucleus. This alters gene expression by activation or inactivation of

transcription factors that regulate the expression of downstream target genes (Hirt

1997).

3

In many plants the hormone abscisic acid (ABA) is an important factor in mediating

the response to environmental stresses, such as drought, salt and cold stress (Bray

1997). ABA is also involved in seed maturation and germination (Skriver & Mundy

1990). Many studies have indicated that endogenous ABA levels increase as a result

of drought stress where ABA induces stomatal closure by activating potassium (K+),

calcium (Ca2+) and anion channels in guard cells (Raschke et al., 1988). Studies of

ABA mutants have helped in the elucidation of the role of ABA in mediating drought

responses in plants. Arabidopsis thaliana ABA-deficient and ABA-insensitive

mutants have been used to characterise the ABA response. These mutants have either

reduced levels of ABA (in the case of ABA deficient A. thaliana) or show abnormal

responses to ABA (ABA-insensitive A. thaliana) (reviewed in Tuteja 2007). The

biosynthesis, signalling and role of ABA are discussed later.

Methyl jasmonate has also been implicated in many stress responses in plants to

pathogens, insects, wounding, as well as being involved in developmental processes

(Pauw & Memelink 2005). In a study by Kim et al. (2009) methyl jasmonate has been

shown to accumulate in drought stressed tissue of rice, which stimulates the

production of ABA leading to grain yield loss. An increase in intracellular

concentration of jasmonic acid has also been shown in pear leaves during water stress,

which lead to an increase in the production of the osmoprotectant, betaine (Gao et al.

2004).

4

1.1.2 Drought responsive element (DRE) and ABA-response element (ABRE)

In Arabidopsis the expression of drought responsive genes has been classified as

either ABA-independent expression (involving the drought responsive element (DRE)

(Agarwal et al., 2006) or ABA-dependent (involving the ABA responsive element

(ABRE) (Hattori et al., 2002) (Figure 1.1). The DRE is a 9bp conserved sequence

(TACCGACAT) which is an essential cis-acting element for the regulation of ABA-

independent drought, cold and high salinity responsive genes. There are similar cis-

acting elements, namely the C-repeat (CRT) and a low temperature responsive

element, which both contain an A/GCCGAC motif that forms the core of the DRE

sequence. There are three known DRE binding proteins encoded in Arabidopsis

(CBF1, DREB1A and DREB2A), which all show sequence similarity in the conserved

DNA binding domain to that found in the ERF/AP2 protein families. Both CBF and

DREB1A genes are induced by cold and salt stress, and the DREB2A gene is induced

by drought stress (reviewed in Sakuma et al. 2006).

ABA-induced gene expression generally relies on the ABRE (ABA-responsive

element) cis-acting element. ABRE has a consensus sequence of RYACGTGGYR

(where R is a purine based nucleotide and Y is a pyrimidine based nucleotide) which

is defined by the core element of ACGT (Hattori et al., 2002). Most ABREs contain

this ACGT core, and similarly G-boxes also contain this core which is present in a

number of genes that are responsive to light, anaerobiosis, salicyclic acid and

jasmonic acid. Although there is this similarity between the ABRE and G-box core

sequence, ABA specific gene induction occurs through the use of an ABA response

complex when the plant has an increase in intracellular ABA concentration (Shen et

al. 1996).

While both the DRE and ABRE pathways in Arabidopsis can act independently of

each other, there is some cross talk between these signal transduction pathways

(Ishitani et al. 1997). For example the response element responsible for the initial

activation of the gene rd29A from Arabidopsis seems to be ABA independent and is

induced by high salinity, cold and dehydration stress via the DRE element, but also

5

contains an ABRE, which influences later stages of expression in response to ABA,

dehydration and high salt stress (Narusaka et al., 2003).

Figure 1.1: Cellular pathways leading to dehydration induced gene expression.

The ABA dependent and ABA independent pathways of gene expression, based on

Arabidopsis, are depicted here (from Bray 1997).

6

1.2 The involvement of ABA in abiotic stress responses

The phytohormone ABA co-ordinates various abiotic stress signalling pathways (such

as drought, cold and salt stress) by activating genes involved in adaptation responses,

osmotic adjustment, root hydraulic conductivity, root/shoot growth and ion

compartmentalisation (Ruggiero et al., 2004). In addition, ABA reduces transpiration

rate (Popisilova et al., 2009), which helps reduce water loss. The biosynthesis of ABA

during stress is primarily localised to the vascular tissues, including guard cells

(Kuromori et al., 2010). ABA then undergoes intercellular transport via two ABC

(ATP-binding cassette) transporters (Kang et al., 2010), as well as nitrate transporters

into neighbouring tissues (Kanno et al., 2012).

The expression of many genes that respond to cold-, salinity-, and drought-stress are

up-regulated by exogenous ABA application, These overlapping gene expression

patterns suggests that various stress signals and ABA signalling share common

elements and cross-talk in order to maintain cellular homeostasis (Shinozaki &

Yamaguchi-Shinozaki 2000). Studies of mutant Arabidopsis deficient in ABA (aba1,

aba2 and aba3) revealed that although the mutant’s growth is comparable to wild-

type under optimal conditions, under prolonged drought conditions (Koornneef et al.,

1998) and prolonged salinity conditions (Xiong et al., 2002) the mutants display poor

growth. ABA also has a role in the establishment of freezing-tolerance in Arabidopsis,

with freezing-tolerance requiring both ABA-dependent and ABA-independent

processes (Mantyla et al., 1995). Studies have indicated that many components of

ABA-dependent and independent pathways cross-talk and converge on signalling

pathways in which calcium acts as a key mediator (Chinnusamy et al., 2004).

Examples of the regulation of gene expression through both ABA-dependent and

independent signalling pathways occur in the regulation of Arabidopsis RD29A

(Yamaguchi-Shinozaki & Shinozaki 1993) and proline accumulation (Mahajan et al.,

2005).

7

1.3 ABA biosynthesis and signalling

The phytohormone ABA is a key plant hormone in the regulation of responses to

dehydration stress as well as having roles in seed dormancy, germination, plant

transpiration and seedling growth (Urano et al., 2009). ABA biosynthesis and

degradation is influenced by environmental and developmental factors and determines

the level of cellular ABA (Cutler & Krochko 1999). Biosynthesis of ABA involves

one of two possible precursors: farnesyl pyrophosphate, a C15 compound, or β-

carotene, a C40 carotenoid. Depending on the precursor used, the ABA biosynthesis

pathway is considered either direct (using farnesyl pyrophosphate precursor) or

indirect (using the β-carotene precursor). Studies of ABA deficient mutants have

suggested that the indirect pathway is the main pathway used by plants to synthesise

ABA. The regulation of ABA biosynthesis is complex, and many factors such as post-

transcriptional regulation and alternative pathways in the final steps of ABA synthesis

contribute towards the ABA hormone end product (reviewed in Seo & Koshiba 2002).

ABA signalling involves several receptors, some of which are bound to the plasma

membrane and others which are intracellular. Some of the reports of ABA interaction

with various cellular components remain controversial (McCourt & Creelman 2008).

ABA receptors that have been identified are the ChlH (Mr-Chelatase H subunit), G-

protein coupled, and PYR/PYL/RCAR (PYRABACTIN RESISTANCE 1/PYR-

LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS) receptors. In a

study by Razem et al. (2006), the RNA-binding protein FCA, binds to ABA and

controls flowering time and RNA metabolism. However, this was refuted by Risk et

al. (2008) where it was found that FCA does not bind ABA. Studies in broadbean

suggested that an ABA-binding protein (ABAR) was involved in stomatal signalling

and codes for ChlH (Zhang et al., 2002). In Arabidopsis, the ChlH binds specifically

to ABA and is expressed in green and non-green tissues suggesting that it may be

involved in ABA perception on the whole plant level. Furthermore, ChlH acts as a

positive regulator of seed germination, growth and stomatal movement (Shen et al.,

2006). However, in, barley, ChlH was not found to be an ABA receptor (Muller and Hansson

2009) and hence the finding that ChlH acts as an ABA receptor is controversial, and requires

further investigation.

8

G-protein-coupled receptors (GPCR) are heterodimeric proteins consisting of a α, β

and γ subunits which link ligand perception by GPCRs with downstream effectors,

GPCRs have been associated with many phytohormone signalling pathways as well as

the control of cell division (Assmann 2002). Two GPCR ABA receptors have been

isolated in Arabidopsis: GCR1 (G-protein coupled receptor 1) (Chen et al., 2004) and

GCR2 (G-protein coupled receptor 2) (Liu et al., 2007). Interestingly, GCR1 has

shown pleitropic effects in phytohormone signalling, where it is also associated with

the signalling of brassinosteriods and gibberellins (Chen et al., 2004). The work

completed by Liu et al. (2007) on GCR2 has been questioned by researchers

(Illingworth et al., 2008, Gao et al., 2007) where it was noted that GCR2 had no role

in ABA control of seed germination in Arabidopsis. The characterization and isolation of

GTG1 and GTG2 in Arabidopsis has shown than these proteins may act as receptors of ABA

(Pandey et al., 2009). However an independent study has shown that these proteins do not

bind ABA (Risk et al., 2009), leading to controversy as to the function of these receptors in

ABA signaling. Phenotypes of gtg1 and gtg2 mutant Arabidopsis also show conflicting results

where Pandey et al. (2009) show altered ABA sensitivity, whereas when the same mutants

were generated independently, gtg1 and gtg2 Arabidopsis show normal ABA sensitivity

(Jaffe et al., 2012). Despite this, it is still likely that GTG1 and GTG2 have some role in ABA

signaling as GTG1 and GTG2 mutant plants display altered ABA sensitivity (Pandey et al.,

2009), and more recent proteomics work shows GTG proteins have a significant role in ABA

responses (Alvarez et al., 2013)

The structure of the ABA perception and signalling network has recently been

elucidated by the discovery of the PYR/PYL/RCAR soluble ABA receptor and the

downstream protein phosphorylation system (Ma et al., 2009, Umezawa et al., 2009,

Park et al., 2009, Vlad et al., 2009). By impairment of ABA perception through

PYR/PYL/RCAR pathway in Arabidopsis vegetative growth and seed production is

reduced, stomata remain open and the phenotype displayed is ABA-insensitive

despite other mechanisms of ABA perception remaining functional (Gonzalez-

Guzman et al., 2012). PYR/PYL/RCAR ABA receptor appears to directly regulate

PP2C phosphatases, which directly regulate SnRK2 kinases (reviewed in Cutler et al.,

2010) suggesting that the PYR/PYL/RCAR receptor is the primary cellular

perception/signal in the ABA perception/signalling pathway (Figure 1.2).

9

Figure 1.2: Summary of ABA signalling factors when ABA is absent (left) and when ABA is

present (right) (from Cutler et al., 2010).

10

1.4 ABA in vegetative development

ABA is regarded as an inhibitor of shoot growth as exogenous ABA application can

result in reduced stem and leaf growth in wheat (Raikhel et al., 1986) and grapevine

(Zhang et al., 2001). However, some studies have shown that A. thaliana, deficient in

both biosynthesis and/or sensitivity to ABA, produces plants which are stunted in

comparison to the wild type (Meurs et al., 1992). This suggests that ABA may have

both growth-promoting and growth-inhibitory roles in plant development. Tomato

mutants deficient in ABA, flacca, notabilis and sitiens, display reduced stem and leaf

growth, which can be restored by the application of exogenous ABA, by up to 75% in

some cases (Nagel et al., 1994, Neill et al., 1986, Sharp et al., 2000). The study by

Sharp et al. (2000) suggests that the reduction in shoot growth in flacca is partially

attributed to ethylene, and flacca mutants have previously been shown to have an

increased rate of ethylene production (Tal et al., 1979). By inhibiting ethylene

production with silver thiosulfate in flacca mutants, shoot growth was partially

restored (Sharp et al., 2000). These studies suggest that shoot and leaf growth

reduction in ABA-deficient mutants may be associated with endogenous ethylene

levels, which are influenced by the reduction in endogenous ABA. Interestingly, the

exogenous application of GA3 has been shown to reverse the inhibition of shoot

growth caused by ABA in rice (Tsai et al., 1997), and research has since identified

that ABA and gibberellins act antagonistically in many aspects of plant development

(Woodger et al., 2010), which suggests that the involvement of ABA in shoot and leaf

growth may be controlled in a secondary manner in vegetative development.

11

1.5 ABA in seed development

Seed maturation, and the establishment and maintenance of seed dormancy involve

the phytohormone ABA. Endogenous ABA levels peak during the mid-stages of seed

development (Audran et al., 1998) and is suggested to induce the dormant state in the

later stages of seed development to prevent precocious germination and establish

desiccation tolerance (Seo & Koshiba 2002). Double-mutant Arabidopsis, both

deficient in ABA (aba) and ABA insensitive (abi3), produce seed that fails to

desiccate, remains green, and loses viability upon drying. These mutants have been

shown to accumulate low stores of carbohydrates and storage proteins in the seed,

suggesting a role of ABA in the accumulation of storage carbohydrates and proteins

required for desiccation tolerance (Meurs et al., 1992). Embryonic gene expression

and seed sensitivity to ABA have been said to be controlled by the B3-type (ABI3)

and AP2/ERF-type (ABI4) transcription factors in Arabidopsis. Analysis of abi3

Arabidopsis mutants have shown that these seeds have a reduced dormancy and

display vivipary (reviewed in Finkelstein 2002). Similarly, in maize, VIVIPAROUS 1

(VP1), homologous to ABI3 in Arabidopsis, binds to the Sph/RY elements in the

promoters of seed maturation genes (Luerssen et al., 1998) suggesting a primary role

in seed development. AB14 has also been implicated in sugar signalling, and its

expression increases in response to glucose. ABI4 mediates ABA- and sugar-inducible

gene expression by binding to cis-acting elements (Bossi et al., 2009; Reeves et al.,

2011) suggesting its involvement in the accumulation of carbohydrate stores in seed

tissue essential for establishing desiccation tolerance.

12

1.6 ABA in germination

Seed germination is regulated by a multitude of cues including light, water,

temperature and endogenous hormonal signals (Finch-Savage & Leubner-Metzger

2006). The transition from seed dormancy to germination is largely controlled by the

balance of the antagonistic hormones ABA and GA, which is controlled by the

reciprocal regulation of their metabolic genes (Seo et al., 2006). ABA catabolism is

required to break dormancy of imbibed seeds and only some of the key enzymes in

ABA-catabolism pathways in Arabidopsis have been discovered to date (Holman et

al., 2009, Kushiro et al., 2004). These key enzymes are the ABA 8’-hydroxylases

CYP707A1, CYP707A2 and CYP707A3. CYP707A1 is expressed during mid

maturation, and CYP707A2 during late maturation and germination. CYP707A3,

along with CYP707A1, have been shown to be involved in post-germination growth.

These results suggest that CYP707A1 has a key role in determining ABA content in

dry seeds, and CYP707A2 has a key role in the rapid catabolism of ABA during early

seed imbibition (Okamoto et al., 2006). More recently, it has been shown that the N-

end rule pathway, which targets specific protein degradation through the amino-

terminal residue of protein substrates, is involved in germination. Specifically, it has

been proposed that in Arabidopsis, the N-end rule pathway inactivates unidentified

components of ABA signalling to remove ABA sensitivity (Holman et al., 2009).

13

1.7 Regulation of plant growth by strigolactone

Recent research has shown that a novel class of carotenoid-derived hormones called

strigolactones (SL), exuded from plant roots can inhibit shoot branching in

conjunction with auxin. Auxin is a key plant hormone involved in apical dominance,

and overall plant growth and development (reviewed in Zhao 2010). In addition, SL

compounds in root exudates trigger the germination of some parasitic weeds, such as

Striga and Orobanche (Cook et al., 1972), as well as promoting arbuscular

mycorrhizal fungi hyphal branching (Akiyama et al., 2006). During plant growth, SL

moves upward in plant stems acting as a long distance messenger for auxin to control

bud outgrowth (reviewed in Dun et al., 2009). The carotenoid cleavage dioxygenase 7

(CCD7) and CCD8 enzymes, also known as MAX3 (More Axillary Branching 3) and

MAX4, respectively, facilitate the conversion of β-carotene into an apocarotenoid

after producing an unknown intermediate within the chloroplast. The cytochrome

p450 MAX1 then converts the apocarotenoid into another unknown intermediate to

ultimately produce strigolactone (Booker et al., 2005). Mutant Arabidopsis with

reduced MAX3 and MAX4 expression present phenotypes of reduced apical

dominance and increased shoot branching (Auldridge et al., 2006). The pea ccd7 and

ccd8 mutants have been shown to have root exudates deficient in SL in comparison to

wild-type (Gomez-Roldan et al., 2008). In rice plants, the iron-containing protein

DWARF27 (D27) is required for SL biosynthesis. D27 is localised to the chloroplast,

and is expressed in root and shoot vascular cells. The phenotype of d27 mutants show

a reduced SL content in root exudates and have enhanced bud outgrowth. Double

mutants of d27 and d10 (a CCD8 mutant) display the same phenotype as d27 and d10

single mutants suggesting that these genes act in the same pathway (Lin et al., 2009).

The biological and biochemical function of D27 has not been elucidated.

SL levels have been shown to be affected by auxin signalling through the phenotype

of bodenlos (bdl) a dominant mutant of IAA12, an Aux/IAA family protein from

Arabidopsis, which are deficient in SLs (Hayward et al., 2009). Grafting experiments

have shown that perception and feedback of SL levels in different species, has been

suggested to involve MAX2 in Arabidopsis (Stirnberg et al., 2002), RMS4

(RAMOSUS4) in Pea (Beveridge et al., 1996) and D14 in Rice (Arite et al., 2009) as a

14

signalling component. These genes encode F-box proteins, which are involved in

ubiquitin-mediated proteolysis (Somers & Fujiwara 2009). This suggests that the

degradation of negative regulators may be a key step in the SL signalling pathway

(Beveridge & Kyozuka 2010).

Brassinoteriods, a class of hormones involved in the promotion of cell expansion and

elongation, vascular differentiation and acceleration of senescence (reviewed in Zhu

et al., 2013), have recently been suggested to act with SLs to regulate developmental

processes. The brassinosteroid transcriptional effector, BES1 (brassinazole-resistant

2), has been shown to act as a substrate for MAX2. In the presence of SL, AtD14

activates the degradation of BES1 by MAX2 to inhibit shoot branching (Wang et al.,

2013).

In addition to the effects on shoot branching, promoting parasitic weed germination

and promoting arbuscular mychorizal fungi hyphal branching mentioned previously,

SL also effects plant senescence. Plants with defects in the SL biosynthesis exhibit

delayed senescence that is thought to be mediated by reduced activity of

ORE9/MAX2 (Snowden et al., 2005). Hormonally-induced senescence utilising

ABA, ethylene or MeJA, as well as age-induced senescence was delayed in

Arabidopsis ORE9 mutants. This study suggests that ORE9 limits leaf longevity by

removing proteins that delay leaf senescence via ubiquitin-dependent proteolysis

(Woo et al., 2001). ORE9 has also been shown to repress hypocotyl elongation in the

light and is also involved in oxidative stress and drought responses (Woo et al., 2001;

Stirnberg et al. 2002; Woo et al. 2004; Tang et al. 2005). More recently, the

Arabidopsis MAX2 mutant was shown to be hypersensitive to drought, salinity, and

glucose, while other SL biosynthesis mutants (max1, max3 and max4) did not display

any responses to these stressors. Hypersensitivity to ABA, another carotenoid-derived

plant hormone (section 1.4) was also displayed by max2 (Bu et al., 2014). This

suggests that MAX2 may interact in a stimulus specific manner to provide pivotal

regulation and feedback for the synthesis of carotenoids and carotenoid-derived

compounds.

15

1.8 Senescence

Senescence is a developmentally regulated process of programmed cell death and

occurs during the final stages of leaf development. Senescence is thought to have

evolved to remobilise nutrients from older leaves to younger, actively growing areas

of the plant (Nam 1997). Senescence can be divided into two age-related processes:

the first being mitotic senescence, associated with the inability of the cell to undergo

further division upon aging, or post-mitotic senescence, which is associated with the

degenerative process of cellular dismantling and nutrient remobilisation occurring

after organ maturation (Lim et al., 2003). Genes up-regulated during senescence have

been referred to as senescence associated genes (SAGs), a term coined by Lohman et

al. (1994), and their expression patterns during leaf aging and roles during senescence

have been reviewed in detail by Buchanan-Wollaston (1997). Cellular respiration is

maintained during senescence, as is transcriptional machinery, which enables the

plant to transcribe senescence-associated genes (SAGs).

Many SAGs, that are thought to be imperative for the successful implementation of

senescence, have been identified in Arabidopsis, maize and tomato (Guo & Gan

2005). However, it has been noted that several SAGs are transcribed at low levels in

non-senescent tissue, and increase in expression as the leaf ages (Weaver et al., 1998).

As well as being developmentally-regulated process, leaf senescence can also be

induced by environmental signals such as salinity, drought and herbivory (Lim et al.,

2003). During senescence of Brassica napus, genes encoding two types of protease,

glutamine synthase, ATP sulphurylase, two types of metallothionein, ferritin, catalase

and an antifungal protein (Buchanan-Wollaston & Ainsworth 1997) as well as

Chitinase, and PR1 (pathogenesis-related 1) are up-regulated (Guerrero et al., 1990).

The action of proteases during senescence has been suggested to be involved in

senescence-related protein degradation (Lohman et al., 1994), which is one of the first

cellular responses to senescence induction. The remobilisation of nutrients may be

attributed to the action of glutamine synthase and metallothioneins, although

metallothioeneins as well as catalase may also provide a protective role by scavenging

free ions to prevent redox damage (Buchanan-Wollaston & Ainsworth 1997). The

16

potential roles of antifungal and pathogenesis related proteins during senescence are

not understood.

The regulatory genes thought to be important in control of leaf senescence include

transcription factors and several signal perception and transduction proteins (reviewed

by Lim & Nam 2005). WRKY53, a MYB protein and a zinc finger protein are

thought to be involved in the early stages of senescence induction (Buchanan-

Wollaston et al., 2003). Also shown to be expressed in the early stages of leaf

senescence is the senescence-associated receptor kinase (SARK) (Hajouj et al., 2000)

that functions to perceive and transduce leaf senescence signals. The activation of

eukaryotic translation factor 5A (eIF-5A) might also be required to facilitate the

translation of mRNA subsets needed for senescence once activated post-

translationally by deoxyhypusine synthase (DHS). These proteins are highly

expressed during the early stages of senescence in Arabidopsis (Thompson et al.,

2004).

1.8.1 Hormonal involvement in senescence processes

Studies have shown that several plant hormones can act differentially to delay or

induce senescence (Weidhase et al., 1987, Zeevaart & Creelman 1988, Abeles et al.,

1988). Hormone-induced senescence and age-related senescence may share a

common senescence pathway, this was demonstrated by the affects of the mutation of

the ORE9 protein. Senescence induced by ABA, ethylene and MeJA-as well as age-

induced senescence was delayed in Arabidopsis when ORE9 was mutated. This study

suggests that ORE9 limits leaf longevity by removing proteins that delay leaf

senescence through ubiquitin-dependent proteolysis (Woo et al., 2001).

Cytokinin, a known inhibitor of senescence has been a key hormone in the subject of

senescence research. The increased presence of cytokinins during this process inhibits

chlorophyll and photosynthetic tissue degradation (Richmond & Lang 1957). It has

been concluded that not only does the production of cytokinins influence senescence,

but also sugars and light have a significant effect on the decline of photosynthetic

17

metabolism (Wingler et al., 1998). The inhibition of leaf senescence has been

accomplished by engineering increased production of cytokinin in senescing tissues

(Smart et al., 1991, Gan and Amasino 1995). Increasing cytokinin levels in tobacco

during senescence was performed by the SAG12 promoter driving expression of IPT

(isopentyltransferase), a rate limiting enzyme in cytokinin synthesis. This resulted in

a delay of senescence-associated declines in nitrogen, protein, photosynthetic rate and

RubisCO (Jordi et al., 2000). Although cytokinins have been shown to play a pivotal

role in the progression of senescence, it has been concluded that it does not trigger

senescence onset. This was shown in a study by Werner et al. (2003), where

cytokinin-deficient Arabidopsis displayed delayed rather than accelerated senescence.

The expression of specific SAGs has been shown to differ depending on the

conditions inducing senescence. Hormone treatments including ABA and ethylene,

and dark-induced senescence increased the expression of a number of SAGs but not to

the same levels as occurs during senescence, cytokinin treatment reduced the

expression of SAGs (Weaver et al., 1998). Interestingly, dehydration produced a

quick response, with half of the SAGs tested being up-regulated after three hours of

dehydration. The results of this study suggests that the expression of some SAGs may

be associated with a general response to stress induced by darkness and drought

(Weaver et al., 1998). This research also provides some of the first data that links age-

related SAG gene expression with drought.

1.8.2 Drought induced senescence

Leaf senescence has been associated with increased plant survival under drought

stress. Remobilisation of nutrients to younger leaves and reduced canopy size may

allow for survival during drought (reviewed by Munné-Bosch & Alegre 2004). The

information on drought-induced senescence in non-resurrection plants revolves

around three key mechanisms: the accumulation of sugars, endogenous cytokinin

levels and endogenous ABA levels. The focus of research into the inhibition of

drought-induced senescence has for the most part been directed at increasing

production of cytokinins. Agriculturally, avoiding senescence during drought is a

18

valuable trait, which is at the forefront of crop science research worldwide. Increasing

endogenous levels of ABA within tissues has been shown to enhance drought

tolerance in tomato (Thompson et al., 2007). It has been shown that enhancing

cytokinin synthesis reduced the drought-induced ABA responses in tobacco.

Cytokinin synthesis during senescence was increased in these plants by expressing the

rate-limiting enzyme for cytokinin synthesis IPT using the senescence associated

receptor kinase (SARK) promoter (PSARK-IPT). This increased cytokinin production,

prevented the degradation of protein complexes involved in photosynthesis (Rivero et

al., 2010). Furthermore, the increased cytokinin synthesis during drought stress

produces a plant with enhanced drought tolerance. This increased drought tolerance

displayed in tobacco plants allows the plant to survive water deprivation for fifteen

days while retaining photosynthetic and water content in comparison to the wild type

plants, which did not survive a fifteen day period of water-deprivation (Rivero et al.,

2007). Abiotic stress often results in sugar accumulation, which is generally

considered to provide cellular protection by aiding in the stabilisation of membranes

and proteins. Senescence has been shown to be delayed in cut Gladiolus spikes by the

immersion of spike in trehalose, which increased the vase-life by two days (Otsubo &

Iwaya-Inoue 2000). This study suggested that trehalose delays water-loss in cut

Gladiolus spikes. Sugar accumulation has also been suggested to be involved in sugar

or redox signalling (Hare et al., 1998). In addition, ABA has been associated with

sugar signalling by the observation that Arabidopsis mutants deficient in ABA

synthesis or ABA signalling show no growth arrest when exposed to high sugar

concentrations (reviewed by Rolland et al., 2006). A link between ABA, senescence

and drought stress has been suggested (Yang et al., 2003) and is thought to be

involved in the activation of the stress-induced senescence pathway.

19

1.8.3 Role of endoplasmic reticulum stress during senescence processes

Endoplasmic reticulum (ER) stress has been linked with osmotic stress-induced cell

death (Alves et al., 2011), where integrated signals from both ER stress and osmotic

stress can activate cell death. In all eukaryote cells, including plants, ER stress

triggers the unfolded protein response (UPR). The ER located molecular chaperone

binding protein (BiP) assists in folding newly synthesized proteins in the ER lumen

and also acts as a sensor of ER stress and stress-related signal generation (Reis et al.,

2011). Environmental stress can lead to an accumulation of unfolded proteins in the

ER. It is thought that a high level of unfolded proteins sequesters BiP, releasing and

activating several ER associated receptors, such as PERK (protein kinase RNA-like

ER kinase in animals), IRE1 (inositol-requiring protein-1) and ATF6 (activating

transcription factor-6). Activation of these receptors triggers the UPR, leading to a

down-regulation of translation and an increase in ER protein-folding components and

protein processing components, including BiP (Reis and Fontes 2012). Several key

components of the plant UPR have been identified although the precise roles of some

of these components remain to be clarified (Fanata et al., 2013). Whilst no homologue

of PERK has been identified in plants, GCN2 may play a similar role in

phosphorylating eIF2α to inhibit protein synthesis (Zhang et al., 2003). bZIP60 acts as

a plant homologue of ATF6 and up-regulates the BiP chaperone as well as SKP1, a

component of the SCF-type E3 ubiquitin ligase complex, that degrades misfolded

proteins via the 26S proteasome (Ye et al., 2011). When the stress is severe enough to

prevent restoration of ER homeostasis, a signal is activated that involves activation of

asparagine-rich protein (NRP) genes (Irsigler et al., 2007) and other downstream

components that result in cell death (Wang et al., 2008, Costa et al., 2008). NRP

expression causes chlorophyll loss, ethylene production and senescence (Reis and

Fontes 2012). The transcription factor, Early Responsive to Dehydration 15,

(ERD15), (Kiyosue et al., 1994) has been identified in several plant species and in

soybean has been shown to bind to the NRP promoter and drive expression of the

NRP-B gene and is proposed to link osmotic stress and ER stress to cell death (Alves

et al., 2011). There may be differences in the roles of ERD15 from Arabidopsis and

soybean. Both the Arabidopsis and soybean ERD15 protein contain PAM2 (Poly-A

20

binding protein (PABP) associated domain 2) domains. PAM2 domains bind PABP

and may be involved in regulating mRNA translation. ERD15 has been proposed to

attenuate ABA signalling in A. thaliana lines, with silenced ERD15 expression

showing hypersensitivity to the exogenous application of ABA during seed

germination, and the over-expression of ERD15 resulting in tolerance to exogenous

ABA (Kariola et al., 2006). In Arabidopsis ERD15 does not appear to directly drive

NRP-B expression but may act through attenuation of the ABA antagonistic effect on

SA signalling, since the SA hypersensitive response appears to induce NRP

expression (Ludwig and Tenhaken 2001).

1.9 Anhydrobiotic organisms

The viability and continuation of an organism’s life is generally reliant on water

availability, however, anhydrobiotic organisms have evolved to tolerate desiccation

and remain viable in extremely dry conditions, with some species capable of

withstanding up to when up to 99% depletion of total water content (Crowe et al.,

1992). Anhydrobiotic organisms are able to suspend their cellular processes and

remain in a state of suspended animation (anhydrobiosis), which can last between 3

months to two years in angiosperm resurrection plants, and in other organisms up to

several decades in some instances. Once the water source is replenished, the organism

can restore full metabolism (Goyal et al. 2005). There are a variety of anhydrobiotic

organisms across all kingdoms, including bacteria, fungi, animals and plants. Some

examples of anhydrobiotic organisms are brine shrimp embryos, the baker’s yeast

Saccharomyces cerevisiae, nematodes, tardigrades, bdelliod rotifers, seeds from most

higher plants (Crowe et al., 1992), as well as ‘resurrection plants’ such as

Craterostigma plantagineum (Bartels et al., 1990) Xerophyta viscosa (Mundree &

Farrant 2000) and Sporobolus stapfianus (Gaff & Ellis 1974). Most desiccation

tolerant organisms can enter an anhydrobiotic state at any stage in their life cycle

(reviewed in Goyal et al. 2002).

21

1.9.1 Resurrection plants

Approximately 330 resurrection plants have been identified among the vascular plant

classes, with gymnosperms the only vascular plant category which does not have a

documented desiccation-tolerant representative (Alpert 2000). Resurrection plants

have evolved desiccation tolerance mechanisms so they can live in areas of stressful

environmental conditions where soil is scarce and rainfall is sporadic (Gaff 1972). C.

plantagineum as well as S. stapfianus are classified as modified desiccation-tolerant

plants as desiccation-tolerance is inducible rather than constitutively expressed

(Oliver et al. 1997). In order for many resurrection plants to survive water loss and

enter a state of anhydrobiosis the water loss to the plant must be slow enough in order

for genes associated with desiccation tolerance to be activated. Some primitive

desiccation-tolerant organisms, including some mosses, can survive rapid dehydration

as the protective genes appear to be constitutively expressed (reviewed in Oliver et

al., 2005, Bartels & Salamini 2001).

In non-resurrection plants, the pollen and seeds of some species are the only tissues

capable of instituting the desiccation-tolerance pathway(s). Many of the genes

associated with desiccation of seeds are similar to those used in vegetative tissues of

resurrection plants for desiccation tolerance. This has lead to the hypothesis that

desiccation tolerance in vegetative tissue is under the control of regulatory genes

associated with desiccation tolerance in pollen and seeds that are ectopically

expressed in vegetative tissue in resurrection plants (reviewed in Ingram & Bartels

1996).

22

1.10 Molecular response to water deficit in plants

Plants undergo morphological and physiological changes in response to water deficit.

Several approaches have been used to investigate the molecular mechanisms involved

in plant responses to water deficit. By manipulating the environmental conditions of

these plants as well as manipulating them genetically, many discoveries regarding

water stress response in plants have been made, for the most part, in desiccation

sensitive A. thaliana and Oryza sativa (rice). This has allowed the identification of

many drought responsive genes and their protective functions. Comparisons of

physiological responses of water deficit in desiccation tolerant species suggest

responses are likely to be similar in most plants during early stages of dehydration

(Bartels & Salamini 2001).

The ability of resurrection plants to survive in the state of anhydrobiosis relies on

several cell stabilizing factors including changes in cellular concentrations of

particular sugars, proteins and lipids. Viability in an anhydrobiotic state in some

species also relies on the ability of the cell walls to fold (Crowe et al., 2002; Farrant

2000), or for the cell walls to be constitutively plastic (Moore et al., 2013). The

protection of cellular components during dehydration is enhanced by the up regulation

of non-reducing sugars, with the types of sugar varying between desiccation-tolerant

species. The sugar that accumulates in “higher” resurrection plants such as C.

plantagineum and S. stapfianus is sucrose (Bartels & Salamini 2001, Ghasempour et

al., 1998) with trehalose preferred by “lower” resurrection plants and small

anhydrobiotic organisms such as yeast and brine shrimp embryos (Crowe 2002). At

low cellular water content disaccharides will often form glasses, or vitrify, preventing

cellular damage through inhibiting molecule diffusion as well as preventing

membrane fusion and protein degradation (Crowe et al., 2002). During vitrification an

increase in cellular lipid content occurs in conjunction with the accumulation of

sugars. The glass-like matrix within the cell is formed by interaction of phosphate

groups in the phospholipids forming a hydrogen bond with the hydroxyl group within

the trehalose or sucrose molecule. This conjunction of lipid and sugar allows for the

decrease in van der Waal’s interactions between acyl chains in the phospholipid

(Crowe 2002). This ultimately leads to stability of membranes and protein structure,

23

as well as preventing crystallisation of embedded chemical compounds (Hoekstra et

al., 2001).

Although sugars are predominantly involved in vitrification, proteins may also be

involved in the formation of a glassy state during water deficit. In Arabidopsis seeds

it was found that the molecular density suggests that the presence of proteins help

form sugar glasses to confer desiccation tolerance (Wolkers et al., 1998). Proteins that

have been shown to be involved in intracellular glasses include some late

embryogenesis abundant (LEA) proteins (section 1.11.1), which are highly abundant

during the maturation of seeds and pollen. Along with sugars it is hypothesised that

they work together in forming a glassy state (Buitink and Leprince 2008).

The accumulation of sugars and the formation of glasses within tissue undergoing

dehydration is important in the survival of desiccation, but it is not sufficient to render

tissue desiccation-tolerant. The glass content is unable to completely decrease

molecular mobility within the organism during the desiccation period to sufficiently

prevent cellular damage by loss of protein structure and membrane degradation

(Crowe et al., 2002). Membrane damage allows solutes to escape the tissue and

decreases cellular viability. Although the ability of desiccation-tolerant tissues to form

glasses during water deficit is characteristic of desiccation tolerance, desiccation

sensitivity of tissues is not related to an absence of a glassy state during water deficit

(Buitink et al., 1996).

Other osmolytes that increase cellular protection during dehydration include glycine,

betaine and proline that accumulate to reduce efflux of water from the cells. These

osmolytes have been proposed to protect cells during drought stress through ion

sequestration, increased water retention and the stabilisation of cytoplasmic

constituents (Hara et al., 1998). It has been suggested that the presence of osmolytes

and oligosaccharides in desiccating tissue creates a preferential exclusion, where the

osmolytes are excluded from the protein surface, keeping the proteins preferentially

hydrated while the cell undergoes desiccation (Hoekstra et al., 2001). Furthermore

the accumulation of arbutin, a glycosolated hydroquinone, results in extremely high

concentrations in particular resurrection plants that are able to survive in a desiccated

state for extended periods of time. In a study conducted by Oliver et al., (1996)

24

arbutin has structural similarity to the PLA2 (phospholipase A2) inhibitor p-

bromophenacyl bromide and it is thought that arbutin may inhibit PLA2 in dehydrated

cells through steric exclusion of the enzyme from its substrate. This action would

prevent the break down of fatty acids, reducing cellular damage associated with

dehydration. High concentrations of arbutin are observed in dehydrated tissue,

however, arbutin has no effect on PLA2 inhibition in fully hydrated conditions. Some

evidence suggests that arbutin interacts with the phospholipid bilayer by the insertion

of its phenol moiety into the hydrophobic region of the bilayer (Oliver et al., 1996;

Hincha et al., 1999).

1.11.1 Late Embryogenesis Abundant (LEA) proteins

The induction of a large set of specific proteins are known to reduce and prevent

damage to cells at low water contents (Goyal et al. 2005). These protective proteins

include chaperones and proton-regulated ATPases and some proteins with enzymatic

functions such as aldehyde dehydrogenase, heat shock proteins and late

embryogenesis abundant (LEA) proteins. LEA proteins a major representative of

protective proteins and can be divided into six functional classes based on sequence

domain motif conservation (reviewed in Shih et al., 2008) LEA proteins are generally

hydrophilic, and have an amino acid composition mostly devoid of cysteine and

tryptophan. In many cases the function of LEA proteins is not known (Bartels &

Salamini 2001). LEA proteins are expressed in both resurrection plants and non-

desiccation-tolerant plants under dehydration stress, and are found in most cell types,

accumulating for the most part in plastids, mitochondria and cytoplasm (reviewed in

Tunnacliffe & Wise 2007). The distribution of LEA protein correlates with a

protective function where the LEA proteins may form anchors in a structural network,

which stabilizes cytoplasmic components during dehydration. Grelet et al., (2005)

showed that a group 3 LEA that was localised to the mitochondrial matrix was able to

prevent damage to mitochondrial proteins in vitro during desiccation.

In further studies, transgenic plants and yeast have been used to investigate the effects

of LEA protein’s protective role against cellular stress (Xu et al., 1996; Mowla et al.,

25

2006); Wang et al., 2003). It has been suggested that LEA proteins exert their

protective role by replacing water to maintain hydration of proteins and other cellular

components. LEA proteins may act in conjunction with carbohydrates to perform this

role and may also bind to ions thereby decreasing ion concentration in dehydrated

cells. Biochemical evidence is lacking for these hypotheses (Bartels 2005).

1.11.2 Dehydrins and rehydrins

One particular group of LEA proteins called dehydrins, undergo post-translational

modification in the form of phosphorylation. This phosphorylation may increase

hydrophilicity of dehydrins and allow the cell to dehydrate slowly (Bartels 2005).

Dehydrins can have one or more lysine-rich motifs that form an amphiphilic alpha-

helical structure. This structure may interact with other macromolecules in the cell.

Drought, salt and cold stresses as well as exogenous application of ABA (abscisic

acid) all result in the accumulation of dehydrins in plants (Hara et al., 2004). In

particular, the accumulation of dehydrins in cowpea has been directly linked to cold

stress (Ismail et al., 1999). In addition, dehydrin-like proteins from spinach and citrus

have been shown to increase cold tolerance in transgenic tobacco. Citrus dehydrin has

been shown to possess free radical scavenging activity, which prevents harmful

oxidation of cellular components in transgenic plants (Hara et al., 2004). In a recent

study, the expression patterns of three dehydrin genes, QrDhn1, QrDhn2 and QrDhn3

were examined in zygotic and somatic embryos of the recalcitrant Oak Quercus

robur. In somatic embryos the expression of QrDhn1 was up-regulated during

osmotic stress and chilling, while QrDhn2 and QrDhn3 were expressed only in

somatic embryos during osmotic stress and in leaves exposed to desiccation stress

(Sunderlíková et al., 2009).

The desiccation-tolerant moss Tortula ruralis, expresses a group of proteins called

rehydrins, which are up-regulated during rehydration of the moss following

desiccation (Velten & Oliver 2001). The activation of rehydrin genes in T. ruralis is

dependent on the speed of desiccation. If the moss is desiccated from a fully hydrated

state within 1 hour, the accumulation of rehydrin transcripts become highly abundant

26

during the first 2 hours of rehydration. If T. ruralis undergoes desiccation over a

period of 3 to 6 hours, the accumulation of rehydrins occurs during the drying phase.

One particular rehydrin, Tr288, which may have a role in reducing and repairing

cellular damage, was found to be abundant in the mRNP fraction of the gametophyte

undergoing slow dehydration, and during rehydration, suggesting that Tr288 may be

stored in mRNP for use during rehydration (Velten & Oliver 2001).

1.12 Physiological changes during dehydration and rehydration in the resurrection plant S. stapfianus

Although a vast amount of information on water-deficit tolerance has been gained

from the study of Arabidopsis and other crop plants, the vegetative tissues of these

plants are desiccation-sensitive, consequently studies involving resurrection plants

exposed to drought stress may provide information important for enhancing drought

tolerance. The molecular basis of desiccation tolerance has been extensively studied

in a small number of resurrection plants which include the dicot C. plantagineum

(Bartels & Salamini 2001), the monocot S. stapfianus (Neale et al., 2000), the moss

Tortula ruralis (Oliver 1996) and the fern Polypodium virginianum (Reynolds &

Bewley 1993). More recently other desiccation-tolerant monocots including

Xerophyta humilis (Collet et al., 2004), Xerophyta viscosa (Sherwin & Farrant 1998),

and the dicot Craterostigma wilmsii (Farrant et al. 2003) have also been the subject of

desiccation tolerance studies. The physiological responses of dehydration and

rehydration in S. stapfianus and other desiccation tolerant species will be discussed in

the following text.

The resurrection grass S. stapfianus displays protoplasmic desiccation tolerance which

allows the plant to reduce its metabolism whilst retaining viable root and leaf tissue in

the desiccated state (Ghasempour et al., 2001). During the dehydration of S.

stapfianus a number of physiological changes occur which are outlined in Table 1.1.

The induction of desiccation tolerance is observed at 60% RWC (Gaff & Loveys

1993). The drying of S. stapfianus to 60% RWC will generally take 2-5 days, and

relatively slow water loss is required for the onset of desiccation-tolerance. This

27

induction of desiccation-tolerance will only occur in intact leaves attached to the

plant. If the leaves are detached whilst drying and before desiccation-tolerance is

induced, desiccation-tolerance is not achieved. This suggests that S. stapfianus

requires a signal, yet to be identified, from the roots of the plant for desiccation

tolerance to develop (Gaff et al., 1997).

Table 1.1: Summary of changes occurring during dehydration in the desiccation tolerant

S.stapfianus

Changes in protein expression, sugar content and ABA levels are summarised in this table

(Gaff 1989).

RWC Level of

drought stress

Physiological events and changes

100% - 90% Hydrated • normal photosynthesis and growth

89% - 80% Mild • reduction of photosynthesis

• appearance of some novel proteins on 2 D gels

(including dehydrins)

79% - 60% Moderate • reduction in starch content

• desiccation-tolerant state attained at 60% RWC

59% - 40% Strong • maximum content of glucose and fructose

• ABA level increases 7-fold

39% - 20% Severe • synthesis of numerous novel proteins

19% - 11% Extreme • maximum ABA level attained

• fragmentation of vacuole into sub-vacuolar

vesicles

≤ 10% -

Air Dry

Anabiosis • high sucrose content (100 µmole/g dried weight)

• elevated content of raffinose and stachyose

28

1.12.1 Sugar content

Glucose, fructose and galactose are present in large amounts in fully hydrated S.

stapfianus leaves. The sucrose content rises to relatively high levels during

dehydration. Sucrose comprises the majority of the total sugar content within the leaf

tissue in the air-dry state and plays a vital role in tissue preservation in this state

(Ghasempour et al., 1998b). The increased amount of sucrose during dehydration is

due to the conversion of starch to sucrose by sucrose phosphate synthase (Toroser &

Huber 1997; Whittaker et al., 2001;2007). After rehydration, glucose forms the

dominant sugar in leaf tissue (Murelli et al., 1996). In several resurrection plants other

than S. stapfianus¸ sucrose is also the predominant sugar accumulated. In C.

plantagieum, sucrose accumulation is thought to occur by the conversion of octulose

which is present at high levels in fully hydrated plants (Bianchi et al., 1991). The

sugar trehalose has also been controversially associated with desiccation tolerance in

plants, however, findings are variable and its accumulation is not significant for the

conferral of desiccation tolerance in comparison to sucrose (Scott 2000). Trehalose is

a sugar involved in signalling thought to be imperative in the coordination of plant

metabolism and development (reviewed in Paul et al., 2008). Trace levels of trehalose

in dehydrating S. stapfianus (Ghasempour et al., 1998b) could be associated with

signalling rather than used to form glasses during vitrification.

1.12.2 Hormonal induction of desiccation tolerance

In the resurrection plant C. plantagieum there is clear evidence that ABA plays a

major role in the induction of desiccation tolerance (Furini et al., 1997). However, in

S. stapfianus, the ABA level increases 6-fold during the latter stages of drought stress

and reaches a maximum during severe desiccation (10-20% RWC). The

accumulation of ABA during early dehydration stages is minimal, and in a study by

Ghasempour et al. (1998a) the exogenous application of ABA did not induce

desiccation-tolerance. This study showed that ABA does not play a key role in the

induction of desiccation tolerance of S. stapfianus. Exogenous hormones applied to S.

29

stapfianus protoplasts including methyl jasmonate, ethylene and brassinosteriods

induce protoplasmic drought tolerance to a slightly higher degree than ABA, but not

to a level that would allow the plant to survive desiccation (Ghasempour et al.,

1998a).

1.12.3 Cell structure during dehydration

Both hydrated and dehydrated leaves of S. stapfianus, as well as those of most other

non-resurrection plants, produce epicuticular waxes, particularly on the adaxial leaf

side, that reflect light and protect the plant from solar radiation and overheating.

These waxes aid in decreasing the rate of water loss and therefore slowing the rate of

plant dehydration (Dalla Vecchia et al., 1998). It has been suggested that a slow rate

of dehydration may be required to allow preservation of the structural integrity of

thylakoid membranes in chloroplasts (Sherwin & Farrant 1999). Large bulliform cells

in S. stapfianus leaves may act as water reservoirs (Dalla Vecchia et al., 1998). In

extreme desiccation, S. stapfianus cells do not show many major changes in structural

components in comparison to fully hydrated cell structure. Upon rehydration cell

organisation and metabolism in S. stapfianus is regained after 48 hours (Dalla

Vecchia et al., 1998).

1.12.4 Antioxidant systems and photosynthesis

Like many resurrection plants, S. stapfianus can survive in a desiccated state for

several months (Gaff et al., 2009). During the desiccation process the release of

reactive oxygen species (ROS) can cause damage to cellular membranes and lipids.

However, S. stapfianus has a robust and efficient antioxidant system to combat

oxidative damage, which can adversely affect tissue viability over these long periods

(Kranner et al., 2002). S. stapfianus induces the expression of ROS scavenging

enzymes, namely glutathione reductase, ascorbate peroxidase and dehydroascorbate

reductase. The activity of antioxidant enzymes to combat ROS is important during the

30

rehydration stage, when high levels of oxidative stress occurs in reviving tissues

(Sgherri et al., 1994).

Photosynthesis is adversely affected by low water availability. During dehydration

some resurrection plants lose chlorophyll and others do not (Oliver et al., 2000). S.

stapfianus lowers photosynthetic activity in a regulated manner during dehydration

and exhibits partial loss of chlorophyll content (Quartacci et al., 1997).

Photosynthetic activity decline could represent a mechanism that protects the cell

against ROS production and maintains membrane integrity to ensure protoplast

survival (Di Blasi et al., 1998). After rehydration, S. stapfianus regains normal

photosynthetic activity. In S. stapfianus, dehydration causes the fragmentation of

vacuoles and starch levels decline, however, chloroplasts only undergo slight

modifications. Thylakoid membranes and photosynthetic pigments are retained during

dehydration, and during rehydration the structure of the chloroplast is recovered

(Quartacci et al. 1997).

1.12.5 Changes in gene expression during dehydration in S. stapfianus

Changes in leaf gene expression during dehydration in S. stapfianus was first

examined by Gaff et al. (1997), where mRNA was isolated from air dry, dehydrating

and fully-hydrated leaf tissue and translated in vitro. A comparison of in vivo protein

extracts with the in vitro translated proteins suggested that some dehydration-related

transcripts may not be translated until the rehydration stage. These products may be

required for protection against cellular damage during the rapid rehydration generally

occurring within twenty four hours after a rainfall event (Gaff et al., 1997) or to allow

for the rapid growth that occurs during the short wet periods when conditions are

favourable (Blomstedt et al., 2010).

The generation and screening of S. stapfianus cDNA libraries resulted in the isolation

of genes up-regulated during the initial stages of dehydration of S. stapfianus leaf

tissue. This resulted in the identification of a number of genes encoding dehydrins, a

group 3 LEA, glyoxylase I, thiol proteases, an eIF1A protein translation initiation

31

factor, glycine- and proline-rich proteins, a tonoplast intrinsic protein and an early

light inducible protein (Blomstedt et al., 1998a,b; Neale et al., 2000; Le et al., 2007).

An extensive list of S. stapfianus dehydration-responsive genes is available in Gaff et

al. (2009). Many of these genes have strong homology to genes associated with

protection from dehydration in many non-resurrection plants as well as homology to

genes expressed in desiccated seeds of higher plants. Some of these proteins have

been suggested to be involved in the rapid translation of proteins during dehydration,

stabilisation of membranes during desiccation, solute flow during

dehydration/rehydration and reducing photosynthetic apparatus damage (Neale et al.,

2000). Interestingly, these genes were isolated on the basis of being differentially

expressed in desiccation-tolerant tissue and not in desiccation sensitive tissue (tissues

dehydrated following excision from the hydrated plant), or in the related

S. pyramidalis, a desiccation-sensitive relative (Gaff et al., 1997). This indicates that

an elevated and/or persistent drought response occurs in desiccation-tolerant tissues.

In some cases the desiccation-tolerance related genes were specific sequences from

multi-gene families although the significance of this is not understood. In severely-

dehydrated or desiccated resurrection grass tissue, the profile of the transcript pool is

different in comparison to that of early dehydration stages. Several of the transcripts

present in high levels in desiccated resurrection plant tissues encode growth-related

protein products for utilization upon rehydration (Blomstedt et al., 2010; Islam et al.,

2013).

While this research has uncovered some of the key genes being up-regulated during

dehydration of S. stapfianus and has set up the framework for understanding the

molecular basis of desiccation tolerance, the pivotal molecular mechanisms

responsible for the successful negotiation of a complete desiccation and rehydration

cycle remain to be identified.

32

1.13 Aims of the current study

Previous experiments at Monash University has identified a number of Sporobolus

desiccation genes (SDGs) and preliminary analysis of the expression of these genes

throughout the dehydration processes in both desiccation-tolerant and desiccation-

sensitive tissue was determined by Le et al. (2007) (Figure 1.3). Based on these

preliminary results the current project focussed on elucidating the functions of three

specific genes considered pertinent to the establishment of desiccation tolerance. This

was conducted by addressing the following:

Figure 1.3: Northern analysis of S. stapfianus desiccation tolerance genes.

Tissues were dried to varying stages of dehydration in intact (desiccation-tolerant) and

detached (desiccation sensitive) S. stapfianus leaf tissue. These are: stage A (≥90% RWC,

fully hydrated), B (89-80% RWC), C (79-60% RWC), D (59-40% RWC), E (39-20% RWC), F

(19-11% RWC) and G (≤11% RWC, air-dried) (from Le et al., 2007).

33

1. Further investigation of gene expression patterns and sequence analysis of

Sporobolus Desiccation Genes (SDGs).

Experiments were undertaken to establish the gene expression patterns of the genes

SDG3i, SDG4i and SDG8i during dehydration and rehydration phases in S. stapfianus

leaf tissue to observe the possible requirements of these gene products during these

stages. The induction of these genes by plants hormones: ABA, cytokinin, methyl

jasmonate, gibberellin, ethylene and salicylic acid was also investigated to further

elucidate the regulation of these genes. A detailed analysis of SDG3i and SDG4i

amino acid sequence was completed, and intracellular localisation of SDG4i was

determined.

2. Phenotype analysis of transgenic plants constitutively expressing SDG4i.

The transformation and regeneration of S. stapfianus has not been possible, so the

SDG4i knockdown analysis of S. stapfianus could not be performed. Instead, a gain-

of-function approach was undertaken to analyse the effect of SDG4i over-expression

in A. thaliana. Developmental phenotype analysis of homozygous 35S:SDG4i A.

thaliana lines during long day and short day conditions was undertaken to observe

growth effects of SDG4i over-expression. Furthermore, the effects on osmotic, salt,

cold and heat tolerance as well as exogenous ABA application on 35S:SDG4i

A. thaliana throughout germination and seedling growth was analysed. To investigate

the potential of SDG4i over-expression in a crop species, a transformation and

regeneration protocol to produce transgenic Sorghum bicolor was explored.

3. Identification of proteins that interact with SDG4i.

Sequence analysis indicated that SDG4i may be involved in a protein-protein

interaction. A Yeast 2-hybrid protocol was utilised to obtain proteins that interact with

SDG4i in cDNA expressed in desiccation S. stapfianus and the interactions confirmed

using a bioflorescence complementation assay. The interaction lead to further analysis

of 35S:SDG4i A. thaliana tolerance to endoplasmic reticulum stress and hormone-

induced leaf senescence.

34

4. Investigation of UDP-glycosyltransferase activity of SDG8i on plant hormones.

Previous work at Monash University has indicated that SDG8i is homologous to a

UDP-glycosyltransferase and A. thaliana over-expressing SDG8i have a bushy

phenotype and appear to have altered hormone homeostasis (Islam et al., 2013). To

assess the ability of SDG8i to glycosylate plant hormones a coupled enzyme assay

using pyruvate kinase and lactate dehydrogenase was utilised.

35

2.0 General materials and methods

2.1 General Solutions and Reagents

3M Sodium Acetate: 24.6% (w/v) anhydrous CH3COONa, pH adjusted to 6.0 with

acetic acid before autoclaving.

5M/3M Potassium Acetate: 60% (v/v) 5M CH3COOK, 11.5% (v/v) glacial acetic

acid.

50X TAE buffer: 242 g/L Tris-base, 0.05M EDTA (pH 8.0), 57.1 ml/L glacial acetic

acid.

Coomassie blue solution: Coomassie blue R-250 (0.25% w/v), 40% (v/v) methanol,

7% (v/v) acetic acid.

Denaturing solution: 1.5M NaCl, 0.5M NaOH, pH adjusted until over 12.0.

MOPS buffer (1×): 0.2M 3-[N-morpholino] propanesulfonic acid (MOPS), 0.05M

CH3COONa, 0.01M EDTA, pH 7.0

Neutralising solution: 1.5M NaCl, 1M Tris, pH adjusted to 8.0 with HCl.

PBS: A 10X stock solution was made with 80g/L NaCl, 2g/L KCl, 14.4g/L Na2HPO4,

2.4g/L KH2PO4, pH 7.4.

Proteinase K: 20mg/ml of proteinase K (Promega) solution was made in TE buffer

(pH 8.0).

RNase A: 10mg/ml of RNase A (Sigma) solution was made in 10mM Tris (pH 7.5),

15mM NaCl solution. The solution was boiled for 15 minutes and then allowed to

cool slowly to room temperature. Aliquots were stored at –20oC.

TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

TLES Buffer: 100 mM Tris-HCl (pH 8.0), 100 mM LiCl, 10 mM EDTA, 1% (w/v)

SDS.

SSC: A 20X stock solution was made with 175.3 g/L NaCl, 88.2 g/L sodium citrate,

pH 7.0.

36

2.2 Bacterial methods

2.2.1 Solutions and media

Antibiotics

Antibiotics were dissolved in sterile distilled water (except where indicated) at

concentrations shown below. The solutions were sterilised through a 0.45 µm filter

unit attached to a syringe and stored at -20o C.

Ampicillin: ampicillin sodium salts (Sigma-Aldrich) dissolved at 50 mg/ml

concentration.

Hygromycin: hygromycin B powder (Sigma-Aldrich) dissolved at 50 mg/ml

concentration.

Kanamycin: kanamycin monosulfate salts (Sigma-Aldrich) dissolved at 50 mg/ml

concentration.

Timentin: timentin (Smith Kline Beecham) dissolved at 100 mg/ml concentration.

Rifampicin: rifampicin (Sigma-Aldrich) dissolved in ethanol at 20mg/ml

concentration.

Luria Bertani (LB) broth: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.5.

The broth was sterilised by autoclaving.

Solid agar medium: 1.5% (w/v) agar was added to the above solutions before

autoclaving.

37

2.2.2 Bacterial transformation

Competent E. coli cells were thawed on ice and 50µl aliquot placed into a chilled

electroporation cuvette. 1µl of ligated plasmid was added and the tube was subject to

electrical charge at 2.45 Watts, 25µFD and 200Ω using a Bio-Rad gene pulser. After

electric charge was applied, 950ul of LB broth was added to the cuvette and the

mixture was co-incubated at 37°C for 1 hour prior to being spread on LB plates

containing the appropriate antibiotic and incubated overnight at 37°C.

Transformation of competent A. tumefaciens cells was completed using the same

approach as above with the incubation temperature of 28°C, co-incubation time of 3

hours and incubation on plates for 3 days.

Stocks of all bacterial cultures carrying the plasmid of interest were made after

transformation was completed by mixing bacterial culture and 50% glycerol at a 1:1

ratio in Eppendorf tubes, snap frozen in liquid nitrogen and stored at -80ºC.

2.2.3 Preparation of E. coli and A. tumefaciens electrocompetent cells

A vial of E. coli cells were spread on LB agar and incubated overnight at 37°C. A

single colony was then incubated in 5ml LB broth for 4-6 hours, this was then

transferred to 400ml of LB broth and incubated overnight at 37ºC with shaking at

150rpm. When the culture reached an OD600 of 0.4 the cells were chilled on ice. The

culture was split into 4 centrifuge tubes, cells were harvested by centrifugation at

1000g for 20 minutes at 4°C, and the supernatant was discarded. Each pellet was

resuspended in 100ml of ice-cold sterile distilled water, and cells were harvested as

mentioned previously. This process was repeated with a further 100ml of ice-cold

sterilised distilled water. The pellet was resuspended 40ml of ice-cold sterile distilled

water and cells were harvested by centrifugation as described previously. Each pellet

was resuspended in 1ml of ice-cold sterile 10% glycerol, and 50µl aliquots were

placed in 0.5ml Eppendorf tubes, snap frozen in liquid nitrogen and stored at -80°C.

38

2.2.4 Colony PCR

After growth of bacteria on LB agar the colonies to be tested for the presence of an

insert were spotted onto a 10µl micropipette tip. The tip was used to inoculate a

standard PCR reaction mix containing the appropriate primers. The reaction tube was

placed in an Eppendorf thermocycler, heated to 95°C for 2 minutes to lyse the cells,

followed by 35 cycles of the appropriate reaction conditions. PCR products were

viewed using 1.5% agarose gel electrophoresis (section 2.5.2).

2.2.5 Small scale isolation of plasmid DNA

Overnight cultures of E. coli in 10ml of LB broth were subject to plasmid extraction

using the Wizard® Plus Miniprep kit (Promega) using the manufacturers protocol.

2.2.6 Medium scale isolation of plasmid DNA

100mL of LB broth containing the appropriate antibiotic was inoculated with a single

bacterial colony and grown overnight at 37oC with shaking at 150rpm. Cells were

harvested by centrifugation at 1500g for 10 minutes, resuspended in 2.5 ml of ice-cold

freshly prepared plasmid extraction buffer (25 mM Tris-Cl pH 8, 5 mM EDTA, 0.1M

NaOH and 0.5% (w/v) SDS) and incubated on ice for 5 minutes. Cells were

equilibrated with 3.75 ml of ice-cold 5M/3M potassium acetate and incubated on ice

for 10 minutes. Cell debris was removed by centrifugation at 10000g for 15 minutes.

The supernatant was transferred to a new tube and RNAase A was added to a final

concentration of 100µg/ml and incubated at 37oC for 30 minutes. Proteins were

removed by the addition of proteinase K solution to the concentration of 100µg/ml

and incubated at 37oC for 30 minutes. A half-volume of ice-cold isopropanol was

added to the supernatant to precipitate the DNA. The DNA pellet was collected at

8000g for 15 minutes at 4°C and dissolved in 500µl of TE buffer. The DNA solution

was extracted twice with phenol and chloroform/IAA (25:24:1 v/v). DNA from the

aqueous phase was precipitated with a 1/10 volume of 3M sodium acetate and 2.5

volumes of ethanol, centrifuged at 13000 rpm for 15 minutes at 4°C, washed with

39

70% ethanol and dissolved in 50-100µl of sterile distilled water. Plasmid extracts

were stored at -20°C.

2.3 Plant materials and methods

2.3.1 Solutions and media

½ MS medium: 0.443% (w/v) Murashige and Skoog basal medium (Sigma), 1.5%

(w/v) sucrose, pH 5.8-6.0. For solid medium, 0.8% (w/v) phytogel (Sigma) was

added.

Arabidopsis nutrient solution: The following solutions were added to tap water to

make 20L of nutrient solution; 100ml of 1M KNO3, 50ml of 20mM FeEDTA, 40ml

of 1M MgSO4.7H2O, 40ml of 1M Ca(NO3)2.4H2O, 40 ml of micronutrient solution

(70mM H3BO3, 14mM MnCl2.4H2O, 0.5mM CuSO4.5H2O, 1mM ZnSO4.7H2O,

10mM NaCl, 0.01mM CoCl2.6H2O) and 50 ml of 1M K Phosphate Buffer (1M

KH2PO4 and 1M K2HPO4 pH 5.5).

2.3.2 Plant material

2.3.2.1 Sporobolus stapfianus

Plants of S. stapfianus Gandoger originally from the Eastern Transvaal province of

South Africa were grown in 15cm pots containing a seed raising mix, potting mix and

perlite mixture (1:1:1) in a controlled environment growth room with the conditions

of 28ºC, 16 hour day/8 hour night and under light of 250µmol photons/m2/s. Before

use plants were kept well watered and to ensure full hydration, plants were covered

with supported plastic bags. The plants were fertilised with Osmocote® when

required.

All leaf tissues were harvested by cutting with sharp scissors mid-photoperiod unless

otherwise stated. RWC was determined by measuring the fresh weight (FW), turgid

weight (TW) and dry weight (DW) of a subsample of harvested material at the time of

harvesting using the equation: RWC (%) = (FW-DW)/(TW-DW) x 100. TW was

40

measured after leaves had been soaked in water for 24 hours, and DW after leaves has

been oven-dried at 80 oC for 24 hours.

2.3.2.2 Arabidopsis thaliana

Plants of A. thaliana ecotype Columbia-0 (referred to throughout this thesis as WT-

COL) were grown in either soil or in vitro conditions. For soil grown plants, A.

thaliana were sown in either 5cm pots or 15 x 8 punnets containing seed raising mix,

potting mix and perlite (1:1:1) and stratified for 3 days in the dark at 4ºC. For in vitro

grown plants, seeds sterilised with 70% ethanol, placed on ½ MS solid medium and

stratified for 3 days in the dark at 4ºC. After stratification of both soil and in vitro

grown plants, the plants were placed in a controlled environment growth room with

the conditions of 23ºC, 16 hour day/8 hour night and under light of 250µmol

photons/m2/s unless otherwise indicated. Plants grown in soil were watered from the

base with Arabidopsis nutrient solution.

2.3.3 Agrobacterium-meadiated transformation of Arabidopsis thaliana

A. thaliana ecotype Columbia-0 plants were transformed using the floral spray

method (Chung et al., 2000). Plants were grown in soil (section 2.4.2.2) under

constant light at a density of 10-15 plants per punnet. Once the primary inflorescence

bolt emerged, it was removed by cutting with sharp scissors. When the plants had

begun flowering, the flowers were sprayed with an A. tumefaciens strain AgL1

containing the appropriate vector at an OD600 of 1 suspended in a medium consisting

of 10 mM MgCl2, 5% (w/v) sucrose, B5 vitamins (10-6 x concentration), 10µM

acetosyringone and 0.03% (v/v) Silwet L-77 surfactant. The punnet was laid on its

side in a tray and covered in clear plastic overnight. The plastic cover was removed

and the plants were allowed to stand upright. Seeds were collected approximately 3-4

weeks after transformation. Transgenic A. thaliana lines were selected using the

appropriate antibiotic to homozygosity before being used in experimentation.

41

2.4 Nucleic Acid methods

2.4.1 Genomic DNA isolation

Plant material was harvested to the weight of 500mg and was ground for 15-30

minutes under liquid N2 with a mortar and pestle until a fine powder was obtained.

Glass sand was used to facilitate the grinding process in S. stapfianus samples. The

powder was transferred and transferred to a 50ml Falcon tube containing DNA

extraction buffer (2% CTAB, 2% PVP, 1.4M NaCl, 20mM EDTA pH 8.0, 100mM

TrisHCl pH 8.0, 2% β-mercaptoethanol) heated to 60ºC and homogenised by

inversion. The sample was incubated for 1 hour at 60ºC and mixed several times

throughout the incubation period. Cellular debris was removed by centrifugation at

12000g for 10 minutes. The supernatant was removed and extracted with an equal

volume of chloroform:isoamylalcohol (24:1 v/v), mixed by inversion for 5 minutes

and centrifuged at 10000g for 10 minutes. The upper aqueous phase was placed into a

new tube and a 0.6 volume of ice-cold isopropanol was added and mixed thoroughly.

The mixture was centrifuged at 10000g for 20 minutes at 4ºC to pellet the DNA. The

DNA pellet was dissolved in 700µl of TE buffer and transferred to a new

microcentrifuge tube. RNase A was added to a concentration of 100µg/ml and

incubated at 37ºC for 3 minutes. The DNA was precipitated by addition of 1/10

volume of 7M potassium acetate and 2 times the volume of ice-cold 100% ethanol

and incubated at -20ºC for 20 minutes. The DNA was pelleted by centrifugation,

supernatant discarded and the pellet rinsed with ice-cold 70% ethanol. The DNA

pellet was dried upside-down on a KimWipe™ and then dissolved in 100µl of TE

buffer.

2.4.2 Agarose gel electrophoresis

DNA samples were mixed in loading buffer (0.05% bromophenol blue, 0.05% xylene

cyanol FF, 3% Ficoll Type 400) at a 1:6 ratio (loading buffer:sample) and separated at

80V on 1% (w/v) agarose gels made with 1× TAE buffer. The gels contained 1µl of a

10mg/ml stock of ethidium bromide per 50 ml of agarose solution. Electrophoresis of

DNA was terminated when the bromophenol blue dye had migrated half the length of

42

the gel. The gels were viewed on a UV-transilluminator and recorded with a CCD

camera.

2.4.3 Sequencing and bioinformatic analysis

DNA sequencing reactions were completed and cleaned-up using Applied Biosystems

BigDye terminator mix using protocol prescribed by the Micromon DNA sequencing

facility at Monash University, Melbourne, Australia. Capillary electrophoresis was

completed by the Micromon DNA sequencing facility. Sequence data was analysed

using the ABI sequence scanner software, databases on the National Centre for

Biotechnology Information (NCBI) website, and the European Molecular Biology

Open Software Suite (EMBOSS) website.

2.4.4 Isolation of RNA

Total RNA from plant tissue was isolated using a LiCl/phenol extraction method

(Verwoerd et al., 1989). Leaf tissue (500mg) was ground to a fine powder in a pre-

chilled mortar and pestle in liquid nitrogen. Grinding of tissue was aided with glass

sand. The powder was added to 50ml Falcon tube containing 1.5ml TLES buffer and

1.5ml phenol that had been pre-warmed to 80ºC. Samples were mixed for 30 seconds

using a vortex. The mixture was added to 750µl of chloroform/IAA (24:1) and

centrifuged at 10000g for 5 minutes in a microcentrifuge at 4ºC. The supernatant was

transferred to a microcentrifuge tube and an equal volume of 4M LiCl was added and

mixed by inversion. Samples were incubated overnight at 4oC in the dark and then

centrifuged at 10000g for 30 minutes at 4oC. The RNA pellet was dissolved in 250µl

of sterile distilled water. RNA was precipitated with 25µl of 3M sodium acetate (pH

6.0) and 500µl of chilled 100% ethanol and incubated at -20oC overnight. The RNA

precipitate was centrifuged at 10000g for 20 minutes at 4ºC, washed with 70%

ethanol and dissolved in 50µl of sterile distilled water.

43

2.4.5 Denaturing agarose gel electrophoresis

RNA samples were diluted in loading buffer at the concentration of 50% glycerol,

1mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol FF, formaldehyde (8%

v/v), formamide (50% v/v) and 1X MOPS. The samples were incubated at 65oC for

15 minutes, and then samples were separated on a 1.5% (w/v) agarose gel containing

formaldehyde (8% v/v) and 1X MOPS.

2.4.6 cDNA synthesis

First strand cDNA synthesis was completed using the SensiFAST™ cDNA synthesis

kit (Bioline) by following the manufacturer’s instructions.

2.4.7 Restriction digestion of DNA

Restriction digestion of DNA was completed in either a 20µl (plasmid digestion) or

50µl (genomic DNA) reaction volume containing the appropriate restriction enzyme

buffer to a 1X concentration and10 units of restriction enzyme, The DNA was

digested in a water bath at the recommended temperature for 1 hour (plasmid

digestion) or overnight (genomic DNA digestion). The restriction enzyme was

inactivated by incubation at 65oC for 10 minutes after digestion is complete.

2.4.8 Ligation of DNA

Ligation reactions were conducted in a 10µl total volume containing 1X DNA ligase

buffer (Promega), 5 units of T4 DNA ligase (Promega) and 150ng of vector DNA and

insert DNA to a 3:1 (insert:vector) ratio. The ligation reaction mixture was incubated

at 4ºC overnight.

44

2.4.9 Purification of DNA fragment from agarose gel

DNA fragments that were separated by agarose gel electrophoresis were purified from

the gel using the Promega Wizard® SV Gel and PCR Clean-Up System using the

manufacturer’s protocol.

2.5 Nucleic acid hybridisation methods

2.5.1 Transfer of nucleic acids from agarose gels to nylon membranes

Nucleic acids were transferred to Hybond-N+ nylon membranes (GE Healthcare). For

Southern analysis, DNA was separated by electrophoresis and the gel was rinsed in a

0.125M HCl solution until the bromophenol blue dye changed colour. The gel was

subsequently washed in denaturing solution until the dye returned to its original

colour. The gel was submerged in neutraliser solution for 30 minutes with gentle

shaking. For northern analysis, RNA was separated by electrophoresis and the gel was

washed in 2× SSC buffer to remove formaldehyde. The washed gel was placed on a

wick platform consisting of three sheets of Whatman™ 3MM filter paper saturated in

20×SSC. The nylon membrane was soaked in distilled water and placed on top of the

gel. Three sheets of Whatman™ 3MM filter paper (soaked in 2X SSC) and a stack of

absorbent paper; all cut to the size of the gel, were placed on top of the membrane.

Cling film was placed over the exposed wick to prevent short-circuiting. A perspex

plate and a weight were placed on top of the paper stack. The transfer was allowed to

proceed overnight in transfer buffer 20X SSC. After transfer, the nylon filter was

removed and dried. Nucleic acids were cross-linked to the nylon membrane by

exposing the nylon membrane to UV light for 5 minutes.

45

2.5.2 Radiolabelling of probe

DNA fragments were labelled with radioactive α-32P-dATP nucleotides using the

DECAprime™ II DNA Labelling Kit (Life Technologies) according to the

manufacturer’s instructions.

2.5.3 Northern blot hybridisation

Following transfer of RNA, the nylon membrane was pre-hybridised in 5ml of

ExpressHyb™ Hybridisation Solution (Clontech) at 68oC for 30 minutes.

Hybridisation was performed in 5 ml of fresh ExpressHyb™ solution containing the

radioactively labelled DNA probe with continuous shaking at 68oC for 1 hour. The

radioactively labelled probe was denatured by boiling for 5 minutes prior to

hybridisation. The membrane was rinsed in wash solution 1 (2X SSC, 0.05% SDS) at

room temperature for 40 minutes with continuous shaking with the wash solution

changed every 10 minutes. The membrane was then washed in wash solution 2 (0.1X

SSC, 0.1% SDS) with continuous agitation at 50oC for 40 minutes, the washing

solution was changed every 20 minutes. The membrane was then wrapped in cling

film to prevent drying.

2.5.4 Southern blot hybridisation

Southern hybridisation was performed as described above, however hybridisation was

performed overnight at 60oC.

2.5.5 Phosphoimaging

Radioactive signals on blots were detected by exposing membranes to a

phosphoimaging screen overnight. The phosphoimaging screen was scanned using the

Typhoon Trio™ Scanner (GE Healthcare) and the resulting image quantified using

the ImageQuant TL V7.0 software.

46

2.6 Protein methods

2.6.1 Isolation of Protein from plant tissue

500mg of plant leaf tissue was ground in ice-cold protein extraction buffer (100mM

potassium phosphate, 1mM EDTA, 1% Triton X-100, 10% glycerol, 1mM DTT) until

homogenised and centrifuged at 10000g for 10 minutes at 4ºC to remove cell debris.

The supernatant was transferred to a fresh tube and stored at -20ºC. Protein

concentration was quantified by Bradford’s assay.

2.6.2 SDS-PAGE

Proteins were separated using the Protean II xi Cell (Bio-Rad) gel apparatus.

Approximately 10 µg of protein in loading buffer (40% glycerol, 240mM Tris.HCl

pH 6.8, 8% SDS, 0.04% (w/v) bromophenol blue, 5% v/v β-mercaptoethanol) at a 1:5

(loading dye:sample volume) ratio was loaded on a 10% SDS polyacrylamide gel.

After electrophoresis, gels were and fixed and stained with Coomassie blue solution.

2.6.3 Transfer of proteins to nitrocellulose membranes

Proteins were transferred to Hybond-C Extra (GE Healthcare) nitrocellulose

membranes using a Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad). After

SDS-PAGE, the stacking gel was removed using a scalpel blade, a Hybond-C Extra

nitrocellulose membrane was cut to the size of the running gel, as well as 6

Whatman™ 3MM filter papers. The nitrocellulose membrane was soaked in distilled

water, and the filter papers were soaked in transfer buffer (25mM Tris, pH 8.3,

200mM glycine, 0.1% SDS and 20% (v/v) methanol). Two sponges were soaked in

transfer buffer and the cassette was loaded in the order of sponge, then 3 Whatman™

3MM filter papers, polyacrylamide gel, nitrocellulose membrane, 3 Whatman™ 3MM

filter papers (stacked on top of each other exactly) followed by another sponge. The

cassette was stacked so that the gel was nearest to the cathode. The cassette was

47

placed in the tank and the tank was filled with transfer buffer. Proteins were

transferred at 60mA overnight at 4ºC.

2.6.4 Western blotting

A custom primary polyclonal-antibody against the S. stapfianus SDG4i protein was

raised in rabbits by the Life Research custom antibody service to be used in western

blotting analysis. The nitrocellulose membrane was blocked for 30 minutes with

gentle agitation in blocking solution (PBST (1X PBS, 0.1% Tween-20), 5% skim milk

powder) and was then washed for 5 minutes with agitation with PBST three times.

The membrane was then sealed in a plastic bag containing a 1:5000 (v/v,

antibody:PBS) concentration of the custom polyclonal antibody and incubated for 1

hour at room temperature with gentle agitation. The membrane was then washed 3

times for 15 minutes in PBST with agitation. The membrane was then sealed in a

plastic bag containing a 1:20000 (v/v antibody:PBS) secondary antibody (HRP

conjugated Goat anti-Rabbit IgG, Promega) solution and incubated for 1 hour at room

temperature with gentle agitation. The membrane was then washed 3 times for 15

minutes in PBST with agitation. The membrane was then sealed in a plastic bag and

incubated for 5 minutes in the dark with Novex® ECL Chemiluminescent substrate

(Invitrogen), the substrate was drained and the membrane was exposed to X-ray film

for 5-15 minutes and developed using the AGFA X-ray developer apparatus.

48

3.0 Dehydration and hormonal responses of genes that are transcribed during the desiccation of Sporobolus stapfianus

3.1 Introduction

A previous study of dehydration related genes in S. stapfianus identified several

cDNA sequences of genes expressed highly during desiccation of S. stapfianus leaf

tissues (Le, 2004, Le et al., 2007). Three of the genes were designated SDG3i

(Accession number: AM268212, partial coding sequence), SDG4i (accession number:

AM268211, full coding sequence) and SDG8i (accession number: AM268210, full

coding sequence). Previous work has suggested that SDG8i has homology to a UDP-

glycosyltransferase, SDG3i is homologous to plant pore proteins, and SDG4i is

homologous a B. napus protein, which has been reported to be involved in an

interaction with histone deacetylase 19 (Le et al., 2007). Some of the characteristics

of SDG8i and its proposed function have been studied by previous PhD students,

Tuan Le (Le et al., 2007) and Sharmin Islam (Islam 2013), however, the

characteristics and function of SDG3i and SDG4i have not been examined.

Molecular studies of resurrection plants can reveal genes that are up-regulated to aid

the plant in the survival of severe dehydration and the role these genes have in the

desiccation tolerance program. An approach to deducing the function of these genes is

to determine basic characteristics of the gene, such as copy number, gene structure,

amino acid homologies, hormonal regulation and their location within the cell. This

basic information can be used to design further experiments to test gene function. This

chapter details the molecular characterization and hormonal responses of SDG3i and

SDG4i as well as the hormonal responses of SDG8i and expression profiles during

plant dehydration.

49

3.2 Experimental methods

3.2.1 Genome walking of SDG3i

Sporobolus stapfianus gDNA was digested separately with four restriction

endonucleases which generate blunt ends: DraI, StuI, PvuII and EcoRV, to create

representative fragments of S. stapfianus gDNA. These fragments were then purified

using the phenol/chloroform method and were ligated with an adaptor (Figure 3.1)

using T4 DNA ligase according to the manufacturers instructions (Promega). A

primary PCR amplification was conducted using Pfu DNA polymerase (Promega)

with standard cycling conditions, and an annealing temperature of 50°C. The primers

utilised were the AP1 (adaptor primer 1) (forward) (5’

GTAATACGACTCACTATAGGGC 3’) and the gene-specific SDG3i1 (reverse)

primer (5’ GCAACGACTCCTTGCTAACGTTCTC 3’). A nested PCR reaction

followed using the same conditions as mentioned above with forward primer AP2

(adaptor primer 2) (5’ ACTATAGGGCACGCGTGGT 3’) and a nested gene-specific

reverse primer SDG3i2 (5’ GACGCGTTCTTCGAGCCGGTGCTGCC 3’). The

fragment obtained from the gDNA PvuII fragment template was then purified from

the gel and sequenced using the AP2 forward primer and SDG3i2 reverse primer

using methods stated in 2.4.3.

Figure 3.1: Genome walking adaptor

The adaptor which was ligated with digested S. stapfianus gDNA fragments. The arrows

depict each adaptor specific forward primer binding site. This allowed amplification of SDG3i

by use of the SDG3i specific reverse primers. This adaptor sequence is provided in the

protocol given in GenomeWalker™ Universal Kit (Clontech).

5' GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT 3' 3' CCCGACCA 5'

AP1 AP2

50

3.2.2 Confirmation of SDG3i coding region by RT-PCR

The 5’ SDG3i sequence obtained from genome walking was used to create a forward

primer at the beginning of the coding region (5’

ATGAGCAACTTGGAGAACCAGG 3’). The previously known SDG3i sequence

(Accession number: AM268212) was used to design a reverse primer (5’

TCAGAATACGCCCCAGAGGACGTTG 3’) at the end of the coding region. These

primers were then used to amplify the full length coding region from cDNA

synthesised utilizing mRNA from dehydrated (~60%RWC) S. stapfianus leaf tissue.

3.2.3 Dehydration/rehydration of S. stapfianus

Fully hydrated (>90% RWC) S. stapfianus plants, growing at 28°C under a 14h

light/10h darkness regime were dehydrated over a period of 14 days with a sample of

leaf tissue taken daily. Leaf RWC was measured for each sample. The plants were

grouped into 7 dehydration stages based on the leaf RWCs: ≥ 90%, 89-80%, 79-60%,

59-40%, 39-20%, 19-10% and <10% and leaf tissue harvested. For rehydration

studies, air-dry plants (RWC <10%) were submerged in water for 12 hours, and

samples taken at 12, 24 and 48 hours after initial submersion. All of these tissue

samples were used in subsequent experiments. RNA was extracted from the leaf

samples representing the 7 dehydration stages and 3 rehydration stages and used for

northern analysis, using one replicate per dehydration stage. The northern blot was

probed with gene-specific fragments consisting of: SDG4i CDS, SDG3i 3’ region and

SDG8i 3’ region. Expression was quantified using a Triticum aestivum 28S ribosomal

RNA subunit CDS probe that was previously shown to hybridise to S. stapfianus

rRNA (Blomstedt et al., 1998).

51

3.2.4 Exogenous application of hormones to S.stapfianus

Fully hydrated S. stapfianus plants were treated separately with the hormones; methyl

jasmonate (150µM), abscisic acid (100µM), cytokinin (100µM), ethylene (150µM),

salicyclic acid (100µM), gibberillic acid (GA3) (1mM), auxin (IAA) (100µM).

Control plants were treated with water and the solvent (ethanol) solution at equivalent

concentrations. Leaf samples were taken at 0, 1, 3, 8 and 24 hours after the

commencement of treatment with the RWC of each sample also recorded. The leaf

tissue samples were subjected to RNA extraction (three replicates per treatment were

pooled into one extraction) for a northern blot which was then probed with the SDG4i

CDS and SDG3i 3’ region and SDG8i 3’ region. Expression was quantified using a

Triticum aestivum 28S ribosomal RNA subunit CDS.

3.2.5 Construction of SDG4i GFP fusion vectors

SDG4i-N-terminal and C-terminal GFP fusion constructs were made using Invitrogen

Gateway® cloning compatible vectors for plant transformation (Curtis &

Grossniklaus 2003). SDG4i fragments were amplified by PCR using Pfu DNA

polymerase from a pBluescript-SDG4i template (50ng) with primers that incorporate

attB1 sites (Table 3.1, underlined) generating a fragment suitable for a BP

recombination reaction with pDONR/Zeo (Invitrogen) donor vector using the

manufacturers protocol (Invitrogen Gateway® cloning system). Transformants were

selected using the antibiotic ZeocinTM (Invitrogen). A stop codon was included in the

amplified SDG4i sequence into the N-terminal GFP fusion construct. Vector maps of

pDONR/Zeo can be seen in Figure 3.2.

52

Table 3.1: Primers used for GFP fusion vector generation

Name Sequence (5’ – 3’)

SDG4i attB1 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGACCAACTT

GGAGAACCAGGC

SDG4i attB1 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCGAATACGCCGG

AGAGGACGTTG

SDG4i attB1 Reverse

(stop codon)

GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGAATACGCC

GGAGAGGACGTTG

Figure 3.2: pDONR/Zeo Gateway® Donor vectors containing SDG4i used to create GFP

fusion destination vectors.

Figure showing feature maps of two donor vectors either with a stop codon (A) for the N-

terminal GFP fusion construct, or without a stop codon (B) for a C-terminal GFP fusion

construct.

EM7 promoter

pDONR/Zeo-SDG4i STOP 2787 bp

Zeo(R)

SDG4ii

T7 promoter

rrnB T1 transcription terminator


Stop codon

pDONR/Zeo-SDG4i 3084 bp

Zeo(R)

SDG4i

EM7 promoter

T7 promoter


rrnB T2 transcription terminator A: B:

53

The donor vectors were then used in two separate LR recombination reactions using

LR clonase (Invitrogen) with the destination vectors pMDC43 (N-terminal GFP) and

pMDC83 (C-terminal GFP) (Figure 3.3) (Curtis & Grossniklaus 2003) following the

manufacturers instructions.

Figure 3.3: pMDC43 (N-terminal) and pMDC83 (C-terminal) GFP fusion vectors

containing the SDG4i insert.

Figure showing the features of the GFP fusion destination vectors with either the SDG4i with

an N-terminal GFP fusion (A) or a C-terminal GFP fusion (B).

54

3.2.6 Intracellular localisation of SDG4i in Allium cepa (onion) epidermal peels

Cultures of A. tumerfaciens containing 35S:GFP (control), pMDC43:SDG4i and

pMDC83:SDG4i GFP-fusion constructs were pelleted by centrifugation and diluted to

OD600 of 0.5 with infiltration medium (10 mM MgCl2, 5 mM MES-KOH (pH 5.6),

and 200 µM Acetosyringone) and mixed 1:1 with P19. P19 was used to prevent the

onset of post-transcriptional gene silencing of the GFP constructs (Vionnet et al.,

2003) by the recipient onion cells. The A. tumerfaciens cultures were placed in Petri

dishes and onion epidermal peels were immersed in the solution for 3 days in the dark

at 28°C. The peels where then washed in distilled water and mounted onto

microscope slides and a coverslip placed on top. The cells were then viewed under a

UV fluorescence microscope.

55

3.3 Results

3.3.1 Sequence analysis of SDG3i

The SDG3i transcript isolated from S. stapfianus in previous studies (Le et al., 2007)

gave a partial sequence of 704bp. Genome walking was undertaken to isolate the full

length CDS of SDG3i in order for the function to be analysed in transgenic A.

thaliania as well as to uncover clues to SDG3i regulation through the analysis of the

promoter sequence. A DNA fragment containing the 5’ region of SDG3i was obtained

using PvuII digested S. stapfianus gDNA as a template. The DNA sequence was

aligned with the known cDNA sequence of SDG3i revealing a 100bp overlapping

region, with 100% identity to the previously known cDNA sequence (Figure 3.4,

shaded grey). However a non-identical sequence of 88bp was contained within this

region and was confirmed by sequencing four individual reactions. This non-identical

region was predicted to be an intron. To analyse the intron-exon structure of the entire

SDG3i gene, intact gDNA was used as a template for PCR and the products were

sequenced and revealed further introns predicted by both the alignment of obtained

SDG3i gDNA sequence and known SDG3i cDNA as well as with intron prediction

software GeneMark (Lomsadze et al., 2005) using Zea mays as the intron prediction

model. A TATA box is observed at the beginning of the known genomic sequence at

11-14bp and the presence of a Kozak-like sequence representing the beginning of

translation is seen at 175-187bp. The stop codon is shown at 1035bp, and a poly-A

signal motif is observed at 1305-1310bp in the 3’UTR (Figure 3.5a). In summary,

genome walking and sequence of genomic DNA revealed a further 298bp of coding

region, 181bp of promoter region and three introns (represented schematically in

Figure 3.5b).

56

1 50 GWF CGGTGTTTAC TATAGGGCAC GCGTGGTCGA CGGCCCGGGC TGGTCTGCTC SsSDG3i ---------- ---------- ---------- ---------- ---------- 51 100 GWF CTCCCAAGTC TTTTTGTCCT TCAGCGCGTC GCGTAATGTT TATCTAGTGA SsSDG3i ---------- ---------- ---------- ---------- ---------- 101 150 GWF CACAGCCGCA CTGCCGTGGC CAGTGTGCGC CTCAGGATAG CAGCAGAGAG SsSDG3i ---------- ---------- ---------- ---------- ---------- 151 200 GWF GAGGAGAGCA GAGCAGGGAA GCGAGGGGAA GATGAGCAAC TTGGAGAACC SsSDG3i ---------- ---------- ---------- ---------- ---------- 201 250 GWF AGGCGCGGGG GTTCGTGGAC GACGTCCACG ACTGCTGGGA GCAGAAGAAG SsSDG3i ---------- ---------- ---------- ---------- ---------- 251 300 GWF AACTGGATGC TGGACCTCGG CCACCCGCTC CTCAACCGCA TCGCCGATAG SsSDG3i ---------- ---------- ---------- ---------- ---------- 301 350 GWF CTTCGTCAAG GCTGCCGGGG TACGTAGTGC TAGTGCTGTT GTCTTCTGTC SsSDG3i ---------- ---------- ---------- ---------- ---------- 351 400 GWF CACCTCTGCT TTCGGCTAAA CCTCTCCCTA GTTTAAGGTA CGCCCCGGCC SsSDG3i ---------- ---------- ---------- ---------- ---------- 401 450 GWF CCGGGGATGT TGACTGACCC AAGTCCGTCA CTTCACTTGT GTTATGTAGA SsSDG3i ---------- ---------- ---------- ---------- ---------- 451 500 GWF TCGGCGCGGC CCAGGCGCTC GCAAGGGAAT CGTACTTCAT GGCTATCGAT SsSDG3i --GGCGCGGC CCAGGCGCTC GCAAGGGAAT CGTACTTCAT GGCTATCGAT 501 550 GWF GGTAAGGAGT ATTTTCCTTT GTTGCTGCTG CATGTAGAAC ACGTTCTTGA SsSDG3i ---------- ---------- ---------- ---------- ---------- 551 600 GWF TTACTCTGCT CACGGAACGG GTGTTGCTGT GACTGACAGC AGGGGAGGGA SsSDG3i ---------- ---------- ---------- --------GC AGGGGAGGGA 601 650 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i GGGTCTGTGT CAGGCAGCAC CGGCTCGAAG AAGCGGTCTT TCCCGGACCT 651 700 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i CAATGGCTCG AACAGTAGCA AGTCGGCTGA GGCCTTGGTG AAGAACGTTA 701 750 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i GCAAGGAGTC GTTGCAGTGG GGGCTTGCGG CTGGGGTGCA TTCCGGCCTG 751 800 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i ACCTACGGCC TCACGGAGGT GCGCGGGACG CACGACTGGA GGAACAGCGT 801 850 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i GGTGGCCGGC GCCGTCACGG GCGCCGCGGT TGCGCTGACG TCGGACCGTG 851 900 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i CGTCGCACGA GCAGGTCGTG CAGTGCGCCA TCGTCGGCGC CGCGTTGTCC 901 950 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i ACGGCGGCCA ACGTCCTCTC CGGCGTATTC TGAGCACCCT TTCCCGGGGC

57

951 1000 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i CTAAGCGTTA GAATGGCGTG CGCCAATTGA GGTTCGGAAC TATAGTAGTT 1001 1050 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i TGTAATGGCA AACCATCATA TTGTATCCCT TTTCATGATA AATTTGGAAT 1051 1100 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i GTCGTACCCA TGCTTTCAGC CTTCCTGTTA TTGACTAGTG GGTGTTATTT 1101 1150 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i ACGCTACATG GTACTATCAT ATTAAATGGA CGTATGATAG TGCTCAAGAG 1151 1200 GWF ---------- ---------- ---------- ---------- ---------- SsSDG3i TTCATTACTG TCATCATCTA TTCTTATTGT GAATCTATTC TTATTGTGAG 1201 1244 GWF ---------- ---------- ---------- ---------- ---- SsSDG3i AATAAACACA ACTGTTATTT TGGCCAAAAA AAAAAAAAAA AAAA Figure 3.4: SDG3i sequence alignment from genome walking experiment Alignment of DNA sequences obtained through genome walking (GWF) and the known partial

cDNA sequence of SDG3i (Accession number: 268212). Homology between the known

sequence and genome walking generated sequence is shaded grey, TATA box and ATG start

codon are in bold and underlined. The gap between bases 501-588 was predicted to be an

intron. This alignment was performed using CLUSTALW (Thompson et al. 1994)

bioinformatics program.

58

A: 1 50 SDG3i GW CGGTGTTTAC TATAGGGCAC GCGTGGTCGA CGGCCCGGGC TGGTCTGCTC SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA CGGTGTTTAC TATAGGGCAC GCGTGGTCGA CGGCCCGGGC TGGTCTGCTC TATA box 51 100 SDG3i GW CTCCCAAGTC TTTTTGTCCT TCAGCGCGTC GCGTAATGTT TATCTAGTGA SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA CTCCCAAGTC TTTTTGTCCT TCAGCGCGTC GCGTAATGTT TATCTAGTGA 101 150 SDG3i GW CACAGCCGCA CTGCCGTGGC CAGTGTGCGC CTCAGGATAG CAGCAGAGAG SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA CACAGCCGCA CTGCCGTGGC CAGTGTGCGC CTCAGGATAG CAGCAGAGAG 151 200 SDG3i GW GAGGAGAGCA GAGCAGGGAA GCGAGGGGAA GATGAGCAAC TTGGAGAACC SDG3i RT ---------- ---------- --------AA GATGAGCAAC TTGGAGAACC SDG3i gDNA GAGGAGAGCA GAGCAGGGAA GCGAGGGGAA GATGAGCAAC TTGGAGAACC Kozak sequence 201 250 SDG3i GW AGGCGCGGGG GTTCGTGGAC GACGTCCACG ACTGCTGGGA GCAGAAGAAG SDG3i RT AGGCGCGGGG GTTCGTGGAC GACGTCCACG ACTGCTGGGA GCAGAAGAAG SDG3i gDNA AGGCGCGGGG GTTCGTGGAC GACGTCCACG ACTGCTGGGA GCAGAAGAAG 251 300 SDG3i GW AACTGGATGC TGGACCTCGG CCACCCGCTC CTCAACCGCA TCGCCGATAG SDG3i RT AACTGGATGC TGGACCTCGG CCACCCGCTC CTCAACCGCA TCGCCGATAG SDG3i gDNA AACTGGATGC TGGACCTCGG CCACCCGCTC CTCAACCGCA TCGCCGATAG 301 350 SDG3i GW CTTCGTCAAG GCTGCCGGGG TACGTAGTGC TAGTGCTGCT GTCTTCTGTC SDG3i RT CTTCGTCAAG GCTGCCGGG- ---------- ---------- ---------- SDG3i gDNA CTTCGTCAAG GCTGCCGGGG TACGTAGTGC TAGTGCTGCT GTCTTCTGTC 351 400 SDG3i GW CACCTCTGCT TTCGGCTAAA CCTCTCCCTA GTTTAAGGTA CGCCCCGGCC SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA CACCTCTGCT TTCGGCTAAA CCTCTCCCTA GTTTAAGGTA CGCCCCGGCC 401 450 SDG3i GW CCGGGGATGT TGACTGACCC AAGTCCGTCA CTTCACTTGT GTTATGTAGA SDG3i RT ---------- ---------- ---------- ---------- ---------A SDG3i gDNA CCGGGGATGT TGACTGACCC AAGTCCGTCA CTTCACTTGT GTTATGTAGA 451 500 SDG3i GW TCGGCGCGGC CCAGGCGCTC GCAAGGGAAT CGTACTTCAT GGCTATCGAT SDG3i RT TCGGCGCGGC CCAGGCGCTC GCAAGGGAAT CGTACTTCAT GGCTATCGAT SDG3i gDNA TCGGCGCGGC CCAGGCGCTC GCAAGGGAAT CGTACTTCAT GGCTATCGAT 501 550 SDG3i GW GGTAAGGAGT ATTTTCCTTT GTTGCTGCTG CATGTAGAAC ACGTTCTTGA SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA GGTAAGGAGT ATTTTCCTTT GTTGCTGCTG CATGTAGAAC ACGTTCTTGA 551 600 SDG3i GW TTACTCTGCT CACGGAACGG GTGTTGCTGT GACTGACAGC AGGGGAGGGA SDG3i RT ---------- ---------- ---------- --------GC AGGGGAGGGA SDG3i gDNA TTACTCTGCT CACGGAACGG GTGTTGCTGT GACTGACAGC AGGGGAGGGA 601 650 SDG3i GW GGGTCTGTGT CAGGCAGCAC CGGCTCGAAG AAGCGGTCT- ---------- SDG3i RT GGGTCTGTGT CAGGCAGCAC CGGCTCGAAG AAGCGGTCTT TCCCGGACCT SDG3i gDNA GGGTCTGTGT CAGGCAGCAC CGGCTCGAAG AAGCGGTCTT TCCCGGACCT 651 700 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT CAATGGCTCG AACAGTAGCA AGTCGGCTGA GGCCTTGGTG AAGAACGTTA SDG3i gDNA CAATGGCTCG AACAGTAGCA AGTCGGCTGA GGCCTTGGTG AAGAACGTTA

59

701 750 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT GCAAGGAGTC GTTGCAGTGG G--------- ---------- ---------- SDG3i gDNA GCAAGGAGTC GTTGCAGTGG GGTATACTCA TACTATTTCT GAACTTTCAT 751 800 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA TGCAGACTTG GCTTCTCGCC TCTTCTCACG ACAAGTGACT GACCCTCGCT 801 850 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- -----GGCTT GCGGCTGGGG TGCATTCCGG SDG3i gDNA GCCTGACTGA TCTTATGCGA TGCAGGGCTT GCGGCTGGGG TGCATTCCGG 851 900 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT CCTGACCTAC GGCCTCACGG AGGTGCGCGG GACGCACGAC TGGAGGAACA SDG3i gDNA CCTGACCTAC GGCCTCACGG AGGTGCGCGG GACGCACGAC TGGAGGAACA 901 950 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT GCGTGGTGGC CGGCGCCGTC ACGGGCGCCG CGGTTGCGCT GACGTCGGAC SDG3i gDNA GCGTGGTGGC CGGCGCCGTC ACGGGCGCCG CGGTTGCGCT GACGTCGGAC 951 1000 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT CGTGCGTCGC ACGAGCAGGT CGTGCAGTGC GCCATCGTCG GCGCCGCGTT SDG3i gDNA CGTGCGTCGC ACGAGCAGGT CGTGCAGTGC GCCATCGTCG GCGCCGCGTT 1001 1051 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT GTCCACGGCG GCCAACGTCC TCTCCGGCGT ATTCTGAGCA CCCTTTC--- SDG3i gDNA GTCCACGGCG GCCAACGTCC TCTCCGGCGT ATTCTGAGCA CCCTTTCCCG 1051 1100 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA GGGCCTAAGC GTTAGAATGG CGTGCGCCAA TTGAGGTTCG GAACTATAGT 1101 1150 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA AGTTTGTAAT GGCAAACCAT CATATTGTAT CCCTTTTCAT GATAAATTTG 1151 1200 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA GAATGTCGTA CCCATGCTTT CAGCCTTCCT GTTaTTGACT AGTGGGTGTT 1201 1250 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA ATTTACGCtA CATGGTACTA TCATATTAAA TGGACGTATG ATAGTGCTCA 1251 1300 SDG3i GW ---------- ---------- ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- ---------- ---------- SDG3i gDNA AGAGTTCATT ACTGTCATCA TCTATTCTTA TTGTGAATCT ATTCTTATTG 1301 1330 SDG3i GW ---------- ---------- ---------- SDG3i RT ---------- ---------- ---------- SDG3i gDNA TGAGAATAAA CACAACTGTT ATTTTGGCC Poly-A signal

60

B:

Figure 3.5: SDG3i gene structure in S. stapfianus Alignment of predicted SDG3i coding region from genome walking (SDG3i GW), RT-PCR

confirmed coding region (SDG3i RT), and genomic DNA sequence (SDG3i gDNA) (A) and

predicted gene structure of SDG3i (B). Features highlighted in A were predicted using the

Gene Runner software v3.05, and intron/exons were predicted using the GeneMark algorithm

(exons highlighted yellow) (Lomsadze et al. 2005). Start and stop codons are in bold and

underlined.

ATG TGA 5’ sequence

3’ sequence

Exons

Introns

0bp 1500bp

534bp

103bp 83bp 129bp

61

Southern blotting was used to analyse the copy number of the SDG3i gene in S.

stapfianus. The probing of a Southern blot of digested S. stapfianus gDNA with the

SDG3i CDS at high stringency reveals only a few bands in the genome (figure 3.6a).

In the BamHI lane, one band corresponding to ~3800bp in size is present, while an

absence of banding is seen in the DraI lane. As these lanes show the digestion of the

S. stapfianus genome that will not cut the known SDG3i sequence, it suggests that the

copy number of SDG3i is low. Interestingly, the StuI digest, known to cut within the

SDG3i genomic sequence once, reveals multiple bands of sizes ranging between

~1000bp-6000bp. The multiple bands may represent a partial digest or may indicate

that homology between SDG3i and other sequences in S. stapfianus is likely and that

SDG3i may belong to a small gene family. A partial StuI digest seems more likely as

in the ClaI digest only one band at ~5000bp is observed. The 3’ UTR of SDG3i was

also used as a probe and in high stringency conditions only one band is seen in the

blot (Figure 3.6b). Like the ORF probed blots, this suggests that SDG3i is a low copy

number gene in the S. stapfianus genome. The Southern blots were repeated in low

stringency conditions, which showed smearing, and no additional information was

obtained (data not shown).


S. stapfianus gDNA was digested with 4 enzymes, BamHI, DraI (which do not cut the SDG3i

sequence), ClaI and StuI (which cuts SDG3i once) and probed at a high stringency with the

SDG3i ORF (A) or the SDG3i 3’ UTR (B).

62

Sequence data obtained from genome walking revealed no in-frame ATG sequences

between the putative TATA box and the proposed methionine translation start site.

Taking into account the three intronic regions in the SDG3i gene, the SDG3i amino

acid sequence was derived using a six-frame translation tool (EMBOSS Sixpack)

(appendix I). The most likely ORF of 177 amino acids contains a Tim17-like domain

found in the inner mitochondrial membrane translocase. This polypeptide motif

contains three transmembrane helicies (Figure 3.7) and in mitochondrial proteins

forms part of a tonoplast inner membrane channel protein involved in protein import

into the mitochondria (Murcha et al. 2005). The amino acid sequence of SDG3i was

compared with other plant proteins in the NCBI database and showed that SDG3i has

high homology to plant pore proteins. The homologous sequences were aligned and

are presented in Figure 3.8. There is strong homology of SDG3i to pore proteins from

Bromus inermis and the resurrection plant Xerophyta humilis. SDG3i also has some

similarity to the chloroplastic outermembrane channel proteins from Pisum sativum

and Hordeum vulgare as well as sequence similarity to hypothetical proteins from rice

and Arabidopsis. However, the strongest sequence similarity is to the bromegrass B.

inermis sequence which has recently been identified as encoding a peroxisome

channel protein (Wu et al. 2005).

MSNLENQARGFVDDVHDCWEQKKNWMLDLGHPLLNRIADSFVKAAGIGAAQALARES YFMAIDAGEGGSVSGSTGSKKRSFPDLNGSNSSKSAEALVKNVSKESLQWGLAAGVH SGLTYGLTEVRGTHDWRNSVVAGAVTGAAVALTSDRASHEQVVQCAIVGAALSTAAN VLSGVF* Figure 3.7: Amino acid motifs in SDG3i

This figure shows the predicted amino acid sequence of SDG3i containing a Tim17-like

domain that is present in the 3’ region (shaded grey). This was predicted using the Pfam

algorithm (www.pfam.sanger.ac.uk). Three transmembrane helices predicted to occur within

the TIM17 domain have been underlined and bolded. Asterisk indicates the C-terminus of the

protein.

63

Figure 3.8: Amino acid sequence alignment of predicted full SDG3i sequence to known

proteins in the NCBI database.

Alignment showing the homology between the full length SDG3i sequence (SDG3i) and the

most similar known pore proteins in the NCBI database from the plants Bromus inermis

(BiPore) (Accession: AAL23749.1), Xerophyta humilis (XhPore) (Accession: AAV84280.1),

Orzya sativa (OsPore) (Accession: BAB20636.1), Hordeum vulgare (HvPore) (Accession:

CAA09867.1), Arabidopsis thaliana (AtPore) (Accession: NP180456) and Pisum sativum

(PsPore) (Accession: AEV46836.1). Dashes indicate gaps inserted in the sequence to

facilitate alignment. Homology between residues is indicated with black (consensus) and grey

(similar residues). The alignment was completed using T-Coffee (www.tcoffee.org). Asterisk

indicates the C-terminal end of the protein sequence.

64

3.3.2 Sequence analysis of SDG4i

The cDNA sequence of SDG4i and the putative protein product encoded by SDG4i

has been previously identified (Le et al., 2007). The SDG4i protein contains a

putative nuclear localisation signal indicating that SDG4i maybe localised to the

nucleus. In addition, the predicted protein contains a serine rich region which suggests

that SDG4i may be involved in a protein-protein interaction (Figure 3.9). A BLASTn

search of the putataive SDG4i amino acid sequence against the NCBI database reveals

homology of SDG4i to a wound induced Medicago truncatula protein, MTD1, of

unknown function as well as similarity to a protein in the rice genome and to the

protein bnKCP1 from Brassica napus (Figure 3.10) In M. truncatula the MTD1

transcript increases following plant decapitation and fungal infection, indicating the

MTD1 gene may have a function during pathogen attack. The bnKCP1 protein has

been shown to interact with histone deacetylase HDA19 from Arabidopsis.

MPVDVARASVFEVAMPASACSSSSIGKDSDECTPPGKEDEEVQSAYMGEKGGAGEEG

GLVGLEALEEALPIRRSISKFYSGKSKSFSCLKEAVTSSGSAKDISKAENAYSRKRK

NLLVYSIMYENSNETATAEVFETGPPKRPSSLSRSSLVTMASSSSRSSSSFSIEDNQ

LHEQLHYPCSPDHSENCGPPKSPIPPPASCAYRTPSAPMTAMRSFSMMDLPGLHSSS

SSVCLKDKKVDS*


The predicted nuclear localisation signal within the SDG4i amino acid sequence is shaded

grey. The serine rich region that predicts a protein-protein interaction is underlined and

bolded. These features were predicted using the WoLF PSORT algorithm

(http://wolfpsort.seq.cbrc.jp/). Asterisk indicates the C-terminus of the protein.

65

Figure 3.10: Amino acid sequence alignment of SDG4i sequence to known proteins in

the NCBI database.

Alignment showing the homology of SDG4i (Accession: CAK26793.1) to other plant proteins

in the NCBI database: a hypothetical protein from Orzya sativa (Rice) (Accession:

NP001061970.1), and known proteins in Medicago truncatula (MTD1) (Accession:

AAF86687.1) and Brassica napus (bnKCP1) (Accession: AAO53442.1). Dashes indicate

gaps inserted in the sequence to facilitate alignment. Homology between residues is indicated

with black (consensus) and grey (similar residues). The alignment was completed using T-

Coffee (www.tcoffee.org). Asterisk indicates the C-terminal end of the protein sequence.

66

To identify introns in the SDG4i sequence, SDG4i was amplified using PCR based on

primers derived from the known cDNA sequence. Figure 3.11a shows the sequence

alignment between SDG4i cDNA sequence and the SDG4i genomic sequence. No

differences between the cDNA and genomic sequence were found indicating that

there are no introns present in this gene. These results are also represented pictorially

in Figure 3.11b. Nucelotides around the ATG conform to Kozak sequences suggesting

that the ATG indicated encodes the initiating methionine.

A: 1 Translation initiation sequence 50

SDG4i cDNA CCCGGTCGGA CGGATGCCGG TTGACGTCGC ACGCGCCTCC GTCTTCGAGG SDG4i gDNA ---------- --GATGCCGG TTGACGTCGC ACGCGCCTCC GTCTTCGAGG 51 100 SDG4i cDNA TGGCGATGCC GGCGTCGGCG TGCAGCTCGT CGTCGATAGG GAAGGACAGC SDG4i gDNA TGGCGATGCC GGCGTCGGCG TGCAGCTCGT CGTCGATAGG GAAGGACAGC

101 150 SDG4i cDNA GACGAGTGCA CACCGCCGGG GAAGGAGGAT GAGGAGGTGC AGAGCGCGTA SDG4i gDNA GACGAGTGCA CACCGCCGGG GAAGGAGGAT GAGGAGGTGC AGAGCGCGTA

151 200 SDG4i cDNA CATGGGAGAG AAAGGCGGAG CAGGGGAAGA GGGCGGCCTT GTGGGATTGG SDG4i gDNA CATGGGAGAG AAAGGCGGAG CAGGGGAAGA GGGCGGCCTT GTGGGATTGG

201 250 SDG4i cDNA AGGCATTAGA GGAGGCGCTT CCCATCAGAC GTAGCATCTC AAAATTCTAC SDG4i gDNA AGGCATTAGA GGAGGCGCTT CCCATCAGAC GTAGCATCTC AAAATTCTAC

251 300 SDG4i cDNA AGTGGTAAAT CCAAATCATT TTCTTGCCTC AAAGAAGCTG TTACTTCTTC SDG4i gDNA AGTGGTAAAT CCAAATCATT TTCTTGCCTC AAAGAAGCTG TTACTTCTTC

301 350 SDG4i cDNA TGGCTCTGCA AAGGATATCT CTAAGGCAGA GAATGCTTAC TCCAGAAAAC SDG4i gDNA TGGCTCTGCA AAGGATATCT CTAAGGCAGA GAATGCTTAC TCCAGAAAAC

351 400 SDG4i cDNA GGAAGAATCT CCTTGTCTAC AGCATCATGT ATGAAAATTC AAACGAAACA SDG4i gDNA GGAAGAATCT CCTTGTCTAC AGCATCATGT ATGAAAATTC AAACGAAACA

401 450 SDG4i cDNA GCAACAGCTG AAGTTTTTGA AACTGGACCT CCTAAAAGGC CTTCTAGTTT SDG4i gDNA GCAACAGCTG AAGTTTTTGA AACTGGACCT CCTAAAAGGC CTTCTAGTTT

451 500 SDG4i cDNA GAGCAGAAGC TCTCTTGTGA CAATGGCTAG TAGCAGTTCG AGGAGCAGCA SDG4i gDNA GAGCAGAAGC TCTCTTGTGA CAATGGCTAG TAGCAGTTCG AGGAGCAGCA

501 550 SDG4i cDNA GCAGCTTCAG CATTGAGGAT AATCAATTGC ATGAACAGCT CCATTATCCT SDG4i gDNA GCAGCTTCAG CATTGAGGAT AATCAATTGC ATGAACAGCT CCATTATCCT

551 600 SDG4i cDNA TGTTCACCTG ATCATAGTGA GAATTGTGGC CCTCCAAAGA GTCCCATACC SDG4i gDNA TGTTCACCTG ATCATAGTGA GAATTGTGGC CCTCCAAAGA GTCCCATACC

601 650 SDG4i cDNA GCCACCAGCA TCTTGCGCAT ATAGGACACC GTCTGCACCT ATGACTGCTA SDG4i gDNA GCCACCAGCA TCTTGCGCAT ATAGGACACC GTCTGCACCT ATGACTGCTA

651 700 SDG4i cDNA TGAGGTCATT CTCGATGATG GATTTGCCAG GTCTCCATAG TTCAAGTTCC SDG4i gDNA TGAGGTCATT CTCGATGATG GATTTGCCAG GTCTCCATAG TTCAAGTTCC

67

701 750 SDG4i cDNA TCGGTTTGTC TCAAAGATAA GAAGGTGGAC TCTTAGATCA GTGTTCAGAG SDG4i gDNA TCGGTTTGTC TCAAAGATAA GAAGGTGGAC TCTTAGATCA GTGTT

751 800 SDG4i cDNA GATCTTCCGT TTCCTTTTAC GAACTTGTTG CTTCTGGTTG CTCCCACCAC SDG4i gDNA

801 850 SDG4i cDNA CTTGTCTATA CTGAATTTGG CGTCAAGGTT CAGTATACAT AAGACGCTTC SDG4i gDNA

851 900 SDG4i cDNA AAGCATTGGG CTGTGTGATT GTGTGTATGT GTGTAAAATA GTCAGATCAC SDG4i gDNA

901 950 SDG4i cDNA TCAGTTGTAA ATTAGCATAA TGTACTAAAA CTGATGGTGT ATATGCTAAC SDG4i gDNA

951 1000 SDG4i cDNA CTACACTCAT GTAATTATTT TCATCTTGAA ATTGACTATC TTCTAGTCGC SDG4i gDNA 1001 1033 SDG4i cDNA TCATGAGTCA TGACCCAAAA AAAAAAAAAA AAA SDG4i gDNA

B:


Alignment of SDG4i coding region (SDG4i cDNA, accession number: AM268211) and

genomic DNA sequence (A) and predicted gene structure of SDG4i (B). Features highlighted

in A were predicted using the Gene Runner software v3.05, and potential intron/exon splice

sites were analysed using the GeneMark algorithm (exons highlighted yellow) (Lomsadze et

al. 2005). Start and stop codons are in bold and underlined.

ATG TGA 5’ sequence

3’ sequence

Exons

Introns

0bp 1000bp

723bp

68

Southern blotting was used to analyse SDG4i copy number in S. stapfianus. Digested

S. stapfianus gDNA was subject to Southern blot analysis and probed with the SDG4i

ORF (Figure 3.13). The suggestion of single bands in the lanes digested with BamHI

and DraI, which do not cut within the SDG4i sequence, in high stringency conditions

indicates that SDG4i may be a low copy number gene in S. stapfianus. Lanes

containing S. stapfianus gDNA digested with EcoRV or StuI reveal multiple bands

suggest incomplete digestion. This banding pattern also suggests that SDG4i is a low

copy number gene in S. stapfianus. The Southern blot was also probed with the

SDG4i ORF in low stringency conditions, as well has the SDG4i 3’ UTR in both low

and high stringency conditions. These blots showed smearing and no further

information was obtained (data not shown).


S. stapfianus gDNA was digested with 4 enzymes, BamHI, DraI (which do not cut the SDG4

sequence), EcoRV and StuI (which cuts SDG4i once) and probed at a high stringency with

the SDG3i ORF.

69

3.3.3 Intracellular localisation of SDG4i Bioinformatic analysis suggested that the SDG4i protein may contain a nuclear

localisation signal. GFP fusion constructs were generated to observe the intracellular

localisation of SDG4i in A. cepa (onion) epidermal cells. Confirmation of nuclear

localisation was obtained using these GFP fusion constructs (Figure 3.13). Both the

N-terminal and C-terminal GFP fusion of SDG4i can be seen to be present in the

nucleus (Figure 3.13A, B). Some GFP can also be seen in the cytoplasm of A. cepa

epidermal cells using these constructs. A control construct containing GFP-only can

be seen to be confined to the cytoplasm (Figure 3.13C).

Figure 3.13: SDG4i intracellular localisation

Localisation of SDG4i with an N-terminal GFP fusion (A), a C-terminal GFP fusion (B), and

35S:GFP control (C) in A. cepa epidermis.

100µm 100µm

50µm

50µm

50µm

50µm

UV Light

A: GFP-SDG4i

B: SDG4i-GFP

C: 35S:GFP

70

3.3.4 Transcript accumulation of dehydration-related genes during dehydration and rehydration

Transcript levels of SDG3i, SDG4i and SDG8i during the dehydration of S. stapfianus

leaf tissue was examined in the publication by Le et al. (2007), however, transcript

levels during rehydration has not been investigated. Figure 3.14 shows the transcript

levels of these genes during various stages of dehydration and rehydration. The leaf

transcript levels have been quantified relative to the amount of 28S rRNA transferred

to the filter. The transcripts of SDG3i appear to be present in fully hydrated tissue at a

low level, and gradually increase to the highest relative abundance at 59-40% RWC

(Figure 3.13A, B). The transcripts then decrease in abundance and remain at a low

level in air dried tissue (<10% RWC). After rehydration the transcripts appear to be

maintained at low levels at 12, 24 and 48 hours post watering (Figure 3.14A, B)

SDG4i transcripts are present at very low levels in fully hydrated and mildly

droughted tissue, however, begin to increase in abundance at 79-60% RWC and peak

at 19-10% RWC (Figure 3.13A, C). After this peak, the transcript abundance then

falls slowly during rehydration. Quantification suggests the transcript may still be

slightly elevated at 48 hours post watering as compared to the levels present before

plant treatment (Figure 3.14A, C). SDG8i transcripts accumulate later than other

transcripts and are present at a minimal level in fully hydrated and mildly droughted

tissue and begin to increase in abundance at 59-40% RWC. The peak relative level of

transcripts is observed at 39-20% RWC, then decreases upon further dehydration

(Figure 3.14A, D). The results suggest a low level of SDG8i transcript may be present

during rehydration.

71

Figure 3.14: Transcript accumulation of SDG3i, SDG4i and SDG8i during the

dehydration and rehydration of S. stapfianus

Phosphoimages of SDG3i, SDG4i and SDG8i transcript accumulation during dehydration

stages represented by relative water content (RWC) and stages of rehydration (RH) at time

intervals post-watering (A). This abundance of transcripts were then quantified relative to 28s

rRNA for SDG3i (B), SDG4i (C) and SDG8i (D).

3.3.5 Transcript accumulation of dehydration related genes after exogenous hormone application

72

To explore the effect of hormones on the induction of SDG3i, SDG4i and SDG8i

expression, northern blotting was used to detect transcript accumulation in fully

hydrated S. stapfianus plants over 24 hours of exposure to different hormones (Figure

3.15). The results show that SDG3i is induced by cytokinin, SDG4i is induced by

ABA and SDG8i responds positively to both ABA and MeJa (Figure 3.15). SDG3i,

SDG4i and SDG8i were not shown to be induced by the other tested hormones.

Transcript accumulation was quantified relative to 28S rRNA (Figure 3.16). The

accumulation of SDG4i transcripts occurs at 8 hours post application of ABA and

increased further at 24 hours post application. The transcript accumulation of SDG8i

after ABA application can be observed 1 hour post application but decreases again at

3 hours and 8 hours post application. There is the suggestion that a small second stage

of accumulation occurs at 24 post application. Cytokinin treatment induced SDG3i

transcript accumulation at 8 hours and 24 hours after application. Lastly, the

application of methyl jasmonate increases transcript abundance of SDG8i at 3 hours

post application, and decreases by the 8 hour time point, before increasing slightly at

24 hours. Whilst there is considerable variation in the 28S signal obtained from these

blots, in most instances the positive signals were obtained with equal or reduced RNA

loading in the treated samples compared with controls, suggesting the results have

some validity.

73

Figure 3.15: Transcript accumulation after exogenous hormone application in S.

stapfianus leaves.

Northern blot phosphoimages of Abscisic acid, Gibberellin (GA3), Cytokinin (Kinetin), Auxin

(IAA), Ethylene, Methyl Jasmonate and Salicylic acid treated S. stapfianus plants (leaves).

Each blot was probed with a radiolabelled SDG3i, SDG4i and SDG8i fragment, and was

quantified relative to 28S rRNA (T. astevium 28S rRNA probe). Each lane in the northern blots

represents a different time point ranging from 0 to 24 hours with (+) and without (-) exogenous

hormone application.

SDG3i

SDG4i

SDG8i

28s rRNA

Abscisic acid

0h 1h 3h 8h 24h

- + - + - + - + - Gibberellin

SDG3i

SDG4i

SDG8i

28s rRNA

0h 1h 3h 8h 24h

- + - + - + - + -

Cytokinin SDG3i

SDG4i

SDG8i

28s rRNA

0h 1h 3h 8h 24h

- - + - + - + - +

Ethylene SDG3i

SDG4i

SDG8i

28s rRNA

0h 1h 3h 8h 24h

- + - + - + - + -

Methyl Jasmonate

SDG3i

SDG4i

SDG8i

28s rRNA

0h 1h 3h 8h 24h

- + - + - + - + -

Salicylic acid

SDG3i

SDG4i

SDG8i

28s rRNA

0h 1h 3h 8h 24h

- + - + - + - + -

SDG3i

SDG4i

SDG8i

28s rRNA

Auxin

0h 1h 3h 8h 24h

- + - + - + - + -

74

Figure 3.16: Relative expression levels of SDG3i, SDG4i and SDG8i transcripts in

hormone treated tissues.

Quantification of SDG3i, SDG4i and SDG8i transcripts relative to 28S rRNA in hormone

treated tissues of S. stapfianus that gave a positive result from figure 3.16. SDG4i transcript

accumulation post ABA treatment (A), SDG3i transcript accumulation post cytokinin

treatment, (B), SDG8i transcript accumulation post abscisic acid (ABA) treatment and SDG8i

transcript accumulation post methyl jasmonate (MeJa) treatment (D).

75

3.4 Discussion

3.4.1 SDG3i, SDG4i and SDG8i transcript accumulation during dehydration and rehydration

In response to desiccation S. stapfianus alters the metabolism of sugar and other

molecules within the cell in a manner thought to be unique, or peculiar to resurrection

plants. Changes in metabolism allow cells to vitrify, and enter a state of suspended

animation without major damage (Martinelli 2008). Several genes have been

identified in resurrection plants that may have a role in this process (Oliver et al.,

2010). Previous study on gene expression in S. stapfianus during dehydration showed

that the expression of SDG3i, SDG4i and SDG8i were up-regulated in leaf tissue

during dehydration (Le et al., 2007). The expression of these genes in leaf tissue

during dehydration and rehydration where examined in this chapter. The increased

expression of SDG3i, SDG4i at approximately 60% RWC correlates with the

induction of the desiccation tolerance program (Gaff et al., 1997) and supports

evidence of expression during dehydration in the previous study (Le et al., 2007),

whilst SDG8i expression is elevated at a slightly later stage, which was not found in

previous work. New evidence of expression of these genes has been noted in this

chapter where it was shown that the expression of SDG4i and SDG8i remain elevated

during the early rehydration stages. SDG3i transcript levels peak during the mid-

stages of drying then decreases to the levels found in fully hydrated plants. Together

this evidence suggests that these genes may be important for cellular survival both

during the stages of dehydration and rehydration.

While the study into the accumulation of transcripts is an important element into

deciphering the requirements of S. stapfianus for the activity of these genes, the

dehydration/rehydration stage at which these gene transcripts are being translated to

form a biologically active protein products is yet to be determined. It is possible that

transcripts are being produced and stored during dehydration, as the gene products

may be required for rapid plant growth immediately following rehydration. Western

analysis could be undertaken to determine when translation of these SDG transcripts

occurs. Two attempts have been made to produce antibodies to peptides from SDG8i,

however, the peptides showed low antigenicity and the antibodies produced cross-

reacted non-specifically to S. stapfianus protein extracts. In addition, while SDG4i

76

antibodies were generated, an proved successful in Arabidopsis SDG4i transgenic

extracts (chapter 4), they produced a lot of background in S. stapfianus extracts. In

addition the transcript accumulation of dehydration-related genes has only been

examined in the leaves, which indicates the requirement for their gene products in this

area of the plant. The root tissue of any plant is important for the sequestration of

water and it would be beneficial to determine if the SDG proteins have a function in

the roots of S. stapfianus.

3.4.2: SDG3i has strong homology to plant pore proteins and expression is induced by exogenous cytokinin application

The study by Le et al. (2007) provided a partial sequence of SDG3i and the full

sequence was required so a functional analysis of the gene could be undertaken.

Genome walking provided the upstream region of SDG3i, revealing the complete

coding sequence. The genomic sequence of SDG3i indicates the gene contains three

introns and four exons. Southern blotting has shown that SDG3i does not appear to

part of a large gene family. The SDG3i protein has homology to pore proteins that

have been isolated from non-resurrection plants. Computational analysis identified a

putative TIM17-like domain in the 3’ region of SDG3i and the transmembrane

helicies associated with the TIM17 domain were found to be present in SDG3i.

TIM17 is an essential component of the TIM23 complex which mediates the

translocation of transit peptides containing proteins across the mitochondrial inner

membrane. Recently, research has shown that TIM17 in plants may have the ability to

bind mRNA and DNA (Carrie et al., 2010). This suggests that TIM17 and TIM17-like

proteins may have a role in the binding of nucleic acids and importing nucleic acids

into the mitochondria. This could be an important factor in allowing desiccation-

related mitochondrial proteins to be transported into the organelle during dehydration

or possible storage of desiccation-related transcripts in the mitochondria for

translation post-rehydration. The role of mitochondria in facilitating desiccation

tolerance is unknown, however, in drought tolerant species the mitochondria is said to

have channels that are activated by reactive oxygen species (ROS) which may act to

decrease ROS production, and dissipate energy by reoxidation of NADH during stress

77

conditions (Pastore et al., 2007). It’s possible that SDG3i may be creating channels

across the mitochondrial membrane to facilitate this process.

Interestingly, SDG3i shows homology with other pore proteins associated with the

peroxisome. Peroxisomes are able to differentiate into three different classes

depending on the cell type and are organelles responsible for the

production/degradation of ROS and fatty acid metabolism (Mano & Nishimura 2002).

These classes of peroxisomes are leaf peroxisomes, glyoxysomes or unspecialised

peroxisomes. The differentiation of peroxisomes depends on both the transcription of

nuclear genes and the import of proteins into the peroxisome (Minorsky 2002). The

role of perioxisomes in drought-related processes is not well understood, however, it

has been suggested that they play a role in ROS scavenging during drought (reviewed

in de Carvalho et al. 2004). Environmental stresses have been shown to increase the

production of ROS in peroxisomes while inhibiting antioxidant mechanisms (del Rio

et al., 1996). More recently, peroxisomal polyamine oxidase has been shown to

contribute to the regulation of drought-responsive genes (Kamada-Nobusada et al.,

2008). Little is known about the role of peroxisomes in desiccation tolerance. It is

possible that transcriptional changes associated with desiccation may be influencing

the function of perosisomes in ROS production and scavenging or inducing changes

in fatty acid metabolism. The SDG3i protein product has homology to a B. inermis

pore protein identified as a peroxisome channel protein. This protein has been

suggested to be associated with succinate export or fatty acid import from the

peroxisome during desiccation allowing for increased sugar production (Wu et al.,

2005). The breakdown of fatty acids and increase in sugar is similar to that seen in

seed undergoing desiccation (reviewed in le Prince et al., 1993). Many studies on

desiccation tolerance show that the accumulation of sugars during desiccation allows

for plant survival under extreme conditions (le Prince et al., 1997, Hoekstra et al.,

2001). This could mean that SDG3i may have a role in providing stability of cellular

components during desiccation of S. stapfianus. Ghasempour et al., (1996) identified

that a sharp increase in the leaf sugar pool of S. stapfianus during desiccation occurs

at mild dehydration stages, where the plant begins to produce glucose, fructose and

sucrose. Glucose and fructose content peaks at 50% RWC. From this point the

production of sucrose accelerates and sucrose become the predominant sugar in

78

desiccated leaves (<10% RWC). The expression pattern of SDG3i shows that its

expression is high through the initial stages of desiccation, with highest expression at

59-40% RWC. This expression pattern of SDG3i correlates with the increase in leaf

sugar pools during the initial stages of desiccation. The correlation between leaf sugar

pool content and SDG3i raises the possibility that SDG3i may be acting to aid in

sugar production during desiccation. To investigate these possibilities it would be

useful to look at the localisation of SDG3i in the cell by expressing SDG3i-GFP

constructs in A. thaliana or Nicotiana benthamiana tissues as well as generating

transgenic A. thaliana over-expressing SDG3i and measuring the content of cellular

sugars.

SDG3i appears to be induced by exogenous cytokinin application with transcripts

appearing at 8 hours post-application, the highest increase in relative abundance is

observed 24 hours post hormone application. Plants may increase levels of cytokinins

within their tissues to antagonise the reduced growth rate caused by increased

intracellular levels of ABA during osmotic stress (Vyroubalova et al., 2009).

Cytokinins have been shown to interact with light and sugars in the regulation of

senescence (Wingler et al., 1998) and in general, an increased abundance of

cytokinins delay senescence onset, and can provide increased drought tolerance to the

plant (Rivero et al., 2007). Evasion of drought-related senescence could be considered

an important aspect in the development of desiccation tolerance. It is possible that in

the later stages of desiccation, cytokinins accumulate within the cellular tissues to

delay senescence of desiccating tissues, and in turn up-regulate desiccation tolerance

genes such as SDG3i, to aid in the accumulation of sugars, preparing the leaf tissues

for desiccation. It must be noted, that the evidence gained from hormone treatments in

this chapter are speculative, and the physiological response of S. stapfianus to

exogenous hormones may not reflect the same response as endogenously secreted

hormones. More work looking into the endogenous content of plant hormones during

the dehydration/rehydration cycle is key to understanding this regulation.

79

3.4.3 SDG4i has strong homology to bnKCP1 and expression is induced by exogenous ABA application

The SDG4i amino acid sequence previously isolated and reported by Le et al., (2007)

shows homology with the annotated sequence bnKCP1, a cold stress inducible protein

that interacts with HDA19 and may act as a transcription factor to regulate gene

expression in B. napus (Gao et al., 2003). SDG4i also shows strong homology to a

hypothetical protein in rice, and MTD1 in M. truncatula which is a transcript up-

regulated in nodules after wounding by decapitation (Curioni et al., 2000). SDG4i

contains a serine rich region which indicates a possible protein-protein interaction.

The SDG4i protein product was shown to be localised to the nucleus and cytoplasm of

onion epidermal peels using SDG4i-GFP fusion constructs.

The drought stress-related expression pattern and localisation of SDG4i to the nucleus

suggests the protein may have a role in controlling gene expression during plant cell

drought stress response. The interaction of bnKCP1 with HDA19 suggests that

bnKCP1 has a role in regulating gene expression via chromatin remodelling (Gao et

al., 2003). In Arabidopsis, HDA19 has been associated with plant responses to

environmental stress (Zhou et al., 2005), and HDA19 may interact with a

transcription factor, AtERF7, in response to ABA and drought stress, which modifies

ABA responses (Song et al., 2005). It is yet to be determined if SDG4i interacts with

HDA19, however, this homology raises the possibility that SDG4i may affect gene

expression during dehydration. Deacetylation is associated with repressing gene

expression during plant development and environmental changes (Tian et al., 2005),

therefore, SDG4i may be repressing HDA19 activity or may be down-regulating

genes whose expression is detrimental to surviving dehydration.

Many drought-stress related genes are also up-regulated in response to exogenous

ABA application (Busk and Pages 1998, Shinozaki & Yamaguchi-Shinozaki 1997).

Transcripts of SDG4i appear to show no substantial increase in abundance at 1 and 3

hours post ABA application, but a high level of expression is seen at 8 hours and 24

hours in fully hydrated leaves. This may mean that SDG4i requires earlier ABA

responsive genes to drive SDG4i expression, such as the translation of a specific ABA

80

transcription factors. This hypothesis could be tested using a protein synthesis

inhibitor such as cyclohexamide, at specific stages post-ABA application, to observe

its effects on SDG4i expression. In addition, SDG4i expression is induced in

dehydration S. stapfianus at 79-60% RWC. This appears to be before the substantial

increase in endogenous ABA levels (Gaff & Loveys 1984) and suggests that SDG4i

may also be induced by an ABA-independent dehydration pathway at an earlier stage

of RWC.

3.4.4 SDG8i expression is induced by exogenous application of abscisic acid and methyl jasmonate

Previous work indicated that SDG8i is a UDP-glucosyltransferase and was reported to

be induced in an ABA-independent manner, as increased expression at 3 hours and 8

hours post ABA treatment was not observed (Le 2004). However, SDG8i shows an

interesting induction pattern with transient peaks in expression induced by exogenous

hormone application. The ABA transient peak occurs at 1 hour, and the methyl

jasmonate transcript peak occurs at 3 hours post-treatment. Recent studies have

suggested that jasmonate signal transduction is integrated into an elaborate network of

signalling, and helps mediate stress signals, including the induction of ABA

biosynthesis (Kim et al., 2009). Microarray analysis of Arabidopsis genes indicated

almost 200 genes were regulated by both drought and JA (jasmonic acid), of which

over 100 were also ABA-regulated (Huang et al., 2008). Within a few hours of

drought stress in citrus roots, endogenous JA concentration increases, and the onset of

ABA accumulation occurs immediately after JA accumulation, and it was concluded

that early JA accumulation was required for the subsequent ABA accumulation (De

Ollas et al., 2013). This suggests that the transient expression peak in SDG8i

expression, induced by methyl jasmonate at 3 hours post application, may be an effect

of this JA/ABA relationship. This suggestion is supported by the quick response (1

hour post-application) of SDG8i to ABA. The possibility for a role of jasmonic acid in

desiccation tolerance in S. stapfianus was reported in Ghasempour et al. (1998b)

where JA induced an improvement in desiccation tolerance of S. stapfianus

protoplasts. As an increase in SDG8i transcript levels does not necessarily indicate the

81

level of an increase in the SDG8i protein product, the presence of SDG8i in the cell

needs to be assessed in the first instance through western blot analysis.

82

4.0 Effect of over-expression of SDG4i on the developmental phenotype of Arabidopsis thaliana

4.1 Introduction

The introduction and analysis of gene activity in a model plant species is a useful tool

for discovering gene function. An indication of function can be obtained by observing

the phenotypic effects of over-expression of the gene, preferably in the plant that the

gene has been isolated from, or in a closely related species that has been well

characterised. S. stapfianus has no known method for transformation, so the analysis

of SDG4i over-expression or knockout is not possible in this species. Hence, this

study focussed on the introduction of SDG4i into the monocot Sorghum bicolor and

the dicot Arabidopsis thaliana. The transformation and regeneration of S. bicolor is

laborious and technically difficult. The method that is widely accepted for the

transformation of S. bicolor involves inducing callus from immature embryos, and

employing either a biolistic or Agrobacterium tumerfaciens approach to transforming

the callus (Gurel et al. 2009, Liu & Godwin 2012). Due to the limitations of growing

S. bicolor to produce seed and immature embryos in southern regions of Australia, a

new protocol was to be developed based on a rapid, high-throughput method of rice

transformation (Toki et al., 2006) that had worked successfully on rice in the

laboratory at Monash University in Melbourne. In addition, the model plant A.

thaliana was used to provide information on the phenotypic effects of SDG4i over-

expression during the development of the plant in both long day and short day

conditions. The effects on growth and yield in Arabidopsis SDG4i transgenic plants

were examined in detail.

83


4.2.1 Callus induction and tissue regeneration in S. bicolor

Mature seed from three varieties of Sorghum: S. sudanese, S. bicolor (82532) and a S.

sudanese/S. bicolor (paternal/maternal) cross “Sweet Jumbo” provided by Pacific

Seeds (Australia), were used to generate callus using a modification of the seed-based

callus generation protocol from rice (Toki et al. 2006). The protocol was modified by

placing the seeds on N6D media containing 4mg L-1 2,4-dichlorophenoxy acetic acid

(2,4-D) and callus was grown in the dark at 32ºC or 28ºC for 2 weeks. The addition of

tryptophan (50mgL-1) to the media to induce the growth of embryogenic callus was

utilised (Siriwardana & Nabors 1983).

Embryogenic callus that had proliferated from the seed was then cut away and placed

on regeneration medium containing a range of concentrations of indole acetic acid

(IAA) (0-2.5mg L-1) and kinetin (0-2.5mg L-1) to determine the best conditions for

inducing root and shoot formation. The media was supplemented with 150ug L-1 citric

acid, 100µg L-1 ascorbic acid and 150µg L-1 proline to inhibit the production of

phenolics in the callus. The regeneration plates containing the calli were placed at

28ºC with 14h/10h day/night light cycles.

4.2.2 Agrobacterium mediated transformation of S. bicolor calli

The transformation of Sorghum calli from mature seed was based on a method by

Gurel et al. (2009), which used embryogenic callus obtained from Sorghum immature

embryo explants. Sorghum calli that had been induced for 2 weeks was removed from

the seed, placed in 5 mL of AAM broth and incubated at 42ºC for either 3, 5, 10 or 20

minutes. The calli was then removed and incubated at either room temperature or

placed on ice for 2 minutes. Media was drained and A. tumerfaciens GV3101

containing pBIN19-GUSINT (pBIN19 vector containing β-Glucuronidase (GUS)

with the potato IV2 intron (Szabados et al., 1990) at an OD600 of 0.5 (co-incubated for

1 hour with 200µM of acetosyringone in AAM broth) was added and the calli and

culture were left to incubate at 28ºC in the dark for 15 minutes. The A. tumerfaciens

84

broth was drained, and the calli were placed on filter paper soaked with AAM media

on AAM plates and co-cultivated for 3 days at room temperature. The calli were

subsequently rinsed with AAM media containing 200µg L-1 timentin and placed on

N6D plates containing 4µg L-1 2,4-D and 50µg L-1 kanamycin and incubated at 28ºC

in the dark for 2 weeks. After this period, callus was either transferred to regeneration

plates, or subject to GUS staining.

4.2.3 Growth conditions of A. thaliana and developmental phenotype analysis

Six individual Arabidopsis 35S:SDG4i over-expression lines (generated as described

in section 2.4.3), homozygous for the transgene, and wild-type plants two wild-type

Columbia lines and one SDG4i-like homolog knockout line (SALK_026166C,

referred to as T-AT3G43850 throughout this chapter) were sown in individual pots in

replicates of 10 (long day analysis) or 5 (short day analysis). After 3 days

stratification at 4°C in darkness the pots were placed in growth cabinets under either

long day (LD) (16h light/8h dark, 23ºC/16°C) or short day (SD) (8h light/16h dark

23ºC/16°C) conditions for the entire life cycle of the plants. Light intensity was

250µmol photons m-2 s. The plants were watered by sub irrigation every 2-3 days.

Measurements, in accordance with Boyes et al. (2001), were taken from plants in long

day conditions every 2 days, and in short day conditions every 4 days. Measurements

included: cotyledon length, rosette leaf length, rosette leaf number, appearance of

flower bud, time taken to bolt, plant height, bolts/shoots, branches, flowers, siliques,

senescence, seed yield and seed viability. Statistical analysis of the data was

completed using GraphPad Prism v6.0 software.

85

4.3 Results

4.3.1 Callus induction of Sorghum bicolor

S. bicolor calli induced on N6D medium, supplemented with 4µg ml-1 2,4-D, and

incubated at 32ºC grew more rapidly than calli induced at 28ºC (Figure 4.1). The

addition of 50mg L-1 tryptophan to the media had no effect on the growth of

embryogenic callus (data not shown). Embryogenic callus was identified as the more

brittle white callus whereas callus that appeared watery and spongy was thought to be

somatic callus. Somatic callus comprised the majority of calli formed. The

temperature at which calli was generated from seed also appeared to have an effect on

the level of embryogenic calli growth, with calli growing at 32ºC producing less

embryogenic calli than at 28ºC (Figure 4.1). However, somatic calli grown at 32ºC

did appear to proliferate faster than at 28ºC. Embryogenic callus growth from seed

was seen in both S. sudanese and S. bicolor. As S. bicolor callus appeared to

proliferate faster, this calli was chosen for further work.

Figure 4.1: Calli grown from differing varieties of Sorghum seed

Callus growth in Sorghum varieties S. sudanese, S. bicolor and the maternal/paternal cross

Sweet Jumbo at 28ºC and 32ºC.

S. sudanese S. bicolor Sweet Jumbo

28°C

32°C

86

4.3.2 Transformation of callus with GUS reporter gene

The proliferated callus containing both embryogenic and somatic callus was co-

cultivated with A. tumerfaciens containing the pBIN19-GUSINT construct, and

subsequently GUS stained to reveal the extent of transformation. The success of

transformation in different pre-treatments of callus can be seen in figure 4.2.

Treatment of the calli at 42ºC for 3 and 5 minutes followed by 2 minutes on ice

resulted in minimal transformation, with only a few small blue sections appearing

after GUS staining. In contrast, the 2 minute room temperature treatment following a

42ºC incubation reveals slightly more staining indicating improved transformation

efficiency. The most transformation was seen following incubation at 42ºC for 10 and

20 minutes with a 2 minute room temperature recovery as demonstrated by blue

staining in figure 4.2. Overall, the most successful transformation technique was a 10

minute 42ºC incubation, followed by a 2 minute room temperature recovery period.

87

Figure 4.2: Gus staining of S. bicolor calli

S. bicolor calli were GUS stained to reveal sites of calli transformation by A. tumerfaciens

carrying the pBIN19-GUSINT construct. Each calli were treated with differing pre-innoculation

incubation treatments of either 3, 5, 10 or 20 minutes at 42ºC followed by a recovery period of

2 minutes on either ice or at room temperature (RT).

20 minutes

Ice RT

10 minutes

5 minutes

3 minutes

Recovery conditions (2 minutes)

Pre-

trea

tmen

t tim

e at

42°

C

88

4.3.3 Regeneration of callus into S. bicolor plantlets

Untransformed S. bicolor callus was used to optimise a plantlet regeneration protocol

for callus proliferated from seed tissues. Preliminary experiments determined that the

optimum level of kinetin was 1.5mg L-1, and this concentration was used as the base

line level of cytokinin when auxin was added. Differing concentrations of auxin

(indole acetic acid, IAA) added to media containing 1.5mg L-1 kinetin, produced

plantlets with different ratios of root and shoot growth from callus. Figure 4.3 shows

regenerated plantlets arising from differing combinations of cytokinin and auxin. At

0.5mg L-1 and 1.25mgL-1 IAA, no root formation is displayed, however, shoot tissue

is abundant. The plantlet generated from 0.75mg L-1 IAA shows healthy shoot

growth, and the appearance of few short roots, however, the plantlet generated from

1.0mg L-1 IAA shows the opposite. The best regeneration of a whole plantlet was seen

in 1.75mg L-1 IAA where both long shoot and long root formation was apparent.

When transplanted into pots, the regenerated plantlets did not survive. When

regeneration of S. bicolor plantlets was attempted using transformed callus with and

without antibiotic selection, the regeneration was not successful.

Figure 4.3: Regeneration of S. bicolor calli

Untransformed proliferated calli placed on regeneration medium containing 1.5mg L-1 kinetin

and varying concentrations of indoyl acetic acid (IAA) for 4 weeks. Their consequent

capability to regenerate into whole plantlets under these conditions is displayed.

1.5 mg/L Kinetin

1.75 mg/L IAA 1.25 mg/L IAA 0.75 mg/L IAA 1.0 mg/L IAA 0.5 mg/L IAA

89

4.3.4 Expression of SDG4i in transgenic A. thaliana under the 35S promoter

The expression levels of SDG4i in homozygous 35S:SDG4i transgenic A. thaliana

was determined using northern blotting (section 2.6.3) and western blotting (section

2.7.4) techniques. The SDG4i transcript is expressed in all six transgenic lines (figure

4.4). The transcript level of SDG4i in A. thaliana lines varies with the highest level of

expression seen in 35S:SDG4i line 5 and the lowest expression level observed in

35S:SDG4i line 1. A band in the WT-COL lane at a similar size to SDG4i can be seen

and is likely to be the SDG4i-like A. thaliana homolog. Protein extracts from each

35S:SDG4i line, as well as WT-COL, T-AT3G43850 and 35S empty vector controls

were run on SDS-PAGE and the coomassie blue stain of the separated proteins can be

seen in figure 4.5A. The subsequent western blot analysis of these extracts (figure

4.5B) shows that each 35S:SDG4i transgenic line is expressing the SDG4i protein

product at approximately 25kDA in size. In addition, no band was seen in the WT-

COL protein sample, suggesting that the transcript present in the WT-COL does not

produce a protein product that cross-reacts with the SDG4i antibody.

Figure 4.4: Transcript levels in individual A. thaliana 35S:SDG4i transgenic lines

SDG4i transcript accumulation was analysed by northern blot and the phosophoimage of

SDG4i and 28S rRNA transcripts (A), and the relative level of SDG4i transcripts (B) is shown.

Relative transcript levels were calculated using ImageQuant TL v4.0 software based on the

level of 28S rRNA.

90

Figure 4.5: Western blot of A. thaliana 35S:SDG4i transgenic lines

Protein levels of SDG4i as determined by western blot following SDS-PAGE. The Coomassie

blue stain of each protein sample is shown in A, and the western blot autoradiograph is

shown in B.

91

4.3.5 Identification of an A. thaliana T-DNA insertion line homologous to SDG4i

Throughout this project, A. thaliana is used as the model species for elucidating the

function of SDG4i. To aid in the exploration of SDG4i function, T-AT3G43850 from

A. thaliana was found to be the amino acid sequence with the strongest homology to

SDG4i from S. stapfianus, with a homology level of 40% (figure 4.6). Subsequently,

the T-DNA insertion line SALK_026166C was ordered from the Arabidopsis

Information Resource (TAIR) to use experimentally (Chapters 4 and 5). This line

contains a T-DNA insertion in the second intron of T-AT3G43850. The predicted

secondary structure of SDG4i and T-AT3G43850 does differ (depicted in figure 4.7).

However, the significance of the predicted model is low, with a good model generally

demanding a p value below 10-4 which is not reached in the SDG4i and T-

AT3G43850 models. The percentage of sequence representing predicted secondary

structures in table 4.1 shows that despite the differences seen in figure 4.7 between the

two proteins, the percentages of amino acids forming helix, beta-sheets and loop are

similar. Interestingly, the level of disorder between the two proteins differs vastly,

with SDG4i at 53% and T-AT3G43850 at 36%. In addition, AT3G43850 does not

appear to contain a serine-rich region whereas SDG4i does. This may indicate

differences in functionality between the two proteins.

92

SDG4i MPVDVAR-----ASVFEVAMPASACSSSSIGKDSDECTPPGKEDEEVQSAYMGEK 50 T-AT3G43850 MFTAIDKIDRTFTVDQEFACLSSSTSSDSIGENSDDDE--GG-ENEIESSYNG-- 50 * . : : : *.* :*: **.***::**: * ::*::*:* * SDG4i GGAGEEGGLVGLEALEEALPIRRSISKFYSGKSKSFSCLKEAVTSSGSAKDISKA 105 T-AT3G43850 -------PLDMMESLEEALPIKRAISKFYKGKSKSFMSLSE--TSSLPVKDLTKP 96 * :*:*******:*:*****.****** .*.* *** ..**::*. SDG4i ENAYSRKRKNLLVYSIMYENSNETATAEVFETGPPKRPSSLSRSSLVTMASSSS- 159 T-AT3G43850 ENLYSRRRRNLLSHRICSRG------------GISKKPF----KSVLAMSQREGD 135 ** ***:*:*** : * .. * .*:* .*:::*:. .. SDG4i -------------------------RSSSSFSIEDNQLHEQLHYPCSPDHSENCG 189 T-AT3G43850 SSSSGDDSLPTLRQHHKTLTPRLRKGSFEAFTFQDKSETDE-------DHKKATK 183 * .:*:::*:. :: **.: SDG4i PPKSPIPPP---------------------------------------------- 198 T-AT3G43850 GATKPVNQTWSSSGHSIKTRQWPRSTIAEARRAGESMASIDKLMAENFLDNVNLD 238 ...*: . SDG4i --ASCAYRTPSAPMTAMRSFSMMDLPGLHSSSSSVCLKDKKVDS 240 T-AT3G43850 PISIKKSRDPCVPKSSCCLFLNTSLYF------------LGRTD 270 : * *..* :: * .* .

Figure 4.6: Amino acid sequence alignment of S. stapfianus SDG4i and A. thaliana

AT3G43850

A ClustalW alignment between S. stapfianus SDG4i (accession: CAK26793) and A. thaliana

AT3G43850 (accession: AEE77835) is depicted and homology between the two amino acid

sequences is shown below with asterisks.

93

Figure 4.7: Predicted secondary structure of SDG4i and T-AT3G43850

The secondary structure of the SDG4i (p < 8.59 x 10-3) and T-AT3G43850 (p < 4.81 x 10-2)

proteins are shown in A and B respectively. Structure prediction was completed using the

RaptorX web server (Källberg et al., 2012).

Table 4.1: Percentage of predicted secondary structure types and level of disorder

within of SDG4i and T-AT3G43850

Helix Beta-sheet Loop Disorder

SDG4i 23% 0% 76% 53%

T-AT3G43850 24% 2% 72% 36%

94

4.3.5 Developmental phenotype of 35S:SDG4i A. thaliana in long day conditions

The developmental phenotype in long day conditions was recorded and analysed

using the A. thaliana: WT-COL, T-AT3G43850 (T-DNA insertion line), and the six

35S:SDG4i over expression lines. Throughout the life cycle of the transgenic plants,

there were no obvious detrimental effects on plant growth, the plants looked green,

healthy, and were able to complete their life cycle without any impact on plant health.

There was no significant difference in the time taken to germinate between transgenic

and control plants, with fully expanded cotyledons being visible in WT-COL, T-

AT3G43850 and the six 35S:SDG4i lines after 5 days (figure 4.8). At the floral-

initiation stage, the rosette leaf number was counted. Little difference is seen between

the transgenic, wild type or knockout lines (Figure 4.9), which indicates that the over-

expression of SDG4i has no significant effect on rosette leaf number at the floral-

initiation stage. The length of the blade of the fifth rosette leaf at floral-initiation stage

was measured and found to be significantly reduced in 35S:SDG4i lines 1, 2, 3 and 6

(figure 4.10) The slight reduction in lines 4 and 5 was not statistically significant.

Conversely, the T-AT3G43850 line was observed to have a longer fifth rosette leaf,

however, the effect was not statistically significant. The petiole size of the rosette

leaves was also significantly different between the transgenic, knockout and wild type

A. thaliana, with the T-AT3G43850 line showing petiole length of 2-3mm larger (p <

0.05) than that of WT-COL, and all 6 35S:SDG4i lines show smaller petiole lengths (-

p < 0.05) than the WT-COL with petioles 5-8mm shorter (figure 4.11).

95

Figure 4.8: Germination time for A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines under LD conditions.

The time taken for each A. thaliana line to display 2 fully expanded cotyledons in soil with

growth conditions of 16h light/8h dark, at 22°C. Bars represent ±SE of 10 replicates.

Figure 4.9: Rosette leaf number of A. thaliana wild-type Columbia, 35S:SDG4i and

SDG4i homolog knockout lines under LD conditions

Rosette leaf number was counted from when the leaf reached > 5mm in size, with final

number taken at floral-initiation in soil with growth conditions of 16h light/8h dark, at 22°C.

Bars represent ±SE of 10 replicates.

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

2

4

6

Day

s

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

5

10

15

20

Leaf

num

ber

96

Figure 4.10: Leaf length of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


The length of the 5th leaf was measured at floral-initiation from the base of the leaf blade to

the end of the leaf along the midrib. Measurements were taken of soil grown plants with

growth conditions of 16h light/8h dark, at 22°C. Significance of p < 0.05 is indicated by an

asterisk. Bars represent ±SE of 10 replicates.

Figure 4.11: Petiole length in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

10

20

30

Leaf

leng

th (m

m)

* * * *

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

5

10

15

Petio

le le

ngth

(mm

)

*

*

* *

* *

97

Petiole length measured at floral-initiation of the 5th rosette leaf in millimetres. Measurements

were taken of soil grown plants with growth conditions of 16h light/8h dark, at 22°C

Significance of p < 0.05 is indicated by an asterisk. Bars represent ±SE of 10 replicates.

The typical appearance of each variety of A. thaliana (WT-COL, T-AT3G43850 and

35S:SDG4i) is depicted in figure 4.12, at the stage of complete flowering,

transitioning into silique maturity. The T-AT3G43850 line appears taller, and the

35S:SDG4i typical transgenic line appears shorter than WT-COL. The overall height

of each line was measured at the completion of flowering, and all 35S:SDG4i lines

had a shorter overall plant height ranging from 20-22cm (p < 0.05) in comparison to

WT-COL, whilst the T-AT3G43850 line showed no difference in plant height in

comparison to WT-COL (figure 4.13). Branch architecture in flowering A. thaliana

lines was analysed by observing the inflorescence, rosette branches and cauline

branches throughout the life cycle of the plants. A diagram referring to the A. thaliana

branch architecture can be seen in figure 4.14. One primary inflorescence developed

per plant with multiple primary rosette branches, and figure 4.15a shows the total

number of primary inflorescence plus primary rosette branches. Across most lines

tested this number was similar, however, the 35S:SDG4i lines 2 and 4 had

significantly less than WT-COL. The timing of flower bud appearance, inflorescence

and secondary rosette branches across wild type and transgenic lines did not alter

significantly (figure 4.15b). The number of secondary branches from the inflorescence

and primary rosette branches was less in both the T-AT3G43850 line and 35S:SDG4i

transgenic lines (figure 4.15c) with the T-AT3G43850 line and transgenic lines

presenting with 2-3 secondary branches, and the wild type presenting with 4-5

secondary branches (p < 0.05). The timing of secondary branch appearance in the

transgenic lines did not appear to be significantly different to that of the wild type

(figure 4.15d).

98

Figure 4.12: Appearance of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines after the completion of flowering under LD conditions

The typical appearance of wild-type, knock out and SDG4i transgenic lines at the completion

of flowering, and beginning of silique maturity. Plants were grown in soil, under the conditions

of 16h light/8h dark at 22ºC.

Figure 4.13: Plant height in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


WT-COL 35S:SDG4i SDG4i KO

5cm 5cm 5cm

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

10

20

30

40

Hei

ght (

cm)

* * * * * *

99

The final plant height is depicted in this figure was measured at the point completion of

flowering in each line and was calculated by measuring from the base of the plant (above

ground) to the tip of the inflorescence. Plants were grown in soil, under the conditions of 16h

light/8h dark at 22ºC.Significance of p < 0.05 is indicated by an asterisk. Bars represent ±SE

of 10 replicates.

Figure 4.14: Branch architecture of A. thaliana.

The branch architecture of A. thaliana throughout long day conditions was analysed according

to this diagram adapted from Aguilar-Martinez et al. (2007).

Secondary rosette-leaf branch (SRB)

Primary rosette-leaf branch (PRB)

Primary cauline leaf branch (PCB)

Secondary cauline leaf branch

Primary Inflorescence (Inf)

100

Figure 4.15: Inflorescence development and growth in A. thaliana wild-type Columbia,

35S:SDG4i and SDG4i homolog knockout lines under LD conditions

Total inflorescence (Inf) and primary rosette branch (PRB) number (A), inflorescence and

primary rosette branch emergence timing (B), Secondary cauline leaf branch and secondary

rosette leaf branch number (per inflorescence/primary rosette branch) (C), and emergence

timing of secondary cauline leaf branching and secondary rosette leaf branching (D). Plants

were grown in soil, under the conditions of 16h light/8h dark at 22ºC. Significance of p < 0.05

is indicated by an asterisk. Bars represent ±SE of 10 replicates.

101

The time taken to complete floral development of wild type and transgenic lines was

measured from the time of the first flower opening, to the completion of flowering,

where all flowers had opened on the plant. The timing of the floral induction was not

significantly different between any of the 35S:SDG4i, T-AT3G43850 and WT-COL

lines (figure 4.16a) with floral induction starting between 31-34 days, and the

completion of flowering occurring between 45-50 days (figure 4.16a). In addition, the

morphology of the flowers in each line (figure 4.16b) was not notably different.

However, the formation of siliques across WT-COL, T-AT3G43850 and 35S:SDG4i

lines was significantly different, with the beginning of silique ripening (silique

yellowing) occurring between 43-45 days across all 35S:SDG4i lines, and at 46 days

in the T-AT3G43850 line, both being significantly different to the WT-COL line

where ripening commenced at 48 days (figure 4.17a). The length of siliques produced

by transgenic lines was significantly shorter by 3-4mm than that of WT-COL (figure

4.17b). The total number of siliques was observed to be slightly higher in many of the

transgenic lines, compared to the WT-COL, but the difference was not found to be

statistically significant Interestingly, the T-AT3G43850 plant had a significantly

smaller number of siliques when compared to the wild type (figure 4.17c). The total

seed yield (measured as number of seeds per plant) (figure 4.18a) shows that the

35S:SDG4i transgenic lines numbers 2-6, have a higher seed yield than WT-COL and

T-AT3G43850 (p < 0.05) with the 35S:SDG4i lines producing up to 1000 more seed

per plant. Although 35S:SDG4i 1 shows a slightly higher seed yield, it is not

significant. The viability of the seed was measured across 60 day post collection

(figure 4.18b) and the viability of all six 35S:SDG4i lines shows significantly lower

viability in comparison to WT-COL with a 40% reduction in germination at 60 days

post collection. T-AT3G43850 displayed no difference in seed viability. The seed

weight (measured per 1000 seeds, figure 4.19) remains unaffected across all lines.

The time taken for plants to senescence across WT-COL, T-AT3G43850 and

35S:SDG4i lines showed no significant difference, although it appears that the

35S:SDG4i lines shows a slightly delayed senescence time than WT-COL (figure

4.20).

102

Figure 4.16: Floral development in A. thaliana wild-type Columbia, 35S:SDG4i and


The progression of flower opening over time (first flower opened to last flower opened) (A)

and the typical appearance of an open flower (B) is represented in this figure. Plants were

grown in soil, under the conditions of 16h light/8h dark at 22ºC. Significance of p < 0.05 is

indicated by an asterisk. Error bars represent ±SE of 10 replicates.

103

Figure 4.17: Silique morphology in A. thaliana wild-type Columbia, 35S:SDG4i and


Days taken for siliques to ripen (A), the final length of siliques at the completion of ripening but

before shattering (B) and the number of siliques observed per plant (C). Plants were grown in

soil, under the conditions of 16h light/8h dark at 22ºC. Significance of p < 0.05 is indicated by

an asterisk. Bars represent ±SE of 10 replicates.

104

Figure 4.18: Seed yield and viability in A. thaliana wild-type Columbia, 35S:SDG4i and


Number of seeds produced per plant (A), and seed viability expressed in % germination over

time (B) in WT-COL, SDG4i KO and six 35S:SDG4i transgenic lines. Plants were grown in



Figure 4.19: Seed weight per 1000 seeds in A. thaliana wild type Columbia, 35S:SDG4i

and SDG4i homolog knockout lines

1000 seed weight (expressed as mg/1000 seeds) of WT-COL, SDG4i KO and six 35S:SDG4i

transgenic lines. Plants were grown in soil, under the conditions of 16h light/8h dark at 22ºC.


WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

18.0

18.5

19.0

19.5

20.0

1000

see

d w

t (m

g)

105

Figure 4.20: Senescence in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines

The completion of senescence (all pedestals brown, rosette leaves >80% yellow) measured in

days for each line of transgenic and wild-type A. thaliana. Plants were grown in soil, under the

conditions of 16h light/8h dark at 22ºC. Bars represent ±SE of 10 replicates.

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

40

45

50

55

60

Day

s

106

4.3.6 Developmental phenotype of 35S:SDG4i A. thaliana in short day conditions

The developmental phenotype of WT-COL, T-AT3G43850 and six 35S:SDG4i lines

in short day conditions was analysed to observe any differences with WT-COL and to

compare with the developmental differences seen between wild type and transgenic

lines in long day conditions. The germination time (figure 4.21) indicates no

difference across all lines. Germination took on average five days to produce two

fully expanded cotyledons. The rosette leaf number (figure 4.22) produced by all lines

was measured during the vegetative phase of the floral-initiation stage. Leaf numbers

varied only slightly, with 35S:SDG4i lines 1 and 3 displaying leaf numbers which

were one tenth less than WT-COL although the difference was not significant. The

length of the rosette leaves (figure 4.23) across all six 35S:SDG4i lines were 20%

shorter (significantly different, p<0.05), with an average rosette leaf length of 33 mm,

in comparison to WT-COL with a rosette leaf length of 40mm, with the standard leaf

used for measurement being the 5th rosette leaf. Similarly, the length of the petioles in

all six 35S:SDG4i lines were also significantly different (p<0.05), being on average

15% shorter than those of the WT-COL (figure 4.24).

107

Figure 4.21: Days taken for A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines to present two fully expanded cotyledons under SD

conditions

Time taken for each transgenic line to display 2 fully expanded cotyledons signifying that

germination is complete. Plants were grown in soil, under the conditions of 8h light/16h dark

at 22ºC.

Figure 4.22: Rosette leaf number of A. thaliana wild-type Columbia, 35S:SDG4i and

SDG4i homolog knockout lines under SD conditions

Rosette leaf number was counted from when the leaf reached > 5mm in size with final

number taken at floral-initiation. Plants were grown in soil, under the conditions of 8h light/16h

dark at 22ºC. Bars represent ±SE of 5 replicates.

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

2

4

6

Day

s

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

10

20

30

40

Leaf

num

ber

108

Figure 4.23: Leaf size of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


The size of the 5th leaf was measured floral-initiation from the base to the end of the leaf along

the midrib. Plants were grown in soil, under the conditions of 8h light/16h dark at 22ºC.

Significance of p < 0.05 is indicated by an asterisk. Bars represent ±SE of 5 replicates.

Figure 4.24: Petiole length in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Petiole length of the 5th leaf was measured prior to bolting. Plants were grown in soil, under

the conditions of 8h light/16h dark at 22ºC. Significance of p < 0.05 is indicated by an asterisk.


WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

10

20

30

40

50

* * * * * *Le

af le

ngth

(mm

)

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

5

10

15

20

* * * * * *

Petio

le le

ngth

(mm

)

109

The typical appearance of each line used in this experiment is shown in figure 4.25 at

the flowering stage. The differences in plant height can be observed, as well as

differences in bolt number, branching and rosette leaf length. At the completion of

flowering, the height (figure 4.27), measured from the base to the tip of the

inflorescence, of all six 35S:SDG4i lines was up to 35% taller (p > 0.05) than WT-

COL, similarly, T-AT3G43850 was 25% taller than WT-COL . Branch architecture of

A. thaliana lines was analysed by observing the inflorescence, rosette branches and

cauline branches throughout the life cycle of the plants. A diagram referring to the A.

thaliana branch architecture can be seen in section 4.3.5, figure 4.14. One primary

inflorescence was developed per plant, figure 4.26A indicates that the T-AT3G43850

and five 35S:SDG4i lines developed a greater amount of primary rosette branches.

Figure 4.26B indicates the average number of secondary rosette branches and

secondary cauline branches in T-AT3G43850 are significantly greater than WT-COL

but there is no significant difference between the number in 35S:SDG4i lines

compared to WT-COL.

110

Figure 4.25: Appearance of A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i

homolog knockout lines after the completion of flowering in SD conditions

The typical appearance of wild-type, knock out and SDG4i transgenic lines at the completion

of flowering, and beginning of silique maturity. Plants were grown in soil, under the conditions

of 8h light/16h dark at 22ºC.

WT-COL 35S:SDG4i SDG4i KO

5cm 5cm 5cm

111

Figure 4.26: Bolt development in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


Total number of inflorescence (Inf) and primary rosette branches (PRB) (A) and average

primary cauline branches and secondary rosette branches (per inflorescence/primary rosette

branch) number (B) was taken upon the completion of flowering. Plants were grown in soil,

under the conditions of 8h light/16h dark at 22ºC. Significance of p < 0.05 is indicated by an

asterisk. Bars represent ±SE of 5 replicates.

112

Figure 4.27: Plant height in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


The final plant height was measured at the point of complete flowering in each line and was

calculated by measuring from the base of the plant to the tip of the inflorescence. Plants were


indicated by an asterisk. Bars represent ±SE of 5 replicates.

WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

10

20

30

40

50

* * * ** * *

Hei

ght (

cm)

113

The timing of floral induction and subsequent floral development of 35S:SDG4i lines

and T-AT3G43850 line was unaffected, and followed a similar pattern to WT-COL

(figure 4.28A). A typical open flower of each A. thaliana line is displayed in figure

4.28B. It can been seen that in the 35S:SDG4i line, flower petals appear slightly

larger than that of WT-COL and T-AT3G43850, while all lines display the same

petal, stigma and stamen number (figure 4.28b). The number of days taken for

siliques to ripen across all A. thaliana lines was between 88 and 90 days (figure

4.29A). In short day grown plants, the length of siliques did not appear to vary

between WT-COL, T-AT3G43850 and 35S:SDG4i lines (figure 4.29B) with the

silique length being between 16-17mm. In addition, the number of siliques per plant

appeared to be similar across all lines (figure 4.29C). Interestingly, despite these

findings, the total number of seeds per plant is slightly, but significantly, larger in the

35S:SDG4i lines in comparison to WT-COL and T-AT3G43850 (figure 4.30A) with

the 35S:SDG4i lines producing up to 100 seeds more than WT-COL and T-

AT3G43850. The viability of the seed produced is significantly lower (figure 4.30B)

across all six 35S:SDG4i lines with a 40% reduction in the number of germinating

seeds at 60 days post collection when compared to WT-COL. T-AT3G43850 seeds

showed no difference in viability. The seed weight did not vary significantly across all

lines (figure 4.31) with grouped seed weight being approximately 19mg/1000 seed.

Days taken for whole plant senescence in each line were significantly different in

comparison to WT-COL, with both T-AT3G43850 and 35S:SDG4i lines (figure 4.32)

taking 5 days longer to senesce than WT-COL

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Figure 4.28: Floral development in A. thaliana wild-type Columbia, 35S:SDG4i and


The progression of flower opening over time (first flower opened to last flower opened) (A)

and the typical appearance of an open flower (B). Plants were grown in soil, under the

conditions of 8h light/16h dark at 22ºC. Bars represent ±SE of 5 replicates.

115

Figure 4.29: Silique morphology in A. thaliana wild-type Columbia, 35S:SDG4i and

SDG4i homolog knockout lines in SD conditions Days taken for siliques to ripen (A), the final length of siliques at the completion of ripening but

before shattering (B) and the number of siliques observed per plant (C). Plants were grown in

soil, under the conditions of 8h light/16h dark at 22ºC. Bars represent ±SE of 5 replicates.

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Figure 4.30: Seed yield and viability in A. thaliana wild-type Columbia, 35S:SDG4i and


Number of seeds produced per plant (A), and seed viability expressed in % germination over

time (B) in WT-COL, SDG4i T-AT3G43850 and six 35S:SDG4i transgenic lines. Plants were


indicated by an asterisk. Bars represent ±SE of 5 replicates.

Figure 4.31: Seed weight per 1000 seeds in A. thaliana wild type Columbia, 35S:SDG4i

and SDG4i homolog knockout lines in SD conditions

Seed weight (expressed as mg/1000 seeds) of WT-COL, SDG4i KO and six 35S:SDG4i

transgenic lines. Plants were grown in soil, under the conditions of 8h light/16h dark at 22ºC.


WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

18.0

18.5

19.0

19.5

20.0

1000

see

d w

t (m

g)

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Figure 4.32: Senescence in A. thaliana wild-type Columbia, 35S:SDG4i and SDG4i


The number of days taken for each line of transgenic and wild-type A. thaliana to reach

senescence completion (entire plant >80% yellow, measured by eye). Plants were grown in



WT-COL

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

50

70

90

110

130

150

* * * * * * *D

ays

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4.4 Discussion

4.4.1 Transformation of Sorghum species using an Agrobacterium based method Cereal crops play a fundamental role in forage and grain production worldwide and

efforts to improve them for human and animal feed have been made (White &

Broadley 2005). The use of genetic engineering has the potential to enhance crop

productivity through increasing biomass production, or enhancing tolerance to abiotic

stresses. The rapid Agrobacterium-mediated transformation protocol (Toki et al.,

2006) has been previously been performed successfully in rice in this laboratory, and

was utilised in an attempt to develop a rapid transformation protocol for sorghum.

Rapid transformation would be a useful tool for introducing resurrection grass S.

stapfianus genes into monocotyledonous Sorghum plants. Callus generation from

Sorghum mature seed has not been previously published. Although all three Sorghum

species (S. bicolor, S. sudanese and “Sweet Jumbo”) demonstrated callus growth, the

growth of embryogenic callus was minimal. The addition of tryptophan to the callus

inducing medium has been demonstrated to increase embryogenic callus is rice

cultivars (Siriwardana & Nabors 1983), but was not successful when applied to the

protocol in Sorghum. The growth of embryogenic callus is strongly influenced by the

type of explant used, traditionally in Sorghum transformations, immature embryos and

shoot apices are used (Liu & Godwin 2012, Maheswari et al., 2006). While

Agrobacterium-mediated transformation of callus was moderately successful in all

Sorghum species used in this study, the most promising callus transformation

occurred in S. bicolor. The success of callus transformation via an Agrobacterium

based method of gene delivery can be influenced by several factors including explant

type, cultivar, growth conditions, co-cultivation medium and sensitivity to

Agrobacterium infection (Carvalho et al., 2004). S. bicolor callus, although

demonstrating successful transformation, was susceptible to Agrobacterium

overgrowth. This could possibly be rectified by changing the cultivar, co-cultivation

medium or explant used. The benefits of Agrobacterium-mediated callus

transformation are that it is a simple and low-cost method for gene delivery in

comparison to other methods such as biolistics. Recently, the biolistic transformation

of S. bicolor TX430 immature embryo generated callus and subsequent regeneration

(Liu & Godwin 2012) proved successful, and is being explored for future Sorghum

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transformation at Monash University. Successful plantlet regeneration of

untransformed callus was displayed in S. bicolor, with minimal phenolic production.

The regenerated plantlets once transferred to soil, did not survive. Several factors can

affect this transfer from in vitro growth to soil, including the susceptibility of plantlets

to temperature, humidity and light changes. Future work will involve a more gradual

transition to soil and greenhouse conditions for regenerated plantlets.

4.4.2 General health of Arabidopsis SDG4i transgenic lines

The over-expression of proteins localised to the nucleus of the cell changes the size

and shape of the nucleus (Chytilova et al., 2013) and may adversely affect normal

plant processes, however, overall growth of the SDG4i transgenic lines was found to

be fairly normal, with germination time, flowering time, organ number and fertility

largely unaffected. The major phenotypic differences are observed in leaf and petiole

length, plant height, seed yield, seed viability and plant senescence. In several, but not

all cases, the phenotype of the AT3G43950 SDG4i homolog T-DNA insertion line

displays opposing characteristics to that of the SDG4i over-expression lines,

suggesting that the A. thaliana homolog may be a protein of similar function to

SDG4i. The observation of developmental phenotype in 35S:SDG4i and T-

AT3G43850 transgenic lines and their comparison to the wild-type in both long day

and short day conditions indicate that SDG4i activity may be directly or indirectly

affecting hormone homeostasis, and may be influenced by photoperiod. These

observations are discussed in the following sections.

4.4.3 35S:SDG4i lines have a reduced leaf length and petiole size

From a genetic perspective, leaf blades and leaf petioles can be considered separate

organs as mutations in some specific genes have been shown to have opposite effects

on petiole and leaf blade length (Kim et al., 2002). The overall appearance of the

rosette leaf is affected across all six SDG4i over-expression transgenic lines. The leaf

blade length in a long day photoperiod shows a reduction across 35S:SDG4i lines 1,

2, 3 and 6, and in a short day photoperiod shows a reduction in all six 35S:SDG4i

lines when compared to that of wild type. Furthermore, a reduction in petiole size

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across all six 35S:SDG4i lines in comparison to the wild type is observed across both

long and short day photoperiods. In the case of the SDG4i homolog knockout line,

both the petiole and leaf blade length appears longer than the wild type. While the

difference in length compared to wild-type is not statistically significant, it suggests

that the T-AT3G43850 gene in Arabidopsis may have a similar function to SDG4i.

The apparent reduction in leaf blade and petiole size can be attributed to a possible

decrease in cell size, cell number, or a combination of both. The morphology of the

35S:SDG4i over-expression lines do not demonstrate an overall decrease in cell size

or cell number in other organs of the plant, which suggests that the reduction in leaf

size by the action of SDG4i occurs specifically in these cells.

Alterations in hormone homeostasis can effect organ growth. While jasmonic acid

(JA) has been implicated in biotic and abiotic stress-mediated responses, as well as

senescence, reproduction and secondary metabolism (reviewed in Avanci et al.,

2010), it was recently shown that methyl jasmonate delays the switch from the mitotic

cell cycle to the endoreduplication cycle in leaf growth of Arabidopsis. The mutant

altered in JA perception coi1-16B (coronatine insensitive 1) has a reduced cell size

and cell number in leaves when JA is exogenously applied (Noir et al., 2013). It may

be possible that SDG4i is influencing JA perception/signalling/accumulation which

may result in a reduced leaf size in transgenic Arabidopsis by reducing cell size or

number. To address this, it would be useful to observe cell size and cell number in

35S:SDG4i plants.

The regulation of petiole and leaf blade length is controlled by many genes (Tsukaya

et al., 2002, Berna et al., 1999). GA is known to stimulate the growth of plant organs

(Tomomasu et al., 1998). Studies have shown that interfering with the expression of

GA biosynthesis and perception genes has an effect on leaf blade and petiole size.

Specifically, plants with a reduction in the expression of GA biosynthesis gene GAI

(GA INSENSITIVE), and mutations in genes involved in the regulation of cell

elongation, rot3 (rotundifolia 3) and acl2 (acaulis 2), displayed a reduced petiole and

leaf blade size. Mutant Arabidopsis line rot3 displays reduced cell elongation and

proliferation, which reduced leaf and petiole length, acl2 displays reduced cell

elongation, which leads to the decreased petiole and leaf length and the gai mutant

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displays a reduction in leaf and petiole length, due to a decreased cell number,

although the mutant also displays an increase in cell elongation (Tsukaya et al., 2005).

In S. stapfianus, SDG4i expression is induced by ABA (section 3.3.5). It is possible

that the over-expression of SDG4i in A. thaliana induces an altered

perception/synthesis of ABA within the cells. In many aspects of plant development,

GA and ABA act antagonistically (Woodger et al., 2010). This could mean that

SDG4i may impact GA biosynthesis/perception and result in the reduced leaf and

petiole size displayed in 35S:SDG4i transgenic lines. It would be interesting to

analyse the endogenous levels of GA/ABA in 35S:SDG4i transgenic lines, or look at

the expression of GA/ABA induced genes in 35S:SDG4i plants to further understand

the possible effects SDG4i may be having on GA/ABA biosynthesis/perception. This

type of hormonal genetic control facilitated by GA and ABA is displayed in the

maturation and germination of seed in maize (White et al., 2000).

In addition to the possible role of SDG4i modifying GA responses, a study recently

published by Holst et al. (2011), showed that enhanced cytokinin degradation in leaf

primordia of A. thaliana plants over-expressing a cytokinin oxidase/dehydrogenase

gene during the initial stages of organ development resulted in a 27% reduction in leaf

size. It is possible that SDG4i may be modifying cytokinin levels in leaf tissue

causing a reduction in leaf size. It may be beneficial to analyses leaf cytokinin content

in 35S:SDG4i lines to find out if cytokinin levels differ from wild-type.

It is a well documented trait that during drought, non-resurrection plants will attempt

to reduce canopy size in order to preserve water, and ultimately, the life of the plant

(Chaves et al., 2003; Munné-Bosch and Alegre 2004). In desiccation tolerant species,

leaves are often preserved during drought and fully recover following a rewatering

event (Ingram and Bartels 1996). In order for S. stapfianus to survive desiccation, the

rate of water loss must be slow enough to allow time for the induction of the

desiccation tolerance program (Gaff & Ellis 1974). It is plausible that S. stapfianus

has a mechanism that reduces leaf growth during drought in order to increase the

chances of surviving drought and initiating the desiccation tolerance pathway. High

levels of SDG4i expression during dehydration has been observed in the leaves of S.

stapfianus (section 3.3.4) and its nuclear localisation (shown in chapter 3) and

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hypothesised function of interacting with HDA19 based on its homology with

bnKCP1 (Gao et al., 2004), suggests that SDG4i may function in control of gene

expression associated with the desiccation tolerance pathway.. It is possible that the

genes being influenced by the presence of SDG4i in A. thaliana are influencing leaf

structure or growth and in S. stapfianus a reduction in leaf growth during dehydration

may aid in the survival of extreme water loss. 35S:SDG4i transgenic lines are seen to

have a reduce petiole and leaf length, which reduces the overall rosette size, possibly

aiding water retention in the leaves in the event of drought, suggesting that SDG4i

may have a role in the reduction of leaf growth during dehydration. It is possible that

these phenotypic differences observed in 35S:SDG4i plants are a non-specific effect

of the over-expression of a nuclear localised protein in the cell, or an indirect effect of

altering hormone homeostasis in the plant. In order to identify what is producing these

developmental effects, more experimentation is required.

4.4.4 35S:SDG4i lines have an increased primary rosette branching in short day conditions, and reduced secondary rosette branching in long day conditions

Secondary axes of growth, including primary rosette branching and secondary rosette

branching, are major aspects in post-embryonic plant development. The development

of axillary shoot apical meristems has been associated with auxin/cytokinin ratio.

When the auxin/cytokinin ratio is high, an inhibitory effect, potentially mediated by

the secondary messenger strigolactone, is seen on axillary bud growth, but when the

ratio is low, axillary bud growth is promoted (Shimizu-Sato & Mori 2001, Brewer et

al., 2009). Strigolactone, a carotenoid based hormone synthesised through the More

Axillary Branching (MAX) pathway and mainly originating in the roots of the plant, is

a negative regulator of shoot branching acting downstream of auxin (Umehara et al.,

2008, Brewer et al., 2009). Complex signalling and regulation of auxin, cytokinin and

strigolactone affects the branching phenotype of the plant (Hayward et al., 2009). The

formation of axillary shoot apical meristems at the junction between the leaf and

inflorescence gives rise to primary rosette branches. Arabidopsis BRANCHED1

(BRC1) and BRC2 mutants display more primary rosette branching, suggesting a key

function for this gene in branching control/formation. High auxin and high cytokinin

levels do not affect the expression of BRC1, and BRC1 expression is down-regulated

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in max mutant lines. It is hypothesised that BRC1 acts downstream of the MAX

pathway to regulate shoot branching (Aguilar-Martinez et al., 2007). In 35S:SDG4i

lines during short day conditions, an increased number of primary rosette branches

was displayed. This suggests that the presence of SDG4i in A. thaliana may be

affecting the auxin/cytokinin ratio and/or influencing strigolactone activity to promote

shoot branching. A trend for reduced primary rosette branching was observed in long

day conditions, suggesting that this phenotype is photoperiod dependent (discussed in

section 4.4.6). The suggestion that SDG4i activity may be affecting an increase in

cytokinin levels and promoting axillary bud outgrowth in short day conditions links to

the idea that SDG4i may be involved in altered cytokinin homeostasis discussed in

section 4.4.3. Although in this section it was suggested that SDG4i has a role in

decreasing the levels of cytokinin in the leaves, it is possible that SDG4i activity in

short day conditions leads to an increase in cytokinin concentration in the axillary

buds, presenting the possibility for a role for SDG4i in altering cytokinin transport.

Alternatively, over-expression of SDG4i may also directly perturb strigolactone

biosynthesis/perception in the A. thaliana transgenic lines.

The control and regulation of secondary rosette branching is also associated with

auxin/cytokinin ratio, acting primarily through strigolactone (Brewer et al., 2009). A

reduced secondary rosette branch and primary cauline branch number was displayed

in 35S:SDG4i lines in long day conditions, however, this was not seen in short day

conditions, where branch number was no different to wild type A. thaliana. This also

suggests photoperiod influences the effect of SDG4i activity in A. thaliana lines

(discussed in section 4.4.6). An increase in secondary rosette branching is indicative

of a perturbation in strigolactone perception/biosynthesis. The strigolactone

biosynthesis genes MAX1 and MAX2 are involved in control of shoot lateral branching

in Arabidopsis (Stirnberg et al., 2002) where MAX1/MAX2 double mutants showed

increased secondary rosette branching resulting in a bushy phenotype. The opposite is

observed in 35S:SDG4i lines under long day conditions suggesting that SDG4i has a

role in altering biosynthesis or perception of strigolactone leading to a lesser

secondary rosette branching phenotype. It is possible, however, that SDG4i is

affecting auxin and cytokinin signalling upstream of strigolactone. The phenotype

observed in 35S:SDG4i plants is similar to the plants obtained from a study on the

control of shoot lateral branching, which showed that when the basipetal flow of

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auxin is increased, cytokinin is reduced in the axillary buds and outgrowth is

prevented (Shimizu-Sato et al., 2009). Interestingly, it was recently found in Pisum

sativum that endogenous gibberellin may be modulating the response of decapitated

plants to the synthetic strigolactone GR24 by changing bud sensitivity to exogenously

applied strigolactone, suggesting that apical dominance may be controlled by

giberellin and strigolactone interactions in pea (Luisi et al., 2011). This information

further suggests that SDG4i activity may be involved in gibberellin

signalling/perception mentioned previously and it would be interesting to observe the

effects of apex removal on SDG4i transgenic plants and exogenous application of

strigolactone on buds.

The AT3G48350 gene is an uncharacterised protein in A. thaliana and only has

homology to 40% to the SDG4i amino acid sequence. It was found through secondary

structure prediction algorithms that T-AT3G43850 and SDG4i have differing protein

structures, however the percentage of amino acids representing helix, beta-sheet and

loop structures were similar. Interestingly, the level of disorder between the two

proteins differs, with 53% disorder predicted for SDG4i and 36% predicted for T-

AT3G43850. Regions of disorder can be associated with protein function and can

encompass activities including protein-protein interactions, enzymatic activity or

binding to metal ions (Dunker et al., 2002). The differences in levels of disorder may

suggest that SDG4i and AT3G43850 have differing functions. If T-AT3G43850 and

SDG4i had identical functions it could be expected that SDG4i over-expression or

AT3G43850 knockout lines would have opposing effects. Since this is not always the

case in this study, the results suggest that they do not have identical functions. The

insertion of T-DNA in the second intron of the T-AT3G43850 T-DNA insertion line

used throughout this chapter is not affecting the reading frame of the gene itself,

suggesting that this line may still be expressing the T-AT3G43850 gene product.

However, it has been shown that modifying introns can affect splicing/recognition

resulting in reduced gene transcription (Luehrsen & Walbot 1992) or translational

activity by affecting nuclear processing (Bourdon et al., 2001). Investigating the

expression of T-AT3G43850 in the T-DNA insertion lines through qPCR will provide

more details of the gene expression in the T-AT3G43850 line, and generating

antibodies against the T-AT3G43850 gene product will elucidate the translation level

to address these possibilities.

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4.4.5 35S:SDG4i lines have a reduced plant height in long day conditions and an increased plant height in short day conditions

Dwarfing of plants is commonly seen in gibberellin insensitive mutants, where stem

elongation is reduced. Mutant plants with reduced sensitivity to GA (GAI

semidominant mutation) or constitutive activation of GA signal transduction (SPY

recessive mutation) also show differing degrees of dwarfism (Jacobsen et al., 1996).

In addition, the application of GA inhibitors in long day conditions reduced stem

elongation in spinach (Zeevart et al., 1993). These studies involving GA insensitivity

in differing degrees display a similar dwarfed phenotype that is seen in 35S:SDG4i

plants in long day conditions, were 35S:SDG4i plants have a 30% reduction in height.

This suggests that 35S:SDG4i lines may be GA insensitive, or that SDG4i may be

influencing the negative regulation of GA responses in the plant. Interestingly, in

short day conditions, 35S:SDG4i plants are taller than the wild type by up to 25%.

Stem growth in short day conditions is promoted by the application of GA (Xu et al.,

1997) and suggests that in short day conditions, SDG4i is promoting the biosynthesis

or signalling pathway of GA causing a taller phenotype. It would be interesting to

cross 35S:SDG4i with the gai mutant to observe if a wild-type phenotype is observed

in short day conditions. Alternatively, differences in plant height could also be an

affect of auxin within the plant. Changes in polar auxin transport have been shown to

affect plant height and photomorphogensis, particularly by BIG (calossin/pushover-

like protein), using doc1-1 (dark over-expression of CAB (chlorophyll a/b binding

protein)) and tir3 (transport inhibitor response 3) mutants (Gil et al., 2001, Ruegger

et al., 1997). It is also possible that the differences seen in phenotype between long

day and short day conditions in the 35S:SDG4i plants is due to SDG4i affecting a

protein that is regulated by photoperiod, and it may be useful to use antibodies to

observe any effects of SDG4i protein stability in long day and short day conditions.

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4.4.6 35S:SDG4i lines appear to be affected by photoperiod The appearance of differing phenotypes in long day and short day suggests that

SDG4i function may be affected by photoperiod perception, or may provide different

functions during conditions inducive of reproductive growth (long day) or vegetative

growth (short day). Photoreceptor proteins, such as phytochromes, detect red light:far-

red light ratio and control many aspects of plant architecture such as branching,

flowering and apical dominance (Finlayson et al., 2010). Phytochrome evolution in

Arabidopsis has enabled the plant to sense light environments and facilitates cross talk

with other signalling systems (reviewed by Franklin & Quail 2009). The role of

phytochrome in detecting light ultimately influences the circadian clock, and plant

growth in long days and short days (Somers et al., 1998). The regulation of branching

by phytochromes was recently demonstrated by Finlayson et al. (2010), where a

mutation in Phytochrome B (PHYB) reduced bud outgrowth by increased correlative

bud inhibition where, effectively, the bud passes into a state of dormancy. This bud

inhibition displayed in phyB Arabidopsis was only apparent when functional AXR1

(auxin resistance gene 1), MAX2 and MAX4, key genes in branching architecture,

were present (Finlayson et al., 2010). AXR1 is required for plant response to auxin in

A. thaliana, and functions with ECR1 to activate ubiquitin-related proteins (del Pozo

et al., 1998). AXR1 has been shown to be required for ubiquitin degradation

processes and for COP1/COP10/COP9 signalosome mediated repression of

photomorphogenesis (Schwechheimer et al., 2002). MAX2 mutant plants lack the

capacity to respond to light signals in a normal manner, have reduced apical

dominance and display delayed senescence and it has been suggested that MAX2

targets multiple factors to control plant responses to photoperception at different

developmental stages (Shen et al., 2007). This information ties in photoperiod

perception and regulation of primary and secondary branching. The opposing

phenotypes of branching displayed in long day and short day conditions in

35S:SDG4i transgenic lines suggests that SDG4i may be regulating plant branching

architecture by influencing auxin or strigolactone synthesis/perception in these

conditions. It would be useful to analyse levels of auxin and strigolactone of the

transgenic lines in both long and short day conditions to observe if the levels of these

hormones are changing according to photoperiod. It has been shown that the

desiccation tolerance gene SDG8i protein product is able to glycosylate the

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strigolactone analog GR24 in vitro, and increases in growth and branching were

observed in SDG8i transgenic Arabidopsis, which suggests that SDG8i may be

modifying the ability of S. stapfianus to perceive or synthesise strigolactone (Islam et

al., 2012). The differential effects on branching in 35S:SDG4i lines under different

photoperiods suggests that SDG4i may provide an integral function in altering the

perception of hormones. How this may operate during desiccation to regulate gene

expression and influence the ability of the plant to tolerate extreme dehydration is

unclear.

AXR1 mutants have been analysed during growth and development and display a

decrease in plant height under constant light conditions (Lincoln et al., 1990).

Functional AXR1 has been shown to be involved in branching architecture in

combination with PHYB (Finlayson et al., 2010), which suggests it may be

photoperiod dependent. The opposing effect on plant height of 35S:SDG4i transgenic

lines compared with wild-type plants grown under long day and short day conditions

suggest that SDG4i may be affecting auxin synthesis/perception to control plant

height in addition to the effects on branching control. The height phenotype of

35S:SDG4i plants also suggest that SDG4i may be affecting GA

biosynthesis/perception/regulation. Photoperiodic control of GA has been

demonstrated in seedlings of Salix pentandra, where in the transition from long day to

short days, GA levels decrease and the plant goes into a state where active growth is

no longer required (Olsen et al., 1997). The data obtained from the phenotypic

analysis of SDG4i in these conditions suggests that SDG4i may act to increase GA

effects in short days, and decrease GA effects in long days. The link between plant

photoperiod and GA pathways has been explored recently by Osnato et al. (2012)

where TEMPRANILLO genes linked photoperiodic effects of GA to flowering in

Arabidopsis. Similarly, Xu et al., (1997) looked at the effects of GA on

inflorescence/primary rosette branching growth in Arabidopsis in differing

photoperiods. These studies both show that the accumulation of endogenous GA

controls flowering and cell elongation in long day and short day photoperiods.

Arabidopsis over-expressing SDG4i have no significant differences in flowering time

when compared to the wild type plants, and display the reverse of typical GA

responses in photoperiods. This suggests that SDG4i may act to deregulate GA

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biosynthesis or signalling or alternatively may interfere with hormonal signalling

pathways that normally act to antagonise GA effects.

4.4.7 35S:SDG4i lines have an increased seed yield and delayed senescence

Increased seed yield is a major goal in agricultural research for grain crops world

wide. A. thaliana over expressing SDG4i display an increased seed yield in both long

day and short day conditions although the increase in yield is considerably higher in

long day, at 28%, in comparison to short day, at 2%. Over-expression of DWARF1 in

the brassinosteriod pathway increases seed yield in Arabidopsis (Choe et al., 2001),

however, this is due to an increased number of silique bearing branches, which is not

displayed in A. thaliana over-expressing SDG4i. Interestingly, seed weight remained

unaffected in the SDG4 transgenics, which suggests that seed yield in a 35S:SDG4i

plant may be due to there being more seeds contained per silique. However, this needs

to be investigated further, as under long day conditions the siliques were shorter than

wild-type. In a recent study by van Daele et al. (2012), a comparative approach of

seed yield was completed where a variety of genes associated with affecting seed

yield in Arabidopsis and showed that modifying the expression of many genes,

particularly those involved in the brassinosteriod pathway, resulted in decreased seed

size and increased seed number, or increased seed size and decreased seed number. It

is not clear that SDG4i activity is modulating brassinosteriod response as the

phenotype displayed by 35S:SDG4i plants of increased seed yield and no change in

seed size/weight has not previously been observed. A reduction in polar auxin

transport caused by mutating TIR3 (TRANSPORT INHIBITOR RESPONSE 3) has

been observed to decrease silique elongation in Arabidopsis (Ruegger et al., 1997),

these mutants also display a reduced plant height (discussed in section 4.4.5). The

phenotype displayed in 35S:SDG4i plants suggests that there may be an alteration in

plant auxin response. In long days 35S:SDG4i silique maturation is also different to

the wild type, where silique ripening is accelerated in the 35S:SDG4i lines.

Acceleration in silique ripening time has also been associated with auxin response,

where ARF2 has been shown to control developmental progression of plant aging,

and mutations in this gene have accelerated organ abscission (Ellis et al., 2005). This

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further suggests that 35S:SDG4i plants may have an altered auxin response in long

days.

Seed viability over time is affected equally in long days and short days in 35S:SDG4i

plants. Seed dormancy and germination are complex processes that are regulated by a

range of external and developmental cues (Koormneef et al., 2002). It is possible that

SDG4i may be affecting the desiccation tolerance of the seed which provides cellular

stability during desiccation (Buitink & Leprince 2008) or that the seed storage is

affected by the level of antioxidant enzymes, as high antioxidant activity is associated

with long term storage of desiccated seed (Bailly et al., 2008).

Light and temperature can also affect seed germination by increasing sensitivity to

GA (Karseen & Lacka 1986) or by inducing GA synthesis (Yamaguchi et al., 1998).

In a study by Toyomasu et al., (1998), the treatment of mature lettuce seed with red

light increased the abundance of GA, suggesting that phytochrome has a role in GA

synthesis in seed. Section 4.4.4 discussed the possibility that 35S:SDG4i plants may

be changing their perception of photoperiod, which may also be a cause of decreased

germination. More recently, cytokinin receptor loss-of-function mutants have

demonstrated that cytokinin functions in seed germination control (Riefler et al.,

2006), raising the possibility that 35S:SDG4i seed have attenuated cytokinin

synthesis/perception (also suggested in section 4.4.3). The reduction of the capacity of

35S:SDG4i seeds to germinate over a period of time may also suggest that these seed

have a reduced testa pigmentation or structure which affects the ability of the seed to

withstand adverse environmental conditions during storage (Debeaujon et al., 2000).

Senescence is delayed in 35S:SDG4i plants in long days and the delay is more

pronounced in short day conditions. Delayed leaf senescence in Arabidopsis may be

associated with increased cytokinin levels within the plant (Gan & Amasino 1995).

Senescence will occur when cytokinin levels are low within the plant, although there

is no evidence that this causes senescence. Interfering with individual cytokinin

receptors (sensor histidine kinases) does not results in senescence being delayed or

accelerated (Riefler et al., 2006). A major symptom of plant senescence is the loss of

chlorophyll, which was used as an indicator in this study. Exogenous application of

ABA degrades chlorophyll, and is associated with inducing senescence (Back &

130

Richmond 1971). ABA and GA are known to have an antagonistic relationship

(Weiss & Ori 2007). In research performed by Li et al. (2010), it was also found that

the exogenous treatment of Paris polyphylla with GA delayed chlorophyll

degradation during senescence. In Pisum sativum photoperiod induced changes in

endogenous GA synthesis show that in short days, senescence is delayed by GA-

promoted shoot growth (Proebsting et al., 1978). This could mean that in 35S:SDG4i

plants senescence is delayed using this mechanism due to the display of taller plants

and delayed senescence in the short day photoperiod or could be due to changes in, or

perception of, cytokinin levels within the plant.

4.5 Conclusion The analysis of developmental phenotype of 35S:SDG4i A. thaliana lines have

revealed some phenotypic changes in comparison to wild-type plants. However, these

changes could be attributed to a variety of processes possibly affected by SDG4i

expression. It has been demonstrated in this chapter that SDG4i may be affecting

hormonal processes within the plant, although the specific mechanism is yet to be

determined. How this may operate during desiccation to regulate gene expression and

influence the ability of the plant to tolerate extreme dehydration is unclear. Further

work needs to be completed to determine the precise function of SDG4i. Investigation

into the effects of SDG4i on abiotic stress, as well as the responses of 35S:SDG4i

lines to growth and stress-associated hormones ABA may provide further information

to elucidate the function of SDG4i. In addition, it would be useful to observe changes

in response to abiotic stress such as osmotic, salt, cold and heat in 35S:SDG4i A.

thaliana. This is explored in the next chapter.

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5.0 Effect of over-expression of SDG4i on abiotic stress tolerance in A. thaliana

5.1 Introduction

The impact of abiotic stresses, most importantly drought, salt, and temperature, on

agricultural production worldwide is increasing. The development of crop cultivars

with capabilities to withstand abiotic stresses is necessary to provide food and fuel for

the world’s current and projected population. The model plant, A. thaliana, provides a

convenient model system to analyse plant response to abiotic stress and has been

extensively studied. Hence A. thaliana provides a good system for exploring the effect

that over-expression of SDG4i may have on plant stress responses. Abiotic stresses

such as drought cause osmotic stress (Boyer 1982) and can be applied in vitro with

agents such as polyethyleneglycol (PEG) or mannitol (Park et al., 2009). Salinity

stress, which can be replicated in vitro using sodium chloride, not only causes osmotic

stress to the plant but also causes ion toxicity, which results in decreased carbon use

efficiency and can lead to a denaturation of membrane and protein structures (Binzel

et al., 1994). The role of genes that allow tolerance to high or low temperatures is also

a growing area of research. Interest also lies within the ability of plants to delay

senescence. Stress can cause senescence and reduce the yield and lifespan of the

plant. By delaying senescence it may be possible to increase plant yield and tolerance

to abiotic stresses (Rivero et al., 2007).

A key plant hormone involved in regulating abiotic stress responses is ABA, with

ABA accumulation acting as an endogenous signal to initiate adaptive responses to

drought and salinity (Zhu 2002). The accumulation of ABA will induce the

transcription of many genes encoding proteins and enzymes involved in providing

cellular protection and osmolyte synthesis to stabilise the cell during a stress event

(Bray 2002).

Responses to osmotic stress in resurrection plants can differ to those in non-

resurrection plants, especially in the later dehydration stages. While many non-

132

resurrection plants will induce pathways to maintain cellular metabolism during stress

conditions (reviewed in Reddy et al., 2004), resurrection plants will induce pathways

associated with preserving cellular tissue and suspending metabolism to allow

tolerance of extreme drought stress, and restore regular metabolism upon rewatering

(Scott 2000). Interestingly, during drought stress S. stapfianus does not appear to

utilise ABA signalling to initiate early stress responses, suggesting that desiccation

tolerance is triggered by an ABA-independent pathway in this plant (Gaff and Loveys

1984). Research is underway to determine if genes associated with desiccation

tolerance mechanisms, ectopically expressed in non-resurrection crop plants, can

provide an increased capacity of crops to tolerate adverse environmental conditions.


5.2.1 General stress assay growth conditions

Wild type, 35S:E (empty vector control line), 35S:SDG4i and T-AT3G43850 lines

were grown in vitro on solid half strength MS medium without sucrose (section

2.4.1). For germination assays, ten seeds of each of the following plant types: WT-

COL, 35S:E, T-AT3G43850 and six 35S:SDG4i lines, were placed on plates

containing a range of mannitol concentrations and stratified at 4°C in the dark for

three days. The plates were then placed at 22ºC in 16 hour light/8 hour dark cycles

and were scored daily for ten days. For root length assays, ten seeds of each of the

following plant types: WT-COL, 35S:E, T-AT3G43850 and six 35S:SDG4i lines

were stratified at 4°C in the dark for three days, then grown vertically for five days on

solid half-strength MS (½ MS) medium supplemented with 3% sucrose. The seedlings

were then moved onto vertically orientated plates containing a range of mannitol

(section 5.2.2), sodium chloride (section 5.2.3) or ABA concentrations (section 5.2.4),

with the position of the tip of the roots marked on the plate. The seedlings were grown

for ten days, with root length measured every two days. Both germination and root

length assays were repeated three times.

133

5.2.2 Osmotic stress assay

The osmotic germination and root growth assays were performed as described in

section 5.2.1, using a range of mannitol concentrations based on the protocol of Park

et al. (2009): 0mM, 50mM, 100mM, 200mM, 300mM and 400mM.

5.2.3 Salt stress assay

Salt stress germination and root growth assays were performed as described in section

5.2.1, using a range of sodium chloride (NaCl) concentrations based of the protocol

by Park et al. (2009): 0mM, 50mM, 75mM, 100mM and 125mM.

5.2.4 ABA stress assay

The ABA stress germination and root growth assay was performed as described in

section 5.2.1, using a range of ABA concentrations based on the protocol by Fujii &

Zhu (2009): 0µM, 1µM, 2µM, 3µM, 4µM and 5µM.

5.2.5 Heat stress assay

The affect of heat shock on Arabidopsis seeds growing in vitro was examined in each

of the six 35S:SDG4i lines and the T-AT3G43850 line and was compared with the

response in the control lines: WT-COL and 35S:E. Ten seeds of each line where

placed on solid ½ MS media supplemented with 3% sucrose. The seeds were then

subject to 3 days stratification at 4ºC, placed at 42ºC for two hours, then allowed to

germinate in the dark at 22ºC. The length of hypocotyl and roots of each seed was

taken over a period of five days. The vegetative heat tolerance test was conducted

similarly, where ten seeds of each six 35S:SDG4i, T-AT3G43850, 35S:E and WT-

COL lines were placed on ½ MS agar supplemented with 3% sucrose. The seeds were

then stratified at 4ºC, and allowed to germinate for two weeks at 22ºC with 16 hour

light/8 hour dark cycles. The seedlings were then placed at 42ºC for 2 hours, then

134

placed back in previous growth conditions, and scored for survival ten days later

(Garwe et al., 2006).

5.2.6 Cold germination assay

An analysis of Arabidopsis seed germination in vitro at a low temperature was

conducted with ten seeds of each of the six 35S:SDG4i lines, T-AT3G43850, 35S:E

and WT-COL lines placed on ½ MS agar supplemented with 3% sucrose. The seeds

were left to germinate in the dark at 4ºC. Germination was monitored by measuring

the time taken for the seed to swell, the radicle to emerge and for the hypocotyl to

reach 2mm in length.

5.2.7 Freezing tolerance assay

The freezing tolerance of Arabidopsis growing vegetatively in vitro was conducted

where ten seeds of each of the six 35S:SDG4i lines, T-AT3G43850, 35S:E and WT-

COL lines were placed on ½ MS agar supplemented with 3% sucrose. The seeds were

subjected to three days stratification at 4ºC followed by two weeks growth at 22ºC in

16 hour light/8 hour dark cycles. The two-week old seedlings were then placed at 4ºC

for 24 hours, the temperature was then lowered to -1ºC for one hour. Temperature was

then lowered by 1ºC every hour until -16ºC was reached. A subsample of the plates

were removed after one hour at -4ºC, -8ºC, -12ºC and -16ºC. The seedlings were

allowed to recover at 4ºC for 24 hours, and then placed at 22ºC with 16 hour light/8

hour dark cycles and survival was scored ten days later.

5.2.8 Senescence assay

Artificial induction of leaf senescence was performed by detaching the 3rd and 4th

rosette leaves of 3 week old soil grown A. thaliana plants (WT-COL, 35S:E, T-

AT3G43850 and six 35S:SDG4i lines). Leaves were excised with a scalpel blade mid-

petiole and floated on distilled water in Petri dishes, ensuring the petiole was

immersed (Hanaoka et al., 2002). Leaf senescence was monitored over 5 days by

135

analysing photosystem II activity (Teaching-PAM) and chlorophyll content daily. To

measure chlorophyll content, tissue samples were taken and weighed immediately.

The tissue was ground in 80% ice-cold acetone in a mortar and pestle. The extract was

centrifuged at 4ºC at 10000rpm for 5 minutes. The supernatant was then kept on ice in

the dark and the pellet re-extracted with 80% ice-cold acetone and centrifuged at 4ºC

for 5 minutes at 10000rpm. This process was continued until the pellet remained

white. The extracted chlorophyll samples were then measured for chlorophyll content

using a spectrophotometer at the wavelengths: 663nm (chlorophyll a), 646nm

(chlorophyll b) and 710nm (impurities). Chlorophyll content was calculated using the

formula (total chlorophyll content): 7.18 * (A663 - A710) + 17.32 * (A646 - A710).

Senescence was monitored over four treatments at a constant temperature of 22ºC in

the dark, or by application with or without 5µM ethylene or 50µM ABA in constant

light.

136

5.3 Results

5.3.1 Germination, root length, lateral root and leaf number of 35S:SDG4i A. thaliana under standard conditions

The germination percentages and root length of each line was assessed in vitro

without exposure to a stress agent, in standard growth conditions of 22°C with 16

hour light/8 hour dark cycles to measure phenotypic differences between each line.

Germination percentages between WT-COL, 35S:E, T-AT3G43850 and the six

35S:SDG4i transgenic lines (figure 5.1) did not vary with 100% germination

displayed across all lines. The typical appearance of each line during a root length

assay can be seen in figure 5.2. The appearance and growth of all of the lines appears

similar. Although the root length of 35S:SDG4i lines 2, 3 and 6 appears longer, no

significant differences were seen between any of the lines (figure 5.3A) after 10 days

root growth. Lateral root number and leaf number (figure 5.3B, 5.3C) also showed no

significant differences.

Figure 5.1: Germination percentage of each A. thaliana line used throughout abiotic

stress assays

Germination was stated to be completed when each seed had produced a plantlet with two

fully expanded cotyledons on ½ MS solid media. Any significant differences (p < 0.05) are

highlighted with an asterisk.

WT-COL 1

35S:E

1

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

50

100

150

% G

erm

inat

ion

137

Figure 5.2: Typical growth of A. thaliana lines used throughout abiotic stress assays

Each WT-COL, 35S:E, T-AT3G43850 and 6 35S:SDG4i transgenic lines were subject to

growth on ½ MS solid media for a period of 10 days at 22°C with 16 hour light/8 hour dark

cycles. The typical appearance of each line is shown in this figure.


used throughout abiotic stress assays

The typical growth of roots (A), lateral root number (B), and leaf number (C) observed over a

10 day period at 22°C with 16 hour light/8 hour dark cycles on ½ MS solid media. No

significant differences (p < 0.05) were observed. Values are the mean ±SE of 10 replicates.

138

5.3.2 Osmotic stress assay

Germination and vegetative root growth and leaf number on mannitol-containing

media was examined in the 35S:SDG4i and T-AT3G43850 A. thaliana lines and

compared to the control lines WT-COL and 35S:E. The germination of each of the six

35S:SDG4i lines was improved under osmotic stress in comparison to the WT-COL,

and 35S:E lines (figure 5.4A). Germination percentage reached approximately 80% (p

< 0.05) in 35S:SDG4i lines, and only 40% in both the WT-COL and 35S:E lines on

200mM mannitol (figure 5.4B). The T-AT3G43850 line appeared to germinate

slightly better than the WT-COL and 35S:E lines, however, the difference was not

significant.

The root growth across all 35S:SDG4i lines showed a high level of variation, with no

discernable difference between all transgenic and control lines observed on mannitol

levels below 200mM (figure 5.5). All six 35S:SDG4i continued root growth after

osmotic stress was applied with 300mM mannitol where the root length of all six

35S:SDG4i transgenic lines were 4-6 times greater than WT-COL and 35S:E (Figure

5.6A). The difference in lateral root number between all lines varied, however

35S:SDG4i lines 2, 3 and 6, showed an increased number of lateral roots in

comparison to the WT-COL and 35S:E lines (Figure 5.6B). The 35S:SDG4i lines also

show an increased leaf number (p < 0.05) in comparison to WT-COL and 35S:E

(figure 5.6C) presenting up to 3 more leaves. The T-AT3G43850 line, presented no

significant differences across root length, lateral root number, or leaf number during

osmotic stress compared with control lines. The typical appearance of each line

subjected to osmotic stress by 300mM mannitol is shows in figure 5.6D. It can be

seen that although the plants appear stunted in morphology, clear differences in root

length can be observed.

139

Figure 5.4: Germination of A. thaliana lines on media containing mannitol

Germination was scored across WT-COL, 35S:E, T-AT3G43850 and 6 35S:SDG4i transgenic

lines 10 days after placement on 0, 50, 100, 200, 300 and 400mM mannitol at 22°C with 16

hour light/8 hour dark cycles. A % germination curve across all mannitol levels (A) and at

200mM mannitol (B) is presented. Any significant differences (p < 0.05) are highlighted with

an asterisk. Values are the mean ±SE of 10 replicates.

0 1 0 0 2 0 0 3 0 0 4 0 00

2 0

4 0

W T -C O L 1

W T -C O L 2

3 5 S :E 1

3 5 S :E 2

T -A T 3 G 4 3 8 50

3 5 S :S D G 4 i 1

3 5 S :S D G 4 i 2

3 5 S :S D G 4 i 3

3 5 S :S D G 4 i 4

3 5 S :S D G 4 i 5

3 5 S :S D G 4 i 6

m M M a n n ito l

Pri

ma

ry r

oo

t le

ng

th (

mm

)

Figure 5.5: Primary root growth of A. thaliana lines on media containing mannitol

Root length was measured across WT-COL, 35S:E, T-AT3G43850 and 6 35S:SDG4i

transgenic lines 10 days post placement on 0, 50, 100, 200, 300 and 400mM mannitol at

22°C with 16 hour light/8 hour dark cycles. Values are the mean ±SE of 10 replicates.

140


subjected to growth under 300mM mannitol

The growth of roots (A), lateral root number (B), and leaf number (C) measured after a 10 day

period on ½ MS solid media supplemented with 300mM mannitol at 22°C with 16 hour light/8

hour dark cycles. Appearance of plantlets growing on 300mM mannitol (D). Any significant

differences (p < 0.05) are highlighted with an asterisk. Values are the mean ±SE of 10

replicates.

141

5.3.3 Salt stress assay

The ability of A. thaliana lines to germinate and grow under salinity stress was

assessed using NaCl in an in vitro assay with concentrations ranging from 0-125mM.

A curve showing the ability of the transgenic and control A. thaliana lines to

germinate under varying levels of salt is shown in figure 5.7A. All six 35S:SDG4i

lines showed an increase in germination at 75mM NaCl over control lines. The

germination percentage of all six 35S:SDG4i lines reached 90% (p < 0.05) in

comparison to 35% for WT-COL and 35S:E at 75mM NaCl (figure 5.7B). The T-

AT3G43850 line germination percentage was 45%, and was not significantly different

to the WT-COL and 35S:E lines.

Interestingly, the root length of 35S:SDG4i lines was greater at 50mM NaCl

compared to that observed in plants grown in salt-free media. The most discernable

difference in root length between transgenic and control lines was seen at 125mM

NaCl (figure 5.8). At 125mM NaCl an increased root length of up to 20mm greater

than controls is observed across all six 35S:SDG4i lines, however, only lines 1-4

shows a significant difference (figure 5.9A). Interestingly, the T-AT3G43850 line

showed a difference in root length (p < 0.05) of 5mm greater than WT-COL and

35S:E controls. The lateral root number (figure 5.9B) showed substantial variation

across all lines tested. A significantly greater number of lateral roots, at 2-3, was

observed in 35S:SDG4i 1, whereas 35S:SDG4i 2 showed significantly less, at 0-1

lateral roots, compared to WT-COL and 35S:E. The T-AT3G43850 line produced a

greater number of lateral roots at 3-4 (p < 0.05). The leaf number across all six

35S:SDG4i lines and T-AT3G43850 was significantly greater than the leaf number

displayed in WT-COL and 35S:E lines (figure 5.9C). These lines presented an

increased leaf number of up to 5-6 leaves in comparison to the WT-COL and 35S:E

lines, presenting 2-3 leaves. The typical appearance of each line during the vegetative

salt stress test is depicted in figure 5.9D. Clear differences in root length and leaf

number are shown.

142

Figure 5.7: Germination percentage of A. thaliana lines on NaCl


lines 10 days after placement on NaCl at 0, 50, 75, 100 and 125mM at 22°C with 16 hour

light/8 hour dark cycles. A % germination curve across all NaCl levels (A) and at 75mM NaCl

(B) is presented. Any significant differences (p < 0.05) are highlighted with an asterisk. Values

are the mean ±SE of 10 replicates.

Figure 5.8: Root length of A. thaliana lines across varying concentrations of NaCl


transgenic lines 10 days after placement on 0, 50, 75, 100, and 125mM NaCl at 22°C with 16

hour light/8 hour dark cycles. Values are the mean ±SE of 10 replicates.

0 50 1000

20

40

60

80

mM NaCl

Prim

ary

oot l

engt

h (m

m)

WT-COL 1WT-COL 235S:E 135S:E 2T-AT3G4385035S:SDG4i 135S:SDG4i 235S:SDG4i 335S:SDG4i 435S:SDG4i 535S:SDG4i 6

143

Figure 5.9: Root length, lateral root number and leaf number of A. thaliana lines

subjected to growth under 125mM NaCl

The typical roots growth (A), lateral root number (B), and leaf number (C) observed over a 10

day period on ½ MS solid media supplemented with 125mM NaCl at 22°C with 16 hour light/8

hour dark cycles. Plantlets growing on 125mM NaCl (D). Any significant differences (p < 0.05)

are highlighted with an asterisk. Values are the mean ±SE of 10 replicates.

144

5.3.4 ABA stress assay

The ability of 35S:SDG4i lines germination potential and vegetative growth upon

exposure to ABA were assessed in vitro. Across the range of ABA levels, all six

35S:SDG4i lines had a higher germination percentage than WT-COL. T-AT3G43850

displayed a similar tolerance to ABA as WT-COL (figure 5.10A). The clearest

difference between the lines was displayed at 3µM ABA. All six 35S:SDG4i lines

germination level was 90-100% (p < 0.05) in the presence of 3µM ABA, in

comparison to 30% germination by the WT-COL and 35S:E lines. The T-AT3G43850

line displayed no significant difference in germination percentage to the controls

(figure 5.10B). The root growth of each A. thaliana line after application of ABA was

variable but showed a general decrease as the ABA concentration was increased

(Figure 5.11). A clear difference between all lines and controls was seen at 4µM

ABA, with the transgenics displaying a substantially increased root length of 50-60%

(p < 0.05) (figure 5.12A). Similarly the lateral root number across all 35S:SDG4i

lines was significantly greater than WT-COL and 35S:E with 35S:SDG4i lines

displaying 2-3 more lateral roots (figure 5.12B). The leaf number across all

35S:SDG4i lines was also larger, presenting 2-3 more leaves than WT-COL and

35S:E (p < 0.05) (figure 5.11C). No significant differences were seen in the T-

AT3G43850 line across root length, lateral root number and leaf number. The typical

appearance of each line during the vegetative growth assay can be seen in figure

5.12D.

145

Figure 5.10: Germination percentage of A. thaliana lines on ABA


lines 10 days supplemented with ABA at 0, 1, 2, 3, 4 and 5 µM at 22°C with 16 hour light/8

hour dark cycles. A germination curve across all ABA levels (A) and at 3µM ABA (B) is

presented. Any significant differences (p < 0.05) are highlighted with an asterisk. Values are

the mean ±SE of 10 replicates.

0 1 2 3 4 50

2 0

4 0

6 0

4M A B A

Pri

ma

ry r

oo

t le

ng

th (

mm

)

W T -C O L 1

W T -C O L 2

3 5 S :E 1

3 5 S :E 2

T -A T 3 G 4 3 8 50

3 5 S :S D G 4 i 1

3 5 S :S D G 4 i 2

3 5 S :S D G 4 i 3

3 5 S :S D G 4 i 4

3 5 S :S D G 4 i 5

3 5 S :S D G 4 i 6

Figure 5.11: Root length of A. thaliana lines across varying concentrations of ABA


transgenic lines 10 days supplemented with 0, 1, 2, 3, 4 and 5 µM ABA at 22°C with 16 hour

light/8 hour dark cycles. Values are the mean ±SE of 10 replicates.

146

Figure 5.12: Root length, lateral root number and leaf number of A. thaliana lines

subjected to growth under 4µM ABA

The typical growth of roots (A), lateral root number (B), and leaf number (C) observed over a

10 day period on ½ MS solid media supplemented with 4µM ABA at 22°C with 16 hour light/8

hour dark cycles. Any significant differences (p < 0.05) are highlighted with an asterisk.

Values are the mean ±SE of 10 replicates.

147

5.3.5 Heat stress assay

Following stratification, the thermal tolerance in the form of excessive heat was

applied to either imbibed seeds or seedlings in vitro at a temperature of 42ºC for 2

hours. Seeds were allowed to germinate in the dark for five days and hypocotyl and

root lengths were measured. The heat shock treatment of seeds produced varied

results across all lines. 35S:SDG4i lines 3, 5 and 6 showed differences in hypocotyl

length (p < 0.05) with an increase of up to 5mm in length compared to WT-COL and

35S:E (figure 5.13A). In root length, 35S:SDG4i lines 1,3,5 and 6 showed significant

differences with root lengths up to 3mm greater than WT-COL and 35S:E (figure

5.13B). For the heat tolerance test during vegetative growth, two week old seedlings

of each A. thaliana line were exposed to heat stress, and percentage survival scored.

The only significant differences observed during this heat stress test were displayed in

lines 35S:SDG4i 3 and 6, where 85% survival was recorded (figure 5.14).

Figure 5.13: Hypocotyl and root length of A. thaliana lines following germination after

the seed was heat shocked at 42ºC

Hypocotyl and root lengths of WT-COL, 35S:E, T-AT3G43850 and 6 35S:SDG4i transgenic

lines were measured after 5 days growth in the dark post seed heat treatment at 42ºC. Any

significant differences (p < 0.05) are highlighted with an asterisk. Values are the mean ±SE of

10 replicates.

A: B:

WT-COL

35S:E

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

5

10

15

20

**

*

Hyp

ocot

yl g

row

th (m

m)

WT-COL

35S:E

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

5

10

15

Roo

t len

gth

(mm

)

** *

*

148

Figure 5.14: Percentage survival of A. thaliana lines in vegetative heat treatment

Survival was scored across WT-COL, 35S:E, T-AT3G43850 and 6 35S:SDG4i transgenic

lines after 5 days at 22°C with 16 hour light/8 hour dark cycles post heat treatment at 42ºC.

Any significant differences (p < 0.05) are highlighted with an asterisk. Values are the mean

±SE of 10 replicates.

WT-COL

35S:E

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

20

40

60

80

100

120%

Sur

viva

l

* *

149

5.3.6 Cold germination assay

The germination of A. thaliana lines was assessed during cold treatment at 4ºC in the

dark. The progression of germination of each line was recorded with reference to seed

swelling, radicle emergence and hypocotyl length. The incidence of seed swelling

occurred 4 days earlier in 35S:SDG4i lines (p < 0.05) in comparison to WT-COL and

35S:E controls. There was a degree of variation when measuring radicle emergence

with only two 35S:SDG4i lines (2 and 3) showing an earlier radicle emergence 3 days

earlier than controls (p < 0.05). When measuring the days taken for the hypocotyl to

elongate beyond 2mm, all six 35S:SDG4i lines displayed this 5 to 6 days earlier than

WT-COL and 35S:E (p < 0.05). The T-AT3G43850 line presented no differences to

the WT-COL and 35S:E lines at each measured stage of germination (figure 5.15).

Figure 5.15: Progression of germination of A. thaliana lines at 4ºC

Germination stages were measured in days post introduction to 4ºC in darkness. Seed

swelling, radicle emergence and a hypocotyl length > 2mm was measured across all A.

thaliana lines. Any significant differences (p < 0.05) are highlighted with an asterisk. Values

are the mean ±SE of 10 replicates.

Seed sw

ollen

Radicl

e emerg

ence

Hypoco

tyl >

2mm

0

5

10

15

20

25 WT-COL35S:ET-AT3G4385035S:SDG4i 135S:SDG4i 235S:SDG4i 335S:SDG4i 435S:SDG4i 535S:SDG4i 6

* * * * * *

* * * * * ** *D

ays

150

5.3.7 Vegetative freezing tolerance assay

Freezing tolerance was measured after exposure to extreme cold, and a 5 day recovery

period at 22ºC. Survival was recorded at -1, -4, -8, -12 and -16ºC (figure 5.16A). All

six 35S:SDG4i transgenic lines exhibited an increased percentage of survival at -8ºC

(figure 5.16B) in comparison to WT-COL and 35S:E by up to 40% (p < 0.05). The T-

AT3G43850 line also displayed some tolerance to freezing at -8ºC, with survival

greater than WT-COL and 35S:E by 15%.

Figure 5.16: Percentage survival of A. thaliana lines after vegetative freezing treatment

at -8ºC

Survival was scored across WT-COL, 35S:E, T-AT3G43850 and six 35S:SDG4i transgenic

lines after 5 days at 22°C with 16 hour light/8 hour dark cycles post freezing treatment at -1, -

4, -8, -12 and -16ºC. A survival curve across all temperatures (A) and freezing tolerance at -8

ºC (B) is presented. Any significant differences (p < 0.05) are highlighted with an asterisk.

Values are the mean ±SE of 10 replicates.

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5.3.8 Senescence assay

Leaf senescence was induced by growing plants under either constant darkness, or

under continuous light with 5µM ethylene or with 50µM ABA, and monitoring the

response over five days. A control test was performed under continuous light without

hormones to observe leaf senescence without hormone application over a five day

period. The observation of total chlorophyll levels prior to senescence induction

across all A. thaliana lines was measured (figure 5.17). 35S:SDG4i lines 1, 2, 3 and 6

show an increased amount of total chlorophyll in comparison to WT-COL and 35S:E

with each line containing between 10-30% more chlorophyll (p < 0.05). During

senescence induction, the total amount of chlorophyll was measured and presented as

a percentage relative to the amount of chlorophyll present at time zero. During

standard conditions at 22ºC under continuous light, the decline in total chlorophyll

across all lines shows no differences (figure 5.18A). During dark induced senescence,

the chlorophyll content across all six 35S:SDG4i lines and 35S:E was significantly

lower after 120 hours than WT-COL and T-AT3G43850 (figure 5.18B). Induction of

senescence using ethylene showed no significant difference in the rate of chlorophyll

loss across all lines (figure 5.18C). Induction of senescence using ABA (figure 5.18D)

showed that all six 35S:SDG4i lines had a significantly greater amount of chlorophyll

after 120 hours in comparison to WT-COL and 35S:E (p < 0.05). The T-AT3G43850

line showed no difference.

The health of photosystem II (measured as Fv/Fm), and total chlorophyll content was

measured daily for the duration of the experiment. Fv/Fm values remained stable

across all lines throughout the control conditions of 22ºC and continuous light, with

slight fluctuations between 0.73-0.83 (figure 5.19A). During the induction of

senescence using total darkness, the Fv/Fm values across all lines decreased at a

similar rate, however, the Fv/Fm values of all six 35S:SDG4i lines appeared lower

than WT-COL 35S:E and T-AT3G43850 at day five, although the difference was not

statistically significant (figure 5.19B). When using ethylene to induce senescence

under constant light (figure 5.19C), 35S:SDG4i lines 1, 2, 4, 5 and 6 retained a higher

Fv/Fm value across 5 days (p < 0.05) in comparison to WT-COL and 35S:E lines.

35S:SDG4i 3 and T-AT3G43850 did not show any difference. During ABA induction

of senescence (figure 5.19D) all six 35S:SDG4i lines showed reduced decline of

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Fv/Fm values over 5 days (p < 0.05) in comparison to the WT-COL and 35S:E. The

T-AT3G43850 showed no difference in Fv/Fm decline.

Figure 5.17: Total chlorophyll content of A. thaliana lines prior to senescence

induction

The chlorophyll content of each line was quantified prior to senescence induction. Chlorophyll

content is displayed as µg total chlorophyll per mg fresh weight tissue. Significant differences

(p < 0.05) are noted with an asterisk. Values are the mean ±SE of 3 replicates.

WT-COL

35S:E

T-AT3G

4385

0

35S:S

DG4i 1

35S:S

DG4i 2

35S:S

DG4i 3

35S:S

DG4i 4

35S:S

DG4i 5

35S:S

DG4i 6

0

100

200

300

400

500

µg/

mg

chlo

roph

yll

* * **

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Figure 5.18: Chlorophyll content of A. thaliana lines during senescence

Total chlorophyll content was measured over five days after senescence induction. Standard

conditions in continuous light (A), Total darkness (B), 5µM ethylene (C) and 50µM ABA (D)

are displayed in this figure as % Chlorophyll over time. Significant differences (p < 0.05) are

noted at 120 hours post senescence induction with an asterisk. Values are the mean ±SE of 3

replicates.

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Figure 5.19: Photosystem II activity of A. thaliana lines during senescence

Photosystem II health of all leaves was measured over five days after senescence induction.

Standard conditions in continuous light (A), Total darkness (B), 5µM ethylene (C) and 50µM

ABA (D) are displayed in this figure as Fv/Fm over time. Significant differences (p < 0.05) are

noted at 120 hours post senescence induction with an asterisk. Values are the mean ±SE of 3

replicates.

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5.4 Discussion

5.4.1 35S:SDG4i A. thaliana lines display increased tolerance to osmotic stress and salinity

Whilst an accurate measure of plant tolerance to drought should be completed using

water withholding assays, osmotic stress similar to that induced by drought can be

explored using the application of mannitol in in vitro assays (Xiong et al., 2006). The

use of mannitol additions in vitro has shown that constitutive expression of XVSAP1,

a dehydration responsive gene from the resurrection plant X. viscosa, increases

osmotic stress tolerance in A. thaliana (Garwe et al., 2006). The ability of SDG4i

transgenic lines to continue root growth on 300mM mannitol indicates that SDG4i

activity can enhance stress tolerance in Arabidopsis. Inhibition of lateral root growth

has been suggested to be an adaptive response of the plant to drought stress mediated

by ABA, however, it is not accompanied by an increase in primary root growth

(Xiong et al., 2006). While primary root growth is enhanced in SDG4i transgenic

lines lateral root growth does not appear to be inhibited in at least three of the SDG4i

transgenic lines (figure 5.6B). This suggests that SDG4i may be affecting the ability

of the plants to sense the drought stress or initiate the adaptation response. A greater

leaf number in SDG4i transgenic lines also provides further evidence of continued

growth and a diminished response to osmotic stress.

Osmotic stress can limit the ability of a seed to germinate. Vallejo et al. (2010)

examined the germination of several A. thaliana accessions under moderate osmotic

and salt stresses and showed that A. thaliana Columbia-0 displays a moderate

tolerance to germination during osmotic stress. The over-expression of SDG4i in this

accession showed a significant improvement in germination under osmotic stress at

200mM mannitol, the germination of 35S:SDG4i lines are 40% more successful than

WT-COL. The experimental data demonstrates that A. thaliana over-expressing

SDG4i have the ability to withstand greater amount of osmotic stress during

vegetative growth and germination. A water withholding soil based drought stress test

to further examine the performance of 35S:SDG4i A. thaliana under drought stress

would help determine the potential of SDG4i to enhance drought stress tolerance.

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Salinity causes osmotic stress in plants by disturbing ion transport of Na+ and Cl-, as

well as other ions including K+ and Ca2+, and the inability of a plant to adapt to a high

salinity environment can result in plant death (Niu et al., 1995). Plants living in saline

environments are exposed to Na+ concentrations between 100-2380mM and to be

considered a halophyte a plant must be able to complete its life cycle in at least

200mM NaCl (Flowers et al., 2010). Under the conditions used in this study, the

growth of WT-COL A. thaliana plants was severely inhibited on media containing

125mM NaCl. This provided a range of Na+ concentrations to assess tolerance to

salinity in SDG4i transgenic plants. Over expression of a wheat dehydrin DHN-5 in

A. thaliana improved germination capacity in media containing 200mM NaCl (Brini

et al., 2007). SDG4i transgenic lines showed a tolerance to salt stress during

germination. At all salt levels tested, the SDG4i transgenic lines showed an increased

germination capacity, with the greatest difference in germination percentage between

WT-COL and SDG4i transgenic lines being seen at 75mM NaCl. In addition to

increased germination capacity under salinity stress the 35S:SDG4i lines also showed

increased vegetative growth across all levels of NaCl tested, however, the greatest

difference was observed at 125mM NaCl, where root length and leaf number where

substantially greater than WT-COL. This is similar to the results obtained in A.

thaliana when XVSAP1 from X. viscosa is over expressed (Garwe et al., 2006). To

assess if 35S:SDG4i plants are exhibiting salt tolerance via an ionic tolerance

mechanism rather than an osmotic tolerance mechanism an experiment utilising a Na+

analogue such as Li+ could be performed. Li+ has been suggested to share the same

toxicity targets and transport systems as Na+ and can be used at concentrations which

have no osmotic effects due to its higher inhibitory potency (Serrano 1996).

Furthermore, it would be interesting to explore whether the osmotic stress tolerance of

seeds during germination could be attributed to genotypic effects of the seed, or if it is

a maternal effect. This could be completed by determining germination capacity of

35S:SDG4i hemizyotes on media containing a range of osmoticum concentrations.

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5.4.2 35S:SDG4i A. thaliana lines display increased tolerance to exogenous ABA application

Many desiccation related genes are up-regulated by the exogenous application of

ABA (Bartels et al, 1990, Michel et al., 1994). Section 3.3.5 showed that SDG4i is

up-regulated by exogenous application of ABA. Characterisation of global responses

to ABA-regulated responses to dehydration identified that ABA-dependent

transcriptional regulation of branched-chain amino acid biosynthesis genes were

required for A. thaliana to tolerate drought stress (Urano et al., 2009). Branched chain

amino acids accumulate during drought stress and may be required by the cell as

compatible solutes and osmprotectants (Bowne et al., 2012, Joshi & Jander 2009).

35S:SDG4i lines appear to be insensitive to ABA and do no show normal inhibition

of growth associated with 35S:E and WT-COL controls. This suggests SDG4i may

have a role in ABA perception/signalling or may attenuate gene expression that is

regulated by ABA. Three classes of ABA insensitive mutants (abi1, abi2 and abi3)

have defined genes that control subsets of ABA responses. Mutants that display

negative regulation of ABA signalling abi1 and abi2 displayed decreased sensitivity

to ABA during seedling growth and increased proline accumulation, while abi3

showed a mild reduction in ABA sensitivity as compared with abi1 and abi2, and no

increase in proline accumulation. However, the abi3 mutant had an increased amount

of storage protein in the seed. The study of these mutants has suggested that ABA

responses may be controlled by two parallel pathways (Finklestein & Somerville

1990). 35S:SDG4i lines, which appear to be ABA insensitive, display similar

characteristics to abi1 and abi2 mutants, which suggests that SDG4i may be enabling

an increase in osmolyte accumulation, thereby protecting the cell from osmotic stress.

The analysis of proline levels in SDG4i transgenic plants may be informative. The

mutant Arabidopsis deficient in ABA biosynthesis aba1, as well as the mutant

Arabidopsis deficient in ABA signalling aba4, shows a wilting phenotype due to

stomatal closure being affected, as well as poor performance during abiotic stress

(reviewed in Zhu 2002). 35S:SDG4i A. thaliana in the current study show not only

ABA insensitivity, but also an increased tolerance to osmotic, salt and cold stress, this

may indicate that changes in ABA biosynthesis/signalling in 35S:SDG4i plants may

not be the same as those displayed in ABA mutant plants.

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In a study on transgenic tobacco expressing an ABA-modulating antibody, which

removed free ABA within seed, the germination capacity of the transgenic tobacco

seed was faster than in wild-type seed (Phillips et al., 1997). Furthermore, precocious

germination of Z. mays seeds deficient in ABA (Robertson 1995) demonstrate that a

reduction in or insensitivity to ABA correlates with an increased germination

capacity. This suggests that the increased capacity of 35S:SDG4i seeds to germinate

in the presence of ABA may be associated with ABA perception/signalling, or have a

reduced accumulation of ABA during development. This possible reduction in ABA

accumulation during seed development may be the cause for reduced seed longevity

discussed in chapter four. The ability of 35S:SDG4i plants to show insensitivity to

ABA is also apparent where 35S:SDG4i lines display an increased leaf number,

lateral root number and root length during vegetative growth on 4µM ABA. Extreme

insensitivity to ABA has been seen in A. thaliana mutants deficient in SnRK2 (SNF1-

related protein kinase subfamily 2) protein kinases associated with ABA signal

transduction, where mutants plants were virtually unaffected by high amounts of

exogenous ABA at all stages of plant development (Fujii & Zhu 2009). This paper

suggested that abscisic acid activated protein kinases were critical for ABA

perception during growth, reproduction and stress. SDG4i over-expression in A.

thaliana appears to restrict the sensitivity of the plant to ABA, suggesting that SDG4i

attenuates the ABA response.

5.4.3 35S:SDG4i A. thaliana lines show altered thermotolerance

The resurrection plant S. stapfianus has provided information on mechanisms

associated with survival of extreme dehydration (Le et al., 2007, Blomstedt et al.

1998), however, genes transcribed in this plant could also provide clues as to how the

plant copes with other abiotic stresses such as heat and cold. The tolerance of

resurrection plants to high temperature stress has not been extensively studied. Of the

six 35S:SDG4i lines, four displayed an increased tolerance to germination under heat

stress and a vegetative heat stress treatment resulted in the survival of two 35S:SDG4i

lines. The results are not conclusive, and while SDG4i may not have a major role in

providing tolerance to heat, the secondary effects of the introduction of SDG4i in A.

thaliana may provide some heat tolerance. When assessing vegetative heat tolerance

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the seedlings were not acclimatised to the heat, rather a shock of extreme heat was

applied to the seedlings. Further information on the possible role of SDG4i in heat

stress tolerance could be provided using an adaptive heat stress assay. In addition, it

would be useful to analyses whether the altered heat tolerance is attributable to the

level of SDG4i expression in each transgenic line. The introduction of SDG4i into A.

thaliana leads to an increased capacity for all six 35S:SDG4i lines to germinate at 4ºC

and enhanced survival of freezing stress at -8ºC. The treatment of seeds to 4ºC for 3

days is used to achieve a uniform germination once the plants are removed from the

cold and placed in normal growth conditions (Nordburg & Bergelson 1999). If seeds

are left at 4ºC, the germination rate slows. Generally, the over expression of cold

shock domain proteins will enhance the ability of the seed to germinate in cold

temperatures (Park et al., 2009). The over-expression of SDG4i in A. thaliana

provides the seed with the ability to germinate more rapidly in cold temperatures. This

ability could be mediated by the possible affect SDG4i has on plant hormone

accumulation (discussed in 4.4 and 5.4.2) or the ability of SDG4i to influence the

perception of abiotic stress. Plant freezing tolerance involves the prevention of freeze-

induced damage to the plasma membrane, which is lethal to the plant (Yamazaki et

al., 2008). This is similar to the effects of osmotic stress on the plant where plasma

membranes are compromised by water loss (Xiong & Zhu 2002). It is possible that

the enhanced stress tolerance exhibited by the transgenic lines to freezing, drought

and salinity are mediated by a similar mechanism. These results provide evidence that

the introduction of genes, involved in extreme dehydration in resurrection plants, into

glycophytic plants can increase tolerance to abiotic stresses and can be useful if

introduced into crop species, which are exposed to abiotic stress during their

germination and growth.

5.4.4 35S:SDG4i A. thaliana lines show delayed senescence in the presence of ABA

The event of senescence involves a highly regulated and ordered degeneration of

molecular structures leading to the death of single cells, organs or whole plants

(Nooden & Penney 2001). As in most monocarpic plants, longevity of A. thaliana is

controlled by the production of reproductive structures. Senescence in plants can be

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accelerated by abiotic stresses (Weaver et al., 1998), the absence of light (Hanaoka et

al., 2002) and traditionally, by an increased exposure to ethylene (Weaver et al.,

1998). Leaf senescence assays are often used to study plant senescence under

differing induction conditions (Hanaoka et al., 2002). Senescence can be gauged by

the ability of the plants to photosynthesize and retain chlorophyll, which is degraded

during senescence (Pruzinska et al., 2005). In section 4.3.6, a delayed senescence of

35S:SDG4i plants were observed, suggesting that SDG4i represses senescence. The

amount of chlorophyll prior to senescence induction was higher in four of six

35S:SDG4i lines by up to 20%. A 7-fold increase in chlorophyll level is seen in A.

thaliana over expressing a seed specific phytoene synthase gene, a rate limiting

enzyme for carotenoid biosynthesis (Lindgren et al., 2003). It is possible that the

action of SDG4i in A. thaliana may be influencing carotenoid biosynthesis, resulting

in the increased amount of chlorophyll seen in SDG4i transgenic plants.

Artificially induced leaf senescence assays showed that 35S:SDG4i plants retained

photosynthetic capacity after 5 days treatment of leaves with ethylene or ABA

however the retention of photosynthetic capacity could be a side effect of the greater

amount of chlorophyll in 35S:SDG4i lines. The amount of chlorophyll lost as a

proportion of the initial total, in ABA treated leaves was significantly less and

retention of photosynthetic capacity it is not seen in the dark induced leaves. Although

photosynthetic capacity was retained in ethylene treated leaves, the reduction in

chlorophyll was observed to be at the same rate as the WT-COL. This demonstrates

that the retention of photosynthetic capacity in ethylene treated leaves may be due to

the increased amount of chlorophyll within the leaf tissue. Although differences

between the control and ethylene treated lines are noted, it has been shown that

response of A. thaliana to ethylene and leaf senescence occurs at a specific age

window (Jing et al., 2002), which was not reached in this study. It would be advisable

to repeat ethylene treatment on older leaves for a definitive result.

Exposure to increased ABA in A. thaliana induces senescence associated genes

(SAGs). Other senescence inducing treatments activate different SAGs some of which

are also induced by ABA, others which are not. This suggests that A. thaliana has a

particular senescence pathway associated with ABA (Weaver et al., 1998). In

addition, the exogenous application of ABA has been demonstrated to degrade

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chlorophyll (Back & Richmond 1971). The ABA-induced senescence experiment in

the chapter has shown that leaf tissues over-expressing SDG4i show a greater

retention of photosynthetic capacity and chlorophyll in the presence of exogenous

ABA in comparison to the wild type control. This suggests that SDG4i may be

involved in attenuating the ABA-induced senescence pathway. It may also be likely

that SDG4i is involved in decreasing sensitivity or perception of the plant to ABA,

which leads to inhibition of ABA-induced senescence. In particular, chlorophyll

degradation is reduced despite the traditional response of chlorophyll reduction upon

exposure to ABA (Back & Richmond 1971). Examining endogenous ABA levels may

indicate if SDG4i transgenic plants accumulate less ABA or are impaired in ABA

signalling.

5.5 Conclusion

The tolerance of 35S:SDG4i A. thaliana to osmotic, salt and cold stress has revealed

the likelihood for a role of SDG4i in S. stapfianus to be related to surviving a stress-

event, such as what is seen during desiccation and the induction of the desiccation

tolerance pathway. In addition, exogenous exposure of these plants to ABA has

revealed that SDG4i may be associated with ABA signalling or perception. The

observation of delayed senescence in 35S:SDG4i A. thaliana in chapter 4 was

explored further in this chapter where it was found that 35S:SDG4i A. thaliana plants

display delayed senescence during exogenous ABA exposure. This chapter has

presented further evidence that SDG4i may be involved in regulation of ABA-

dependent stress-responses in S. stapfianus. The next chapter will look at the potential

protein-protein interaction of SDG4i and other proteins present in dehydrating

S. stapfianus leaf tissue.

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6.0 Interaction of SDG4i with ERD15-like protein using a yeast two-hybrid screen

6.1 Introduction

The ability of a resurrection plant to survive severe dehydration is a complex process,

which is yet to be fully understood (Chaves 2003). The isolation of novel genes that

are expressed during the dehydration process is the target of current investigations

into the molecular basis of the ability of S. stapfianus to survive extreme dehydration

(Le et al., 2007). In previous chapters, the exploration of the effects of SDG4i over-

expression in A. thaliana on the developmental phenotype and abiotic stress

tolerances have provided detailed morphological information, however it does not

provide evidence of SDG4i function at the molecular level.

SDG4i contains a serine rich region, which may be indicative of a protein-protein

interaction. The possibility of a protein-protein interaction between SDG4i and an

unidentified partner was also raised by the homology that SDG4i has with a B. napus

bnKCP1 protein, which is involved in an interaction with histone deacetylase (Gao et

al., 2003). A well established in vivo approach to detecting protein-protein

interactions is provided by the yeast two-hybrid (Y2H) screening protocol. The

potential for finding protein-protein interactions using this method is high; however,

the detection of false negative and false positive interactions can be a problem. When

a positive interaction(s) is detected through a Y2H assay, confirmation of this

interaction is necessary (Bruckner et al., 2009). The confirmation can be obtained

using the bimolecular fluorescence complementation assay (BIFC) which is a

commonly used method of visualising protein-protein interactions in plants (Walter et

al., 2004, Lee et al., 2008). This chapter details the results of an investigation of

potential protein-protein interactions between SDG4i and other proteins expressed

during the desiccation of S. stapfianus. The potential role of SDG4i in S. stapfianus in

light of this information will be discussed.

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6.2.1 Construction of cDNA-GAL4AD library

A cDNA-GAL4AD (GAL4 Activating Domain) library was previously constructed

for a yeast one-hybrid screen (Le, 2004). This GAL4AD library was made by ligating

insert DNA from a desiccated S. stapfianus cDNA library (Blomstedt et al., 1998)

into the pGAD424 vector using EcoR1 and Xho1 restriction sites followed by

transformation into XL-10 GOLD E. coli electro-competent cells (Stratagene) (Le

2004). To test the integrity of this library a 1mL frozen aliquot of the library was

spread onto 20 individual LB agar plates supplemented with 50µg mL-1 ampicillin

and incubated overnight at 37ºC. Ninety colonies were selected for colony PCR

amplification (section 2.3.4) to assess the size of the inserts present in this library.

The following pGAD424 specific primers were used: forward:

5’ CTATTCGATGATGAAGATACCCCACCAAACCC 3’ (ADLDF) and reverse:

5’ GTGAACTTGCGGGGTTTTTCAGTATCTACGAT 3’ (ADLDR) for colony PCR

to assess insert size and library amplification. The library was amplified in 10

individual 100mL LB broths containing 50µg mL-1 amplicillin and the plasmids were

isolated using a plasmid midi-prep protocol as previously described (Section 2.3.6).

The resulting plasmid DNA was pooled and used for transformation of S. cerevisiae

cells. A library scale yeast transformation system (YeastmakerTM Yeast

Transformation system 2, Clontech) was employed to transform Y187 S. cerevisiae

with the DNA from the desiccated S. stapfianus cDNA-GAL4AD library. Competent

yeast cells were made from an individual colony of Y187, inoculated into YPDA

broth and were grown at 30ºC overnight (shaking at 150rpm). The culture was spun

down in a bench top centrifuge at 700g for 5 minutes, resuspended in 100mL fresh

YPDA and allowed to grow for 4 hours at 30ºC with shaking at 150rpm. Once the

OD600 reached 0.8, the culture was centrifuged as described previously, resuspended

in 100mL of distilled water, centrifuged again and resuspended in 2.5mL of

1.1×TE/LiAc (Tris EDTA/Lithium acetate, Clontech). The cell suspension was

divided into two 1.7mL Eppendorf tubes and the cells spun down briefly at 11000g,

followed by resuspension in 600µL of 1.1×TE/LiAC. The cells were used

immediately for transformation with the cDNA-GAL4AD library DNA. In a 15mL

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tube, 15µg of cDNA-GAL4AD plasmid isolate, 20µL of Yeastmaker carrier DNA

(10µg µL-1), 600µL of competent Y187 cells and 2.5mL of PEG/LiAc (Clontech)

were gently mixed and incubated at 30ºC for 45 minutes. Cells were then spun down

at 700g for 5 minutes in a bench top centrifuge and resuspended in 3mL of YPD Plus

(Clontech) medium and incubated at 30ºC for 90 minutes. Cells were spun down as

described previously and resuspended in 0.9% NaCl. Dilutions of the transformed

cells of 1/10 and 1/100 were spread onto SD/-Leu media and transformation

efficiency was calculated after 3-5 days growth at 30ºC. Transformation efficiency

was calculated to be 3.2x105 cfu µg-1. The remaining transformed cells were grown in

SD/-Leu media at 30ºC with shaking at 150rpm and were subcultured every 3 days for

1 week. The library culture was then centrifuged at 1000g for 5 minutes and

resuspended in SD/-Leu with 25% glycerol at a density of 2 x 108 cells mL-1, snap

frozen in liquid N2, and stored at -80ºC.

6.2.2 Construction of SDG4i-GAL4BD bait

The construct used to make the bait construct was the GAL4 binding domain (BD)

containing vector pGBKT7 (Clontech). SDG4i was amplified using pMDC32-SDG4i

as a template with the forward primers containing the EcoRI cleavage site, forward:

5’ CATGGAGGCGGAATTCATGCCGGTTGACGTCGCACGCGCC 3’ and

the reverse primer containing the BamHI cleavage site: 5’

GCAGGTCGACGGATCCCTAAGAGTCCACCTTCTTATCTTT 3’. The

amplification was carried out with Pfu DNA polymerase (Promega). The vector

pGBKT7 and the amplified SDG4i fragment were digested with EcoRI and BamHI

(section 2.5.7) and purified using the Wizard® PCR purification kit (Promega)

(section 2.5.9). The fragments were ligated using a 1:10 vector/fragment ratio (section

2.5.8) producing pGBKT7-SDG4i. This construct was electroporated into TOP10 E.

coli cells (section 2.3.2) and selected by ampicillin resistance (50µg mL-1). The

construct was purified from an E. coli broth (section 2.3.5) and subject to sequencing

using the forward and reverse cloning primers previously mentioned to verify the

reading frame (section 2.5.3). The pGBKT7-SDG4i construct was transformed into S.

cerevisiae Y2HGold (Clontech) using the method stated in YeastmakerTM Yeast

Transformation system 2 (Clontech), described in section 6.2.1, for small scale

165

transformation. Cells were spread on SD/-Trp media and allowed to grow for 3-5 days

at 30ºC.

6.2.3 Bait autoactivation and toxicity test

Prior to mating, pGBKT7-SDG4i containing yeast were tested for autoactivation of

the Y2HGOLD reporter gene for histidine biosynthesis. pGBKT7-SDG4i yeast were

spread onto SD/-Trp/-His plates and incubated at 30ºC for 3 days. Colonies greater

than 2mm in diameter were counted. The addition of 3-Amino-1,2,4-trizole (3-AT)

(Sigma-Aldrich) at concentrations of 1, 5, 10, 25 and 50mM was included to

determine the level of 3-AT required to inhibit auto-activated histidine production

within the yeast. Toxicity of SDG4i expression in yeast was observed by comparing

the growth of yeast containing pGBKT7 (empty vector) and pGBKT7-SDG4i on SD/-

Trp media for 3 days at 30ºC.

6.2.4 Screening of cDNA-GAL4AD library using yeast mating

In order to screen the cDNA-GAL4AD (containing the GAL4 AD) DNA library with

the bait yeast strain pGBKT7-SDG4i (containing the GAL4 BD) yeast mating was

used following the protocol detailed in the MatchmakerTM GOLD Yeast Two-Hybrid

system user manual (Clontech). A fresh colony of pGBKT7-SDG4i was used to

inoculate a flask of SD/-Trp broth and was incubated at 30ºC for 20 hours until OD600

was 0.8. The culture was centrifuged a 1000g for 5 minutes and the cells were

resuspended to a density of 1 x 108 cells mL-1 in SD/-Trp. A 2mL aliquot of the

cDNA-GAL4AD library was thawed and 10µL removed to assess titre by spreading

1/100, 1/1000 and 1/10000 dilutions onto SD/-Leu plates and incubating for 3 days at

30ºC. The thawed cDNA-GAL4AD library was added to a 2L flask and mixed with

5mL of resuspended pGBKT7-SDG4i bait strain. 45mL of 2xYPDA liquid media was

added to the flask and kanamycin added to reach a final concentration of 50µg mL-1.

The flask was then allowed to shake at 50rpm at 30ºC for 24 hours. This process was

repeated using the pGBKT7 vector without an insert as a control.

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6.2.5 Selection of positive diploids

The mating culture was centrifuged at 1000g for 10 minutes and cells were

resuspended in 10mL of 0.5xYPDA containing 50µL kanamycin. To assess the

number of colonies screened, 1/10, 1/100 and 1/10000 dilutions of the mated culture

were spread on SD/-Trp, SD/-Leu and SD/-Trp/-Leu plates. The remainder was

spread on SD/-Trp/-Leu/-His plates and allowed to grow at 30ºC for 3 days. 3.5

million diploids were screened. Colonies greater than 2mm in diameter growing on

SD/-Trp/-Leu/-His plates were streaked onto SD/-Trp/-Leu/-His/-Ade plates and

colonies that continued growth on these plates were subject to yeast colony PCR to

view any possible duplicate clones based on insert size. This involved inoculating

10µL 0.02M NaOH with a yeast colony using a pipette tip and incubating at 99ºC for

10 minutes to lyse the yeast. 1µL of the lysis mix was then used as a template for a

standard PCR using Taq DNA polymerase with the primers ADLDF and ADLDR

(section 6.2.1).

6.2.6 Plasmid rescue from positive diploids

Three subcultures of each positive diploid growing on SD/-Trp/-Leu/-His/-Ade plates

were scraped off, placed in individual 1.7mL Eppendorf tubes and resuspended in

10µL of lyticase solution (5 units µL-1 in TE buffer). The tubes were incubated for 1

hour at 37ºC. After incubation, 10µL of 20% SDS (w/v) solution was added and the

tubes were vortexed, and placed at -20ºC for 30 minutes. The tubes were thawed and

vortexed to ensure complete lysis. TE buffer was added to the tubes to a final volume

of 200µL and extracted once with 25:24:1 phenol:chloroform:isoamyl alcohol

(Sigma-Aldrich). The tubes were vortexed, then centrifuged at 16000rpm for 10

minutes and the aqueous phase transferred to a new tube, 1/10 volume of 10M

ammonium acetate and 2½ volume of 100% ethanol were added, the tubes were

mixed by inversion and placed at -70ºC for 1 hour. The tubes were then centrifuged at

4ºC for 20 minutes and the plasmid DNA resuspended in 20µL of TE buffer. The

resulting plasmids were electroporated into TOP10 E. coli (section 2.3.2) and selected

using 50µg mL-1 kanamycin. The rescued plasmids were amplified and purified from

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E. coli (section 2.3.5) and were sequenced (section 2.5.3) using the ADLDF and

ADLDR primers (section 6.2.1).

6.2.7 Bimolecular fluorescence complementation assay (BIFC) vector construction

A BIFC assay was utilised to confirm positive interactions between SDG4i and

proteins encoded by S. stapfianus cDNA inserts. Constructs for BIFC vectors were

split yellow fluorescent protein (YFP) (defined as N terminal YFP and C terminal

YFP): pNBV-gYN (gene-N terminal YFP), pNBV-YNg (N terminal YFP-gene),

pNBV-gYC (gene-C terminal YFP) and pNBV-YCg (C terminal YFP-gene). The split

YFP was fused at both the N and C terminal of interacting candidate protein to

maximise the potential for interaction. The N terminal YFP expression constructs

were used for fusion with SDG4i, while the C terminal YFP expression constructs

were used for fusion with the positive interaction proteins from the yeast two-hybrid

assay. SDG4i, ERD15, BIRK1 and ADF genes were amplified with the primers listed

in table 6.1 using the appropriate yeast two-hybrid constructs as a template. The

fragments were amplified using PCR with Pfu DNA polymerase and the products

purified from an agarose gel (section 2.5.9). Each fragment and construct was

digested with the appropriate restriction enzymes (table 6.1) (section 2.5.7), purified

from an agarose gel (section 2.5.9) and ligated at a 3:1 insert:vector ratio (section

2.x.x) and electroporated into TOP10 E. coli (section 2.3.2). E.coli containing each

constructed was grown overnight in LB broth containing 50µg mL-1 of ampicillin, and

the plasmids isolated using a miniprep method (section 2.3.5). The plasmids were

then electroporated into A. tumerfaciens GV3101 (section 2.3.2).

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Table 6.1: Primers used for BIFC vector construction. Restriction sites are underlined. Destination

vector

Insert

gene

Primer Restriction

endonuclease

pNBV-gYN SDG4i Forward: 5’ GAGATCTAGAATGCCGGTTGACGTCGCAC 3’

Reverse: 5’ GAGAGGATCCAGAGTCCACCTTCTTATC 3’

XbaI

BamHI

pNBV-YNg SDG4i Forward: 5’ GAGAACTAGTATGCCGGTTGACGTCGCAC 3’

Reverse: 5’ GAGAGGATCCAGAGTCCACCTTCTTATC 3’

SpeI

BamHI

pNBV-gYC ERD15 Forward: 5’ GAGATCTAGAATGGGTGCCACCG 3’

Reverse: 5’ GAGAAGATCTAGATCTCTGCAGG 3’

XbaI

BglII

pNBV-YCg ERD15 Forward: 5’ GAGAACTAGTATGGGTGCCACCG 3’

Reverse: 5’ GAGAAGATCTAGATCTCTGCAGG 3’

SpeI

BglII

pNBV-gYC BIRK Forward: 5’ GAGATCTAGAATGGACACGAGCTGTAA 3’

Reverse: 5; GAGAGGATCCTCATAATAAGC 3’

XbaI

BamHI

pNBV-YCg BIRK Forward: 5’ GAGAACTAGTATGGTGACACGAGCTGTAAT 3’

Reverse: 5’ GAGAGGATCCTCATAATAAGC 3’

SpeI

BamHI

pNBV-gYC ADF Forward: 5’ GAGATCTAGAGTGCAGAAAAGC 3’

Reverse: 5’ GAGAGGATTCCGTGAGCCCGCT 3’

XbaI

BamHI

pNBV-YCg ADF Forward: 5’ GAGAACTAGTGTGCAGAAAAGC 3’

Reverse: 5’ GAGAGGATTCCGTGAGCCCGCT 3’

SpeI

BamHI

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6.2.8 N. bethamiana infiltration, co-incubation and visualisation of positive interactions

Cultures of A. tumerfaciens containing either the BIFC plasmids detailed in section

6.2.7, or an A. tumerfaciens containing P19, to suppress antiviral gene silencing

mechanisms in the host plant (Voinnet et al., 2003), were grown individually

overnight at 28ºC shaking at 150rpm. Each culture was spun down in a bench top

centrifuge at 1000g for 10 minutes and the pellet resuspended to an OD600 of 0.5 in

infiltration medium (10mM 2-N-morpholino-ethanesulfonic acid (MES) pH 5.5,

10mM MgSO4 and 200µM acetosyringone) and incubated at 28ºC with shaking at

150rpm for 1 hour. Each interaction shown in the yeast two-hybrid assay was tested

by mixing an equal amount of each culture, one part containing the P19 construct, one

part N-terminal YFP fused with SDG4i and one part C-terminal YFP fused with the

interacting protein of interest from the yeast two-hybrid screen. Each interaction was

tested by mixing the constructs according to table 6.2. The construct mixture was then

infiltrated using a syringe into the abaxial side of 3 week old N. benthamiana leaves

and left to co-incubate for 3 days. The leaves were then removed and 1cm2 sections

were cut and placed on a microscope slide and a coverslip placed on top. The

fluorescence of YFP was viewed using a UV light emitting microscope. Empty

constructs were included in the experiment as controls to asses the level of

background illumination.

Table 6.2: A. tumerfaciens infiltration mixtures. Interaction tested P19 SDG4i containing

construct used Gene of interest containing construct used

SDG4i-ERD15 P19 P19

pNBV-SDG4i-YN pNBV-YN-SDG4i

pNBV-ERD15-YC pNBV-YC-ERD15

SDG4i-BIRK P19 P19


pNBV-BIRC-YC pNBV-YC-BIRC

SDG4i-ADF P19 P19


pNBV-ADF-YC pNBV-YC-ADF

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6.2.9 Semi-quantitative RT-PCR of SDG4i and ERD15 in dehydrating S. stapfianus tissue

Leaf tissue from dehydrated S. stapfianus plants was subjected to RNA extraction

(section 2.5.4) and cDNA synthesis (section 2.5.6). The expression levels of SDG4i

and ERD15 were examined by semi-quantitative PCR, with SDG1i utilized as a

control, as it has been previously identified that the level of transcription of this gene

does not change throughout dehydration of S. stapfianus (Le 2004). The primers used

to amplify these genes are listed in table 6.3. cDNA (200ng) was used as a template in

each PCR reaction conducted with MyTaq DNA polymerase (Bioline) according to

the manufacturers instructions. Samples were removed at 10, 20, 30 and 40 cycles.

All reactions were duplicated with 200ng of total RNA as a template to determine the

level of gDNA contamination. Five microliters of each sample was loaded onto a

1.5% EtBr agarose gel and subject to electrophoresis (section 2.5.2). Amplification

products were directly quantified from the fluorescence intensity under UV light

using ImageQuantTL v. 4.0 software.

Table 6.3: Primers used for S. stapfianus gene expression study. Amplified gene Use 5’-3’

SDG4i Forward TGTGACAATGGCTAGTAGCAGT

Reverse GCGACTAGAAGATAGTCAATTTC

ERD15 Forward TCTATTGATGAAGATAC

Reverse GTGAACTTGCGGGGTTTTTG

SDG1i Forward AGCCAAGACCGGCGGTTG

Reverse GGTAGGCCGTAGCGGAGTACAG

171

6.2.10 Endoplasmic reticulum stress germination assay The stress tolerance of the endoplasmic reticulum was assessed using an in vitro

method. Basic ½ MS solid media (section 2.4.2.2) without sucrose addition was

supplemented with the following tunicamycin (Sigma-Aldrich) concentrations: 0µg

ml-1, 0.3µg ml-1 and 1.0µg ml-1 (Koizumi et al., 1999). For the germination assay, 10

seeds of each WT-COL, 35S:Empty vector (35S:E), T-AT3G43850, and six

35S:SDG4i lines line were placed on plates containing the appropriate level of

tunicamycin and were stratified at 4°C in the dark for 3 days. The plates were then

placed at 22ºC in 16 hour light/8 hour dark cycles and were scored daily for 10 days.

The assay was conducted 3 times.

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6.3 Results

6.3.1 Construction of cDNA-GAL4AD library

An aliquot of the cDNA-GAL4AD library containing cDNA inserts from desiccated

S. stapfianus leaf tissue was subject to colony PCR to assess the quality of the library

prior to transformation into the S. cerevisiae Y187 strain for use in a yeast two-hybrid

screen. The range of fragment sizes generated following PCR amplification of the

inserts can be seen in figure 6.1. Ninety individual E. coli colonies were screened and

a range of insert sizes from 300-2000bp in length was observed. The variety of insert

sizes was considered to be appropriate for the use of this library in a yeast two-hybrid

screen and the library was subsequently subject to midiprep and transformed into S.

cerevisiae Y187, at a transformation efficiency of 3.2x105 cfu µg-1. The transformed

yeast were also subjected to colony PCR and insert size variability was assessed.

Figure 6.2 shows the insert sizes, ranging from 300-2000bp, of 96 individual yeast

colonies and was considered suitable for use as the “prey” library for the yeast two-

hybrid screen.

173

Figure 6.1: Amplified inserts from the desiccated S. stapfianus cDNA-GAL4AD library

in E. coli

Colony PCR was used to amplify the inserts contained in the desiccated S. stapfianus cDNA-

GAL4AD library. Each lane contains an amplified insert from a single E. coli colony. The lanes

labelled λ/BsteII contain molecular weight markers.

174

Figure 6.2: Amplified inserts from the desiccated S. stapfianus cDNA-GAL4AD library

after transformation into S. cerevisiae Y187

Colony PCR was used to amplify the inserts contained in the desiccated S. stapfianus cDNA-

GAL4AD library following transformation into S. cerevisiae Y187. This library was used as the

“prey” for the Yeast two-hybrid screen. Each lane contains an amplified insert from a single

yeast colony. The lanes labelled λ/BsteII contain molecular weight markers.

175

6.3.2 Construction of Yeast two-hybrid bait construct Construction of the “bait” construct for the yeast two-hybrid screen was completed by

using PCR to amplify SDG4i using primers flanked with restriction enzyme sites,

which were then used to clone SDG4i in frame with the GAL4BD in the

MatchMakerTM vector pGBKT7 (Clontech). The resulting vector is depicted in figure

6.3 where the GAL4BD is located at the N-terminal of SDG4i. Sequencing was used

to verify the reading frame of the SDG4i insert (data not shown). The vector was then

transformed into S. cerevisiae Y2HGOLD strain, which contains the histidine and

adenine biosynthesis reporter genes that are regulated by GAL4 activation.

Figure 6.3: Yeast two-hybrid bait construct

SDG4i was cloned in-frame into pGBKT7 containing the GAL4 binding domain and was used

as the “bait” construct in the Yeast two-hybrid screen in conjunction with the cDNA-GAL4AD

library (prey construct).

pGBKT7-SDG4i8022 bp

GAL4 BD

c-Myc epitope tag

Kanamycin R

SDG4iT7 RNA pol Prom

T7 terminatorADH1 terminator

176

6.3.3 Bait toxicity, auto-activation, and screening of the cDNA-GAL4AD library

Prior to screening the cDNA-GAL4AD library, the yeast Y2HGOLD strain

containing the pGBKT7-SDG4i construct was tested for auto-activation of the

histidine synthesis reporter gene using 3-AT. The pGBKT7-AD construct did not

activate the histidine synthesis reporter gene to a sufficient level to allow colony

growth when 5mM 3-AT was added (data not shown). Therefore 5mM 3-AT was

added to selection media post-yeast mating to prevent false positives. In addition, the

expression of SDG4i in S. cerevisiae Y187 facilitated by the pGBKT7-SDG4i

construct did not appear to be toxic to the yeast, with colonies having similar

appearance to the pGBK empty vector control S. cerevisiae Y187 (data not shown).

The cDNA-GAL4AD library was screened by a yeast mating protocol using the

SDG4i construct (pGBKT7-SDG4i) containing the GAL4BD as bait. The resulting

mating culture was screened using media lacking histidine to observe positive

interactions within diploids marked by the histidine biosynthesis reporter gene.

Colonies growing in this initial screen were selected and streaked onto media lacking

both histidine and adenine to verify the ability of these diploids to induce histidine

and adenine biosynthesis. The activation of the histidine and adenine reporter genes

determined by the growth of diploids on media lacking these amino acids is shown in

figure 6.4. Growth on this media suggests a positive interaction between the SDG4i

protein and the protein encoded by the cDNA-GAL4AD insert is occurring in these

diploids. Eighteen positive diploids were isolated from the yeast two-hybrid screen

and subjected to colony PCR to determine the insert size. Similar sized inserts were

seen across some of the diploids, which suggests some diploids may contain duplicate

cDNA inserts (Figure 6.5). Following plasmid rescue, the cDNA-GAL4AD insert

from each positive diploid was sequenced. Sequenced inserts were subjected to a

BLASTn homology search resulting in two clones with homology to an actin

depolymerising factor 6 (ADF) in Z. mays, two clones with homology to

brassinosteriod insensitive 1-associated receptor kinase (BIRK) in Z. mays, three

clones with homology to the transcription factor, ‘early response to dehydration”

factor 15-like protein (ERD15) in Z. mays and one clone each with homology to a

type-A response regulator (Z. mays), an aldehyde oxidase (Z. mays) and a Ras-related

177

GTP binding protein (O. sativa) (Table 6.4). Duplicate and triplicate clones, with

homology to the same genes in the NCBI database, are fragments overlapping the

same region of the gene that the inserts share the most homology with (appendix II).

Figure 6.4: Yeast diploids growing on SD/-Trp/-Leu/-His/-Ade with 5mM 3-AT

The diploids that exhibited growth on SD/-Trp/-Leu/-His media containing 5mM 3-AT from the

initial screen were streaked onto SD/-Trp/-Leu/-His/-Ade with 5mM 3-AT plates. The colonies

that were able to grow were subject to colony PCR and plasmid rescue.

Figure 6.5: Yeast colony PCR of diploids showing a positive interaction on SD/-Trp/-

Leu/-His/-Ade with 5mM 3-AT plates Diploids growing on on SD/-Trp/-Leu/-His with 5mM 3-AT plates were subject to yeast colony

PCR to observe variations in insert sizes. Each lane contains an amplified insert from a single

yeast colony. The lanes labelled λ/BsteII contain molecular weight markers.

178

Table 6.4: Results from a BLASTn analysis of library inserts interacting with SDG4i in the yeast two-hybrid assay at the nucleotide level Clone number

Insert size (bp)

Species Gene % Homology

E value Accession Number

23 478 Zea mays Actin-depolymerising factor 6

76% 1 x 10-107 NM_001155189

20 521 Zea mays Actin-depolymerising factor 6

71% 2 x 10-34 NM_001155189

10 177 Zea mays Brassinosteriod insensitive 1-associated receptor kinase

81% 2 x 10-35 NM_001155673.1

26 181 Zea mays Brassinosteriod insensitive associated receptor kinase 1

84% 4 x 10-31 NM_001155673.1

3 457 Zea mays Early response to dehydration 15-like protein

84% 2 x 10-58 EU967244.1


84% 2 x 10-58 EU967244.1


85% 2 x 10-33 EU967244.1

1 850 Zea mays Type-A response regulator

86% 8 x 10-4 NM_001156396.1

21 439 Zea mays Aldehyde oxidase

84% 3 x 10-133 NM_001111838.1

33 472 Oryza sativa

Ras-related GTP binding protein

81% 8 x 10-71 AY103999.1

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6.3.4 Construction of BIFC vectors Positive interactions between SDG4i and proteins encoded by the selected library

inserts were verified using a BIFC assay. Positive interactions, indicated through the

yeast two-hybrid screen that were rescued two or more times, were chosen for further

analysis. Thus, the interactions investigated through BIFC assay were SDG4i with the

largest fragment encoding ADF, BIRK or ERD15 (Table 6.4 clone numbers 20, 26

and 3, respectively). Each library insert was cloned into the BIFC assay vectors by

using PCR to amplify the inserts from the cDNA-GAL4AD library vector with

primers flanked by restriction enzyme sites. The restriction sites were then used to

clone each fragment into the BIFC assay vectors in frame with the C-terminal half of

YFP. SDG4i was amplified by PCR in a similar approach and cloned in frame with

the N-terminal half of YFP. Each vector made for the BIFC assay is depicted in figure

6.6, with each gene fused with YFP at the N-terminal or C-terminal end of the gene of

interest. The reading frame of each vector used in the BIFC assay was verified by

sequencing, and transformed into A. tumerfaciens GV3101 for use in the assay.

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Figure 6.6: BIFC assay vectors

Vectors were constructed for the BIFC assay using a split YFP system where the YFP was

fused at either the N terminal or C terminal of the gene of interest. The SDG4i expressing

constructs are fused with the N terminal of YFP (A) and the predicted interacting proteins

identified by the yeast two-hybrid screen are fused to the C terminal of YFP. The genes of

interest are ADF (B), BIRK (C) and ERD15 (D).

pNBV-YC-BIRK4600 bp

LinkerYC

BIRK1

Amp RpNBV-YC-BIRK

4600 bp

LinkerYC

BIRK1

Amp R

pNBV-SDG4i-YN5335 bp

YN

SDG4i

Amp R

pNBV-SDG4i-YN5335 bp

YN

SDG4i

Amp R

pNBV-ADF-YC4573 bp

YC

ADF

Amp RpNBV-ADF-YC

4573 bp

YC

ADF

Amp R

pNBV-BIRK-YC4519 bp

YC

BIRK1Amp R

pNBV-BIRK-YC4519 bp

YC

BIRK1Amp R

pNBV-ERD15-YC4633 bp

YC

ERD15Amp R

pNBV-ERD15-YC4633 bp

YC

ERD15Amp R

pNBV-YC-ADF4654 bp

YC

ADF

Amp RpNBV-YC-ADF

4654 bp

YC

ADF

Amp R

pNBV-YC-ERD154714 bp

LinkerYC

ERD15

Amp RpNBV-YC-ERD15

4714 bp

LinkerYC

ERD15

Amp R

pNBV-YN-SDG4i5365 bp

YN

SDG4iAmp R

pNBV-YN-SDG4i5365 bp

YN

SDG4iAmp R

A:

B:

C:

D:

N terminal fusion C terminal fusion

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6.3.5 Transient expression of BIFC vectors in N. benthamiana

Each BIFC vector pair (section 6.2.7) was transiently expressed in N. benthamiana

leaf tissue and viewed under a UV emitting microscope to visualise YFP florescence,

indicating an interaction between SDG4i and the insert. SDG4i and ADF did not show

evidence of interaction in the BIFC assay (Figure 6.7A) since there was a lack of

fluorescence when infiltrated leaf tissue was exposed to UV light in both N and C

terminal YFP fusions. A similar outcome was observed when SDG4i and BIRK was

tested in the BIFC assay, with no fluorescence detected (Figure 6.7B) for either N and

C terminal YFP fusions. The interaction between SDG4i and ERD15 tested though

BIFC assay yielded fluorescence in the cytoplasm of N. benthamiana leaf cells only in

the YFP fusion to the C terminal of ERD15 signifying that the interaction between

SDG4i and ERD15 found through the yeast two-hybrid assay was genuine (Figure

6.7C) No fluorescence was observed through the YFP fusion to the N terminal of

ERD15 (data not shown). Infiltrating tissue with empty vectors (figure 6.7D) showed

very little background fluorescence indicating that that the fluorescence presented in

the interaction between ERD15 and SDG4i is a product of that interaction, and not

fluorescence produced by the vectors alone.

182

Figure 6.7: BIFC visualisation of SDG4i interactions with ADF, BIRC and ERD15.

Epifluorescence and bright field images of interactions between SDG4i and ADF (A), SDG4i

and BIRK (B) and SDG4i and ERD15 (C) and split YFP empty vectors (D).

200µm

200µm

200µm 200µm

200µm

200µm

Epifluorescence Bright field

SDG4i-YFPN/ADF-YFPC

SDG4i-YFPN/BIRK-YFPC

SDG4i-YFPN/ERD15-YFPC

A:

B:

C:

YFPN/YFPC

D:

200µm 200µm

183

6.3.6 Sequence analysis of ERD15-like

The S. stapfianus cDNA sequence showing homology to the transcription factor,

ERD15 in the BLASTn analysis (section 6.3.3), displayed a positive interaction with

SDG4i through the yeast-2 hybrid screen and BIFC assay. To enable a comparison to

similar proteins in the NCBI database using a BLASTp search, the sequence was

translated to obtain the amino acid sequence. Following translation into six reading

frames (figure 6.8), the CDS is predicted to be in the +3 frame with the initiation

codon at 167bp, due to the presence of a Kozak sequence. The sequence upstream of

the predicted initiation codon is predicted to form part of the 5’ UTR based on the

alignment of S. stapfianus ERD15 with ERD15 from other species. The predicted stop

codon is located at 561bp, with the region downstream representing the start of the 3’

UTR. A poly-A tail was not present suggesting that the S. stapfianus ERD15-like

CDS has not been completely sequenced. S. stapfianus ERD15-like protein is

predicted to be 131 amino acids in length, has a molecular weight of 14.3kDa and an

isoelectric point of 4.87 as predicted by ExPASy (Gasteiger et al., 2005). The S.

stapfianus ERD15 is predicted to localise to the nucleus using the Plant-mPLoc

webserver (Chou & Chen 2010).

The BLASTp search of the S. stapfianus ERD15 sequence indicates the sequence has

homology to an ERD15 protein from A. thaliana, and ERD15-like proteins from S.

italica, Z. mays and O. sativa. The search also revealed a PAM2 (poly(A)-binding

protein (PABP) interacting motif 2) and PAE1 (PAM2-associated element 1) domains

(figure 6.9). The level of homology between S. stapfianus ERD15-like and other

ERD15 and ERD15-like proteins is at its highest throughout the PAM2 and PAE1

domains. PAM2 domains have been found to interact with poly(A)-binding proteins

across many eukaryotes, and the PAE1 domain is found exclusively in plant ERD15

proteins, however, no function has been assigned to this motif (Aalto et al., 2012).

184

A P A D P S L K E A N P R K Q P W V P P F1 R R P I H L S R K Q T R G N N H G C H R F2 A G R S I S Q G S K P E E T T M G A T A F3 1 GCGCCGGCCGATCCATCTCTCAAGGAAGCAAACCCGAGGAAACAACCATGGGTGCCACCG 60 ----:----|----:----|----:----|----:----|----:----|----:----| 1 CGCGGCCGGCTAGGTAGAGAGTTCCTTCGTTTGGGCTCCTTTGTTGGTACCCACGGTGGC 60 A G A S G D R L S A F G L F C G H T G G F6 L A P R D M E * P L L G S S V V M P A V F5 R R G I W R E L F C V R P F L W P H W R F4 R C R R R R * T R T R H S S S R R R S C F1 G V V V D V E P E R A T L H P G G V P A F2 V S S S T L N P N A P L F I P A A F L H F3 61 CGGTGTCGTCGTCGACGTTGAACCCGAACGCGCCACTCTTCATCCCGGCGGCGTTCCTGC 120 ----:----|----:----|----:----|----:----|----:----|----:----| 61 GCCACAGCAGCAGCTGCAACTTGGGCTTGCGCGGTGAGAAGTAGGGCCGCCGCAAGGACG 120 R H R R R R Q V R V R W E E D R R R E Q F6 A T D D D V N F G F A G S K M G A A N R F5 P T T T S T S G S R A V R * G P P T G A F4 M W R T S R R S G G T S S P P P H G S A F1 C G G L L A A V V E P R H H H R M V P R F2 V E D F S P Q W W N L V T T T A W F R D F3 121 ATGTGGAGGACTTCTCGCCGCAGTGGTGGAACCTCGTCACCACCACCGCATGGTTCCGCG 180 ----:----|----:----|----:----|----:----|----:----|----:----| 121 TACACCTCCTGAAGAGCGGCGTCACCACCTTGGAGCAGTGGTGGTGGCGTACCAAGGCGC 180 M H L V E R R L P P V E D G G G C P E A F6 C T S S K E G C H H F R T V V V A H N R F5 H P P S R A A T T S G R * W W R M T G R F4 T T G P A S T P N S T T W Q S A R R R R F1 L L V L R A H P T R R H G R A L D A A A F2 Y W S C E H T Q L D D M A E R S T P P L F3 181 ACTACTGGTCCTGCGAGCACACCCAACTCGACGACATGGCAGAGCGCTCGACGCCGCCGC 240 ----:----|----:----|----:----|----:----|----:----|----:----| 181 TGATGACCAGGACGCTCGTGTGGGTTGAGCTGCTGTACCGTCTCGCGAGCTGCGGCGGCG 240 V V P G A L V G L E V V H C L A R R R R F6 S * Q D Q S C V W S S S M A S R E V G G F5 S S T R R A C G V R R C P L A S S A A A F4 S P P R R * * P T R T R T S P T T R A C F1 L L P D D D D R R G R G P R L R P E R A F2 S S P T M M T D E D E D L A Y D P S V L F3 241 TCTCCTCCCCGACGATGATGACCGACGAGGACGAGGACCTCGCCTACGACCCGAGCGTGC 300 ----:----|----:----|----:----|----:----|----:----|----:----| 241 AGAGGAGGGGCTGCTACTACTGGCTGCTCCTGCTCCTGGAGCGGATGCTGGGCTCGCACG 300 E G G R R H H G V L V L V E G V V R A H F6 S E E G V I I V S S S S S R A * S G L T F5 R R G S S S S R R P R P G R R R G S R A F4 S R T S A R A R T A R R S T P A R R A A F1 Q E P Q Q E P A Q R E E V R Q P E E Q R F2 K N L S K S P H S E K K Y A S P K S S V F3 301 TCAAGAACCTCAGCAAGAGCCCGCACAGCGAGAAGAAGTACGCCAGCCCGAAGAGCAGCG 360 ----:----|----:----|----:----|----:----|----:----|----:----| 301 AGTTCTTGGAGTCGTTCTCGGGCGTGTCGCTCTTCTTCATGCGGTCGGGCTTCTCGTCGC 360 E L V E A L A R V A L L L V G A R L A A F6 S L F R L L L G C L S F F Y A L G F L L F5 * S G * C S G A C R S S T R W G S S C R F4 * S T A A R A G A P P S A A R R A P T A F1 D P R Q P E Q G R R P Q R R E E H R Q Q F2 I H G S P S R G A A L S G E K S T D S S F3 361 TGATCCACGGCAGCCCGAGCAGGGGCGCCGCCCTCAGCGGCGAGAAGAGCACCGACAGCA 420 ----:----|----:----|----:----|----:----|----:----|----:----| 361 ACTAGGTGCCGTCGGGCTCGTCCCCGCGGCGGGAGTCGCCGCTCTTCTCGTGGCTGTCGT 420 H D V A A R A P A G G E A A L L A G V A F6 T I W P L G L L P A A R L P S F L V S L F5 S G R C G S C P R R G * R R S S C R C C F4

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A A P A G T S S V D M Q X F1 Q H L Q G P L A S T C R F2 S T C R D L * R R H A X F3 421 GCAGCACCTGCAGGGACCTCTAGCGTCGACATGCAGA 457 ----:----|----:----|----:----|----:-- 421 CGTCGTGGACGTCCCTGGAGATCGCAGCTGTACGTCT 457 A A G A P V E L T S M C F6 L L V Q L S R * R R C A S F5 C C R C P G R A D V H L F4

F1: +1 F4: -1

F2: +2 F5: -2

F3: +3 F6: -3

Figure 6.8: Six-frame translation of S. stapfianus ERD15-like

The cDNA sequence obtained from the interacting partner of the SDG4i yeast 2-hybrid diploid

was translated into six reading frames using EMBOSS sixpack software (www.ebi.ac.uk). A

kozak sequence was found using GeneRunner software (www.generunner.net) and is

highlighted in yellow. The predicted initiation ATG and termination TAG are highlighted in blue

and the predicted reading frame is in bold. The predicted amino acid sequence is highlighted

in grey.

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Figure 6.9: Amino acid sequence alignment of S. stapfianus ERD15-like with homologs

in the NCBI database

The amino acid sequence of the S. stapfianus cDNA ERD15-like (Ss ERD15-like) was subject

to blastp analysis and showed similarity to five known proteins in the NCBI database:

Arabidopsis thaliana ERD15 (At ERD15) (accession: NP_181674.1), Setaria italica ERD15-

like protein isoform X1 (Si ERD15-like) (accession: XP_004984352.1), Zea mays ERD15-like

protein (Zm ERD15-like) (accession:ACG25626.1), Oryza sativa ERD15-like protein (Os

ERD15-like) (accession: BAC78564.1) and Glycine max ERD15 (Gm ERD15) (Alves et al.,

2011) and were aligned using the Clustal Omega algorithm (Sievers et al., 2011). High

homology is indicated by black shading, scaling down to poor homology in grey. PAM2

(PABP-interacting motif 2) and PAE1 (PAM2 associated element 1) domains are overlined.

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6.3.7 Expression levels of ERD15 and SDG4i transcripts during dehydration

Semi-quantitative RT-PCR was used to observe the pattern of SDG4i (figure 6.10a)

and ERD15 (figure 6.10b) gene expression throughout the desiccation of S. stapfianus

leaf tissues. SDG1i was used as a control, as expression of this gene does not change

throughout dehydration (Le 2004). Both ERD15 and SDG4i are present at the lowest

relative levels at 90% RWC, and expression of both increases throughout dehydration.

The highest relative expression of SDG4i is observed at 19-10% RWC, while the

highest relative expression of ERD15 is observed at 79-60% RWC. In general, both

SDG4i and ERD15 gene expression throughout dehydration is similar, with relative

expression of both increasing at 89-80% RWC and remaining abundant throughout

the remaining stages of dehydration.

Figure 6.10: Relative expression levels of SDG4i and ERD15-like transcripts in

dehydrating S. stapfianus leaf tissue

Semi-quantitative RT-PCR was used to calculate the expression levels of SDG4i (A) and

ERD15-like (B) in dehydrating leaf tissue, relative to SDG1i. Relative SDG4i/ERD15-like

expression is represented in the Y-axis, and stages of desiccation expressed as RWC (%) are

identified on the X-axis. Bars represent the ±SE of 2 replicates.

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6.3.8 Endoplasmic reticulum stress germination assay Endoplasmic reticulum (ER) stress was simulated by the addition of tunicamycin to ½

MS media during the germination of transgenic 35S:SDG4i and control A. thaliana

lines. Tunicamycin induces endoplasmic reticulum stress through the unfolded protein

response (Liu et al., 2007). In the absence of tunicamycin induced ER stress, all lines

exhibited a 100% germination capacity (figure 6.11a). The induction of ER stress at

0.3µg/ml tunicamycin (figure 6.11b) reduced the germination capacity of WT-COL,

35S:E and SDG4i KO lines by 90%, while all six 35S:SDG4i lines show only a 40-

50% reduction in germination capacity (p < 0.05). Similarly, at a tunicamycin level of

1.0µg/ml, WT-COL, 35S:E and SDG4i KO lines show no apparent ability to

germinate, while the 35S:SDG4i lines show a significantly increased ability to

germinate under these conditions (figure 6.11c).

Figure 6.11: Germination of A. thaliana lines under endoplasmic reticulum stress

Endoplasmic reticulum stress was induced by the addition of tunicamycin to ½ MS media and

germination was scored across WT-COL, 35S:E, T-AT3G43850 and six 35S:SDG4i lines.

Germination percentages of each line under 0µg/ml (A), 0.3µg/ml (B) and 1.0µg/ml (C)

tunicamycin is presented. Any significant differences (p < 0.05) are highlighted with an

asterisk. Bars represent the ±SE of 10 replicates.

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6.4 Discussion

6.4.1 Attenuation of drought related senescence by SDG4i

For resurrection plants to survive desiccation the plants must be able to negate the

effects of dehydration stress, such as cellular breakdown as well as down-regulating

dehydration-induced senescence responses and up-regulating genes involved in

desiccation tolerance (Griffiths et al. 2014). The developmental regulation of

senescence is under the control of several genes, many of which are induced by

reproductive development in monocarpic plants (Noonden 1980). Abiotic stresses

negatively affecting plant growth can also influence the senescence of the plant

regardless of life cycle stage. Dehydration stress-induced senescence of leaves is one

of the primary responses as a plant attempts to reduce canopy size and preserve

cellular nutrients (Fischer & Turner 1978). In resurrection plants, the genetic control

of avoiding senescence during dehydration stress as the plant desiccates has not be

been well studied. In S. stapfianus the process is complicated by the observation that

during desiccation, not all green leaf blades survive, suggesting the propensity for an

individual leaf to senesce may be dictated by chronological age (unpublished

observation). This observation is supported in Vander Willigen et al. (2001) in the

resurrection species Eragrostis nindensis.

Endoplasmic reticulum (ER) stress has been associated with osmotically-induced cell

death (Alves et al., 2011). ER stress triggers the unfolded protein response (UPR) in

all eukaryotic cells. The molecular chaperone BiP, located in the ER, facilitates the

folding of newly synthesised proteins and this protein can also act as a sensor for ER

stress and stress-related signal generation (Reis et al., 2011). Severe cases of ER

stress can affect the processing capacity of the ER resulting in accumulation of

unfolded proteins and triggering of the UPR, which ultimately leads to induction of

UPR genes and attenuation of translation to restore ER homeostasis (reviewed in

Fanata et al., 2013). Under severe ER stress where the UPR fails to restore ER

homeostasis, a signal can be generated to activate asparagine-rich protein (NRP)

genes (Irsigler et al., 2007) and other downstream components, which results in cell

death (Wang et al., 2005; Costa et al., 2008). The activation of NRP gene expression

results in chlorophyll loss, ethylene production and senescence (Reis & Fontes 2012).

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A recent study into the activation of osmotic and endoplasmic reticulum (ER) stress

induced senescence shows that the transcription factor ERD15 is responsible for the

activation of the N-rich protein (NRP) genes in soybean (Alves et al., 2011). The

NRPs which are up-regulated by ERD15 require both an osmotic stress and ER stress

signal for full activation, however, NRPs are moderately up-regulated by either

stressor (Costa et al., 2008). ERD15 expression is thought to be regulated post-

translationally by the interaction of poly-A binding proteins (PAB), specifically

PAB2, PAB4 and PAB8, to the PAM2 domain of ERD15. The binding of ERD15 to

PAB proteins was identified in a split ubiquitin yeast 2-hybrid assay (Belostotky et

al., 1996, Chekanova et al., 2001, Palanivelu et al., 2000).

The attenuation of NRP-mediated cell death signalling has been observed following

over-expression of the ER-resident molecular chaperone binding protein (BiP) in

soybean. During the exposure of these transgenics to drought and ER stress the

induction of NRP transcripts was delayed, implicating BiP as a negative regulator of

the NRP-mediated cell death response (Reis et al., 2011). The up-regulation of NRP-

mediated cell death signalling by ERD15 has been suggested to be modified by a

protein-protein interaction, however, the mechanism is currently unknown (Wang &

Grumet 2004). However, it has been shown that ERD15 interacts with the NRP-B

promoter, to direct expression of NRP-B during osmotic and ER stress (Alves et al.,

2011). The possibility of SDG4i involvement in a protein-protein interaction was

suggested by the presence of a serine rich region identified and discussed in section

3.4.3 and a yeast two-hybrid screen was utilized to explore the possible interaction of

SDG4i with proteins present in desiccating S. stapfianus leaf tissue. In this chapter,

results from the yeast 2-hybrid assay, and confirmed by BIFC assay, suggest that

SDG4i is able to interact with an ERD15-like protein in vitro. SDG4i sequence

analysis does not indicate any homology to any known PAB proteins or proteins

known to interact with PAB proteins. It is not known if SDG4i interacts with the

PAM2 domain or another separate domain in ERD15. It is possible that SDG4i may

be blocking the binding of PAB2/4/8 to ERD15 to modify the downstream effects of

ERD15-mediated expression of NRPs, as SDG4i activity appears to modifying the

tunicamycin-induced UPR in transgenic A. thaliana over-expressing SDG4i. By

inference, the action of SDG4i in S. stapfianus may have a role in preventing

senescence during dehydration. In order to help elucidate the potential interacting

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domain between ERD15 and SDG4i, a specific yeast 2-hybrid assay between SDG4i

and a deletion series of ERD15 could provide more information on the interaction

between the two proteins. In A. thaliana the ERD15 gene produces four differentially

processed transcripts. Three of the transcripts produce the same amino acid sequence

and the other produces a protein lacking three amino acids (QPR) at the C-terminus

(or terminal end). This form of the transcript is highly expressed in comparison to the

others (Aalto et al., 2012). It is hypothesised that the presence of these transcripts is

related to post-transcriptional regulation of ERD15 (Aalto et al., 2012). Arabidopsis

express both the truncated and fuller length versions of ERD15 transcripts, however,

the significance of this is not yet understood (Aalto et al., 2012). Differences in

ERD15 sequences between species are apparent. Across species examined, the N

terminus remains highly conserved, and the C-terminus diverges (Alves et al., 2011).

This is also the case in the S. stapfianus ERD15-like protein where the N-terminus,

containing the PAM2 and PAE1 domains, has a high homology to other ERD15-like

proteins, with a lower level of homology at the C-terminal end.

During dehydration, resurrection plants can suppress the cell death signalling

processes, which could be expected to occur in leaf tissue of dehydrating non-

resurrection plants. In the desiccation-sensitive plant Solanum pennellii, ERD15 is up

regulated at one hour post introduction of drought and salinity stress, one hour post

cold stress and 3 hours post ABA application (Ziaf et al., 2011). The transgenic

studies of abiotic-stress tolerance presented in chapter 5, suggest that SDG4i has the

potential to interact with ERD15 produced in Arabidopsis. It would be useful to

analyse transcript levels of ERD15 in 35S:SDG4i plants during abiotic stress. In

Arabidopsis, ERD15 has been suggested to be a negative regulator of ABA signalling

(Kariola et al., 2006), however, it is not known if this is the same in soybean. ERD15

in soybean has been shown to interact with the NRP-B promoter, and has a putative

DNA binding domain, this has not been demonstrated for the Arabidopsis ERD15

(Alves et al., 2011). In the S. stapfianus ERD15-like sequence, a DNA binding

domain has not been identified. This may indicate that ERD15 proteins present in

various species may have differences in biological function, and may control gene

expression through differing pathways. In Arabidopsis ERD15 does not drive NRP

expression directly, rather ERD15 down-regulated the ABA response, which in turn

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up-regulates the salicylic acid response and activates the senescence pathway (Kariola

et al., 2006).

The enhanced tolerance of 35S:SDG4i A. thaliana plants during germination and

vegetative growth under the imposition of osmotic and cold stresses (section 5.3.2,

5.3.3) support the proposal that SDG4i is interacting with ERD15 to increase the

tolerance to these stresses. A germination assay of 35S:SDG4i A. thaliana lines under

tunicamycin-induced ER stress showed that the over-expression of SDG4i allows the

plant to become more tolerant to ER stress, further supporting the contention that

SDG4i may be associated with the attenuation of the UPR and subsequent cell death

signal associated with ERD15 activity.

While SDG4i transcription is up regulated in S. stapfianus during the early stages of

desiccation as demonstrated by northern blot (section 3.3.4) and through a semi-

quantitative RT-PCR approach in this chapter, these experiments have also shown that

SDG4i transcript is present at lower levels in fully hydrated (>90% RWC) leaves.

This suggests that SDG4i may be expressed in leaves during regular growth (prior to a

desiccation cycle) in readiness to combat some of the traditional responses to

dehydration stress normally found in non-resurrection plants. The expression of

ERD15 is up-regulated in response to drought stress in S. pennelli relatively quickly,

with transcript abundance accumulating 1 hour post stress introduction, and is also

expressed at low levels in non-droughted tissue (Ziaf et al., 2011). The expression

pattern of ERD15 in dehydrating S. stapfianus leaf tissue is similar to the SDG4i

expression pattern with minimal expression in fully hydrated leaves, and with a

marked increase in expression induced by dehydration. Co-expression lends some

support to the hypothesis that the transcript abundance of SDG4i accumulates to

reduce ERD15 activity and combat the activation of the cell death signal mediated by

ERD15. Over-expression of ERD15 in A. thaliana increases the sensitivity of the plant

to drought stress (Kariola et al., 2006) and it would be interesting to see if crossing a

35S:SDG4i A. thaliana with an ERD15 over-expression line would restore wild type

tolerance to drought stress. In order to further observe the effects of SDG4i on NRPs

and other senescence associated gene (SAG) expression it would be useful to observe

the expression of these SAG genes in SDG4i transgenic lines. This may help elucidate

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how SDG4i regulates the expression of these genes, and genes further downstream of

the drought and ER induced programmed cell death pathway.

6.4.2 ERD15, SDG4i and the modification of abiotic stress and ABA responses

Kariola et al. (2006) have suggested that ERD15 is a negative regulator of early ABA

responses to stress. Reduction in ERD15 activity in ERD15-RNAi plants leads to

ABA hypersensitivity and results in an increased tolerance to freezing and drought

stress. The increase in tolerance to cold and osmotic stress exhibited by SDG4i plants

supports the contention that the interaction between SDG4i and ERD15 can reduce

ERD15 activity in the transgenic plants. In ERD15-RNAi plants, germination and

growth is more sensitive to inhibition by exogenous ABA than controls (Kariola et

al., 2006). However, the germination phenotype of SDG4i transgenics is opposite to

that of ERD15-RNAi plants, where they display an increased ability to germinate, and

an increased ability to grow under the presence of exogenous ABA (section 5.3.3).

This indicates that the phenotype of 35S:SDG4i A. thaliana does not exactly match

that of A. thaliana ERD15-RNAi plants, where the activity of ERD15 has been

reduced. This may mean that SDG4i can modulate the ABA signalling response

pathways differentially where SDG4i may repress ERD15 signalling under stress

conditions without making the plants ABA hypersensitive.

ERD15 over-expression A. thaliana lines have an increased tolerance to exogenous

ABA. Furthermore, in non-stressed conditions an increase in endogenous ABA levels

is seen in ERD15 over-expressing plants in comparison to the wild type (Kariola et

al., 2006). A similar increase in endogenous ABA content is seen in ABA insensitive

dominant mutants, abi1-1 and abi2-1, as a result of a feedback loop between ABA

synthesis and sensing (Verslues et al., 2006). This suggests that ERD15 is decreasing

the sensitivity of the plants to ABA, by affecting either signalling or perception,

however, it is unclear how ABA signalling is affected. ABA increases the expression

of ERD15 (Ziaf et al., 2011), and A. thaliana over-expressing ERD15 have an altered

ABA-responsive gene expression profile (Kariola et al., 2006). Upon exposure to

exogenous ABA, 35S:SDG4i A. thaliana plants show insensitivity to ABA during

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germination and in root length assay in vitro. The leaves of 35S:SDG4i A. thaliana

plants also exhibit a reduced rate of senescence upon ABA application (section 5.3.3,

5.3.7). This suggests that the interaction of SDG4i and ERD15 are involved in a

feedback loop between ABA synthesis and perception and raises the possibility that

SDG4i has more than one role in S. stapfianus during dehydration that may include

both attenuation of senescence and disruption of ABA signalling. The interaction of

SDG4i with ERD15 supports the possibility of SDG4i interacting in an ABA

feedback loop. As discussed in section 6.4.1, a pool of alternatively spliced ERD15

transcripts are present in A. thaliana plants. It is unknown as to whether the

35S:SDG4i plants have an increased or decresed pool of ERD15 transcripts. If it is

assumed that the 35S:SDG4i plants have an unchanged pool of ERD15 transcripts

such as what it seen in wild-type A. thaliana, it could be argued that in 35S:SDG4i

plants the pool of transcripts may act as a resource for the quick production of ERD15

during stressed conditions, and in the case of 35S:SDG4i plants, SDG4i may be

interacting with ERD15 and repressing the ability of the transcription factor to

activate NRPs. This may increase the amount of ERD15 transcripts in the pool, which

may be continually transcribed during stress conditions, and this increased production

of ERD15 transcripts decreases the sensitivity of the plant to ABA (figure 6.12),

involving SDG4i in a feedback loop with ERD15 activity.

Figure 6.12: Schematic diagram of potential effect of SDG4i on ERD15 in biological

processes (adapted from Aalto et al., 2012). ERD15 expression is modulated by abiotic

stress and ABA signals. The biological role of ERD15 is attenuated by SDG4i, causing an

increase in the transcript pool of ERD15 which influences ABA processes.

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Recent advances in the understanding of the core ABA signalling module has

provided further insight into how plants respond to ABA. There is strong evidence to

implicate PYR/PYL/RCAR (Pyrabactin resistance 1/Pyrabactin resistance 1-

like/Regulatory component of ABA receptor) receptors functioning at the beginning

of a negative regulatory pathway to, in turn, regulate PP2C (protein phosphatase 2C)

phosphatases. The PP2C phosphatases directly regulate SnRK2 (SNF1-related protein

kinase) kinases, which by and large, regulate ABA related gene expression by

phosphorylating ABFs (ABA-responsive element binding factor) (reviewed in Cutler

et al., 2010). ERD15 expression in A. thaliana has been shown to affect the

expression of ABA-responsive element binding factors/ABA-responsive element

(ABF/AREB) genes (Kariola et al., 2006), which are under the direct control of the

ABA signalling module. It is still unclear whether ERD15 may act in parallel or

interacts with the ABA signalling module. The interaction between SDG4i and

ERD15 provides insight into how this may occur, and suggests that the active ERD15

may not influence the ABA signalling module, but the pool of alternatively spliced

transcripts may provide a regulatory signal. Further analysis of this transcript pool in

35S:SDG4i A. thaliana as well as NRPs expression analysis would be advantageous

to investigate this hypothesis.

6.4 Conclusion

The work in this chapter shows that SDG4i interacts with, and potentially attenuates

the activity of the transcription factor ERD15, of which ERD15 is involved in abiotic

stress responses and initiation of senescence during dehydration. However, sequence

analysis and phenotypic characteristics of 35S:SDG4i A. thaliana suggests that in

addition to the role this interaction plays, that SDG4i may have additional roles in the

plant, specifically modification of the ABA signalling pathway. It may be possible

that the role of SDG4i in the plant is dependent on the state of dehydration. Further

detailed investigation of the role of SDG4i in the senescence pathway as well as in the

ABA signalling module will increase our understanding of SDG4i function, and the

role this protein plays in allowing resurrection plants to survive desiccation.

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7.0 Glycosyltransferase activity of SDG8i

7.1 Introduction

Several genes have been previously identified to be up-regulated during the

desiccation of S. stapfianus (Blomstedt et al., 1998, Neale et al., 2000, Le et al.,

2007). Ongoing studies at Monash University have been analysing the function of

these genes. Several of these genes are thought to play protective roles during the

early dehydration stages (Blomstedt et al., 1998a,b, Neale et al., 2000) and some

genes isolated from late dehydration stages appear to be associated with growth

control (Blomstedt et al., 2010). The transcript SDG8i has been shown to have strong

sequence homology to glycosyltransferases (Le et al., 2007). In plants,

glycosyltransferases can regulate the bioactivity, metabolism and intracellular

localisation of small lipophilic compounds. These compounds include many

hormones, secondary metabolites and foreign compounds, such as herbicides (Lim &

Bowles 2004). Physiological studies of the effect of SDG8i over-expression in A.

thaliana by Sharmin Islam (a PhD student in this laboratory) has revealed the

possibility of SDG8i glycosyltransferase activity on hormones that have a role in

regulating branching, in particular, strigolactones. The biosynthesis of strigolactones

involves the carotenoid biosynthesis pathway, and utilises more axillary branching 1

(MAX1), MAX3 and MAX4 genes for biosynthesis, and MAX2 for perception

(Booker et al., 2005). The phenotype of A. thaliana over expressing SDG8i shows

increased shoot branching, which is similar to the phenotype observed in strigolactone

biosynthesis and signalling mutants (MAX mutants) (Islam 2013). The mechanism

whereby auxin controls shoot branching has recently been shown to be affected by

strigolactone levels within the plant (Bennet et al., 2006). SDG8i is highly expressed

during the later stages of dehydration in S. stapfianus (section 3.3.4) and it is possible

that SDG8i may be functioning in the regulation of hormone activity during

dehydration or rehydration. The increase in shoot branching in A. thaliana lines over-

expressing SDG8i leads to an increase in above-ground biomass production,

implicating this S. stapfianus dehydration-responsive gene in growth regulation. This

chapter aims to examine SDG8i glycosyltransferase activity on plant hormones

utilising an in vitro coupled enzyme assay approach. The effects of dehydration on the

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expression of the strigolactone biosynthesis gene MAX4 will be examined and the

expression of SDG8i in response to exogenous auxin application will be explored.

This will build on the knowledge gained in chapter three on the regulation of SDG8i

expression. In addition, an examination of leaf tissue regrowth following the

rehydration of S. stapfianus from the desiccated state will be conducted to initiate an

exploration of the potential for using resurrection plant genes to increase plant

biomass production.


7.2.1 S. stapfianus leaf regrowth assay

The length of leaf regrowth was measured by removing the leaf tissue just above the

meristem of the plant, and monitoring leaf regrowth in length (mm) every day for 10

days and by measuring final biomass at 10 days. The plants utilized were five fully-

hydrated S. stapfianus plants that had not undergone a recent dehydration/rehydration

cycle, as well as five S. stapfianus plants 24 hours post-rehydration of completely

dehydrated plants (<10% RWC).

7.2.2 ICON vector construction

The viral based MagnICON vector system (Icon Genetics GmbH, Germany) was

utilised to produce a transient expression system for the production of the SDG8i

protein under the control of the A. thaliana ACT2 (actin 2) promoter. Three provectors

were used in this system: (i) pICH12190 (containing the 5’ module of the viral vector

including the chloroplast signal sequence, (ii) pICH11599 (containing the 3’ module

of the viral vector including SDG8i CDS) and (iii) pICH14011 (encoding the

integrase) (Marillonnet et al., 2005). The full length SDG8i cDNA sequence was

amplified using RT-PCR from total RNA isolated from S.stapfianus leaf tissue at

RWC 60% with Pfu DNA polymerase (section 2.5.4). The primers used for the

amplification of SDG8i were iconSDG8i forward

(5’GAGAGAATTCATGACGAAGACCGTGGTTCTGTAC3’) and iconSDG8i

reverse (5’GAGAGGATCCTCACGGACGACCGAC3’). The PCR products were

198

then digested with the enzymes EcoR1 and BamH1 (section 2.5.7) where the

recognition sites were included in the primers (underlined). The construct pICH11599

was digested with these same restriction enzymes and ligated (section 2.5.8) to

generate pICH11599-SDG8i. The plasmid was electroporated into E. coli TOP10

(section 2.3.2) and subject to sequencing to confirm the presence of the insert.

pICH12190, pICH14011 and pICH11599-SDG8i were separately electroporated into

Agrobacterium tumerfaciens GV3101 (section 2.3.2).

7.2.3 Transient SDG8i expression in N. benthamiana leaves

Each A. tumerfaciens strain containing each provector was cultured overnight at 28°C

in liquid LB media with shaking at 150rpm. Each strain was then pelleted by

centrifugation at 400g for 10 minutes and resuspended to an OD600 of 0.5 in

infiltration medium containing 10mM 2-N-morpholino-ethanesulfonic acid (MES) pH

5.5, 10mM MgSO4 and 200µM acetosyringone and incubated at 28ºC at 150rpm for 2

hours. Leaves were infiltrated with equal amounts of A. tumerfaciens containing each

of the three constructs using a syringe into the abaxial side of leaves on three week

old N. benthamiana that had been covered in aluminium foil for three days. The

infiltrated leaves were re-covered in aluminium foil and left to co-incubate for 5 days

on the plant. The leaves were harvested and ground in liquid nitrogen and were

resuspended in 500µL cold protein extraction buffer (5mM sodium phosphate buffer

pH 7.5, 10mM EDTA, 0.1% Triton X-100). The homogenate was then centrifuged for

10 minutes at 13500g at 4ºC to remove cell debris. The total protein content was

determined by Bradford assay and extracts analysed by SDS-PAGE (section 2.7.2).

Protein was extracted from both infiltrated and un-infiltrated leaves.

199

7.2.4 SDG8i coupled enzyme assay

A coupled enzyme assay (Jackson et al., 2001) was utilized to examine the ability of

SDG8i to glycosylate a range of hormone substrates. The enzyme assay involved the

coupling of the activity of pyruvate kinase and lactate dehydrogenase (figure 7.1).

Figure 7.1: Coupled enzyme assay between pyruvate kinase and lactate

dehydrogenase activity.

The activities of both pyruvate kinase and lactate dehydrogenase are dependent on the ability

of the SDG8i protein to transfer the glucose molecule from UDP-Glucose to the hormone

tested. This generates free UDP which is used by pyruvate kinase to begin the coupled

assay.

200

The reaction rate was monitored by the change in NADH absorbance at 340nm. The

assay was conducted in a 1mL volume in a quartz cuvette containing the following

components: 50mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH

7.6, 2.5mM MgSO4, 10mM KCl, 2mM PEP (phosphoenolpyruvic acid), 10mM UDP-

glucose, 0.15mM NADH, 3 units pyruvate kinase, 1 unit lactic dehydrogenase (rabbit

muscle), 25µL of infiltrated N. benthamiana leaf protein extract and 10mM of the

hormone substrate being tested (GR24 (Chiralix B.V. Nijmegen, The Netherlands)

gibberillin (GA3), salicylic acid, kinetin, auxin (IAA), methyl jasmonate, ABA([±]-

cis, trans-abscisic acid (Sigma, St Louis, MO)). The assay was set up at room

temperature, and the hormone was added last to start the reaction. The change in

absorbance at 340nm was monitored over a period of 30 minutes on a Metertech

UV/Vis SP8001 spectrophotometer. The activity of SDG8i was calculated in µmol

NADH min-1 mg-1 using the extinction coefficient 6.22 x 10-3 M-1 cm-1 for NADH.

Background activity of extracts, monitored by measuring the absorbance change over

a 30 minute period without substrate addition, was subtracted from the reaction rate.

The rates obtained using SDG8i infiltrated N. benthamiana extracts were compared

with the rates obtained when the assay was conducted using control N. benthamiana

protein extracts infiltrated with vectors not containing the SDG8i CDS.

7.2.5 SDG8i enzyme kinetics The kinetic analysis of SDG8i enzyme activity was calculated by monitoring the

glycosyltransferase activity of SDG8i using the same method outlined in section 7.2.3

with concentrations of GR24 in individual reactions as follows: 0, 0.2, 0.8, 1.0 and

1.2mM. The results were plotted using the Lineweaver-Burke double reciprocal plot

and the Km and Vmax were calculated.

201

7.2.6 SDG8i and MAX4 expression in S. stapfianus leaf tissue

Dehydrated S. stapfianus leaf tissues at the RWCs ≥ 90%, 89-80%, 79-60%, 59-40%,

39-20%, 19-10% and <10%, as well as IAA treated (while attached to the plant) fully-

hydrated S. stapfianus leaf tissue (section 3.2.3) were subject to RNA extraction

(section 2.5.4) and cDNA synthesis (section 2.5.6). The genes assayed for expression

included SDG8i (in IAA treated tissue), MAX4 (in dehydrating tissue) and SDG1i as a

control. SDG1i was utilized as a control as it has been previously identified that the

level of transcription of this gene does not change throughout dehydration of S.

stapfianus (Le 2004). The primers used to amplify these genes are stated in table 7.1.

200ng of cDNA was used as a template in each PCR reaction conducted with MyTaq

DNA polymerase (Bioline) according to the manufacturers instructions. Samples were

removed at 10, 20, 30 and 40 cycles. All samples were repeated with 200ng of total

RNA as a template to detect possible gDNA contamination. 5µL of each sample was

loaded onto a 1.5% EtBr agarose gel and subject to electrophoresis (section 2.5.2).

Amplification products were directly quantified from the intensity under UV light

using ImageQuantTL v. 4.0 software.

Table 7.1: Primers used for S. stapfianus gene expression study.. Amplified gene Use 5’-3’

SDG8i Forward GTGCTCCACCACCGTGCG

Reverse GCAAAAATGACCATGACTAACTTCAGG

MAX4 Forward TCGCTCACCGACAACTCCAAC

Reverse CCGCTGGCCTTGCACATGAC

SDG1i Forward AGCCAAGACCGGCGGTTG

Reverse GGTAGGCCGTAGCGGAGTACAG

202

7.3 Results

7.3.3 S. stapfianus leaf regrowth assay

The capability of S. stapfianus to grow leaf tissue either under standard conditions

(fully hydrated plant) or at twenty four hours post-rehydration was assessed by

measuring the leaf regrowth following leaf excision from just above the meristem.

Growth was measured over 10 days, and a final measurement of regenerated leaf fresh

biomass at 10 days. Over a 10 day period, the rehydrated plant (represented in figure

7.2 as dehydrated) produced a leaf length three-fold longer than a hydrated plant

(figure 7.2a) and at time points from three to ten days the regrowth of dehydrated

plants was significantly longer (p < 0.05). In addition, the total fresh weight biomass

at ten days was greater in the dehydrated/rehydrated plant than the hydrated plant (p <

0.05) (figure 7.2b).

Figure 7.2: S. stapfianus leaf regrowth.

The leaf regrowth rate of fully hydrated S. stapfianus (hydrated) was compared with the leaf

regrowth rate of S. stapfianus (rehydrated) leaf tissue 24 hours after a

dehydration/rehydration cycle. Measurements were taken of leaf length in mm over 10 days

(a) and final leaf biomass at 10 days (b). Statistical significance of p < 0.05 is indicated with

an asterisk.

H yd rate

d

R e h y d rate

d

0

2 0

4 0

6 0

8 0

B io m a s s

mg

fre

sh

we

igh

t *

1 2 3 4 5 6 7 8 9 1 00

2 0

4 0

6 0

L e a f le n g th

D a y s

Le

af

len

gth

(m

m) H y d ra te d

R e h y d ra te d

**

*

*

*

*

**

A: B:

203

7.3.1 ICON vector construction

The MagnICON vector expression system utilises three provectors in order to

assemble a virion within infiltrated N. benthamiana tissue (Marillonnet et al., 2005).

The SDG8i containing provector pICH11599 was generated using the EcoR1 and

BamH1 restriction sites (figure 7.3). The sequence of the SDG8i insert was verified by

sequencing (data not shown). The SDG8i protein was targeted to be expressed in the

chloroplast by infiltrating the N. benthamiana leaf tissue with the three provectors:

pICH12190 (chloroplast signal), pICH14011 (integrase) and pICH11599-SDG8i

hosted individually in A. tumerfaciens GV3101.

Figure 7.3: MagnICON pICH11599 provector containing the SDG8i insert.

This provector was used in an A. tumerfaciens mediated transient expression system in N.

benthamiana leaves for rapid SDG8i protein production.

pICH11599SDG8i7906 bp

3' intronAttB

SDG8i Nos Term

204

7.3.2 Transient SDG8i expression in N. benthamiana leaves Once N. benthamiana leaves were infiltrated with the MagnICON vectors they were

covered in aluminium foil and left to co-incubate for five days. The typical

appearance of infiltrated leaves is shown in figure 7.4. The leaves remained attached

to the plant throughout the co-incubation period (figure 7.4a). A control infiltration

was conducted using the empty pICH11599 construct designed so that the assembled

virion does not produce a recombinant protein. The leaf infiltrated with the control

construct did not change in appearance (figure 7.4b). The over expression of SDG8i

in the assembled virion produced leaves that changed in appearance which was

thought to be associated with increased protein production (figure 7.4c). Antibodies

generated specifically for the SDG8i protein were not able to successfully detect

SDG8i on a western blot and the presence of SDG8i in the protein extracts had to be

estimated visually. The SDG8i protein has a molecular weight of approximately

50kDa and the expression can be masked on a coomassie stained SDS-PAGE gel by

the large subunit of RubisCO. Decreasing the production of RubisCO by covering the

leaves with foil aided in the visualisation of SDG8i. Figure 7.5 shows the coomassie

stained SDS-PAGE gel containing lanes of total protein extracts from un-infiltrated

and control and SDG8i virion infiltrated leaves. The wells were loaded equally with

50µg of total protein extract and the presence of an intense ~50kDa protein can be

seen in the lane containing protein extracted from leaves infiltrated with the SDG8i

construct in comparison to the control lanes suggesting that SDG8i is present in this

extract.

205

Figure 7.4: N. benthamiana infiltrated tissue.

Three week old N. benthamiana leaves were covered in aluminium foil for three days (a) prior

to infiltration. The appearance of leaves infiltrated with the empty MagnICON vector system

(b) and leaves infiltrated with the MagnICON vector system expressing SDG8i (c) after five

days co-incubation are pictured.

A: B:

C:

206

Figure 7.5: N. benthamiana total protein extracts

The total protein content of N. benthamiana leaf tissue extracts on 10% SDS-PAGE with the

following treatments: Un-infiltrated control (C), SDG8i expressing MagnICON vector system

(SDG8i) and empty MagnICON vector system (E). 50µg of total protein extract was loaded

into each lane. Lane M represents the Invitrogen Benchmark™ molecular weight marker. The

SDG8i protein is approximately 50kDa in size.

M C SDG8i E

~50 kDa

207

7.3.4 SDG8i coupled enzyme assay and kinetics

The protein extracts from infiltrated N. benthamiana leaves of either the recombinant

SDG8i protein or the empty provector control were used for the glycosyltransferase

activity assay. The activity of the enzyme was calculated by monitoring the reduction

of NADH content in the enzyme assay mixture and is represented as µmol NADH

min-1 mg-1 of total protein extract. The glycosyltransferase activity of SDG8i was

tested using a variety of hormones as substrates. The hormones were chosen for their

known ability affect plant growth. The small amount of background activity observed

without substrate addition, presumably due to endogenous NADH oxidase activity,

was subtracted when calculating the activity of control and sample extracts. The

majority of hormones tested showed no statistically significant difference in activity

in extracts from control and SDG8i infiltrated tissues. However, the extract from

leaves infiltrated with the SDG8i construct showed a significantly increased activity

over the control extract when GR24 was used as a substrate (p < 0.05) (figure 7.6).

The Km and Vmax of the SDG8i enzyme were calculated using a Lineweaver-Burke

plot with different concentrations of the GR24 substrate. The SDG8i total protein

extract has a Km of 0.348mM and a Vmax of 5.67 (Figure 7.7).

208

Figure 7.6: SDG8i activity in vitro

The ability of SDG8i recombinant protein extract to display glycosyltransferase activity using

the following plant hormones: Gibberllin (GA3), Salicyclic acid (SA), Kinetin, Auxin (IAA),

Methyl Jasmonate (MeJa), [±]-cis, trans-abscisic acid (ABA), and GR24 (a synthetic

strigolactone analogue) at pH 7.4. Statistical significance of p < 0.05 is indicated with an

asterisk.

Figure 7.7: Lineweaver-Burke plot of SDG8i activity

A Lineweaver-Burke plot of SDG8i protein extract at varying concentrations of GR24

indicating a Km of 0.348mM and a Vmax of 5.67.

GA

3S A

K ine t in IA

A

Me J a

A B A

GR 2 4

0

2

4

6

8

1 0

4m

ol

NA

DH

/min

/mg

S D G 8 i

C o n tro l

*

-6 -4 -2 2 4 6

-0 .2

0 .2

0 .4

0 .6

1/[4

mo

l/NA

DH

/min

]

1 /[m M G R 2 4 ]

209

7.3.5 SDG8i and MAX4 expression in S. stapfianus leaf tissue

Endogenous auxin levels within plants has been demonstrated to be a regulator of

branch formation in conjunction with strigolactones (Hayward et al., 2009), and the

response of SDG8i transcript levels to application of exogenous auxin was observed

to help elucidate the possible role for SDG8i in S. stapfianus shoot branching. The

capacity of the SDG8i protein to glycosylate GR24 in vitro, suggests SDG8i activity

may be affecting the strigolactone homeostasis in S. stapfianus during dehydration.

Alterations in strigolactone homeostasis can affect strigolactone biosynthesis via a

feedback mechanism (Hayward et al., 2009). Evidence for increased SDG8i activity

having an affect on the strigolactone feedback mechanism was analysed by observing

the levels of MAX4, a strigolactone biosynthesis gene (Mashiguchi et al., 2009), in

response to dehydration in S. stapfianus leaves. Auxin increases the expression of

MAX4 (Brewer et al., 2009), if SDG8i decreases the bioactivity of strigolactone, it is

possible that auxin could regulate SDG8i expression. The expression level of SDG8i

after exogenous IAA application to fully hydrated S, stapfianus was calculated using

semi-quantitative RT-PCR. Expression levels of SDG8i were monitored over twenty

four hours. The expression level of SDG8i was the highest at both zero hours and

twenty four hours post application and a drop in expression levels are seen at one and

three hours (figure 7.8), however, the change in the levels are not statistically

significant. The relative expression levels of MAX4 were analysed by semi-

quantitative RT-PCR throughout dehydration of S. stapfianus leaf tissue. The

expression levels of MAX4 did not appear to change throughout dehydration (figure

7.9).

210

Figure 7.8: SDG8i expression after exogenous auxin treatment.

The expression level of SDG8i relative to SDG1i expression was calculated over time post

exogenous auxin (IAA) treatment using semi-quantitative RT-PCR.

Figure 7.9: MAX4 expression in dehydrating S. stapfianus leaf tissue.

The expression level of MAX4 was calculated relative to SDG1i expression at dehydration

stages of S. stapfianus leaf tissue using semi-quantitative RT-PCR.

0 1 3 8 2 40

5 0

1 0 0

H o u rs p o s t IA A a p p lic a t io n

% R

ela

tiv

e e

xp

res

sio

n o

fS

DG

8i

> 9 0%

8 9 -80%

7 9 -60%

5 9 -40%

3 9 -20%

1 9 -10%

< 1 0%

0

5 0

1 0 0

% R

ela

tiv

e e

xp

res

sio

n o

fM

AX

4

211

7.4 Discussion

7.4.1 SDG8i glycosylates GR-24 in vitro Recent research into the function of plant glycosyltransferases has made considerable

progress into the biochemistry and roles of these enzymes within the plant. Enzymes

with structural similarity to SDG8i form part of the large multi-gene family classed as

family 1 UDP-glucose glycosyltransferases (Le 2004). Plant genomes typically

encode more than 100 of these enzymes, which function to transfer sugars to

molecules, such as hormones, secondary metabolites and biotic/abiotic chemicals.

Glycosylation often affect the bioactivity of these acceptor substrates (Bowles & Lim

2010). The over-expression in A. thaliana of a UDP-glycosylatransferase (UGT),

which glycosylates IAA (UGT84B1), produces a dwarfed and bushy phenotype that

has altered auxin homeostasis (Jackson et al., 2002) and demonstrates the role of

UGTs in hormone regulation/perception. The over-expression of A. thaliana

UGT85A5, of unknown target substrate, in tobacco produces plants that are tolerant to

salt stress, and results in an increased expression of carbohydrate metabolism related

genes. This suggests a role for this UGT in the protection of plants against salt stress

(Sun et al., 2013). Continuing work has suggested that the activity of SDG8i

ectopically expressed in A. thaliana confers abiotic stress resistance to freezing, salt

and drought, as well as delayed leaf senescence, a bushy phenotype and altered

hormone homeostasis (Islam, Griffiths et al., 2014).

The germination of Phelipanche ramose (Orobanchaceae) can be stimulated by root

exudates of A. thaliana (Kohlen et al., 2011) and a germination bioassay of

Orobanchaceae has been used for determining the level of strigolactone being

secreted from the host plant roots (Lopez-Raez et al., 2008). The decreased capacity

of the root exudates of A. thaliana over expressing SDG8i to stimulate germination of

Orobanchaceae has suggested that SDG8i may be glycosylating strigolactone or

strigolactone like compounds, which interferes with the biosynthesis or secretion of

strigolactone from A. thaliana roots (Islam 2013). The hormone strigolactone is a

carotenoid-based signal originating in the roots and acts as a second messenger for

auxin in the control of branching (Dun et al., 2009). The synthetic strigolactone

analog GR24 has been used in studies to uncover the roles of strigolactones within the

212

plant (Ruyter-Spira et al., 2011). The growth-related phenotypic changes observed in

A. thaliana over-expressing SDG8i suggested that SDG8i may be glycosylating a

growth-related hormone. The analysis of the in vitro UGT activity of SDG8i against a

variety of hormone substrates was completed using a coupled enzyme assay. It was

determined that SDG8i has an affinity for glycosylating GR24. The Km and Vmax of

SDG8i indicates a lower affinity for GR24 than that of some other glycosyltranferases

for their acceptor substrates (Noguchi et al., 2007, Offen et al., 2006). This could be

attributed to the synthetic nature of GR24 in comparison to natural strigolactones. It

should also be noted that no attempt was made to optimise the assay by varying the

pH or temperature. In addition, the SDG8i protein produced extracted from whole N.

benthamiana leaf tissue was not purified, leaving the possibility of inhibitors in the

protein extract interfering with SDG8i activity, resulting in lower affinity

measurements. However, no activity above background was observed for any of the

other hormones tested, suggesting that the phenotypic changes in 35S:SDG8i

transgenic plants are mediated via activity against strigolactone-like compounds.

Recently, the ability of plant UGTs to glycosylate hormones has been demonstrated in

A. thaliana where UGTs were shown to glycosylate brassinosteriods (Poppenberger et

al., 2005), salicyclic acid (Lim et al., 2002), cytokinins (Hou et al., 2004) and IAA

(Tognetti et al., 2010). In addition, A. thaliana UGTs have also been shown to

glycosylate benzoates (Lim et al., 2002). SDG8i is the first known UGT to glycoslate

a strigolactone-like compound, however, activity against synthetic GR24 does not

prove that the glycosylation of natural strigolactone compounds is occuring in planta.

Further experimentation on the ability of SDG8i to glycosylate natural strigolactones

in planta would be advantageous. It is also not possible to rule out the UGT activity

of SDG8i on other plant hormones or compounds as some UGTs have been shown to

glycosylate several structurally-related compounds (Poppenberger et al., 2005). For

this reason it would be advantageous to screen SDG8i affinity for untested hormone

or compounds that have structural similarity to GR24, for example karrikin (Janssen

& Snowden 2012).

The strigolactone biosynthesis pathway is carotenoid based and is known to involve

several proteins in Arabidopsis including, More axillary branching 1 (MAX1), MAX3

and MAX4. Signal perception is regulated by MAX2, which is an F-box protein

213

(reviewed in Dun et al., 2009). The protein MAX2 acts downstream of

MAX1/MAX3/MAX4 and is suggested to be required for feedback regulation in the

production of strigolactones by the MAX3 and MAX4 biosynthesis genes (Booker et

al., 2005). The ability of SDG8i to glycosylate GR24 and the increased branching

phenotype of A. thaliana over-expressing SDG8i indicate that the

signalling/perception of strigolactone is affected. It would be interesting to look at the

expression levels of MAX3/MAX4 in these plants. However, during the dehydration

of S. stapfianus leaf tissue, where SDG8i is highly expressed, the relative expression

levels of MAX4 remain unchanged. As the expression levels were calculated for leaf

tissue only as previous experiments have shown that successfully extracting RNA

from S. stapfianus roots is difficult. It cannot be conclusively stated that MAX4

expression does not change throughout dehydration as strigolactone synthesis occurs

mostly in the root tissue (Dun et al., 2009). Furthermore, it is unknown if SDG8i is

translated during dehydration or rehydration, which may mean that its affects on

MAX4 expression may not occur during dehydration which was the only stage of

expression explored in this chapter. It is possible that strigolactone may be inhibiting

growth during dehydration, and may be the signal originating in the roots that initiates

the desiccation tolerance program. Both strigolactone and auxin have the ability to

modulate the effect of each other in a feedback loop for the control of axillary

branching (Hayward et al., 2009). In A. thaliana over-expressing SDG8i, auxin levels

are elevated in the vascular tissues, root tips and leaf margins (Islam 2013). This data

suggests that SDG8i may be affecting auxin production and possibly other hormone

responses within the plant. However, it is not known if auxin levels are affected in

dehydrating S. stapfianus. The level of expression of SDG8i in fully hydrated leaves

of S. stapfianus is very low (Le et al., 2007). Since auxin is known to up-regulate

strigolactone biosynthesis genes (Hayward et al., 2009) an examination of the effect

of auxin on SDG8i expression was conducted. Exogenous application of auxin to

fully hydrated S. stapfianus leaves has not been shown to up-regulate the expression

of SDG8i in a northern blot (section 3.3.4), however, using semi-quantitave RT-PCR,

exogenous application of auxin appeared to reduce the expression of SDG8i up to

eight hours post application, and baseline SDG8i levels were restored at twenty four

hours. The difference in expression is minimal, so it cannot be concluded that SDG8i

expression is influenced by auxin levels or that SDG8i is not directly involved in an

auxin signalling/feedback to influence strigolactone levels.

214

7.4.2 S. stapfianus has an increased leaf growth rate 24 hours post rehydration

Increased growth rates and biomass production in plants is considered a valuable trait

in crop sciences and an understanding of the genes involved in regulating biomass

production and growth rate will aid in enhancing these properties in crops. The idea

that genes associated with desiccation tolerance in S. stapfianus may act to regulate

plant growth was addressed recently by Blomstedt et al., (2010) where the results

from the S. stapfianus leaf regrowth experiment detailed in this chapter appears.

Desiccation tolerance is a well regulated process involving induction and repression

of many genes. In vitro translated proteins produced by S. stapfianus mRNA

harvested at several stages of dehydration have been compared to in vivo translated

proteins extracted from dehydrated plants. The results suggest that many transcripts

up-regulated during dehydration are not translated during this process (Gaff et al.,

1997) and may be stored for rapid translation during the rehydration stageThe

importance of protein synthesis in rehydration stages has been noted in the desiccation

tolerant moss, Tortula ruralis (Oliver et al., 2009). Recent research efforts have gone

into understanding the transcriptome and proteome of resurrection plants during

dehydration and/or rehydration (Ingle et al., 2007, Jiang et al., 2007, Abdalla &

Rafudeen 2012, Suarez Rodriguez et al., 2010, Oliver et al., 2010) and these studies

have helped identify genes and proteins that are being up-regulated/down-regulated in

plant tissues undergoing desiccation. The production of increased biomass in S.

stapfianus leaf tissue upon rehydration following dehydration, suggests that a role of

some genes/proteins that become active during rehydration are associated with

generating biomass. The molecular mechanisms surrounding this hypothesis are not

clearly understood, however, recent research has indicated that by modulating

hormone biosynthesis/perception an increase biomass production can be achieved. For

instance, regulated modulation of brassinosteriod biosynthesis has been shown to

increase both grain and biomass production in rice (Sakamoto et al., 2006), and

increasing gibberillin biosynthesis in trees promoted the growth and production of

biomass (Eriksson et al., 2000). The over-expression of SDG8i in A. thaliana

produces plants with a greater biomass and seed yield in comparison to wild-type

(Islam et al., 2013) and SDG8i has been shown to glycosylate GR24, suggesting that

215

the phenotype seen in SDG8i A. thaliana is a symptom of attenuating the

signalling/perception of strigolactone.

The ability of genes from resurrection plants to enhance the phenotype of crop species

has been recently discussed (Toldi et al., 2009, Moore et al., 2009), and the ability of

S. stapfianus to increase biomass production post-rehydration could be considered a

valuable trait to apply in crop species. The mechanisms that allow this rapid regrowth

in S. stapfianus post-rehydration are not clear, and proteomic analysis of tissues

undergoing this process could provide information on possible proteins involved.

Ongoing research efforts at Monash University plan to assess the ability of S.

stapfianus desiccation tolerance associated genes to enhance crop plants.

7.5 Conclusion

This chapter presents evidence that regrowth rate of leaf tissues is increased after a

dehydration cycle, suggesting that the phenomenon of desiccation facilitates a faster

growth rate in leaf tissue. In addition, this chapter presents the first case of GR-24, a

strigolactone analog, being a glycosyltransferase substrate. This has created a new

line of inquiry as to hormonal pathways that may be involved in establishing

desiccation tolerance. Although in its infancy, this research may be valuable to

establishing these traits in desiccation-sensitive species, particularly in crops.

216

8.0 General discussion

8.1 Desiccation tolerance genes for improving biomass production It has been predicted that the current and projected global population’s demand for

food will increase for at least another four decades, and ability of crops to produce

this food may be adversely affected by climate change (Godfray et al., 2010).

Increased biomass production in plants is considered a valuable trait in crop sciences

and efforts to produce plants exhibiting this trait are underway (Eriksson et al., 2000,

Ragauskas et al., 2006, Sakamoto et al., 2005). Approaches for increasing plant

biomass have included the exploitation of plant-microbe partnerships (Weynens et al.,

2009), manipulating starch synthesis (Smidansky et al., 2003) and reducing

photorespiratory flux (Kebeish et al., 2007). The manipulation of hormone

metabolism may also have an impact on plant biomass production. In tobacco, the

over-expression of GA20-oxidase (Gibberellin 20-oxidase) resulted in an increased

photosynthetic rate, which correlated with an increased biomass production (Biemelt

et al., 2004).

The present study is one of the first of its kind that raises the possibility of using

genes from resurrection grasses to improve biomass production. The regrowth of S.

stapfianus leaf tissue in plants that have undergone a dehydration/rehydration cycle is

significantly greater than plants that have not (chapter 7) and these findings were

published in Blomstedt et al. (2010). The molecular mechanisms that S. stapfianus

employs to increase plant growth after a dehydration/rehydration cycle is not known.

However the results presented here, suggest a pathway involving the SDG8i gene that

may be facilitating the rapid regrowth that is observed in rehydrated S. stapfianus.

The S. stapfianus gene SDG8i, whose transcripts are highly elevated in desiccated leaf

tissue, has UDP-glucosyltransferase activity with GR24 strigolactone analogue (see

chapter 7; Islam et al. 2013). This raises the possibility that SDG8i attenuates

strigolactone levels in planta. When SDG8i is overexpressed in Arabidopsis the

shoot biomass production of transgenic plants is increased substantially. The

phenotype of SDG8i transgenics is consistent with a reduction in MAX2 activity

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(Islam et al. 2013) and show similarities to the phenotype of Arabidopsis plants

engineered to overproduce brassinosterioids (Li et al., 2007). The results presented in

the current study, when coupled with the recent results by Wang et al (2013), suggest

that a positive regulator in the brassinosteroid (BR) signalling pathway, BES1

(brassinosteroid insensitive1-EMS-suppressor), may be involved with promoting

increased branching in the SDG8i transgenics. BES1, a positive promoter of

branching, is a degradation target of MAX2. Since SL binding to the SL receptor

AtD14 activates MAX2, attenuation of SL bioactivity by SDG8i glycosylation could

lead to increased BES1 activity and increased shoot branching. In grasses, the

environment also plays a role in regulation of branching. When grown at high

density, the branching of grasses is reduced (Kebrom & Brutnell 2007). The data

presented in this study suggests that the activity of SDG8i in S. stapfianus may be

aiding in the modulation of hormone-related growth processes during dehydration,

rehydration or both. Considering that S. stapfianus leaf biomass production is

increased after a dehydration cycle, the argument can be made that SDG8i has a role

in mediating this increased biomass production. Although the transcript accumulation

of SDG8i (detailed in chapter 3) is reduced during the rehydration stages, it is

possible that SDG8i is not translated, or is not biologically active, until rehydration

occurs. Further analysis is required to determine when SDG8i protein production and

activity occurs.

Although the effect is not as substantial as occurs in the 35S:SDG8i A. thaliana

transgenic plants (Islam et al. 2013), the SDG4i transgenic A. thaliana lines also

display an increased branching phenotype, indicating that the SDG4i resurrection

gene also has a role in branching control and biomass production. Manipulation of

starch synthesis (Smidansky et al., 2002) and brassinosteroids (Choe et al., 2001) has

resulted in plants with an increased seed yield. A. thaliana over-expressing SDG8i

show an increased seed yield (Islam 2013) and in this study, A. thaliana over-

expressing SDG4i also show an increased seed yield, particularly in long day

conditions. The phenotype of 35S:SDG4i A. thaliana suggests that SDG4i may be

affecting hormone signalling and perception (chapter 4) and the possibility of

modulation of the brassinosteroid pathway influencing seed yield in 35S:SDG4i A.

thaliana was considered (section 4.4.7). However alterations in the activity of a

218

number of hormones apart from brassinosteroids could potentially affect biomass

production and seed yield.

An increase in biomass and seed yield is beneficial for facilitating the production of

food crops, biofuel production and other feedstocks for industrial processes. In the

production of these crops, not only is the production of grain and biomass important,

but the nutritional content and other environmental implications need to be

considered. The increased seed yield conferred by overexpression of SDG4i in A.

thaliana indicates that the gene could potentially be utilized to advantage in the

production of foodstuffs in crop plants, however, in 35S:SDG4i A. thaliana, seed

longevity was also reduced. Hence, the application of this gene to enhance crop

production may be limited as the 35S:SDG4i transgenic seed exhibits decreased

germination after storage. Although no problems have been observed with the

longevity of seed produced by 35S:SDG8i A. thaliana, this needs to be tested more

extensively as this could be a problem from an agricultural perspective. It is not yet

known whether or not the beneficial effects of the SDG4i or SDG8i genes in A.

thaliana would be mirrored in crop species. Although an attempt to design a high-

throughput sorghum transformation and regeneration protocol was included in this

study, it proved time-consuming and unfortunately the introduction of resurrection

genes in monocot crop species was not achieved. The introduction of SDG8i into crop

species canola and rice is currently underway.

8.2 The effect on hormone signalling pathways by SDG4i overexpression

The manipulation of hormone biosynthesis or signalling has the potential to change

the phenotypic characteristics of a plant to be beneficial in particular environments.

This includes interfering with ABA signalling for enhanced plant growth in stressful

conditions (Tuteja 2007) or affecting brassinosteriod production to increase biomass

and seed yield (Choe et al., 2001). Extensive cross-talk between hormone signalling

pathways has been revealed in many studies (Gertman & Fuchs 1972, Kansakar &

Bajracharya 1978, Nemhauser et al., 2006, reviewed by Santner & Estelle 2009). The

modulation of sensitivity to one hormone can cause changes in the sensitivity of an

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unrelated hormone. An example of this is provided by an Arabidopsis ethylene-

response mutant where mutation of ETR1 (ethylene receptor kinase 1) also caused a

reduction in ABA sensitivity in roots (Beaudoin et al., 2000). Further research

indicated that these hormones act antagonistically during germination and additively

during root growth, however during later stages of plant development the interaction

between these hormones is not observed (Hugouvieux et al., 2001). A loss of function

mutation in EIN2, a positive regulator of the ethylene response in Arabidopsis,

induces cytokinin insensitivity. This is due to the rate-limiting enzyme in ethylene

synthesis being positively regulated by cytokinin. Consequently some growth

phenotypes that are usually associated with cytokinin application are mediated by

increased ethylene production (Vogel et al., 1998). EIN2 has also been associated

with jasmonic acid (JA) signalling (Alonso et al., 1999). Similar interactions between

auxin, JA and GA have been noted in Arabidopsis. A mutation in AXR1 (auxin

resistance 1) results in plants that are both insensitive to auxin, JA (Tiyaki &

Staswick 2002) and SCF (SKP1, Cullin/CDC53, F-box protein). SCF protein turnover

is essential in both GA and JA signalling (Xie et al., 1998). Recently, the convergence

of auxin and GA signalling was identified where constitutive activation of GA

signalling suppressed arf2 (auxin response factor 2) mutant phenotypes by repressing

the GATA transcription factors GNC (NITRATE-INDUCIBLE CARBON-

METABOLISM INVOLVED) and GNL (GNC-LIKE) (Richter et al., 2013). These

results illustrate the extensive cross-talk that occurs between the various plant

hormones.

In the current study 35S:SDG4i A. thaliana displayed insensitivity to ABA,

suggesting that SDG4i may be attenuating ABA biosynthesis or signalling (section

5.3.3) or alternatively, modulating the activity of an ABA-interacting hormone. The

long day and short day developmental phenotype analysis of 35S:SDG4i A. thaliana

showed that although flowering and germination time remains unaffected in

35S:SDG4i plants, petiole size, leaf length, plant height, seed yield and branching are

affected. GA and ABA have an antagonistic relationship and interaction between

these two hormones can influence many developmental processes including

germination, growth and flowering (Woodger et al., 2010). In addition, GA is known

to cross talk with other plant hormones, namely auxin, cytokinin and ethylene in a

positive or negative manner (Weiss & Ori 2007). Compared to control plants the

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35S:SDG4i plants display opposing differences in plant height and branching in long

and short day conditions (Chapter 4). While the mode of GA action can be influenced

by environmental cues (Weiss & Ori 2007) the mechanism accounting for the

photoperiodic differences displayed in 35S:SDG4i plants is not understood. Light

responses are dictated by phytochromes in plants (reviewed by Smith 2001) and

research by Toyomasu et al. (1998) has shown that phytochromes regulate GA

biosynthesis in lettuce seed germination. More recently, BZR1 (brassinozale-resistant

1), DELLA and PIF4 (phytochrome interacting factor 4) have been shown to interact,

defining a core transcription module which mediates GA, brassinosteriod, and light

signal growth regulation in Arabidopsis (Bai et al., 2012).If GA has a differential role

in regulation of growth and development under the different photoperiod conditions,

the altered ABA biosynthesis/signalling in 35S:SDG4i A. thaliana might destabilise

the antagonistic ABA:GA balance. This could influence branching and plant height

differentially under opposing photoperiodic conditions.

It should also be noted that changes in ABA signalling can not only affect plant

height, but can also induce changes in brassinosteriod signalling (reviewed in Vert et

al., 2005). The yeast two-hybrid assay, detailed in chapter five, revealed the potential

interaction between SDG4i and brassinosteriod insensitive 1 receptor kinase (BIR1-

RK). Although the interaction between the proteins was not confirmed in a BIFC

(bimolecular fluorescence complementation) assay, it is of interest that the interaction

between BIR1-RK and SDG4i in yeast was observed multiple times. When

brassinosteriods are present, they bind to the extracellular domain of BIR1-RK

(Gendron & Wang 2007) leading to the phosphorylation of BIR1 to initiate a cascade

of cellular events (Vert et al., 2005). Mutant plants deficient in BIR1 display

developmental effects including dwarfed phenotype, delayed senescence, male

infertility, delayed flowering and altered leaf morphology (Vert et al., 2005). A.

thaliana over-expressing SDG4i display some of these effects, notably, delayed

senescence and a dwarfed phenotype. It might be worthwhile exploring the possibility

that SDG4i is interacting with BRI1-RK in planta, to alter the phosphorylation state

of BRI1 and thereby initiating a signalling cascade ultimately leading to changes in

brassinosteriod-regulated gene expression.

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The opposing effects of long day and short day conditions on plant height could also

be caused by alterations in photosynthetic processes. Photosynthesis is a major

producer of reactive oxygen species (ROS) in plants (Mehler 1951) and plants employ

several antioxidant enzymes, such as catalase and ascorbate reductase to detoxify

ROS and avoid cellular damage (reviewed in Asada 2006). The equilibrium between

production and scavenging of ROS can be perturbed by abiotic stressors such as

drought, salt, light and temperature (Malan et al., 1989, Prasad et al., 1994).

Endogenous ABA levels increase during abiotic stress to regulate gene expression

associated with plant survival, and ABA signalling is required to induce antioxidant

enzymes (Galvez-Valdivieso 2009). Photosynthetic organisms can respond to changes

in light intensities by adjusting their chlorophyll antenna complexes (Chow et al.

1990). During stress conditions, ABA also initiates degradation of chlorophyll to

reduce photosynthesis as a mechanism for surviving stress (Back & Richmond 1971).

Extended periods of photosynthesis may result in greater ROS production in plants

growing under long days than in short day grown plants. 35S:SDG4i A. thaliana have

an increased level of chlorophyll in leaf tissues compared to levels in control plants

grown in long-day conditions. The insensitivity of 35S:SDG4i plants to ABA may be

repressing ABA-mediated degradation of chlorophyll. This may mean that during

long day conditions, the potential increase in photosynthesis via an increased amount

of chlorophyll may increase ROS accumulation, causing a stress response in the plant.

Elevated ROS could potentially dwarf the plant in long day conditions. This effect is

seen in the Arabidopsis catalase mutant cat2. Plants grown in high light conditions are

dwarfed due to decreased ROS scavenging (Queval et al., 2007). It has recently been

noted that ABA is required for the adjustment of the plant to high light conditions

(Galvez-Valdivieso 2009). As 35S:SDG4i A. thaliana have been shown to be

insensitive to ABA, it is possible that during long day conditions, the plant fails to

properly adjust to the light, leading to the altered phenotype in these plants during

long day conditions. The regulation of chlorophyll level has been associated with

early light-induced proteins (ELIPS), where ELIPS work to prevent photooxidative

stress by preventing free chlorophyll accumulation (Tzvetekova-Chevolleau et al.,

2007). In resurrection plants, ELIPS are expressed during dehydration in S. stapfianus

(Neale et al., 2000) and during dehydration and following application of exogenous

ABA in C. plantagenium (Bartels et al., 1992). The altered ABA signalling displayed

by 35S:SDG4i A. thaliana may reduce the expression of ELIPS leading to increased

222

chlorophyll production, resulting in plants with higher chlorophyll content.

Conversely during short day conditions, the exposure to light is reduced. Under these

conditions the increased level of chlorophyll may be providing more energy to the

plant for growth, leading to a taller phenotype. In order to further elucidate if these

phenotypic differences are associated with ROS, it would be useful to look at ROS

production in 35S:SDG4i A. thaliana. Some information may also be obtained by

examining the production of ELIPs in SDG4i transgenics under high light stress

The evidence that the phenotypic changes in 35S:SDG4i plants result from the

attenuation of the endogenous ABA biosynthesis/signalling, which may in turn affect

the biosynthesis/regulation/activity of other plant hormones, particularly GA is largely

reliant on the developmental phenotype analysis of 35S:SDG4i plants. ABA is a

carotenoid-derived hormone (Umehara et al., 2008). The rate-limiting enzyme of the

carotenoid biosynthesis pathway is phytoene synthase (PSY), producing the first

carotenoid from the catalysis of two geranylgeranyl diphosphate molecules.

Carotenoids are involved in many processes in plants including photosynthesis,

photoprotection and hormone production (reviewed in Cazzonelli 2011). Some alleles

of PSY are responsive to abiotic stress (reviewed in Li et al. 2009) and the ectopic

expression of PSY increases salt and drought tolerance as well as endogenous ABA

levels in tobacco (Cidade et al., 2012). Over-expression of PSY in Arabidopsis has

also resulted in increased endogenous ABA, carotenoid and notably increased

chlorophyll levels (Lindgren et al., 2003). Abiotic stress-induced ABA formation is

associated with the PSY3 allele, and it has been shown that an increase in endogenous

ABA levels positively regulates PSY3 expression (Welsch et al., 2008). It may be

possible that the over-expression of SDG4i is indirectly affecting the regulation of

phytoene synthase. This is supported by the increased chlorophyll levels and abiotic

stress tolerance in 35S:SDG4i A. thaliana. It would be beneficial to not only measure

endogenous ABA levels in 35S:SDG4i plants, but to also look for changes in ABA

biosynthesis/signalling genes and to measure endogenous levels of other plant

hormones. To observe the effects of SDG4i expression in A. thaliana, a

transcriptomic study would provide information on global changes in gene expression

and may help identify the key affects of SDG4i expression and how this may relate to

the acquisition of desiccation tolerance in S. stapfianus.

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An overlapping expression pattern is observed in some genes that are responsive to

drought, salinity and cold stress (Kreps et al., 2002). The gene expression pattern

observed under these stresses can be mimicked by exogenous ABA application,

suggesting that drought, salinity, cold and ABA induced stress cross-talk with each

other to maintain cellular homeostasis (Shinozaki & Yamaguchi-Shinozaki 2000,

Leung & Giraudat 1998). ABA signalling mutant abi1 and ABA biosynthesis mutant

aba2 show sensitivity to exogenous ABA, salt and osmotic stress (Saaz et al., 2006,

Xiong et al., 2006). The over-expression of ABA2 in A. thaliana results in increased

tolerance to these stresses (Lin et al., 2006). In this study, 35S:SDG4i A. thaliana

display similarities with A. thaliana over-expressing ABA2 as the 35S:SDG4i plants

show tolerance to osmotic, salinity and cold stress. The effect of exogenous ABA on

A. thaliana over-expressing ABA2 has not been tested. Although there are no studies

that directly address the tolerance of plants over-expressing ABA

biosynthesis/signalling genes to heat, it could be hypothesised that the over-

expression of these genes would lead to increased heat stress tolerance as the

Arabidopsis ABA biosynthesis/signalling mutants are heat stress sensitive (Larkindale

et al., 2005). The 35S:SDG4i A. thaliana transgenics in this study display no

difference in sensitivity to heat when compared to the wild type (chapter 5). It is

possible that SDG4i may not attenuate ABA biosynthesis/signalling in the same way

displayed in ABA biosynthesis/signalling gene over-expression plants, but rather

adjust ABA biosynthesis/signalling to allow for changes in plant metabolism during

the desiccation tolerance pathway. As ABA induces the expression of SDG4i in S.

stapfianus (chapter 3), it is possible that SDG4i is involved in an ABA feedback

response to change cellular processes during desiccation. To further study the

potential role for SDG4i during this process, it may be useful to examine changes in

SDG4i expression in S. stapfianus under salinity and cold stress, as well as measuring

the endogenous levels of ABA in 35S:SDG4i A. thaliana.

The current study has suggested that SDG4i may be interfering with ABA-mediated

cell death induced by osmotic stress (chapter 6). Osmotic stress has been linked with

endoplasmic reticulum (ER)-stress-induced cell death where signals from both

osmotic and ER stress are required to activate the cell death pathway (Alves et al.,

2011). ER stress will trigger the unfolded protein response (UPR) which attempts to

restore ER homeostasis by increasing chaperone production and degrading misfolded

224

proteins (Ye et al., 2011). If the UPR fails to restore ER homeostasis, a signal is

activated to express asparagine-rich protein (NRP) genes and other downstream

components to activate cell death (Costa et al., 2008). Osmotic and ER stress have

been linked to NRP-B gene activation by ERD15 in soybean, where ERD15 has been

shown to bind to the NRP-B gene promoter (Alves et al., 2001). However in

Arabidopsis, ERD15 does not appear to directly drive NRP-B expression, but may act

through the attenuation of ABA signalling (Kariola et al., 2006). The attenuation of

ABA signalling is thought to induce the SA signalling pathway, resulting in SA

hypersensitivity that appears to induce NRP expression (Ludwig & Tenhaken 2001).

Protein-protein interaction studies have revealed that SDG4i is interacting with

ERD15, and the transgenic 35S:SDG4i A. thaliana plants display tolerance to

tunicamycin-induced ER stress and ABA-induced senescence (chapter 6). This

suggests that in A. thaliana, SDG4i may be associated with modifying ABA

signalling through ERD15 which results in plants with increased tolerance to osmotic,

salinity, cold and ER stress, and repressed ABA-induced senescence. The results

obtained from the 35S-SDG4i transgenic Arabidopsis plants suggest that SDG4i

activity in dehydrating S. stapfianus may be associated with modulating ABA-

induced gene expression and may be critical for repression of one of the drought-

induced senescence pathways that occurs in resurrection plants (Griffiths et al., 2014).

Without the ability to transform S. stapfianus it is difficult to test this hypothesis,

however, analysis of expression levels of several senescence-associated genes, along

with those of ABA- associated genes during the dehydration of S. stapfianus tissues

may be informative.

8.3 Conclusion and future perspectives

The current study presents evidence that the desiccation-tolerance-related gene SDG4i

is associated with the modification of ABA signalling/biosynthesis when expressed in

A. thaliana and that SDG4i activity represses the osmotic- and ER-stress induced cell

death pathway. Some resurrection plants have been found to induce desiccation

tolerance in an ABA-dependent manner (Bartels 2001). An increased ABA content

also occurs in the desiccation-tolerant tissue of orthodox seed (reviewed in Nambara

& Marion-Poll 2003). The ABA content in dehydrating S. stapfianus leaves does not

225

peak until the later stages of dehydration (40-15% RWC) which is after the induction

of the desiccation tolerance program at 60% RWC, suggesting that in S. stapfianus the

induction of desiccation tolerance may be ABA-independent (Gaff & Loveys 1993).

Peak expression of SDG4i occurs in dehydrating S. stapfianus leaf tissue at 19-10%

RWC. Although SDG4i has some basal expression in fully hydrated and mildly

dehydrated S. stapfianus leaf tissue, the transcript abundance increases substantially at

70-60% RWC and increases further as dehydration continues. Since A. thaliana over-

expressing SDG4i exhibit ABA insensitivity, it could imply ABA insensitivity,

conferred by SDG4i, is required in the later stages of dehydration to allow vegetative

tissue to enter a desiccation-tolerant state.

As S. stapfianus leaf tissue dehydrates to around 60% RWC before the desiccation-

tolerance program is induced, it seems likely that avoidance of drought-related

senescence would be required during the earlier stages of water loss. It is possible that

the interaction of SDG4i with ERD15 may act to prevent drought-related senescence

during the early stages of dehydration. This may allow the tissues to activate the

molecular mechanisms that will ultimately lead to the establishment of desiccation

tolerance.

The rehydration of desiccated tissues is considered the most stressful part of the

resurrection process where substantial damage to tissues can occur (Gaff et al., 1997).

The gradual decrease in SDG4i expression in rehydrating leaf tissues may also serve

to prevent deleterious ABA-responses occurring while the tissue is rehydrating. It has

been observed that senescence is not repressed in older leaves of S. stapfianus

undergoing dehydration (Quartacci et al., 1997). In non-resurrection plants, drought-

related senescence of tissues is thought to be a strategy to survive water-deficit by

remobilising nutrients to surviving tissues and to reduce transpiration and canopy size

to enhance plant survival of the drought event (Chaves et al., 2003). During

dehydration of resurrection plants photosynthesis slows down (reviewed in Dinakar et

al., 2012) and remobilization of resources from senescencing older tissues in S.

stapfianus may provide energy for cellular metabolism during desiccation as

photosynthesis is reduced. An examination of SDG4i expression in the older leaf

tissues that are not expected to survive desiccation, may be informative. Mentioned

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earlier was the possibility that the expression of SDG4i may be increasing carotenoid

biosynthesis through an ABA-mediated feedback loop. Carotenoids are required for

photosystem assembly. Some carotenoids are bound to light harvesting complex II

(LHCII) where they absorb light and transfer the energy to chlorophyll (Pogson et al.,

2005, Polivka & Frank 2010). These carotenoids such as xanthophylls are associated

with the stabilisation and photoprotection of thylakoid membranes (reviewed in

Havaux 1998). S. stapfianus is considered neither a poikilochlorophyllous (total

chlorophyll loss and thylakoid degradation) nor homoiochlorophyllous (chlorophyll

and thylakoid retention) however it does exhibit partial loss of chlorophyll and

thylakoid membranes (Gaff & McGregor 1979; Quartacci et al., 1997). It is possible

that SDG4i activity in dehydrating S. stapfianus leaf tissues may be indirectly adding

to the preservation of chlorophyll and thylakoid membranes.

The phenotypic effects of overexpressing SDG8i in Arabidopsis was undertaken by a

previous PhD student Sharmin Islam (Islam et al., 2013) and the analysis of

enzymatic activity undertaken here has indicated that SDG8i glycosylates the

strigolactone analogue compound GR24 in vitro. Further studies are needed to

confirm that SDG8i glycosylates strigolactone in planta. Like SDG4i, the over-

expression of SDG8i in A. thaliana displays repression of ABA-mediated senescence.

SDG8i transgenic plants also display enhanced growth under several stress conditions

(Islam 2013). If SDG8i activity in planta is glycosylating strigolactone or

strigolactone-like compounds, it may indicate that strigolactone acts downstream of

ABA to mediate ABA responses during stressful conditions. The increased branching

phenotype of SDG8i transgenic plants under non-stressed conditions suggests that

SDG8i may be modifying shoot branching inhibition recently described in the

literature to involve strigolactone (Beveridge & Kyozuka 2010, Islam 2013). The

rapid regrowth of S. stapfianus following rehydration may be required to restore a

healthy, actively growing plant post-rehydration. This increase in active growth after

rehydration could be compared to seedling growth once dormancy is broken in

desiccated seed. The effect of SDG8i over-expression in A. thaliana increasing

biomass and branching has identified SDG8i as a gene potentially associated with this

process. Peak SDG8i transcript accumulation occurs at 39-20% RWC (chapter 3) and

suggests that the SDG8i gene product may be required at this stage. So far, the use of

227

SDG8i antibodies has failed to provide evidence of when translation of SDG8i into a

fully functional protein occurs.

Research has shown that TIM17-containing proteins in plants may have the ability to

bind mRNA (Carrie et al., 2010) and raises the possibility that such proteins may be

facilitate transcript storage. It is possible that SDG3i is associated with the storage of

transcripts that are required to be translated during rehydration, however, more

evidence is needed before this hypothesis could be accepted. Western blotting could

help determine if storage of SDG8i transcripts in desiccating tissue occurs and

subsequent translation upon rehydration contributes to increased regrowth. . The

results presented here, along with those of Islam et al. (2013) suggest that SDG8i may

have a dual role in the desiccation/rehydration of S. stapfianus. One function could be

to suppress dehydration-induced senescence in association with SDG4i. Another

function may be to enable rapid regrowth following rehydration.

Avoiding dehydration-induced senescence is pivotal to the survival of resurrection

plants. The utilisation of the yeast-two hybrid system has uncovered protein-protein

interactions between SDG4i and ERD15 and provided some indication of the role of

SDG4i in regulating osmotic-stress induced senescence and modulating ABA

signalling/biosynthesis. If phenotypic effects of SDG4i over-expression in A. thaliana

are mirrored in crop species, this could provide a mechanism for delaying abiotic

stress-dependent plant death as well as increasing seed yield and increasing the

tolerance to osmotic, salinity and cold stress. The results from this study provide some

verification of the potential of utilising resurrection genes to improve crop plant

performance. In addition, this is the first study that has uncovered a UDP-

glycosyltransferase capable of glycosylating a strigolactone-like hormone. This in

vitro biochemical assay supports previous work in the laboratory and reveals the

potential of SDG8i to modify strigolactone perception. Further work in this area may

help reveal the molecular mechanisms that resurrection plants utilise for surviving

extreme drought stress. These studies may also provide novel approaches to

increasing production capacity of crop plants in adverse environmental conditions.

228

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I

Appendix I Six-frame translation of the SDG3i coding sequence obtained through genome

walking. This was completed using the EMBOSS sixpack algorithm. The predicted

nucleotide coding sequence is highlighted in yellow, and the predicted amino acid

sequence is highlighted in green (frame 2). Start and stop codons are in bold, and the

predicted Kozak sequence is underlined.

R C L L * G T R G R R P G L V C S S Q V F1 G V Y Y R A R V V D G P G W S A P P K S F2 V F T I G H A W S T A R A G L L L P S L F3 1 CGGTGTTTACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTCTGCTCCTCCCAAGTC 60 ----:----|----:----|----:----|----:----|----:----|----:----| 1 GCCACAAATGATATCCCGTGCGCACCAGCTGCCGGGCCCGACCAGACGAGGAGGGTTCAG 60 R H K S Y P V R P R R G P S T Q E E W T F6 X T N V I P C A H D V A R A P R S R G L F5 P T * * L A R T T S P G P Q D A G G L D F4 F L S F S A S R N V Y L V T Q P H C R G F1 F C P S A R R V M F I * * H S R T A V A F2 F V L Q R V A * C L S S D T A A L P W P F3 61 TTTTTGTCCTTCAGCGCGTCGCGTAATGTTTATCTAGTGACACAGCCGCACTGCCGTGGC 120 ----:----|----:----|----:----|----:----|----:----|----:----| 61 AAAAACAGGAAGTCGCGCAGCGCATTACAAATAGATCACTGTGTCGGCGTGACGGCACCG 120 K K D K L A D R L T * R T V C G C Q R P F6 R K T R * R T A Y H K D L S V A A S G H F5 K Q G E A R R T I N I * H C L R V A T A F4 Q C A P Q D S S R E E E S R A G K R G E F1 S V R L R I A A E R R R A E Q G S E G K F2 V C A S G * Q Q R G G E Q S R E A R G R F3 121 CAGTGTGCGCCTCAGGATAGCAGCAGAGAGGAGGAGAGCAGAGCAGGGAAGCGAGGGGAA 180 ----:----|----:----|----:----|----:----|----:----|----:----| 121 GTCACACGCGGAGTCCTATCGTCGTCTCTCCTCCTCTCGTCTCGTCCCTTCGCTCCCCTT 180 W H A G * S L L L S S S L L A P F R P S F6 G T H A E P Y C C L P P S C L L S A L P F5 L T R R L I A A S L L L A S C P L S P F F4 D E Q L G E P G A G V R G R R P R L L G F1 M S N L E N Q A R G F V D D V H D C W E F2 * A T W R T R R G G S W T T S T T A G S F3 181 GATGAGCAACTTGGAGAACCAGGCGCGGGGGTTCGTGGACGACGTCCACGACTGCTGGGA 240 ----:----|----:----|----:----|----:----|----:----|----:----| 181 CTACTCGTTGAACCTCTTGGTCCGCGCCCCCAAGCACCTGCTGCAGGTGCTGACGACCCT 240 S S C S P S G P A P T R P R R G R S S P F6 L H A V Q L V L R P P E H V V D V V A P F5 I L L K S F W A R P N T S S T W S Q Q S F4

II

A E E E L D A G P R P P A P Q P H R R * F1 Q K K N W M L D L G H P L L N R I A D S F2 R R R T G C W T S A T R S S T A S P I A F3 241 GCAGAAGAAGAACTGGATGCTGGACCTCGGCCACCCGCTCCTCAACCGCATCGCCGATAG 300 ----:----|----:----|----:----|----:----|----:----|----:----| 241 CGTCTTCTTCTTGACCTACGACCTGGAGCCGGTGGGCGAGGAGTTGGCGTAGCGGCTATC 300 A S S S S S A P G R G G A G * G C R R Y F6 L L L L V P H Q V E A V R E E V A D G I F5 C F F F Q I S S R P W G S R L R M A S L F4 L R Q G C R D R R G P G A R K G I V L H F1 F V K A A G I G A A Q A L A R E S Y F M F2 S S R L P G S A R P R R S Q G N R T S W F3 301 CTTCGTCAAGGCTGCCGGGATCGGCGCGGCCCAGGCGCTCGCAAGGGAATCGTACTTCAT 360 ----:----|----:----|----:----|----:----|----:----|----:----| 301 GAAGCAGTTCCGACGGCCCTAGCCGCGCCGGGTCCGCGAGCGTTCCCTTAGCATGAAGTA 360 S R * P Q R S R R P G P A R L P I T S * F6 A E D L S G P D A R G L R E C P F R V E F5 K T L A A P I P A A W A S A L S D Y K M F4 G Y R C R G G R V C V R Q H R L E E A V F1 A I D A G E G G S V S G S T G S K K R S F2 L S M Q G R E G L C Q A A P A R R S G L F3 361 GGCTATCGATGCAGGGGAGGGAGGGTCTGTGTCAGGCAGCACCGGCTCGAAGAAGCGGTC 420 ----:----|----:----|----:----|----:----|----:----|----:----| 361 CCGATAGCTACGTCCCCTCCCTCCCAGACACAGTCCGTCGTGGCCGAGCTTCTTCGCCAG 420 P * R H L P P L T Q T L C C R S S S A T F6 H S D I C P L S P R H * A A G A R L L P F5 A I S A P S P P D T D P L V P E F F R D F4 F P G P Q W L E Q * Q V G * G L G E E R F1 F P D L N G S N S S K S A E A L V K N V F2 S R T S M A R T V A S R L R P W * R T L F3 421 TTTCCCGGACCTCAATGGCTCGAACAGTAGCAAGTCGGCTGAGGCCTTGGTGAAGAACGT 480 ----:----|----:----|----:----|----:----|----:----|----:----| 421 AAAGGGCCTGGAGTTACCGAGCTTGTCATCGTTCAGCCGACTCCGGAACCACTTCTTGCA 480 K G P G * H S S C Y C T P Q P R P S S R F6 R E R V E I A R V T A L R S L G Q H L V F5 K G S R L P E F L L L D A S A K T F F T F4 * Q G V V A V G A C G W G A F R P D L R F1 S K E S L Q W G L A A G V H S G L T Y G F2 A R S R C S G G L R L G C I P A * P T A F3 481 TAGCAAGGAGTCGTTGCAGTGGGGGCTTGCGGCTGGGGTGCATTCCGGCCTGACCTACGG 540 ----:----|----:----|----:----|----:----|----:----|----:----| 481 ATCGTTCCTCAGCAACGTCACCCCCGAACGCCGACCCCACGTAAGGCCGGACTGGATGCC 540 * C P T T A T P A Q P Q P A N R G S R R F6 N A L L R Q L P P K R S P H M G A Q G V F5 L L S D N C H P S A A P T C E P R V * P F4 P H G G A R D A R L E E Q R G G R R R H F1 L T E V R G T H D W R N S V V A G A V T F2 S R R C A G R T T G G T A W W P A P S R F3 541 CCTCACGGAGGTGCGCGGGACGCACGACTGGAGGAACAGCGTGGTGGCCGGCGCCGTCAC 600 ----:----|----:----|----:----|----:----|----:----|----:----| 541 GGAGTGCCTCCACGCGCCCTGCGTGCTGACCTCCTTGTCGCACCACCGGCCGCGGCAGTG 600 G * P P A R S A R S S S C R P P R R R * F6 A E R L H A P R V V P P V A H H G A G D F5 R V S T R P V C S Q L F L T T A P A T V F4

III

G R R G C A D V G P C V A R A G R A V R F1 G A A V A L T S D R A S H E Q V V Q C A F2 A P R L R * R R T V R R T S R S C S A P F3 601 GGGCGCCGCGGTTGCGCTGACGTCGGACCGTGCGTCGCACGAGCAGGTCGTGCAGTGCGC 660 ----:----|----:----|----:----|----:----|----:----|----:----| 601 CCCGCGGCGCCAACGCGACTGCAGCCTGGCACGCAGCGTGCTCGTCCAGCACGTCACGCG 660 P R R P Q A S T P G H T A R A P R A T R F6 R A G R N R Q R R V T R R V L L D H L A F5 P A A T A S V D S R A D C S C T T C H A F4 H R R R R V V H G G Q R P L R R I L S T F1 I V G A A L S T A A N V L S G V F * A P F2 S S A P R C P R R P T S S P A Y S E H P F3 661 CATCGTCGGCGCCGCGTTGTCCACGGCGGCCAACGTCCTCTCCGGCGTATTCtgaGCACC 720 ----:----|----:----|----:----|----:----|----:----|----:----| 661 GTAGCAGCCGCGGCGCAACAGGTGCCGCCGGTTGCAGGAGAGGCCGCATAAGactCGTGG 720 W R R R R T T W P P W R G R R R I R L V F6 G D D A G R Q G R R G V D E G A Y E S C F5 M T P A A N D V A A L T R E P T N Q A G F4 L S R G L S V R M A C A N * G S E L * * F1 F P G A * A L E W R A P I E V R N Y S S F2 F P G P K R * N G V R Q L R F G T I V V F3 721 CTTTCCCGGGGCCTAAGCGTTAGAATGGCGTGCGCCAATTGAGGTTCGGAACTATAGTAG 780 ----:----|----:----|----:----|----:----|----:----|----:----| 721 GAAAGGGCCCCGGATTCGCAATCTTACCGCACGCGGTTAACTCCAAGCCTTGATATCATC 780 R E R P R L T L I A H A L Q P E S S Y Y F6 G K G P G L R * F P T R W N L N P V I T F5 K G P A * A N S H R A G I S T R F * L L F4 F V M A N H H I V S L F M I N L E C R T F1 L * W Q T I I L Y P F S * * I W N V V P F2 C N G K P S Y C I P F H D K F G M S Y P F3 781 TTTGTAATGGCAAACCATCATATTGTATCCCTTTTCATGATAAATTTGGAATGTCGTACC 840 ----:----|----:----|----:----|----:----|----:----|----:----| 781 AAACATTACCGTTTGGTAGTATAACATAGGGAAAAGTACTATTTAAACCTTACAGCATGG 840 N T I A F W * I T D R K M I F K S H R V F6 T Q L P L G D Y Q I G K * S L N P I D Y F5 K Y H C V M M N Y G K E H Y I Q F T T G F4 H A F S L P V I D * W V L F T L H G T I F1 M L S A F L L L T S G C Y L R Y M V L S F2 C F Q P S C Y * L V G V I Y A T W Y Y H F3 841 CATGCTTTCAGCCTTCCTGTTaTTGACTAGTGGGTGTTATTTACGCtACATGGTACTATC 900 ----:----|----:----|----:----|----:----|----:----|----:----| 841 GTACGAAAGTCGGAAGGACAAtAACTGATCACCCACAATAAATGCGaTGTACCATGATAG 900 W A K L R G T I S * H T N N V S C P V I F6 G H K * G E Q * Q S T P T I * A V H Y * F5 M S E A K R N N V L P H * K R * M T S D F4 I L N G R M I V L K S S L L S S S I L I F1 Y * M D V * * C S R V H Y C H H L F L L F2 I K W T Y D S A Q E F I T V I I Y S Y C F3 901 ATATTAAATGGACGTATGATAGTGCTCAAGAGTTCATTACTGTCATCATCTATTCTTATT 960 ----:----|----:----|----:----|----:----|----:----|----:----| 901 TATAATTTACCTGCATACTATCACGAGTTCTCAAGTAATGACAGTAGTAGATAAGAATAA 960 M N F P R I I T S L L E N S D D D I R I F6 * I L H V Y S L A * S N M V T M M * E * F5 Y * I S T H Y H E L T * * Q * * R N K N F4

IV

V N L F L L * E * T Q L L F W P K K K K F1 * I Y S Y C E N K H N C Y F G Q K K K K F2 E S I L I V R I N T T V I L A K K K K K F3 961 GTGAATCTATTCTTATTGTGAGAATAAACACAACTGTTATTTTGGCCAAAAAAAAAAAAA 1020 ----:----|----:----|----:----|----:----|----:----|----:----| 961 CACTTAGATAAGAATAACACTCTTATTTGTGTTGACAATAAAACCGGTTTTTTTTTTTTT 1020 T F R N K N H S Y V C S N N Q G F F F F F6 Q S D I R I T L I F V V T I K A L F F F F5 H I * E * Q S F L C L Q * K P W F F F F F4 K K F1 K X F2 K X F3 1021 AAAAAA 1026 ----:- 1021 TTTTTT 1080 F F F6 F F F5 F F F4 Total ORFs in frame 1 : 11 Total ORFs in frame 2 : 9 Total ORFs in frame 3 : 9 Total ORFs in frame 4 : 14 Total ORFs in frame 5 : 12 Total ORFs in frame 6 : 13

V

Appendix II

Clones that showed interaction with SDG4i in a yeast 2-hybrid screen were subject to

BLASTn search of the NCBI database. The following alignments show the homology

of the isolated clones (of arbitrary numbers) to the most homologous nucleotide

sequence in the NCBI database. The alignments are titled as the gene the clones are

most related to. Dots represent homologous bases.

Zea mays actin-depolymerizing factor 6 (accession: NM_001155189)

10 20 30 40 50 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 ---------- ---------- ---------- ---------- ---------- 60 70 80 90 100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 ---------- -----AACCC CGCAGCCAAC AGCCCAACAG GACAGTAGGG 110 120 130 140 150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 GCTCCCTTCT CCAGTTCTCC TTGCTTGCGT CTTCCCGGTC CCGTCTGCGT 160 170 180 190 200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 GCGTGCGATC GACGCAGAGG CGCGGGCGCT CGCACCAAGC CGATCCGATG 210 220 230 240 250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 GCCTTCATGC GCTCCCGCTC AAATGCATCT TCTGGCATGG GAGTTGCTCC 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 TAACATTAGG GAGACGTTTG TCGAGCTCCA AATGAAGAAG ACATTCCGAT 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ---------- ---------- Clone 23 ---------- ---------- ---------- ---------- ---------- Zm actin-depol F6 ATGTTATCTT CAAAATCGAA GAAAAGCAAA AGCAGGTGGA GAAGACAGGG 360 370 380 390 400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ---------- ---------- ---------- ----.A.GAG ....A..... Clone 23 ---------- ---------- ---------- ---------- ------.... Zm actin-depol F6 GCCACTACTG AAAGCTATGA TGATTTTCTG GCCTCTCTCC CAGAGAATGA

VI

410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 T...A....C ..C..C.... .......C.. ......T... .......... Clone 23 T...A....C ..C..C.... .......C.. ......T... .......... Zm actin-depol F6 CTGCCGATAT GCGCTGTATG ATTTTGATTT TGTCACCGGG GAGAATGTGC 460 470 480 490 500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 .......... .......... .......... .T..A..T.. .........T Clone 23 .......... .......... .......... .T..A..T.. .........T Zm actin-depol F6 AGAAAAGCAA GATTTTCTTC ATTGCCTGGT CCCCGTCGAC ATCCCGCATC 510 520 530 540 550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 .....T.... .......... ......C... .......... ..C....A.. Clone 23 .....T.... .......... ......C... .......... ..C....A.. Zm actin-depol F6 CGTGCCAAGA TGCTTTACTC CACCTCGAAG GACCGCATCA AGTATGAGCT 560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 T..T..A..T .......... .......A.. .........A ....T...T. Clone 23 T..T..A..T .......... .......A.. .........A ....T...T. Zm actin-depol F6 CGACGGGTTC CACTACGAGA TCCAGGCGAC CGACCCATCC GAGGCGGACA 610 620 630 640 650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ....T..G.. C..A...... .......... ....A..GA. ..CG..G.-- Clone 23 ....T..G.. C..A...... .......... ....A..GA. ..CG..G..C Zm actin-depol F6 TTGAGGTTCT GCGGGAGCGG GCTCACTGAA GCTGGTTCGC CTTCGAAGAA 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 -------A.. C..AT.CAT. .A..---C.. CA.......C .......... Clone 23 TCT.ATG.A. TGA..CTT.A ..C.GATCCA AAT..TAT.A .CCG.GA.G. Zm actin-depol F6 GAAGGGACCT GTGGAGGGCT GTGAACCTTT TGGTCAGATG CAAATTCTAT 710 720 730 740 750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 ....CG.... ----.T...T TG.A...... .GT.A..A.G AAAGATATAT Clone 23 .C.TTGCATG .T.TTG.TAA TAAGAAAG.. A.A.CCA.GT TTCCTGTAAG Zm actin-depol F6 TACCAATGAT GAAAGCTCCC GCCTTGGTAT TTGTTATTAA G--------- 760 770 780 790 800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 CCATGT.... .........A ..A.....T. .T.TC..A.. .......... Clone 23 CATATCA.TT A..CGTGTC. CATGACTCTA ..TGGTGAA. CG.TATC.GA Zm actin-depol F6 ------TTCC TGTAAGCATG TCGTTTAGGC GCGATGCGTG ACTCTAGCTG 810 820 830 840 850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 .......... ...CG..A.C C.G......T ...A.C.T.G ------.... Clone 23 .ACCCTGTC. G.ATG..ATC T.GGGCC.GT A.ACGAA.CA ACATTT..AA Zm actin-depol F6 GTGAAGCGTT ATCAAAG-CT GTATCTGTAA GAGTTTTGGT TGTCGAGCCT 860 870 880 890 900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 20 .......... ...CA..... A..AC.CA.. ........AT .ACCCT..T. Clone 23 AACTCAT.TA TT-------- ---------- ---------- ---- Zm actin-depol F6 GTAAACGAAG CAAGGTTTGC GAACTTGTTA TATTGTGGTA TTGATGGTAC 910 920 930 ....|....| ....|....| ....|....| .... Clone 20 .......ACA .AA....... .........A CTCG Clone 23 Zm actin-depol F6 TGGATTACTG ACTAAAAAAA AAAAAAAAA

VII

Zea mays brassinosteriod insensitive 1-associate receptor kinase 1 (accession

NM_001155673)

10 20 30 40 50 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 ---------- ---------- ---------- ---------- ---CTACCAC 60 70 80 90 100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 CACTACCAAG CAGGCAAGCA CCAGCCCGCC ACCCGCCCAC CCCACCACTC 110 120 130 140 150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 CACCGGGCAG GCATCCAGCG ACGGACCGGG CAGGCAGAGA AAAGCAGACC 160 170 180 190 200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 AGACGAGACG TCGTGCGTCG CGTCCTCCCT GCCGTTCCCC TTCTTCAGTT 210 220 230 240 250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 CTCCCGCTGC TGCTGCCGCC GCCGCTCTCC GTCCGCGTCC AGATCCCGCC 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 ACCCCAACCG ATCTGCCGGC CTCCAGCATG GCGGCGGGGG CGGGGGCGAG 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 GGCGAAGGCA GCGGGAGCCC TGGCGCTGGC GCTCGTGCTC GCGCTAGCGG 360 370 380 390 400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 GGGCCAACTC CGAGGGCGAC GCGCTCTCGG CGCTGCGCCG CAGCCTCAGG 410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 GACCCCGGCG GCGTGCTGCA GAGCTGGGAC CCCACGCTCG TCAACCCCTG 460 470 480 490 500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 CACCTGGTTC CACGTCACCT GCGACCGCGA CAACCGCGTC ACCCGCCTAG 510 520 530 540 550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 ATCTTGGTAA TTTGAACTTA TCTGGTCATC TGGTGCCTGA GCTTGGGAAA

VIII

560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 TTGGAGCATC TGCAGTACCT GGAACTTTAC AAGAACAGCA TTCAAGGAAC 610 620 630 640 650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 AATCCCTTCT GAGCTTGGTA ATTTGAAGAA TCTAATAAGC TTGGACTTGT 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 ACAAGAACAA CATTTCGGGG ACAATACCTC CTTCCCTTGG AAAGTTGAAA 710 720 730 740 750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 TCCCTTGTAT TCCTGCGGCT CAATGGCAAT CATTTGACTG GGCCGATCCC 760 770 780 790 800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 AAGGGAACTT TCTGGAATAT CTAGTCTCAA AGTTGTCGAT GTTTCAAGCA 810 820 830 840 850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 ATGATTTATG TGGGACGATT CCCACATCTG GACCATTTGA GCACATTCCT 860 870 880 890 900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 CTGAGCAACT TTGAGAAGAA CCCACGCTTG GAAGGTCCAG AGCTACAAGG 910 920 930 940 950 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- ---------- ---------- ---------- Zm BIRK1 CCTGGCCATA TATGACACCA ACTGCTAGAA GGGGCACCAC GAACAAAAGA 960 970 980 990 1000 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ---------- ---------- ---------- ---------- ---------- Clone 26 ---------- ---------- -CC.C.CCAA ...CA.A.AA ..AGA.C.AA Zm BIRK1 ATCGCTAGAA AGTAATGGGC GAACAAGGTT ACCATATATT AGGTCTGGTT 1010 1020 1030 1040 1050 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 -------.GA G......... T......... .....C.... ...T...... Clone 26 T.CGACA.GA G......... T......... .....C.... ...T...... Zm BIRK1 CTGCTGCCAC TCTGTAATCC CCCCTGCATA GCTCTTATTT GTAACTTGCT 1060 1070 1080 1090 1100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 .......A.. .....--..C C.....C... GC.....G.. ..CT...... Clone 26 .......A.. .....--..C C.....C... GC.....G.. ..CT...... Zm BIRK1 GTCTGCACGA AAGTTGGTGG ATGTTTTAGC AGAATGCTTT CTGCCCGTGA 1110 1120 1130 1140 1150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 10 ..T.G..... .......... .......C.. .......A.. ..C..CTG.A Clone 26 ..T.G..... .......... .......C.. .......A.. ..C..CTG.A Zm BIRK1 AAGTAAACTT GAAACTGCTT ATTATGAGTG AAATGATGAT GTTAT---TT 1160 1170 1180 ....|....| ....|....| ....|....| ....|. Clone 10 ..ACGTAA.. TA...A.... .......... ..AACT Clone 26 ..ACGTAA.. TA...A.... .......... ..A Zm BIRK1 CC------CT --TGCCAAAA AAAAAAAAAA AA

IX

Zea mays early response to dehydration 15-like protein (accession: EU953508) 10 20 30 40 50 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- ---------- ---------- ---------- Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- ---------- ---------- ---------- Zm ERD15-like AGCTCACTCG CTGGTCGTTG CCTGCTGCGG ATTGCCCCCA CTCACTCCGC 60 70 80 90 100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- ---------- ---------- ---------- Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- ---------- ---------- ---------- Zm ERD15-like TGCTGATATT TCGTTTGGTT CTCCTTTCCG TCTTGTCCGT CGCCGAGGCC 110 120 130 140 150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- ---------- ---------- ---------- Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- ---------- ---------- ---------- Zm ERD15-like TTCCGCTCGC TCGGTCGCTC CCCAGCTCCA GCAGGCAGAT CAGATCCATC 160 170 180 190 200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- ---------- ---------- ---------- Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- ---------- ---------- ---------- Zm ERD15-like CAACCACGAA CAGGAAGGTG AGCGCCGGGA ACGAGCCCTC CGTCCTCCTA 210 220 230 240 250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- ---------- ---------- ---------- Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- ---------- ---------- ---------- Zm ERD15-like CTAGCTGCGA TCCGTAGCCT TGCGTCGCTT TCGCGCCTAG ATCGCCACGT 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- ---------- ---------- ---------- Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- ---------- ---------- ---------- Zm ERD15-like TCTAGATCTG TGGTGTTTTA CTTTCCCGGG TGCGGGTGGT CGAATCCTTC 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ---------- ---------- TCTA.TCGA. GATGAAGA.A CCCCA.CAA. Clone 15 ---------- ---------- ---------- ---------- ---------- Clone 27 ---------- ---------- TCTA.TCGA. GATGAAGA.A CCCCA.CAA. Zm ERD15-like CATCCACCAG GAGAGCTCGT GGGTTCGTTT TGGTTTTTTC GGTGCCGTGA 360 370 380 390 400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 C...A..A.A ..GATCGAAT TCGGCACGAG CCG.C.CA.A .CA.A..A.C Clone 15 ---------- ---------- -----ACGAG CCG.C.CA.A .CA.A..A.C Clone 27 C...A..A.A ..GATCGAAT TCGGCACGAG CCG.C.CA.A .CA.A..A.C Zm ERD15-like GCCAGAATAC GAACGAAGGG GGCTTGTCTT TGATTCGCTC GGTCTCCCCT 410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 .CCG.G.A.. .A.TT.TCCT C.T.CCCGTC TC.G.GAAC. GCGCCGGCCG Clone 15 .CCG.G.A.. .A.TT.TCCT C.T.CCCGTC TC.G.GAAC. GCGCCGGCCG Clone 27 .CCG.G.A.. .A.TT.TCCT C.T.CCCGTC TC.G.GAAC. GCGCCGGCCG Zm ERD15-like GTGCCCGTGA TCTGATATAC GTCCGGATCT GTCTGCTGTA ATCATCAGGA 460 470 480 490 500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 .TCC.TCTCT .AA.GAA.CA AACC.GAGGA AAC....... G.....AC.. Clone 15 .TCC.TCTCT .AA.GAA.CA AACC.GAGGA AAC....... G.....AC.. Clone 27 .TCC.TCTCT .AA.GAA.CA AACC.GAGGA AAC....... G.....AC.. Zm ERD15-like AGGTAGGAGG CCTGAGGGGC TGGGCTGAAG TTGAACCATG AGTGCCGTCG

X

510 520 530 540 550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ....G..... ....ACGT.. .......... ....A..... .......... Clone 15 ....G..... ....ACGT.. .......... ....A..... .......... Clone 27 ....G..... ....ACGT.. .......... ....A..... .......... Zm ERD15-like CGGTTTCGTC GTCGC---TG AACCCGAACG CGCCGCTCTT CATCCCGGCG 560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ...T.C.... .T........ .......... .........A .....G.... Clone 15 ...T.C.... .T........ .......... .........A .....G.... Clone 27 ...T.C.... .T........ .......... .........A .....G.... Zm ERD15-like GCGCTGCTGC AGGTGGAGGA CTTCTCGCCG CAGTGGTGGG ACCTCATCAC 610 620 630 640 650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 .........A .......... ..T....... .T........ A....A..C. Clone 15 .........A .......... ..T....... .T........ A....A..C. Clone 27 .........A .......... ..T....... .T........ A....A..C. Zm ERD15-like CACCACCGCC TGGTTCCGCG ACCACTGGTC CCGCGAGCAC GCCCACCTGG 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ....C..G.. A....GCTCG AC.CC.CCGC T.TC.TCCC. GACGATGAT. Clone 15 ....C..G.. A...GC.... ..CGCC.C.. .T.....CC. .G.......T Clone 27 ....C..G.. A....GCTCG AC.CC.CCGC T.TC.TCCC. GACGATGAT. Zm ERD15-like ACGAGATAGC CGAGCAGCTC GAGTTGGTCG CCCTCCTTGC CTACGATGAG 710 720 730 740 750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 ACCTCTTC.T CGAC..G.A. A..T.TATGA AT..TA.ATA .T.AAAAACC Clone 15 ..CC Clone 27 ACCTCTTC.T CGAC..G.A. A..T.TATGA AT..TA.ATA .T.AAAAACC Zm ERD15-like GATGAGGACC TCTTCTACGG CGAGCAGGCC CCCGCCGCCG CCGCCCTTAA 760 770 780 790 800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 CCGCA.G.TC AC Clone 15 Clone 27 CCGCA.G.TC AC........ .......... .......... .......... Zm ERD15-like GACAGATTCG GTGCTCAAGG CGCTGAACTT GGGCTCCCCG AAGAGCGGCG 810 820 830 840 850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 .......... ...... Zm ERD15-like ACGCCCCGCG GGGGTTCCGG GAGAAACCCA GGCACTCCGA GAAGCCGACC 860 870 880 890 900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like AAGTACGCCG GCAGCCCCAA GAGCAGCGCT CCTCGCGTGA TCCACCAGCC 910 920 930 940 950 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like TCGCTAGGTT CGCTGGCTGG CCCTGGCCTG ATGGGGACTC AGTGACTCAC 960 970 980 990 1000 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like ATGGAAGGCT GCTGCCTCTC TAGCAGCATC TGCTCCTGGC TGCAGCTGCC 1010 1020 1030 1040 1050 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like GTCGGCGTCG TGGTCTGTCT GTCAAAGTCG AAGTAAAAAA ACGTGAGTAG

XI

1060 1070 1080 1090 1100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like TTGTACTACT GTAGAAGTTT CTTTTGTATA GCATGCACGT AAGAAATGAC 1110 1120 1130 1140 1150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like CAAAAAATAT AACAAGAATG CAGCTGTTAG TTTACTGCTG CTCTCCTCGT 1160 1170 1180 1190 1200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 3 Clone 15 Clone 27 Zm ERD15-like GTGTCTAGGG TGCACTTCTG TAAGCAAACG TATATGATGT GTAATGGCAG 1210 1220 1230 ....|....| ....|....| ....|....| ... Clone 3 Clone 15 Clone 27 Zm ERD15-like ACAGCTTAAT GTTACAAAAA AAAAAAAAAA AAA

XII

Zea mays aldehyde oxidase (accession: NM_001111838) 10 20 30 40 50 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTGCTGTGTT GTGCTGTGCT GCGTGCTGTG GAGGGGGAGG AGGAGATGGG 60 70 80 90 100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GAAGGAGGCA GGGGCAGCGG AGTCGTCGAC GGTGGTGCTG GCCGTCAACG 110 120 130 140 150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GCAAGCGCTA CGAGGCGGCC GGCGTGGCTC CGTCCACGTC GCTGCTGGAG 160 170 180 190 200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TTCCTCCGCA CCCAGACGCC CGTCAGAGGC CCCAAGCTCG GCTGCGGCGA 210 220 230 240 250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AGGTGGCTGC GGTGCATGCG TGGTCCTCGT CTCCAAGTAC GACCCGGCCA 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CGGACGAGGT GACCGAGTTC TCTGCCAGCT CCTGCCTGAC GCTGCTCCAC 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AGCGTGGACC GCTGCTCAGT GACCACCAGC GAGGGAATCG GCAACACCAG 360 370 380 390 400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GGATGGCTAC CACCCCGTGC AGCAGCGCCT CTCCGGCTTC CACGCCTCGC 410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AGTGCGGCTT CTGCACACCC GGCATGTGCA TGTCCATCTT CTCCGCCCTT 460 470 480 490 500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTCAAGGCCG ACAACAAGTC CGATCGCCCG GACCCTCCTG CTGGCTTCTC 510 520 530 540 550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CAAGATCACT ACCTCGGAGG CAGAGAAGGC TGTCTCGGGC AACCTTTGTC 560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTTGCACCGG ATACAGACCC ATTGTTGACA CCTGCAAAAG CTTTGCCTCT 610 620 630 640 650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GATGTTGACC TCGAGGACCT AGGCCTCAAC TGTTTCTGGA AGAAGGGCGA 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AGAACCTGCA GAAGTCAGCA GGCTGCCGGG GTACAACAGC GGTGCCGTCT

XIII

710 720 730 740 750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GCACCTTTCC AGAGTTTCTC AAATCCGAAA TCAAGTCTAC TATGAAGCAG 760 770 780 790 800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTGAACGATG TCCCCATTGC AGCCTCAGGT GATGGCTGGT ACCATCCTAA 810 820 830 840 850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GAGCATTGAA GAGCTTCACA GGTTGTTTGA TTCCAGCTGG TTTGATGACA 860 870 880 890 900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTTCTGTGAA GATTGTTGCT TCAAACACTG GGTCTGGAGT GTACAAGGAT 910 920 930 940 950 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CAGGACCTCT ACGACAAGTA CATTGACATC AAAGGAATCC CAGAGCTTTC 960 970 980 990 1000 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AGTCATCAAT AAAAACGACA AAGCAATTGA GCTTGGATCA GTTGTGTCCA 1010 1020 1030 1040 1050 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TCTCTAAAGC TATTGAAGTG CTGTCAGATG GAAATTTGGT CTTCAGAAAG 1060 1070 1080 1090 1100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATTGCTGATC ACCTCAACAA AGTGGCTTCA CCGTTTGTTC GGAACACTGC 1110 1120 1130 1140 1150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AACCATAGGA GGAAACATAA TGATGGCACA AAGGTTGCCA TTTGAATCGG 1160 1170 1180 1190 1200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATGTTGCAAC CGTGCTCCTA GCTGCGGGTT CGACAGTCAC AGTCCAGGTG 1210 1220 1230 1240 1250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GCTTCCAAAA GGCTGTGCTT CACTCTGGAG GAATTCTTGG AACAACCTCC 1260 1270 1280 1290 1300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATGTGATTCT AGGACCCTGC TGCTGAGCAT ATTTATCCCA GAATGGGGTT 1310 1320 1330 1340 1350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CAGACTATGT CACCTTTGAG ACTTTCCGAG CCGCCCCACG ACCATTTGGA 1360 1370 1380 1390 1400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AATGCTGTCT CTTATGTAAA CTCTGCTTTC TTGGCAAGGA CATCAGGCAG 1410 1420 1430 1440 1450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CCTTCTAATT GAGGATATAT GCTTGGCATT TGGTGCCTAC GGAGTCGATC

XIV

1460 1470 1480 1490 1500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATGCCATCAG AGCTAAGAAG GTTGAAGATT TCTTGAAGGG AAAATCGCTG 1510 1520 1530 1540 1550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AGCTCATTTG TGATACTTGA AGCAATTAAA CTACTCAAAG ATACCGTTTC 1560 1570 1580 1590 1600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ACCATCAGAA GGCACTACAC ATCATGAATA CAGGGTCAGC TTGGCTGTCA 1610 1620 1630 1640 1650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTTTCTTGTT CAGTTTCTTA TCTTCCCTTG CCAACAGTTC GAGTGCACCA 1660 1670 1680 1690 1700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TCAAATATTG ATACTCCCAA TGGGTCATAT ACTCATGAAA CTGGTAGCAA 1710 1720 1730 1740 1750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TGTGGACTCA CCTGAGAGGC ATATTAAGGT TGACAGCAAT GATTTGCCAA 1760 1770 1780 1790 1800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TTCGTTCAAG ACAAGAAATG GTTTTCAGCG ATGAGTACAA GCCTGTTGGC 1810 1820 1830 1840 1850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AAGCCGATCA AGAAAGTCGG GGCAGAGATC CAAGCATCAG GGGAGGCTGT 1860 1870 1880 1890 1900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GTACGTTGAT GATATCCCTG CTCCCAAGGA TTGCCTCTAT GGAGCATTTA 1910 1920 1930 1940 1950 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TCTACAGCAC ACATCCTCAT GCTCATGTGA GAAGTATCAA CTTCAAATCA 1960 1970 1980 1990 2000 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TCCTTGGCTT CACAGAAGGT CATCACAGTT ATAACCGCAA AGGATATTCC 2010 2020 2030 2040 2050 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AAGCGGTGGA GAAAATATTG GAAGCAGCTT CCTGATGCAA GGAGAAGCAC 2060 2070 2080 2090 2100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TATTTGCAGA TCCAATCGCT GAATTTGCTG GTCAAAATAT TGGTGTCGTG 2110 2120 2130 2140 2150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATTGCTGAAA CACAAAGATA TGCTAATATG GCTGCAAAGC AAGCTGTTGT 2160 2170 2180 2190 2200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TGAGTATAGC ACAGAAAATC TGCAGCCACC AATTCTGACA ATAGAAGATG

XV

2210 2220 2230 2240 2250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CCATCCAAAG AAACAGCTAC ATCCAAATTC CCCCATTTTT AGCTCCAAAG 2260 2270 2280 2290 2300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CCAGTTGGTG ACTACAACAA AGGGATGGCT GAAGCAGACC ACAAGATTCT 2310 2320 2330 2340 2350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATCAGCAGAG GTAAAACTTG AATCCCAGTA CTACTTCTAC ATGGAAACTC 2360 2370 2380 2390 2400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO AAGCAGCACT AGCGATTCCT GATGAAGATA ACTGCATAAC AATCTATTCC 2410 2420 2430 2440 2450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TCGACACAAA TGCCTGAGCT CACACAAAAT TTGATAGCAA GGTGTCTTGG 2460 2470 2480 2490 2500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO CATTCCATTT CACAATGTCC GTGTCATCAG CAGAAGAGTA GGAGGAGGCT 2510 2520 2530 2540 2550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TTGGTGGAAA GGCAATGAAA GCAACGCATA CTGCATGTGC ATGTGCCCTT 2560 2570 2580 2590 2600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GCTGCCTTCA AGCTGCGGCG TCCAGTTAGG ATGTACCTCG ATCGCAAGAC 2610 2620 2630 2640 2650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO GGACATGATA ATGGCTGGAG GGAGACATCC AATGAAGGCG AAGTACTCTG 2660 2670 2680 2690 2700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO TTGGGTTCAA GTCAGATGGC AAGATCACAG CCTTGCACCT AGATCTTGGA 2710 2720 2730 2740 2750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- ---------- ---------- ---------- ---------- Zm AO ATCAATGCTG GAATATCACC AGATGTGAGT CCATTGATGC CACGTGCTAT 2760 2770 2780 2790 2800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ---------- --------.. .......... A.A...CTC. ......G... Zm AO CATAGGAGCT CTCAAAAAGT ACAACTGGGG CACTCTTGAA TTTGACACCA 2810 2820 2830 2840 2850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 .......... .........A ..T....... ....TG.A.. ..G....... Zm AO AGGTCTGCAA GACAAATGTC TCATCAAAGT CAGCAATGCG AGCTCCTGGA 2860 2870 2880 2890 2900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 .......... ....A..... T......... .....T.... ....C..G.. Zm AO GATGTGCAGG GCTCTTTCAT CGCTGAAGCC ATCATCGAGC ATGTTGCCTC 2910 2920 2930 2940 2950 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ..AG..TT.G G.C....... .T.T.A.A.. .......... ........C. Zm AO AGCACTCGCA CTAGACACTA ACACCGTCAG GAGGAAGAAC CTTCATGATT 2960 2970 2980 2990 3000 ....|....| ....|....| ....|....| ....|....| ....|....|

XVI

Clone 21 ....G..... ..C...G... .T........ ....C..... .......... Zm AO TTGAAAGCCT TGAAGTTTTC TATGGAGAAA GTGCAGGTGA AGCTTCTACA 3010 3020 3030 3040 3050 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 .......... .CA.T..... C..A.....A ....CT.... ....C...A. Zm AO TACAGCCTGG TTTCCATGTT TGACAAGCTG GCCTTGTCTC CAGAATACCA 3060 3070 3080 3090 3100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 A..A..A... ...GC.G.G. ....C..... C....C.... ..G....... Zm AO GCACAGGGCT GCAATGATTG AGCAGTTCAA TAGCAGCAAC AAATGGAAGA 3110 3120 3130 3140 3150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 .......... ...C...... ...AT...G. .......G.G G.....T... Zm AO AACGCGGCAT TTCTTGTGTG CCAGCCACTT ATGAGGTTAA TCTTCGACCA 3160 3170 3180 3190 3200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ..C.....A. .......T.. .......... ........A. ....T..... Zm AO ACTCCAGGCA AGGTGTCAAT CATGAATGAT GGTTCCATCG CTGTCGAGGT 3210 3220 3230 3240 3250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 ......G Zm AO TGGAGGAATT GAGATAGGTC AAGGATTGTG GACTAAAGTG AAGCAGATGA 3260 3270 3280 3290 3300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 Zm AO CGGCCTTTGG ACTGGGACAG CTGTGTCCTG ATGGTGGCGA ATGCCTTCTG 3310 3320 3330 3340 3350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 21 Zm AO GACAAGGTTC GGGTTATCCA GGCAGACACA TTAAGCCTGA TCCAAGGAGG

XVII

Oryza sativa GTP-binding protein (accession: AF327517)

10 20 30 40 50 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel ACCCGCCGCA GTCCACGCGC CCGCACCGCA CCGCGCGCCG CGCTCGCTGC 60 70 80 90 100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel CGTGCCCGTG TCCCCKTCCT TTCCSGTTTC CCCYTCCYTY TYTCTTTCCC 110 120 130 140 150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel CASGCCGMGA SGCCCCCACC AYTCTYTCGA TCCAAAGCCA GATCGGCACG 160 170 180 190 200 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel ACGAGCAGAG GGCCCCCCGG AGCGGCGCGG AGCAATGGCG GCGGGATACC 210 220 230 240 250 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel GGGCGGAGGA CGACTACGAC TACCTCTTCA AGGTGGTACT GATCGGCGAC 260 270 280 290 300 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel TCCGGCGTCG GCAAGTCCAA TCTGCTCTCA CGCTTCACGC GCAACGAGTT 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel CAGCCTTGAG TCCAAGTCCA CCATCGGCGT CGAGTTCGCC ACCCGCTCTC 360 370 380 390 400 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel TCCAGGTCGA CGGCAAGGTT GTCAAGGCCC AGATTTGGGA CACTGCTGGC 410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel CAGGAACGAT ATCGTGCTAT TACTAGTGCG TATTATCGAG GTGCTGTTGG 460 470 480 490 500 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel GGCGTTGCTT GTATATGACG TCACTCGTCA CTCAACCTTT GAGAACGTTG 510 520 530 540 550 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel AGCGTTGGCT GAAGGAGTTG AGGGACCACA CAGATCCCAA TATAGTTGTT 560 570 580 590 600 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel ATGCTGGTTG GCAACAAATC TGATCTCCGC CATCTTGTAG CAGTTCAAAC 610 620 630 640 650 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- ---------- ---------- ---------- ---------- Os Ras-rel TGATGAAGGA AAAGCATTCG CAGAGAGGGA GTCATTGTAT TTCATGGAGA 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ---------- --CCCACCAA .C.CAAA... .GAG..CGAA TTC..CACG. Os Ras-rel CCTCTGCACT CGAGTCGACT AACGTTGAAA ATGCATTCGC AGAGGTCTTA

XVIII

710 720 730 740 750 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 G......... .T..C..C.. ...T...... .......... .A..T..... Os Ras-rel ACTCAGATCT ACCGTATTGT GAGCAAGAGA GCAGTTGAAG CTGGCGAAGA 760 770 780 790 800 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ......T..A .......... ....T..A.. ...C...... .......... Os Ras-rel TGCAGCATCT GGGCCTGGCA AGGGCGAGAA GATTAACATA AAGGATGATG 810 820 830 840 850 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 .......T.. .......... .......... .T...T...G .--AA..C.. Os Ras-rel TTTCGGCGGT GAAGAAGGGT GGCTGCTGCT C-TAGCTGAC CTGCCCTGCT 860 870 880 890 900 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 .........- ---....G.. -...G..... T.....T..T ...T...G.. Os Ras-rel GTATTTTTTT TTGGAGCTGT GGTGATGTAG CTTATGGTAC GGCATTTCCA 910 920 930 940 950 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 .....T.... A-T.AC.... .......A.. A....A.... .........G Os Ras-rel GCGACCGAGA -ACTGGTAGT GGCATTAGCA GCTTCTATGT TACCTAGAGA 960 970 980 990 1000 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 .......... ..-...---- .....G...- ---....... ..A.CT.... Os Ras-rel GTTTATTGTC AA-ACTGGGG GAAAAAAACT GAAGGTTAGA TCGGTGTAGA 1010 1020 1030 1040 1050 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ...C...AT. .T........ ....G..... .......... ..CC.G..C. Os Ras-rel TCTTTCTGGT ACTGTATTGT GTATCCGGCA ATAGGCATGG TATTGTTTTT 1060 1070 1080 1090 1100 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ...-...... ......T..C A......... G......C.. .C....G... Os Ras-rel TTTGGCTGTG AATCCACTGG TACCATGTTT AAACTTATGA ATTGTTCAAA 1110 1120 1130 1140 1150 ....|....| ....|....| ....|....| ....|....| ....|....| Clone 33 ......ATAT GC.TA..... AG......T. .......... .........C Os Ras-rel ATAGAA---- --AGGTGCTT CATGCTTGCC AAAAAAAAAA AAAAAAAAAA 1160 1170 ....|....| ....|....| . Clone 33 TC Os Ras-rel AAAACAAAAA AAAAAAAAAA A

XIX

Appendix III Published versions of papers containing information detailed in this thesis.

SI PLANT DESICCATION STRESS

The resurrection plant Sporobolus stapfianus: An unlikely modelfor engineering enhanced plant biomass?

Cecilia K. Blomstedt • Cara A. Griffiths •

Dale P. Fredericks • John D. Hamill •

Donald F. Gaff • Alan D. Neale

Received: 18 January 2010 / Accepted: 12 May 2010 / Published online: 27 May 2010

� Springer Science+Business Media B.V. 2010

Abstract The resurrection grass Sporobolus stapfianus

Gandoger can rapidly recover from extended periods of

time in the desiccated state (water potential equilibrated to

2% relative humidity) (Gaff and Ellis, Bothalia 11:305–308

1974; Gaff and Loveys, Transactions of the Malaysian

Society of Plant Physiology 3:286–287 1993). Physiolog-

ical studies have been conducted in S. stapfianus to

investigate the responses utilised by these desiccation-

tolerant plants to cope with severe water-deficit. In a number

of instances, more recent gene expression analyses in

S. stapfianus have shed light on the molecular and cellular

mechanisms mediating these responses. S. stapfianus is a

versatile research tool for investigating desiccation-toler-

ance in vegetative grass tissue, with several useful char-

acteristics for differentiating desiccation-tolerance adaptive

genes from the many dehydration-responsive genes present

in plants. A number of genes orthologous to those isolated

from dehydrating S. stapfianus have been successfully used

to enhance drought and salt tolerance in model plants as

well as important crop species. In addition to the ability to

desiccate and rehydrate successfully, the survival of res-

urrection plants in regions experiencing short sporadic

rainfall events may depend substantially on the ability to

tightly down-regulate cell division and cell wall loosening

activities with decreasing water availability and then grow

rapidly after rainfall while water is plentiful. Hence, an

analysis of gene transcripts present in the desiccated tissue

of resurrection plants may reveal important growth-related

genes. Recent findings support the proposition that, as well

as being a versatile model for devising strategies for pro-

tecting plants from water-loss, resurrection plants may be a

very useful tool for pinpointing genes to target for

enhancing growth rate and biomass production.

Keywords Desiccation tolerance �Growth-related genes � Sporobolus stapfianus

Resurrection plants and the evolution

of desiccation-tolerance

Plants growing in a favourable natural environment gen-

erally experience mild water-deficit during the day. A leaf

relative water content (RWC) of about 90%, representing a

10% reduction from full hydration is common in plants

growing in moist environments (Arvidson 1951). A lower

RWC may invoke developmental programs resulting in

morphological changes enabling improved procurement

and conservation of water to allow survival during dry

periods. Such drought-resistance mechanisms include an

increased root to shoot ratio, thickened cuticles and leaf

surface waxes, leaf abscission and reduced leaf size (Levitt

1980). When water content falls below 40% RWC, many

plants experience extensive cellular damage from which

recovery is not possible (Hofler et al. 1941; Schnepf 1961).

In contrast, desiccation tolerance as exhibited by many

resurrection plants does not rely heavily on water conser-

vation. Desiccation tolerance is considered to be a proto-

plasmic phenomenon whereby the plant institutes changes

at the sub-cellular level which allows cell survival irre-

spective of the level of water loss (Kuang et al. 1995;

C. K. Blomstedt � C. A. Griffiths � J. D. Hamill �D. F. Gaff � A. D. Neale (&)

School of Biological Sciences, Monash University,

Clayton, Vic 3800, Australia

e-mail: [email protected]

D. P. Fredericks

ARC Special Research Centre for Green Chemistry,

Monash University, Clayton, Vic 3800, Australia

123

Plant Growth Regul (2010) 62:217–232

DOI 10.1007/s10725-010-9485-6

Schnepf 1961). Resurrection plants such as S. stapfianus

(Fig. 1) may remain viable in a desiccated state for several

months. The desiccation-tolerance program is believed to

be reversible at any stage of the dry down process. In a

number of resurrection plants much of the pre-existing

tissue can remain viable during desiccation and revive and

resume normal metabolism within hours of rehydration.

In rapidly drying less complex plants such as bryo-

phytes, the protection mechanisms conferring desiccation

tolerance are likely to be constitutive (Bewley et al. 1978),

whereas desiccation tolerance in angiosperms is likely to

be induced during dehydration, e.g. Borya constricta (Gaff

and Churchill 1976). The existence of environmentally-

induced desiccation tolerance in a number of unrelated

plant species suggests this ability has evolved indepen-

dently in desiccation-sensitive progenitors (Oliver 2007)

and supports the hypothesis that the phenomenon is related

to the ability of a large majority of land plants to produce

viable dry seed (Gaff 1980; Oliver 2007). This hypothesis,

argued further by Gaff and Oliver (in preparation) implies

that the genes required are inherent within the genomes of

the majority of land plants, and that desiccation tolerance

in resurrection plants was acquired by an extension of

components of the developmental program used to produce

desiccated seed tissue into vegetative tissues. While further

work is needed to substantiate this hypothesis, it raises the

possibility that key regulatory factors with the ability to

confer desiccation-tolerance on vegetative tissues of non-

resurrection plants may be identified.

Initiation of the desiccation-tolerance program

in vascular resurrection plants requires a signal

from the root system

Induction of the genes necessary for establishment of the

adaptive desiccation-tolerance program is believed to be

initiated, at least in part, by a signal generating from the

root system in response to a drying rhizosphere. The

mechanism of generation or nature of the signal emanating

from the roots is unknown and may involve both xylem-

borne chemical signals and hydraulic signals. As may be

the case in many plant species, chemical signals probably

precede hydraulic signals to provide an ‘early warning’ of

soil water deficit (Davies et al. 2002).

Some evidence for the involvement of a root signal/s

important in desiccation tolerance come from physiological

studies in S. stapfianus which have shown that disturbance

of the root system during the drying period can disrupt the

acquisition of the desiccation-tolerant state (Gaff et al.

1997). In addition, the cells in leaf tissue removed from the

intact S. stapfianus plant before the critical threshold level

of around 60% RWC do not remain viable, whereas leaf

cells removed after the threshold level remain viable and

survive further dehydration (Gaff and Loveys 1993). This

suggests that a signal from roots during the early-to-mid

stages of drying may be required to induce the desiccation-

tolerance transcriptional response.

It has been shown, at least in some plants, that an

increased efflux of ABA from the root systems plays a

central role in root to shoot signalling during soil drying

(Davies and Zhang 1991). Exogenous ABA induces desic-

cation tolerance in the resurrection plants Borya constricta

(leaves) and in Craterostigma plantagineum (callus) but not

in S. stapfianus leaves (Gaff and Churchill 1976; Bartels

et al. 1990; Gaff and Loveys 1993). ABA accumulates in

drought-stressed plants in general, including S. stapfianus,

although the increase in S. stapfianus leaves occurs mainly

in the late stage of dehydration, after the desiccation-tol-

erance program has been induced at 61% RWC (Gaff and

Loveys 1993). This suggests that S. stapfianus has adopted

or emphasised an ABA-independent pathway for induction

of the desiccation-tolerance program and that hydraulic

signals may be required to drive accumulation of ABA in

S. stapfianus leaves during the later stages of drying.

Other chemical signals that have been implicated or

proposed to function in root to shoot signalling under

drought include malate, an ethylene precursor (ACC) and

cytokinins, as well as peptides and microRNAs (Schacht-

man and Goodger 2008). Some recent reports have ascri-

bed a role for jasmonic acid or methyl jasmonate in drought

responses (Catala et al. 2007; Mahouachi et al. 2007). In

rice, drought and salt-stress induce jasmonate biosynthetic

genes leading to increased jasmonate levels in leaves and

roots (Moons et al. 1997; Tani et al. 2008) and Kim et al.

(2009) have postulated that the 19-fold increase in methyl

jasmonate levels in the panicles of drought-stressed rice

plants may be required to stimulate the production of ABA.

Of eleven individual, and nine combinations of, plant

hormones tested for improvement in the protoplasmic

Fig. 1 The resurrection grass S. stapfianus Gandoger in the hydrated

and desiccated state, respectively

218 Plant Growth Regul (2010) 62:217–232

123

drought tolerance of S. stapfianus mesophyll cells, methyl

jasmonic acid was the most effective. However, the

increment of 6 MPa is well below the 500 MPa increase in

protoplasmic drought tolerance induced in intact S. stapfi-

anus plants by drought (Ghasempour et al. 2001). Never-

theless, several S. stapfianus genes thought to be associated

with desiccation tolerance (Neale et al. 2000; Le et al.

2007) are induced by methyl jasmonate (unpublished

result) further supporting a role for methyl jasmonate in

desiccation tolerance in S. stapfianus. Recent transcript

profiling in T. ruralis has also suggested that jasmonate

may be involved in regulating the desiccation tolerance

mechanism in this bryophyte (Oliver et al. 2009).

S. stapfianus has several advantages for research

into desiccation tolerance and growth regulation

S. stapfianus is a perennial resurrection grass (Gaff 1971)

that is easily propagated by subdivision or from seed and is

well placed for research on the family Poaceae that is par-

amount for human nutrition. Desiccation tolerance in

S. stapfianus leaves is not constitutive, but is induced over a

period of 1–2 days during the early-to-mid stage of drying.

Leaf rolling during drying, caused by a greater contraction of

the large thin walled bulliform epidermal cells in rows along

the adaxial (upper) leaf surface than of the abaxial epidermal

cells, is a reliable indicator of the level of dehydration (Gaff

and Loveys 1993). Live air-dry leaves differ in colour from

dead air-dry leaves that have senesced on dry plants,

allowing them to be easily distinguished. When dried in full

sunshine, the surfaces of the inrolling S. stapfianus leaves

develop a purple colour, intensifying to purple–black,

which, based on field observation and over 30 years of

experience of cultivated plants of S. stapfianus (DF Gaff) is

a reliable indicator that the air-dry S. stapfianus leaves are

alive. Dehydrated S. stapfianus undergoes a partial loss of

chlorophyll content to around 40% in dry leaves and returns

to 90% on rehydration (Gaff and McGregor 1979). A com-

parison within the S. stapfianus genotype of the differential

anabiotic state in dehydrated leaves can be made as leaves

become desiccation tolerant while they dry attached to intact

plants in the light, whereas leaves detached and then dried

remain desiccation sensitive (Gaff and Loveys 1993). A

comparison of metabolic processes in S. stapfianus suggests

that the desiccation-sensitive leaves may still be alive when

air-dry but that the recovery process fails during rehydration

(Whittaker et al. 2004).

Probes made from desiccation-tolerant and desiccation-

sensitive leaves can be used to correlate gene expression

with the desiccation-tolerant state and the ability to suc-

cessfully rehydrate and reinitiate metabolism and growth

(Neale et al. 2000; Le et al. 2007). Desiccation-sensitive

and -tolerant Sporobolus species can also be compared to

help pinpoint important genetic response differences

(Blomstedt et al. 1998a, b).

The early responses to dehydration are similar

in resurrection and non-resurrection plants

The strategies employed by both desiccation-sensitive and

desiccation-tolerant plants to cope with mild water deficit

have a number of similarities (Neale et al. 2000), indicating

that drought avoidance mechanisms are required in vascular

resurrection plants to slow down drying rates in the field

sufficiently for the desiccation-tolerance genetic program to

be induced. These so called early responses have been well

documented in a number of desiccation-sensitive model

plants and important crop species and to a lesser extent in

resurrection plants (Neale et al. 2000; Yamaguchi-Shino-

zaki and Shinozaki 2005; Itturriaga et al. 2006; Tardif et al.

2007). A number of specialised proteins are produced, such

as LEAs and other hydrophillins which are believed to

physically associate with and protect critical cellular protein

and membrane structures from de-solvation and denatur-

ation. LEA proteins may also be involved in sequestering

salt ions and scavenging reactive oxygen species (Tunnac-

liffe and Wise 2007). The conversion of stored starch into

mono- and di-saccharides also has the dual effect of pre-

venting de-solvation by coating molecular components

(glaciation theory) as well as increasing osmotic pressure

for enhancement of the ability of cells to retain and take up

water. In vascular plants in general, as well as many des-

iccation-tolerant grasses, including S. stapfianus, and all

mosses, sucrose is the major protectant sugar (Smirnoff

1992; Ghasempour et al. 1998). Increased osmotic pressure

can also be achieved in some plants by the production of

other osmolytes such as glycine betaine and proline

(Holmstrom et al. 1994; Kishor et al. 1995). In both des-

iccation-sensitive and resurrection plants the transcripts of

genes mediating some of these protection processes appear

at quite mild levels of water-loss (Neale et al. 2000).

Recent evidence suggests that expression of some early-

response water-stress genes may be regulated by both

positively–and negatively–acting transcription regulators

(Kariola et al. 2006). However, a number of research

groups have reported that over expression of a single reg-

ulatory protein is sufficient to enhance the stress tolerance

to a modest but significant degree (Zhang et al. 2004). This

strategy has been shown to be effective for drought as well

as cold and salt stress. In specific cases, cross-protectivity

has been demonstrated for all three stresses (Kasuga et al.

1999). This reflects the commonality in plant responses to

these three stresses which all have the ultimate effect of

decreasing water availability in the cell.

Plant Growth Regul (2010) 62:217–232 219

123

Analyses of drought-response mechanisms unique

to resurrection plants

While early drought-tolerance responses to water deficit

appear similar in both desiccation-sensitive and desicca-

tion-tolerant plants, desiccation tolerance and drought

tolerance have differing endpoints. Drought-tolerance

mechanisms act to maintain water content and metabolic

activity under sub-optimal water availability, whereas

desiccation-tolerance mechanisms allow survival of com-

plete desiccation. Hence, it is likely that resurrection plants

contain both drought-tolerance mechanisms, for dealing

with daily mild water-deficit, and reversible desiccation-

tolerance mechanisms for dealing with severe intermittent

water-deficit. We would postulate that the genetic mecha-

nisms regulating drought-tolerance and desiccation-toler-

ance responses are necessarily different.

The unique features that allow resurrection plants but

not non-resurrection plants to survive desiccation have not

been fully elucidated. A pivotal difference could include

the ability to tightly link a rapid (decline in growth rate)

cessation of cell division in the shoot and a controlled

down-regulation of specific metabolic processes to the

level of decreasing water availability. Surviving desicca-

tion appears to require a systematic shutdown of specific

metabolic activities which may be injurious to the cell as

well as establishment of protective mechanisms required

during rehydration from the air-dry state (Di Blasi et al.

1998; Neale et al. 2000).

Efficient mechanisms for protecting against photo-oxi-

dative injury and removing free reactive oxygen species

and maintaining DNA-repair processes are probably

essential. Catalase transcripts in dehydrating S. stapfianus

increase 2-fold between the 60–40% RWC stages then

drop on further dehydration to levels well below those

observed in fully hydrated tissue (Blomstedt et al. 1998a).

This suggests that other antioxidant systems such as glu-

tathione reductase and dehydroascorbate reductase may be

more important in drying S. stapfianus (Sgherri et al.

1994).

In plants exhibiting an environmentally-induced desic-

cation tolerance program, (but not in the moss Tortula

ruralis, Bewley 1979) the ability to maintain both tran-

scriptional and translational activity during dehydration

also appears to be essential. Desiccation-sensitive angio-

sperm plant species cease protein synthesis at mild levels

of drought stress whereas protein synthesis continues in

some inducible desiccation-tolerant species almost until

leaves are air-dry (Bartels et al. 1990; Gaff et al. 1997).

Similarly, accumulation of specific transcripts in S. sta-

pfianus occurs almost throughout the entire dry-down

process (Neale et al. 2000). A detailed analysis of the

transcription patterns during progressive dehydration of

around twenty desiccation-tolerance specific genes from

S. stapfianus has been summarised in Gaff et al. (2009). A

number of these genes exhibit maximum levels of tran-

script accumulation in the air-dry state while some others

have complex bimodal patterns with peaks occurring at

both the earlier and later stages of dehydration. While the

techniques used for isolating desiccation-tolerance genes

from S. stapfianus dehydrated leaf tissue have focussed on

eliminating dehydration-responsive genes expressed in

desiccation-sensitive tissue (Blomstedt et al. 1998a, b;

Neale et al. 2000), a number of the genes identified fall

into similar categories as those isolated from dehydrating

desiccation-sensitive plants. Hence, desiccation tolerance

may require extending the transcriptional activation of

early-response genes into later stages of dehydration. For

example, transcripts from different family members of

group 2 and 3 LEAs accumulate differentially during

dehydration and even transcripts from some genes

required for house-keeping activities, such as eIF1 (Neale

et al. 2000) and eEF1A (see below) accumulate to high

levels in dried tissue. Expression of such genes may be

important for maintaining the translational activity in

dehydrating S. stapfianus and rapidly re-initiating growth

upon rehydration.

A comparison of in vitro translated proteins, produced

from S. stapfianus mRNAs harvested at several dehydra-

tion stages, with in vivo proteins extracted from dehy-

drated plants (Gaff et al. 1997) suggests that several

transcripts produced during dehydration may not be

translated during the dry down process. An analysis of

gene expression during the rehydration in S. stapfianus

(O’Mahony and Oliver 1999) revealed that few novel

transcripts are produced during this phase (M. Oliver. pers

comm.). This suggests that some of the transcripts accu-

mulated to high levels in air-dry tissue may function dur-

ing rehydration when much of the potential damage to

cellular components may occur. Establishment of mecha-

nisms for allowing both survival during rapid rehydration,

and reinstituting rapid regrowth when water becomes

available, before the desiccated state is achieved may be

essential for survival of these plants in environments where

rainfall is sporadic.

An analysis of the growth capacity of S. stapfianus

following a dehydration-rehydration treatment was con-

ducted by comparing the amount of regrowth in treated

plants with that of fully-hydrated healthy plants that had

been kept well-watered (untreated) for several months.

Regrowth was substantially more rapid in the treated

plants (Fig. 2). Plants that had been subjected to a dehy-

dration-rehydration cycle formed approximately twice the

biomass, measured as dry weight, within 10 days follow-

ing removal of mature leaf tissue, compared to well-

watered plants.


123

Isolation of desiccation-tolerance specific genes

We have utilized several techniques to isolate and identify

the drought-responsive genes that are expressed in

S. stapfianus desiccation-tolerant leaf tissue but are not

expressed in S. stapfianus desiccation-sensitive leaf tissue

or in the leaf tissue of the desiccation-sensitive Sporobolus

species S. pyramidalis. These desiccation-tolerance spe-

cific genes from S. stapfianus, designated S. stapfianus

Drought-related Genes (SDG), are listed in Gaff et al.

(2009). A number of genes orthologous to those we have

isolated from S. stapfianus have been successfully utilised

to enhance drought or salt or cold tolerance in model plants

or important crop species. These include glyoxylase1,

catalase, cysteine protease, several group 2 and group 3

LEAs, aquaporins, a myb transcription factor and the

translation initiation factor eIF1 (Mohamed et al. 2003;

Villalobos et al. 2004; Sheokand et al. 2005; Brini et al.

2007; Cui et al. 2008; Diedhiou et al. 2008; Roy et al.

2008; Wang et al. 2009a). Approaches to using genes

associated with desiccation tolerance for improving

drought tolerance in crop species have recently been

reviewed by Toldi et al. (2009) and Moore et al. (2008b).

A number of genome profiling experiments analysing

the transcriptome in dehydrating and rehydrating resur-

rection plants and comparison with desiccation sensitive

plants has been performed (Collett et al. 2004; Oliver et al.

2009). In an attempt to identify pivotal transcriptional

differences between desiccation-tolerant and desiccation-

sensitive tissue and identify transcripts important in pro-

tection during drying and re-establishing metabolic activity

and growth upon rehydration we have recently applied a

subtractive hybridization technique (Diatchenko et al.

1996) using desiccation-tolerant leaf tissue against fully-

hydrated desiccation-sensitive leaf tissue from S. stapfi-

anus and dehydrated S. pyramidalis (unpublished). The

isolated fragments were subjected to a reverse northern

technique with a probe prepared from fully-hydrated plants

and a combined probe from plants dehydrated to 30%

RWC and air-dry plants. A subset of these fragments has

been subcloned, reprobed and sequenced. An example of

the reverse northern results, whereby several fragments that

hybridized to the dehydrated probe but not the fully-

hydrated probe, were selected for further analysis, is

depicted in Fig. 3. The outcome from the sequence analysis

revealed a number of genes of unknown function, however,

some of the sequence fragments, representing mRNAs

accumulated in desiccated S. stapfianus tissue can be

identified from highly homologous sequences in the data-

base of rice and other plants. Interestingly, the majority of

transcripts identified so far appear to originate from genes

that may function in growth-related processes (see

Table 1).

Fig. 2 Leaf regeneration in dehydrated-rehydrated and control

S. stapfianus plants. Leaf length was measured following removal

of mature leaf tissue in 10 treated and 10 control plants. Treatment

involved dehydration to 10% RWC and rehydration for 24 h before

the leaf tissue was removed. Leaf tissue was removed at the same

time from healthy control plants that had not been subjected to a

dehydration/rehydration cycle

Φ/HaeIII

ladder (kb) 1.35

0.87

0.31

1.35

0.87

0.31

1.35

0.87

0.31

A

B

C

Fig. 3 Reverse northern analysis of fragments generated by the

subtraction hybridization technique showing differential hybridization

to the probes prepared from hydrated and dehydrated tissues. A PCR-

amplified and subcloned subtraction fragments probed with: B cDNA

produced from fully-hydrated S. stapfianus and C cDNA produced

from dehydrated (30% RWC) and airdry S. stapfianus plants


123

Transcripts associated with cell wall modification

and signalling accumulate in desiccation-tolerant

S. stapfianus tissue

One such example is the endo-ß-1,4-glucanase or cellulase

encoded by SDG8s. Plant cellulases hydrolyse ß-1, 4-

linkages behind unsubstituted glucose residues. These

enzymes can, in theory, act on cellulose or xyloglucan

although little is known about the endogenous polysac-

charides targeted by these enzymes and in most cases plant

cellulases appear to have no significant activity on crys-

talline cellulose (Mølhøj et al. 2002). Some members of

this plant glycosyl hydrolase (GH) 9 cellulase family are

membrane anchored at the plasma membrane/cell wall

interface, while others are predicted to be cytosolic (Lopez-

Casado et al. 2008). Plant cellulases have a well docu-

mented association of activity with fruit ripening, cell wall

loosening during cell expansion, organ abscission and

cellulose synthesis (Cosgrove 2005).

Transgenic approaches utilising plant GH9 cellulases

show that over-expression results in growth promotion with

significant differences in cell expansion and organ size

(Park et al. 2003; Shani et al. 2004). Similarly, introducing

related enzymes from bacteria into plants accelerates

growth and increases biomass (Safra-Dassa et al. 2006).

Conversely, the Korrigan (kor) mutant of Arabidopsis that

is deficient in a membrane-bound endo-1,4-beta-glucanase

is dwarfed and defective in both cell elongation and cell

division (His et al. 2001). The mechanism whereby endo-ß-

1, 4-glucanases affect cell growth is not clear. They may

act by altering cellulose biosynthesis or microfibril

assembly or cell wall loosening directly, or alternatively

they may change the porosity of the wall to allow the

activity of expansins (Mølhøj et al. 2002; Cosgrove 2005).

The gene product encoded by SDG72s is a chitinase-like

protein that may also have a role in growth-related pro-

cesses. Chitinases encompass a group of enzymes that target

N-acetyl-glucosamine (GlcNAc) containing compounds

(Meins et al. 1994). Various members of the chitinase

family play a role in plant defence but it is now generally

accepted that some chitinases and chitinase-like genes have

roles in plant growth and development (Kasprzewska

2003). Accumulation of cellulase and chitinase and related

proteins in the bean abscission zone and anthers and pistils,

as well as the high levels of chitinase transcripts found in

young leaves, stems, flowers and pollen (Neale et al. 1990;

Campillo and Lewis 1992; Patil and Widholm 1997) sug-

gest that these proteins may be important in cell wall dis-

ruption and loosening in rapidly growing tissues. There is

good evidence for chitinolytic enzymes participating in cell

wall remodelling and daughter cell separation in fungi

(Kasprzewska 2003). In addition, Patil and Widholm (1997)

have reported a correlation between seedling vigour and cell

culture growth in tobacco over-expressing chitinase.

It has been demonstrated that GlcNAc-containing mol-

ecules are important regulatory signalling molecules in both

Table 1 Description of S. stapfianus genes isolated from dehydrated tissue using a subtractive hybridization technique (see text for details)

showing homology to database sequences

Clone Homology Species Reference

SDG71s (SsGH3) IAA- conjugating enzyme GH3 Capsicum chinense 9e-15 Liu et al. (2005)

Arabidopsis thaliana (GH3.3) 4e-12 Staswick et al. (2005)

Oryza sativa (GH3.8) 1e-7

SDG72s Putative endochitinase A Sugarcane 7e-12

Daucus carota 4e-11

Wheat 8e-11 Li et al. (2001)

SDG8s Endo-beta-1,4-glucanase precursor A. thaliana 4e-15

Tobacco 2e-14

Cotton 5e-15 Libertini et al. (2004)

SDG11s (SsGRP3) Glycine rich protein (GRP) A. thaliana AtGRP3 3e-5 Park et al. (2001)

Hordeum vulgare 8e-7 Rohde et al. (1990)

Cucumis sativus 2e-6 Shimizu et al. (2006)

SDG15s Eukaryotic elongation factor (eEF1A) Nicotiana paniculata 3e-38

Maize 3e-38 Carneiro et al. (1999)

SDG16s Cystein proteinase inhibitor A. thaliana AtCys6 2e-5 Martinez et al. (2005)

Medicago sativa 2e-5 Rivard et al. (2007)

SDG20s Origin recognition complex subunit

6—like protein—Orc6 (6e-10)

A. thaliana AtOrc6 2e-28


123

plant and animal development (Lee et al. 2003). GlcNAc

residues are present in secondary plant walls (Benhamou

and Asselin 1989) and it is thought that chitinases secreted

into cell walls may act to cleave chitooligosaccharide

components from glycolipids or GlcNAc containing pro-

teins to produce signal molecules important in cell growth.

A chitinase-related receptor-like kinase localised in

plasma membranes has been shown to bind oligomeric

chitin oligosaccharides potentially derived from GlcNAc-

containing glycoproteins. This receptor, which may act in a

ubiquitin/proteosome signalling pathway, affects plant

development and cytokinin homeostasis in tobacco (Lee

et al. 2003). Arabidopsis containing a mutation in a chiti-

nase-like gene (AtCTL1) shows reduced cell expansion and

increased ethylene production (Zhong et al. 2002). AtCTL1

is thought to localise to the cell wall. Ethylene inhibitors

only partially rescued the mutant phenotype suggesting that

the chitinase activity may be associated with normal cell

elongation processes (Zhong et al. 2002). It is possible that

the presence of normal growth signals coupled with

reduced ability to loosen cell walls may stimulate ethylene

production.

While it should be noted that the SDG72s fragment

containing the chitinase-like sequence is too short to

determine if it is likely to be located in the apoplast, these

results suggest that the chitinase and cellulase genes whose

transcripts are present in desiccation-tolerant tissue may be

involved in growth and development related processes and

perhaps more specifically in cell wall signalling or loos-

ening in rehydrated resurrection plants. It has been shown

that water uptake is stimulated by cell wall loosening

which reduces turgor pressure and increases the water

potential gradient, but it is not clear that wall loosening

would be required during rehydration. However, once a

threshold level of turgor is attained, cell expansion can be

controlled by the yielding capacity of the cell walls (Ter-

maat et al. 1985) and mechanosensory events associated

with the wall loosening could modulate hormone levels,

changing gene expression and inducing leaf development

(Taleisnik et al. 2009).

The small fragment of the SDG11s gene translates into a

section of a putative glycine rich protein (GRP) of 51

amino acids, of which 43% are glycine residues. GRPs

form a large and diverse protein group that have the key

characteristic of comprising 20–70% glycine. Several GRP

families are defined by the repeating glycine-rich motif

(GGGX, GGXXXGG, GXGX, GYPPX) or the presence of

RNA binding consensus sequences, RNP-1 and RNP-2

(Sachetto-Martins et al. 2000; Fusaro et al. 2001; Bocca

et al. 2005). The flexibility of the protein structures con-

ferred by the high proportion of glycine residues has led to

the suggestion that GRPs may be involved in protein–

protein interactions (Sachetto-Martins et al. 2000). Outside

the repeating motifs there is often little sequence homology

between members of a designated family, suggesting that

the proteins have specific, though often obscure functions.

Several GRPs localise to specific tissue or cell types, such

as epidermis, vascular tissue and cell walls (Showalter

1993; Marty et al. 1996; Cassab 1998; Sachetto-Martins

et al. 2000) also suggesting that GRPs are involved in a

wide variety of cellular responses. A dehydration-induced

GRP that is targeted to the cell wall has been isolated from

the resurrection plant Boea hygrometrica (Wang et al.

2009b). The expression of GRPs have been shown to be

modulated by hormones, such as salicylic acid (Park et al.

2001), as well as biotic and abiotic factors, such as cold,

excess water, wounding and pathogenic attack (Sachetto-

Martins et al. 2000; Park et al. 2001). Transcripts encoding

several GRPs and glycine and proline rich (GPRPs) pro-

teins that correlate strongly with desiccation tolerance have

been isolated from S. stapfianus (Blomstedt et al. 1998a, b;

Neale et al. 2000) and it has been suggested that these

proteins may have important roles in maintaining the

integrity of plasma membrane and cell wall.

The SDG11s GRP shows a high degree of similarity to

GRP3 from Arabidopsis thaliana (AtGRP3) (de Oliveira

Fig. 4 ClustalW alignment of S. stapfianus SDG11s to GRP proteins

from Nicotiana glauca [Q9M6T8; Smart et al. (2000)], A. thalianaAtGRP3 [Q9ZSJ6; Park et al. (2001)]; Hordeum vulgare [P17816;

Rohde et al. (1990)], Cucumis sativus [A0P8W6; Shimizu et al.

(2006)] and Zea mays [B6U4A6; Alexandrov et al. (2009)]. Con-

served tyrosine residues and cysteine residues (see text for details) are

indicated by arrows and triangles, respectively


123

et al. 1990) as well as to proteins encoded by a gene from

Nicotiana glauca (NtGRP1) that is predominantly expres-

sed in guard cells and epidermal cells (Smart et al. 2000),

one from Hordeum vulgare (HvGRP1) with homology to

vertebrate cytokeratins (Rohde et al. 1990) and CcGRP1

isolated from cucumber (Cucumis sativus) seedlings, which

is expressed during gravimorphogenesis (Shimizu et al.

2006) (Fig. 4). These proteins contain conserved cysteine

residues in a region called the CD domain with the general

pattern, C-(X)2–3-CC- (X)6-C(X)2–3-CC, which is thought

to be involved in interactions with cell wall proteins

(Domingo et al. 1999; Shimizu et al. 2006). There are also

several tyrosines that may also be involved in cross-linking

with the cell wall (Domingo et al. 1999). The protein

fragment encoded by SDG11s is not large enough to cover

this entire region, but the presence of the tyrosines and

conserved cysteine residues indicate that SDG11s is a

member of this group of GRPs.

AtGRP3 has been shown to regulate the function of a

wall-associated receptor kinase (WAK1) in Arabidopsis.

The cysteine rich domain of the GRP is essential for

binding to WAK1 (Park et al. 2001). WAKs possess an

extracellular domain, a transmembrane domain and a

cytoplasmic protein kinase domain (He et al. 1999;

Anderson et al. 2001; Kanneganti and Gupta 2008). WAK1

is covalently linked to pectin in the cell wall, and associates

with the extracellular AtGRP3 and the cytoplasmic phos-

phatase 2C, KAPP (Park et al. 2001). There are 5 WAK

genes and 22 WAK-like genes in Arabidopsis. It has been

suggested that WAKs physically link the cytoplasm to the

extracellular matrix and transduce signals in response to

changes in the cell wall (Decreux et al. 2006; Kanneganti

and Gupta 2008). WAKs are expressed in shoot and root

apical meristems, in expanding leaves and in response to

wall disturbances (Wagner and Kohorn 2001). Activation

of AtGRP3/WAK1 in Arabidopsis by pathogen attack leads

to phosphorylation of the oxygen evolving enhancer pro-

tein2 (Yanga et al. 2003), whereas anti-sense experiments

have demonstrated that WAK2 and WAK4 are associated

with cell expansion and affect growth and development in

Arabidopsis (Lally et al. 2001; Wagner and Kohorn 2001).

During dehydration, plant cells are susceptible to dam-

age caused by plasmolysis. Desiccation-tolerant plants are

able to maintain cell wall flexibility to allow extensive cell

wall folding which maintains the integrity of the connec-

tions between the plasma membrane and cell wall as the

cells shrink due to water loss (Farrant 2000; Jones and

McQueen-Mason 2004; Moore et al. 2006). This charac-

teristic is probably dependent on changes in cell wall

architecture involving the activity of expansins and pectin

modification and disruption of non-covalent bonds between

wall polysaccharides leading to greater flexibility (Jones

and McQueen-Mason 2004; Moore et al. 2008a). Such

changes may also assist in the rapid expansion of the cell

wall as water becomes available (Jones and McQueen-

Mason 2004). Yanga et al. (2003) have demonstrated that

addition of AtGRP to protoplasts upregulates WAK1 in

Arabidopsis. Expression of SDG11s and changes to the cell

wall during dehydration in S. stapfianus may activate

WAK-mediated mechanisms to protect vital cellular

functions and allow recovery and growth when water

becomes available.

Transcripts with potential roles in cell proliferation

and resource mobilisation are present in dehydrated

S. stapfianus tissue

The GH3 gene family encode proteins that conjugate the

plant hormones auxin, jasmonic acid and salicylate to

amino acids. Conjugation regulates hormone homeostasis

by affecting the storage, transport and metabolism of these

hormones (Ljung et al. 2002). GH3 genes have been iso-

lated from numerous plant species with 20 members in

Arabidopsis and 12 in rice (Jain et al. 2006). The SDG71s

fragment encodes a protein with homology to Arabidopsis

group II GH3.3, AtGH3-5 and Oryza sativa, OsGH3-8.

Several members of this group II GH3 family are IAA-

amido synthases that have been shown to conjugate amino

acids to IAA (Staswick et al. 2005; Park et al. 2007).

Plant growth and developmental processes, including

cell elongation, cell division, gravitropism and apical

dominance are thought to be associated with the amount of

free auxin (IAA) available in various tissues (Hagen and

Guilfoyle 2002). The function of GH3 genes, mainly from

Arabidopsis and rice have been investigated, however, the

physiological roles of GH3 genes remains largely a mys-

tery with altered expression in closely related members

resulting in a complex variation in phenotypes (Terolet al.

2006; Khan and Stone 2007). Mutant analysis has indicated

roles in light signalling, responses to abiotic stress

including drought and cold, and increased tolerance to

bacterial pathogens (Takase et al. 2003; Park et al. 2007;

Zhang et al. 2007).

Interestingly, Ding et al. (2008) have reported that the

rice GH3-8 protein may be associated with reduced cell

wall loosening. Over-expression of the rice OsGH3-8

results in dwarf plants with altered leaf morphology and

reduced fertility (Ding et al. 2008). The OsGH3-8 over-

expressing plants also show enhanced disease resistance to

the rice pathogen Xanthomonas oryzae. A reduction in the

ability of the pathogenic bacteria to invade the plant cell

may result from the suppression of growth-related wall-

loosening expansin proteins. Over-expression of OsGH3-8

suppresses auxin signalling which is required to induce the

rice expansin genes (Ding et al. 2008).


123

In Arabidopsis some members of the group II GH3

family, such as GH3.5 can conjugate both IAA and SA

(Zhang et al. 2007). Most of the IAA and SA in plants are

in conjugated forms with very little accumulation of free

IAA and SA (Ljunget al. 2002; Zhang et al. 2007). Inter-

estingly, over-expression of GH3.5 in transgenic Arabid-

opsis does not substantially alter the level of free IAA or

IAA conjugates although repressors of auxin signalling

(SAUR and Aux/IAA genes) were down regulated in these

plants (Zhang et al. 2007). De-repression of the signalling

pathway may account for the dwarfed, auxin-resistant

GH3.5 over-expression phenotype. Pathogen infection in

Arabidopsis up-regulates GH3.5. Zhang et al. (2007) have

shown that up-regulation of GH3.5 leads to the activation

of genes involved in breakdown of starch, lipids and

nucleotides and in the transport of oligopeptides, amino

acids and sugars which they propose may be associated

with pathogen nutrient acquisition. Alternatively, Park

et al. (2007) have suggested that reallocation of metabolic

resources is required to establish resistance and that the

GH3 mediated growth suppression represents the fitness

cost of induced resistance.

It seems unlikely that the major role of elevated GH3

transcripts in dehydrating S. stapfianus is associated with

suppression of expansin activity. Elevation of expansin

transcripts and activity has been demonstrated in

C. plantagineum during dry down (Jones and McQueen-

Mason 2004). High, expansin activity is thought to be

required to increase flexibility of the cell wall during

dehydration and rehydration in resurrection plants. Ele-

vated GH3 activity in dehydrating S. stapfianus may

function in suppressing cell elongation and division during

dehydration and potentially in mobilisation of metabolic

resources required for protection from water loss and

re-establishing metabolism following rehydration.

The transcript from SDG16s encodes a cysteine protease

inhibitor (phytocystatin). Much interest in protease inhib-

itors has focussed on their use to improve insect resistance

in crop plants (Ferry et al. 2003) or to improve stability of

heterologously expressed recombinant proteins (Rivard

et al. 2007). More recently the role of cysteine protease

inhibitor during cold stress and anaerobiosis has been

investigated (Gaddour et al. 2001; Van der Vyver et al.

2003) and a growing body of evidence suggests devel-

opmentally-regulated phytocystatins function to mediate

fine control of endogenous cysteine protease activity dur-

ing development (Rivard et al. 2007). Proteolysis in gen-

eral in cells plays a vital role in protein homeostasis as well

as controlling enzyme activity and signal transduction

components associated with plant growth and development

(Dreher and Callis 2007). Cysteine proteases in particular

function in nitrogen remobilisation during senescence and

programmed cell death (Grudkowska and Zagdanska

2004). Cysteine proteases may also be involved in degra-

dation of LHC II (Forsberg et al. 2005) and modulating

auxin physiology (Chen et al. 2007). Hence cysteine pro-

tease inhibitors in resurrection plants may participate in

protecting chloroplast components, delaying drought-

stimulated senescence program or play a role in resource

allocation. The cystatin C-terminus encoded by the

SDG16s fragment has extensive homology to a develop-

mentally regulated cystatin from alfalfa, MsCYS1, and

includes the 2nd inhibitory (p/sW) loop, which interacts

with the active site of the target protease. The develop-

mentally regulated expression pattern of this alfalfa cyst-

atin, as well as a preferential inhibitory activity against

endogenous rather than heterologous proteases, suggests

that it plays a regulatory role in the early stages of leaf and

stem growth (Rivard et al. 2007).

The SDG20s gene product, origin recognition complex

subunit 6 (Orc6), also has a role in growth and development.

During the cell cycle, DNA replication origin selection

occurs around late M/early G1phase and is mediated by the

binding of the six-subunit origin recognition complex

(ORC1-6). Recruitment of Cdc6 (cell division cycle 6) and

Cdt1 (cdc10-dependent transcript 1) follows. These proteins

then load the replicative helicase complex, Mcm2-7 onto

the origin (Schultz et al. 2009). Orc6 is required to promote

the initiation of replication. In budding yeast, Orc6 interacts

with the S-phase cyclin, Clb5 and with Cdt1 (Duncker et al.

2009). In metazoans Orc6 appears to have a critical role in

ORC DNA binding through interaction with high mobility

group protein member HMGA1a. Elevated levels of

HMGA1a are associated with highly proliferating cells

(Thomae et al. 2008). Apart from its role in initiating rep-

lication Orc6 has been shown to have roles in chromosome

segregation and cytokinesis in both human and Drosophila

cells (Duncker et al. 2009). In Arabidopsis the majority of

the AtORC genes including AtORC6 are regulated by the

E2F/DP family of transcription factors. Following sucrose

starvation to arrest cell proliferation in Arabidopsis cell

suspension cultures, AtORC6 mRNA increased 29-fold

when the cell cycle was reactivated by sucrose addition.

While Arabidopsis ORC genes are preferentially expressed

in proliferating tissues, high abundance of AtORC6 tran-

scripts in cauline and mature rosette leaves raises the

possibility of additional roles for AtORC6 (Diaz-Trivino

et al. 2005).

Transcripts encoding translation factors associated

with growth related processes are elevated

in dehydrated S. stapfianus

It is well recognized that initiation and elongation factors

play an important role in the control of the translation


123

process (Browning 2004). The report on elevated levels of

the translation initiation factor eIF1A transcript in dehy-

drating S. stapfianus was one of the earliest reports of

stress induction of a translation initiation factor in plants

(Neale et al. 2000). Since then elevated levels of a number

of initiation factors, including eIF1A has been associated

with increased tolerance to drought and salt stress in sev-

eral plant species (Rausell et al. 2003; Diedhiou et al. 2008;

Rangan et al. 2009).

The SDG15s transcript encodes the translation elonga-

tion factor eEF1A. An analysis of SDG15s transcripts in

dehydrating and rehydrating S. stapfianus tissue shows

elevated transcript levels in fully hydrated plants and at

around 60% RWC when the desiccation tolerance program

is initiated, with a smaller peak around 20% RWC (Fig. 5).

This bimodal pattern was observed in two independent

experiments where transcript levels were plotted against

the intensity of the rRNA signal. eEF1A catalyses the

binding of aminoacyl-tRNA to the A-site of the ribosome

and can also stimulate aa-tRNA synthetase activity (Ejiri

2002). Recent reports also suggest that eEF1A may have a

role in extracting tRNAs and specific proteins from the

nucleus (Khacho et al. 2008). Several reports detail accu-

mulation of eEF1A transcripts in response to various

stresses including cold and salt stress (Dunn et al. 1993; Li

and Chen 1999). Many recent studies point to a complex

multifunctional role for eEF1A during stress responses and

cell proliferation in both plants and animals. eEF1A has

been reported as having chaperone-like activity for pro-

tecting aminoacyl-tRNA synthetases and other proteins

(Lukash et al. 2004; Shin et al. 2009) as well as a role in

polyamine biosynthesis (He et al. 2008). The polyamines

spermidine and spermine are elevated in salt-tolerant rice

cultivars (Krishnamurthy and Bhagwat 1989) and have

been reported as regulating peptide chain elongation

(Giannakouros et al. 1990). The report by He et al. (2008)

provides evidence that eEF1A can form a complex with S-

adenosylmethionine synthase and the thermospermine

synthase ACL5. Mutations in ACL5 cause a stunted phe-

notype in Arabidopsis (Kakehi et al. 2008). The connection

between eEF1A and stem elongation has not been fully

elucidated but the finding adds further support to the

growing body of evidence that eEF1A is involved in a

number of growth-related cellular processes not directly

linked to a role in translation. eEF1A can undergo post-

translational modifications, such as methylation and phos-

phorylation, and can interact with various proteins,

including tubulin, calmodulin and actin. It has been sug-

gested that eEF1A may regulate the cytoskeleton by pro-

moting and stabilizing microtubule assembly in plants

potentially in response to auxin signalling or changes in

intracellular calcium levels (Moore and Cyr 2000). There is

evidence that specific eEF1A isoforms, with variation in

actin binding motifs, may be expressed in rapidly elon-

gating maize tissue (Carneiro et al. 1999). The multifunc-

tionality of eEF1A includes a role in signal transduction

Fig. 5 The transcript levels of SDG15s in S. stapfianus at various

stages of dehydration and rehydration. The top panel shows the

northern blot analysis using total RNA (20 lg lane-1) isolated from

S. stapfianus dehydrated to the RWC levels indicated or at sample

times indicated following rehydration. Plants were rehydrated by

immersing in water for 12 h. The histograms in the lower panel

represent the mean (±SE) transcript levels obtained from two

independent experiments. After quantification using the SDG15sprobe, the filters were stripped and re-probed and quantified with

a ribosomal RNA probe. The most intense signal detected was

designated 100%


123

pathways in animals. The mammalian eEF1A1 and

eEF1A2 isoforms have 98% similarity yet show specificity

in their interactions with various signalling proteins in

growth factor pathways and cytoskeleton reorganization.

These findings support a role for eEF1A in cell cycle

regulation and oncogenesis (Panasyuk et al. 2008). The

ability of eEF1A to interact with a number of different

proteins suggests several possibilities for eEF1A function

in proliferating cells. These include stimulation of trans-

lation of growth related proteins, accelerated transport of

tRNAs out of the nucleus, reorganization of the cytoskel-

eton and transport of transcription factors into the nucleus

in response to mitogenic signals (Ejiri 2002).

Conclusion

Resurrection plants are often thought of as being small

slow growing plants with the implication being that both

these features are intricately linked with the capacity to

survive desiccation. We have shown that following har-

vesting of leaf material, rehydrated S. stapfianus plants

rapidly produce new leaf material to attain their former

size. It seems likely that the evolution of an adaptive

desiccation-tolerance mechanism to enable survival in

locations with sporadic and limited rainfall would require a

mechanism to limit plant size to enable full rehydration.

However, the ability to rapidly replace any leaf material

lost during desiccation would also seem likely to be

advantageous. Studies in the resurrection plant Reaumuria

soongorica have indicated the importance of carbon

reserves to support vegetative regrowth following rehy-

dration (Xu et al. 2010). Our analysis of the transcripts that

accumulate to high levels in dried tissues of a resurrection

plant have revealed that several of these transcripts encode

gene products related to proteins that have been implicated

in positively regulating growth associated processes in

plants. This is in stark contrast to the situation reported in

desiccation-sensitive plants such as Arabidopsis where

water-deficit initiates a global decrease in expression of

genes that promote cell expansion (Bray 2004).

Northern analyses indicate that, with the exception of

SDG15s (eEF1A), the transcripts of the genes isolated here

are not expressed at detectable levels in mature leaf tissue

during dehydration or rehydration (data not shown) which

suggest that expression may be largely confined to meri-

stematic or young tissues. Further analysis of the spatial

expression patterns of these genes is required.

It also may be of fundamental importance to determine

how the transcription of the genes isolated here are acti-

vated. An increased understanding of how these genes are

regulated may help elucidate the link between translation

capacity, cell division and desiccation tolerance. For

example it would be of interest to determine if the desic-

cation-tolerance program stimulates a specific member of

the multicopy eEF1A gene in S. stapfianus. Many of the

plant genes involved in fundamental growth processes,

such as the cell cycle, cytokinesis and cell expansion have

been identified and recent advances have begun to identify

pathways controlling growth processes in response to

developmental and environmental signals (Ingram and

Waites 2006). TOR (target of rapamycin) kinase has been

proposed as a link between environmental cues and growth

processes in plants. TOR regulates growth-related pro-

cesses such as protein synthesis, cytoskeleton remodelling

and nutrient uptake in response to stress conditions and

availability of energy resources such as amino acids and

ATP (Deprost et al. 2007). One of the effects of over-

expression of TOR kinase in Arabidopsis is increased

production of ribosomal components and enhanced trans-

lation capacity which has profound effects on growth and

stress resistance. Conversely, silencing of TOR triggers

chlorophyll breakdown and accumulation of high concen-

trations of soluble sugars (Deprost et al. 2007). The activity

of TOR kinase mediates the S6 kinase signal pathway. This

leads to stimulation of ribosome biogenesis, increased

protein synthesis and cell growth (Turck et al. 2004). TOR

mRNA, which appears to be present in all plant tissues, can

be post-transcriptionally regulated to initiate premitotic

cytoplasmic growth during cell proliferation (Robaglia

et al. 2004).

While protein production is important for cell growth,

proteolysis may play a central role in attenuating stress-

induced growth arrest. In Arabidopsis the KEG (KEEP ON

GOING) gene has ubiquitylation activities and can target

ABA-responsive regulatory proteins to allow plants to

resume their growth in response to more favourable con-

ditions (Stone et al. 2006). An analysis of the activity of

these growth regulatory genes and pathways in resurrection

plants during the dehydration and rehydration phases may

be illuminating.

An understanding of the molecular mechanisms driving

plant growth is needed if we are to meet humanities food

and energy requirements by boosting biomass production

while keeping the use of water and fertilizer to a minimum.

The genes we have isolated in dehydrated S. stapfianus

appear to be involved in both proliferative growth, involv-

ing increased protein synthesis, as well as post-mitotic

growth, which mainly involves cell expansion. It would

seem unlikely that the gene products encoded by these

transcripts are active in dehydrating or desiccated tissue and

are more likely to be required during the rehydration phase.

The importance of protein synthesis in the recovery of

T. ruralis has been noted by Oliver et al. (2009).

Separating the coordinated effect of environmental sig-

nals and developmental signals on the basic growth


123

processes may be difficult but necessary if we are to

achieve general growth enhancement without adverse

effects on development and morphology. Further analysis

of the nature of the gene transcripts present in dehydrated

S. stapfianus tissue may be useful for this purpose. S. sta-

pfianus has evolved highly efficient environmental

response mechanisms to down-regulate growth during

water-loss and re-institute rapid growth when conditions

become favourable. Hence an analysis of the transcriptome

and proteome of desiccated S. stapfianus may reveal

physiologically relevant regulatory pathways and down-

stream molecular mechanisms associated with both stress

resistance and growth regulation that could be applied to

enhancing biomass production.

Before being utilized in a program to manipulate bio-

mass production, an analysis of the proteome in desiccated

tissue, along with an investigation of the post-translation

activation mechanisms of the gene products present is

needed to determine which genes analysed so far have a

primary function in growth retardation during dehydration

or are transcribed to prime rapid regrowth capacity fol-

lowing rehydration. Growth related genes active during

dehydration may be useful targets for suppression, whilst

those active during rehydration could be targeted for

overexpression.

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Increased Biomass, Seed Yield and Stress Tolerance IsConferred in Arabidopsis by a Novel Enzyme from theResurrection Grass Sporobolus stapfianus ThatGlycosylates the Strigolactone Analogue GR24Sharmin Islam1, Cara A. Griffiths1, Cecilia K. Blomstedt1, Tuan-Ngoc Le1,2, Donald F. Gaff1, John D.Hamill3, Alan D. Neale1*

1 School of Biological Sciences, Monash University, Melbourne, Victoria, Australia, 2 Biosciences Research Division, Victorian AgriBiosciences Centre,Melbourne, Victoria, Australia, 3 Department of Forest and Ecosystem Science, University of Melbourne, Creswick, Victoria, Australia

Abstract

Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted inthe identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT). Here, we examine the effects ofintroducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana. Resultsshow that Arabidopsis plants constitutively over-expressing SDG8i exhibit enhanced growth, reduced senescence,cold tolerance and a substantial improvement in protoplasmic drought tolerance. We hypothesise that expression ofSDG8i in Arabidopsis negatively affects the bioactivity of metabolite/s that mediate/s environmentally-inducedrepression of cell division and expansion, both during normal development and in response to stress. The phenotypeof transgenic plants over-expressing SDG8i suggests modulation in activities of both growth- and stress-relatedhormones. Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme actingdownstream of ABA to reduce drought-induced senescence. Analysis of the in vitro activity of the UGT recombinantprotein product demonstrates that SDG8i can glycosylate the synthetic strigolactone analogue GR24, evoking a linkwith strigolactone-related processes in vivo. The large improvements observed in survival of transgenic Arabidopsisplants under cold-, salt- and drought-stress, as well as the substantial increases in growth rate and seed yield undernon-stress conditions, indicates that overexpression of SDG8i in crop plants may provide a novel means ofincreasing plant productivity.

Citation: Islam S, Griffiths CA, Blomstedt CK, Le T-N, Gaff DF, et al. (2013) Increased Biomass, Seed Yield and Stress Tolerance Is Conferred inArabidopsis by a Novel Enzyme from the Resurrection Grass Sporobolus stapfianus That Glycosylates the Strigolactone Analogue GR24. PLoS ONE8(11): e80035. doi:10.1371/journal.pone.0080035

Editor: Els JM van Damme, Ghent University, Belgium

Received June 28, 2013; Accepted September 27, 2013; Published November 5, 2013

Copyright: © 2013 Islam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Sharmin Islam was supported by a Monash University Graduate Research Scholarship and Cara Griffiths by a Monash University Faculty ofScience Dean’s Postgraduate Research Scholarship; URL;www.monash.edu. The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Competing interests: Monash University has lodged an Australian Provisional Patent Application No 2012904356, entitled "Method for improving cropproductivity", associated with this work. This does not alter our adherence to all PLOS ONE policies on sharing data and materials.

* E-mail: [email protected]

Introduction

The desiccation tolerant grass Sporobolus stapfianus growsin shallow, nutrient poor soils in regions experiencing intenseseasonal drought. For their persistence these plants rely on theability of the protoplasm of their vegetative tissue to desiccate(loss of ≥ 95% total water content) and rehydrate rapidly. Therehydrated plant restores normal metabolism within 24 hours[1], grows very quickly following rain, and has proven useful forpinpointing genes for increased stress-tolerance [2,3] andenhanced growth rate [4]. Characterization of Sporobolusdrought genes (SDGs) that are specifically expressed in

desiccation-tolerant tissue [5], has the potential to revealmechanisms for coping with stress which are peculiar to, orenhanced in, resurrection plants. Such coping mechanismsinclude the ability to adjust growth rapidly in response tochanges in water availability, to inhibit dehydration-inducedsenescence programs, to protect cellular components duringdehydration and to reinstitute photosynthetic capacity quicklyfollowing a severe dehydration event [1]. The mechanismsrequired for S. stapfianus to exhibit these characteristics mayrely on coordinately regulated plant hormone activity linked toenvironmental cues.

PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80035

http://www.monash.edu

The Sporobolus SDG8i gene encodes a Group 1 UDP-glycosyltransferase (UGT) whose transcript levels increasesubstantially under severe water deficit [5]. Plant genomestypically encode a large number of UGTs that collectively canconjugate sugars to a range of acceptor molecules includingmany plant hormones, secondary metabolites and xenobiotics[6]. UGTs have an important role in cellular metabolism sinceglycosylation can affect the solubility, transport and biologicalactivity of these compounds [7]. Hence glycosylation cancontrol the bioactivity of plant growth regulators crucial toenabling adaption of plants to changing environments [8]. Themajority of the classical hormones occur as glycosides inplanta and UGTs capable of glycosylating auxins, cytokinin,ABA, salicylic acid, jasmonic acid and brassinosteroids or theirsynthetic precursors have been identified [9-15]. The possibilitythat glycosylation of one or more growth regulators may play arole in promoting onset of desiccation tolerance in S. stapfianuswas suggested by the study of Le et al. [5],, but as yet noexperimental evidence for such a role has been reported.

As no protocol for transformation of resurrection grassesexists, functional analysis of the dehydration-induced UGTSDG8i was undertaken in Arabidopsis. Ectopic expression ofSDG8i in Arabidopsis was found to have a profound effect onplant architecture and growth and confer a substantialimprovement in protoplasmic drought tolerance. Here we reportthat SDG8i encodes a functional UGT that can glycosylate thesynthetic strigolactone analogue GR24, and that ectopicexpression of this UGT leads to a substantial enhancement ofplant growth and stress resistance.

Materials and Methods

Plant materials and growth conditionsArabidopsis thaliana (L.) Heynh, Sporobolus stapfianus

Gandoger and Sorghum bicolor L. seed were obtained fromlaboratory stocks. Wild-type (WT) plants refer to Arabidopsisaccession Columbia-0 (Col-0). Orobanche seeds wereobtained from the South Australian Department of Water, Landand Biodiversity Conservation. Arabidopsis plants werestratified at 4°C for 3 days and grown at 22°C under continuouslight unless stated otherwise. Under long day (LD) photoperiodconditions the plants were subjected to a 16 hour light and 8hour dark cycle. Under a short day (SD) photoperiod, the cycleconsisted of 8 hours light and 16 hours dark. Soil grown plantswere placed in a growth cabinet at 22°C, 25% relative humidityand approximately 200 µmole/m2/sec light intensity. For axenicculture, seeds were surface-sterilized in 70% (v/v) ethanol andrinsed with sterile water and cultured at 22°C withapproximately 100 µmole/m2/sec light intensity. Crossing ofArabidopsis plants was performed as described in Weigel andGlazebrook [16].

Generation of transgenic plantsThe SDG8i coding sequence (EMBL/GenBank accession

number AM268210) was amplified and inserted into the donorvector pDONR221 using the Gateway cloning system(Invitrogen) following the manufacturer’s instructions.

5′attB1 Primer;GGGGACAAGTTTGTACAAAAAAGCAGGCTATGACGAAG

ACCGTGGTTCTG3′attB2 Primer;GGGGACCACTTTGTACAAGAAAGGTGGGTCTCACGGAC

GACCGACAGCCTCCA

The pDONR221:SDG8i construct was transferred to theexpression vector pMDC32 containing the 2 × 35S promoter[17] and transformed into Arabidopsis Columbia-0 (Col-0) usingAgrobacterium tumerfaciens (AGL-1strain) by the floral dipmethod [18]. Second generation (T2) transgenic plantshomozygous for SDG8i were generated under hygromycinresistance.

Recombinant UGT productionThe UGT was produced by transient transformation of

Nicotiana bethamiana leaves using a viral MagnICON vectorsystem (Icon Genetics GmbH, Germany). The SDG8isequence was amplified by RT-PCR, using RNA from S.stapfianus, with the primers:

Forward:5′GAGAGAATTCATGACGAAGACCGTGGTTCTGTAC3 ′

Reverse: 5′GAGAGGATCCTCACGGACGACCGAC3 ′.The PCR product was ligated into pICH11599 to generate

pICH11599-SDG8i. The three pro-vector fragments: (i)pICH12190 (containing a chloroplast signal sequence), (ii)pICH11599-SDG8i and (iii) pICH14011 (the integrase) wereseparately electroporated into Agrobacterium tumerfaciensGV3101. For the vector-only control pICH11599 was used with(i) and (iii). Equal amounts of the three A. tumerfaciens strainswere infiltrated into aluminum foil-covered N. benthamianaleaves using the protocol described by Marillonet et al. [19].After 5 days co-incubation, the leaf tissue was ground in liquidnitrogen and mixed with 500 µl cold protein extraction buffer(5mM sodium phosphate buffer pH 7.5, 10mM EDTA, 0.1%(v/v) Triton X-100). The extract was centrifuged (13,500 g) for10 minutes at 4°C and the protein content was determined byBradford assay and extracts analysed by SDS-PAGE [20].

Observation of palisade cells in fully expanded leavesThe second fully expanded rosette leaves from transgenic

and control seedlings were immersed in 0.1% (v/v) TritonX-100, then centrifuged (10,000g) for 1 min to remove air fromintercellular spaces and imaged using a light microscope. Theleaf area was measured with Adobe Photoshop CS5.1 usingthe formula [(No. of pixels of leaves/No. of pixels of physicalunit) × Area of physical unit]. Twenty sub-epidermal palisadecells aligned along the proximo–distal and medio–lateral axeswere used to calculate the average cell area using ImageJsoftware. The total cell number per leaf was calculated bydividing the leaf area by the palisade cell area for each leaf.

Examination of root architecturePlants were grown under SD (8h day/16h night) for 13 days

in vertically-orientated petri dishes on MS media (pH 5.8) with1% (w/v) sucrose and 0.8% (w/v) agar. Roots were then fixedin 4% (v/v) formaldehyde in 0.025 M phosphate buffer (pH 7.2)

Ectopic Expression of SDG8i in Arabidopsis


overnight at 4°C. The fixative was replaced with 30% (v/v)glycerol containing 2% (v/v) DMSO and left for 30 min at roomtemperature. Roots were mounted in a clearing solution (4.2 MNaI, 8 mM Na2S2O3, 2% (v/v) DMSO in 65% (v/v) glycerol) androot primordia and root cell-length were examined 1 h after thesample preparation. The number of primordia was determinedwithin the lateral-root-formation zone between the most-distalinitiated primordium and the most-distal emerged lateral root[21]. Lateral root primordium density (d) was calculated foreach individual primary root as number of primordia per mm.ILRI, (the number of lateral root primordia initiated within aportion of the root that corresponds to the length (l, mm) of 100fully elongated cortical cells in a single file in the same parentroot) was determined as 100dl, where l is the average corticalcell length in mm for each individual primary root.

Histochemical analysis of ß-glucuronidase (GUS)activity

Seedlings were GUS stained at 37°C for 4 h according to theprotocol of Jefferson et al. [22] and then cleared with 70% (v/v)ethanol. Images were taken using a microscope (LeicaMEFLIII).

Salt stress assaysSeedlings, grown for six days on vertically-orientated

germination media plates (½ MS, 0.5% (w/v) sucrose, pH 5.7,0.8% (w/v) agar), were transferred to fresh media containingsalt (0, 50, 100, 125, 150 and 175 mM NaCl) and the positionof root tips noted. The plates were inverted to allow roots togrow downward in the shape of a hook. Root elongation wasmeasured after 7 days on salt media.

Freezing stress assaySeedlings (25/plate) were grown for 3 weeks at 22°C on MS-

agar (½ MS, 0.5% (w/v) sucrose, pH 5.7, 0.8% (w/v) agar)before acclimation at -1°C for 16 hours. The temperature waslowered by 2°C per hour. Seedlings were held at the desiredtemperature for one hour before a plate containing asubsample was removed, and then the temperature waslowered further. Following the one hour exposure to -4°C, -8°Cand -12°C, subsampled plants were transferred to 4°Covernight for recovery then returned to 22°C and survivalscored 7 days later. Plants that were bleached were scored asdead, while green plants were scored as having survived thefreezing test.

Water withholding testPlants in separate pots were kept fully and uniformly watered

by sub-irrigation under optimal growth conditions in LD at 22°Cuntil the 6-7 leaf pre-flowering stage. Water was then withheld,and the plants were observed over a period of 3 weeks.Subsamples of drought treated plants were re-watered atregular intervals and recovery was monitored after 24 hours.Five plants from each line were tested at each sample pointand the experiment was performed twice.

Water vapor equilibrationProtoplasmic drought tolerance (PDT), the water potential at

which 50% of plant tissue survives in equilibrium with the air,was assessed using CaCl2 solutions. This permits waterpotentials (ψw), down to 30% relative humidity (RH) inequilibrium with a saturated solution, to be imposed on theplants via the air phase [23]. The RH in equilibrium with theCaCl2 solutions is calculated from freezing point depressiondata [24]. Chambers containing CaCl2 solutions ranging from98% (-2.8 MPa) to 86% (-20.5 MPa) RH at 25°C were used.Pre-flowering plants were soaked in water until turgid and thenblotted dry. Shoots were detached and the initial turgid weight(TWto) recorded. Shoots were enclosed in insulated chambersfor 3 days until equilibration to differing RH was reached (ψleaf =ψsolution). The shoots were then rehydrated for 24 hours and thefinal turgid weight (TWtF) recorded. Survival of plants wasdetermined by: Method A, the number of leaves alive per plant,judged subjectively by the recovery of the healthy colorationand crisp texture (dead leaves are readily distinguished in thethin fibre-poor tissues of Arabidopsis); Method B, an objectivetest, depends on the loss of semipermeability in dead cellswhich prevents osmotic absorption of water, resulting in anapparent loss in the “turgid” weight per dry weight of deadtissue compared with that of live tissue before vapor-equilibration. The ratio TWtF/TWto was plotted against % RHand the PDT determined as the % RH at which 50% of thetissue is alive. Four shoots from each line were tested for eachsample point and the experiment was performed twice.

Senescence testsFor dark-induced senescence, excised leaves from 12 day

old soil-grown Arabidopsis plants were floated, adaxial side up,on deionized water. For ABA treatment, detached leaves werefloated on 3 mM MES buffer (pH 5.8) solution under continuouslight in the presence or absence of 50 µM abscisic acid.

Chlorophyll analysisLeaf tissue (20 mg) was ground under liquid nitrogen and

extracted twice with 1.5 ml ice-cold 80% (v/v) acetone,centrifuged (14,000 rpm) at 4°C for 3 min and supernatantsstored in the dark. The chlorophyll concentration wascalculated as described by Lichtenthaler [25] . The Fv/Fm ratiowas measured after dark adaptation of the leaves for 15 minusing a PAM-210 (Teaching-PAM) (Heinz Walz GmbH,Germany).

UGT enzyme assayRecombinant UGT activity was measured using the coupled

enzyme assay described by Jackson et al., [9]. The assay wasconducted at pH 7.4 with a final substrate concentration of 1mM using 25 µg of protein extract. GR24 was obtained fromChiralix B.V. Nijmegen, The Netherlands. All other hormoneswere obtained from Sigma St. Louis, MO. Activity in millikatalskg-1 was calculated using the extinction coefficient 6.22 x 10-3

M-1 cm-1 for NADH. Background activity of extracts, monitoredby measuring the rate without substrate addition, wassubtracted from the reaction rate.



Orobanche germination assayOrobanche (Phelipanche ramosa (L.) Pomel) seeds were

surface-sterilized with 2% sodium hypochlorite containing0.02% (v/v) Tween-20, rinsed with sterile ddH2O and dried in alaminar flow cabinet. Seed (~100) on a sterile glass microfiberdisk were placed on ddH2O soaked filter paper in Petri dishesand preconditioned by adding 1ml of GA3 (30 mg/L) andincubation at 20°C in the dark for 7 days. Surface-sterilizedseeds of host plants were added to the microfiber disks andplaced on ¼ MS media with 0.5% (w/v) sugar and 0.8% (w/v)agar. For controls, 0.6 ml of GR24 (0.0001 mg/L) or ddH2O wasapplied to the preconditioned Orobanche seeds. The plateswere covered in foil and incubated at 20°C for 7 days.Orobanche germination was determined by counting the seedswith an emerged radicle.

Effect of GR24 on shoot branchingTo determine the effect of GR24 on shoot branching,

Arabidopsis plants were grown in soil for 23 days to pre-boltingstage. The plants were then treated with 50 µl per plant of 5 µMGR24 applied to the shoot meristem and axillary meristemregion. Control plants were treated with 50 µl of water. Thetreatment was repeated every third day for 20 days when thenumber of rosette branches (>5 mm) was counted.

Statistical AnalysesAll data were examined by analyses of variance using

GraphPad Prism software version 5.0. Tukey’s MultipleComparison Test was used for comparison between means ofwild-type and SDG8i transgenic plants at 5% level ofsignificance.

Results

Ectopic expression of SDG8i affects the shootarchitecture and the growth rate of Arabidopsis

Several Arabidopsis lines (F1aD, D1E, F6bA, D5aA, D2E,D4I, D7c) designed to express the SDG8i protein under thecontrol of the 35S promoter (SDG8i transgenic plants) weretaken to homozygosity and the presence of the SDG8itranscript was confirmed (Fig. S1). The developmentalphenotype of these independent SDG8i transgenic lines wasexamined under long day (LD: 16h day/8h night) and short day(SD: 8h day/16h night) photoperiods (Figure 1). Flowermorphology, seed development and seed weight were normal(Fig. S2). Transgenic plants were not affected in flowering timein LD, with production of a similar number of primary rosetteleaves as formed by the wild-type Col-0 controls. In SD, bothwild-type Col-0 and transgenic plants produced the firstinflorescence 52 days after germination, with wild-type plantsproducing around 30-34 primary rosette leaves compared withapproximately 40-42 primary rosette leaves produced by thetransgenics.

When compared to wild-type plants the transgenic lines weretaller, and had increased branching, shoot biomass and seedyield (p<0.05) (Figure 1 and Fig. S2 and S3). These phenotypicdifferences were more pronounced in SD. One month after

flowering the total number of leaves, including primary rosetteleaves and leaves formed in the rosette from axillary meristemsby transgenic plants was 1.3 to 1.6 times more than wild-typeplants (p<0.01) (Fig. S3A). The average size of the leaves ofthe transgenic plants was significantly larger than that of wild-type plants (p<0.05) (Fig. S3B) and the number ofinflorescences produced was 1.4 to 1.6 times that of wild-typeCol-0 (p<0.01) (Fig. S3C). Despite the increased branching, theheight increases exhibited by SDG8i plants over wild-typeranged from 9% to 16% after 21 days of bolting (Fig. S3D). Therate of growth of the primary inflorescence of transgenic plantswas approximately 20% greater (7.1 cm week-1) than that ofwild-type plants (5.4 cm week-1). After 50 days in SD

Figure 1. A phenotypic comparison of SDG8i transgenic(T) and wild-type Col-0 (WT) plants. Plants were grown at21°C under (A) long day and (B) short day. The figure showsthe increased height, branching, shoot biomass (FW) and seedyield typical of all the SDG8i transgenic lines. Dry seed wascollected following senescence. The biomass was measured16 days (LD) and 50 days (SD) after germination and beforebolting. Values are the means ± SE of 5 replicates. Thetransgenic plant grown in short days has been tied with cottonthread.doi: 10.1371/journal.pone.0080035.g001



photoperiods the average fresh weight (FW) shoot biomass ofall transgenic plants was almost twice that of wild-type (Figure1) and the seed yield of SDG8i transgenic plants was 1.4-1.6times the mass of seed from wild-type plants (p<0.001) (Figure1).

The leaves of SDG8i plants have increased cell size andnumber

The rosette leaves of SDG8i plants are larger than wild-typeCol-0 plants in both LD and SD. When the leaf cells of SD soil-grown plants were examined, it was found that the leaves ofSDG8i transgenic plants contained more cells than comparableleaves from control plants and the cells were larger (p<0.01)(Table 1 and Fig. S4). The increase in the number and size ofleaf palisade cells of transgenic plants indicates that SDG8iactivity promotes both leaf cell expansion and cell division andmay affect the number of meristematic cells allocated to theleaf primordium.

SDG8i expression increases root cell length and lateralroot initiation

When grown in both LD and SD the average fresh weight ofthe root biomass of all SDG8i transgenic plants was almosttwice that of wild-type (Figure 2A, B). A comparison of the rootarchitecture in SDG8i transgenics and wild-type plants grown invitro in SD was conducted using the method described inDubrovsky et al., [21]. The primary root of all SDG8i plants waslonger than wild-type under SD (p<0.001) (Figure 2C). The fullyelongated cortical cells in all the transgenic plants were longerthan wild-type (p<0.05) (Figure 2D). Calculation of the lateralroot primordium density also showed a difference betweenSDG8i transgenic plants and wild-type plants (p<0.005) (Figure2E). The higher estimation of ILRI (root primordia/100 corticalcells) in transgenic plants indicates increased root branching(Figure 2F). These findings demonstrate that SDG8i activitypromotes both primary root growth and lateral root initiation.

Overexpression of SDG8i affects auxin homeostasisThe spatial distribution of auxin controls many aspects of

plant growth and development [26]. The synthetic auxinresponse reporter construct DR5-GUS, has been used in manystudies to examine the patterns of auxin distribution [27]. Toexamine the effect of SDG8i activity on auxin levels and

Table 1. Leaf area, leaf cell size and leaf cell number inwild type (WT) and SDG8i plants.

Plant Type Av. Leaf Area Av. Cell Area Av. Cell (mm2) (µm2) Number/leafWT 47.55 ± 1.98 3160 ± 30 15047 ± 586D5aA 63.92 ± 0.84 3540 ± 159 18129 ± 845D4I 62.0 ± 1.4 3575 ± 113 17392 ± 831F6bA 59.97 ± 3.13 3592 ± 173 16858 ± 885

The leaf area and cell area were calculated in 25-day-old soil-grown plants in SDusing imaging software. Values are the means ± SE of 3 replicates.doi: 10.1371/journal.pone.0080035.t001

distribution, the SDG8i transgenics were crossed with plantscontaining the DR5-GUS reporter construct [28] and theprogeny analyzed for histochemical GUS activity [22]. WhileGUS activity appeared slightly elevated during early growthstages in SDG8i transgenics when compared with controls, thedifference in staining was quite pronounced at the four leafstage with transgenics showing much more GUS activity in theroot tips and the vascular tissue of shoots and high level GUS

Figure 2. Root growth in SDG8i lines and wild-type Col-0plants. Root biomass (FW) of pre-flowering plants growing in(A) long day at 21°C, measured 16 days after germination and(B) short day at 21°C, measured 50 days after germination.Values are the means ± SE of 4 replicates.Root development in 13-d-old Arabidopsis plants grown onvertical plates at 21°C under SD showing; (C) length of primaryroot and (D) length of fully elongated cortical cells and (E)lateral root primordium density and (F) lateral root initiationindex. Values are the means ± SE of 4 replicates.doi: 10.1371/journal.pone.0080035.g002



activity at the leaf margins (Fig. S5). These results suggest thatendogenous auxin levels are elevated in SDG8i plants.

SDG8i plants exhibit salt and freezing toleranceA comparison of the response of SDG8i transgenics and

wild-type plants to growth on high salt media (Figure 3A)showed no substantial difference at salt concentrations below100 mM after 7 days growth. However, at 150 mM NaCl alltransgenic lines showed 2-3 times more primary root growththan that of wild-type plants (p<0.001). At 175 mM NaCl wild-type plants showed severe inhibition of root growth (Figure 3Aand Fig. S6A) whereas the root growth of transgenic lines was5-7 times that of wild-type plants (p<0.001).

Figure 3. Salt and freezing stress tolerance of SDG8ilines. (A) Effect of salt on the root growth of SDG8i lines andwild-type Col-0. Root growth was measured after 7 daysfollowing transfer to NaCl. Values are the means ± SE of 10replicates.(B) Freezing tolerance of SDG8i lines and wild-type Col-0.Plants were exposed to freezing at -12°C after cold-acclimationat -1°C for 16 hours. Survival was scored 7 days after theplants were returned to normal growth conditions at 22°C.Values are the means ± SE of 3 replicates.doi: 10.1371/journal.pone.0080035.g003

A freezing tolerance test was also performed on thetransgenic and wild-type control plants (Figure 3B and Fig.S6B). All transgenic and control wild-type Col-0 plants exposedto -4°C freezing stress survived. At -8°C, all SDG8i transgeniclines survived, whereas the survival rate of wild-type Col-0plants was 83% (p<0.01). At -12°C, control wild-type Col-0plants were affected greatly with a survival rate of only 34%,whereas the survival rate of the transgenic plants ranged from69-72% (p<0.01).

SDG8i transgenics are drought tolerantThe response of SDG8i transgenics to water-deficit was

compared to that of wild-type plants. Plants were kept fullywatered by sub-irrigation until the 6-7 leaf pre-flowering stage.Water was then withheld over a period of 3 weeks, then theplants were re-watered and recovery monitored after 24 hours.In the wild-type Col-0 plants, wilting was visible after 7 daysand after 13 days of drought all control plants were severelydehydrated and showed signs of senescence, whereas allSDG8i transgenic plants were still green and healthy (Figure4A, B). The survival rate of wild-type plants was 50% after 11days (p<0.001) and by the thirteenth day had dropped to zero.On the other hand, the survival rate of transgenic plants was100% after 13 days and then reduced to 50% after 17 days. Allthe transgenic plants were severely dehydrated on day 18 andthey did not recover after one further day of drought treatment.

To quantify the increase in drought tolerance conferred bySDG8i expression, a measure of protoplasmic droughttolerance (PDT; determined as the percent lowest relativehumidity at which 50% of leaf cells survive) was obtained. ThePDT of wild-type plants, as determined by both the subjectivemethod (Figure 4C) and the objective method (Figure 4D) was~97-98% RH (~-2.8 to -4.2MPa). This indicates thatArabidopsis is very sensitive to water deficit, which is notsurprising in the thin tender leaves on well-watered greenhouseplants of an ephemeral species. The three transgenic linesgave closely similar data to each other in both methods. TheirPDT value of ~94-95% RH (~-7.0 to -8.4MPa) is considerablybetter than the value obtained for wild-type plants, indicating asubstantial improvement of PDT (p<0.05).

SDG8i activity delays dark-induced senescence andacts downstream of ABA

To test the effect of SDG8i activity on senescence, detachedleaves from wild-type and transgenic plants were floated onddH2O in the dark and chlorophyll degradation monitored overseveral days.

After five days in darkness the chlorophyll content and thephotochemical efficiency (Fv/Fm) values of wild-type plantsdecreased to around 20% (p<0.001) (Figure 5A, B) and theleaves turned completely yellow. In the SDG8i transgenic linesthe leaves remained green after five days, retaining about55-60% of their chlorophyll, with Fv/Fm values of about 52-53%of the original reading (p<0.05) (Figure 5A, B). These resultsindicate that SDG8i transgenic plants exhibit reduced dark-induced senescence.

To test the relationship between SDG8i activity and thesenescence-promoting phytohormone ABA, we repeated the



Figure 4. Drought tolerance of SDG8i plants. (A) Plantswere deprived of water, re-watered at intervals and monitoredfor recovery as described in the text. Values are the means ±SE of 2 replicates.(B) Wild-type Col-0 (WT) and a transgenic plant (T) after 13days without water.(C) Leaf survival and (D) PDT survival curve [% of finalsaturation weight to the initial turgid weight ratio] showing theeffect of decreasing relative humidity on cell survival of SDG8iand WT shoots. PDT value is the water potential (expressed asthe corresponding equilibrium relative humidity at 20°C) atwhich 50% of cells are dead after 3 days vapor equilibrationover CaCl2 osmotica. Line D represents the lowest value ofTWtf/TWt0 reached by comparable shoots killed by chloroformvapor. Line H is the 50% survival point halfway between line Dand 100% TWtf /TWt0.doi: 10.1371/journal.pone.0080035.g004

assay under continuous light with, and without, the exogenoushormone (Figure 5C, D). Over the five days incubation incontinuous light without ABA, the chlorophyll content of controlwild-type leaves reduced to 54%, whereas SDG8i transgenicleaves retained around 71% of their chlorophyll content. TheFv/Fm values for both transgenic and control plants were notsignificantly different under these conditions. Upon treatmentwith ABA, the chlorophyll content of wild-type leaves decreasedrapidly from day two down to 20% by day five (p<0.001) (Figure5C). On the other hand, the SDG8i transgenic leaves retainedabout 75% of their chlorophyll up to day four and then reducedto around 58% by day five (p<0.05) (Figure 5C). The Fv/Fm

value of SDG8i transgenics was also substantially higher thancontrol plants on day five (p<0.005) (Figure 5D). These resultsshow that SDG8i expression reduces ABA-inducedsenescence and suggests that it functions downstream of ABA.

SDG8i encodes a UGT that glycosylates GR24 in vitroThe phenotype of the transgenic plants suggests that SDG8i

UGT activity is influencing hormone homeostasis. Toinvestigate the catalytic function of SDG8i, the UGT activity ofNicotiana benthamiana leaf extracts infiltrated with an actin(AtACT2)-promoter driven SDG8i construct, using a viral-basedsystem [19], was tested against a number of plant hormones assubstrates and compared with UGT activity in extractsinfiltrated with a vector-only control. The substrates used werechosen for their known ability to affect plant growth and stressresponses. The SDG8i extract showed substantialglycosylation activity of the strigolactone analogue GR24(Figure 6A) with a Km of 0.349 mM and a Vmax of 5.67μmole/min/mg (Figure 6B). The activity observed with the othersubstrates showed very little increase over backgroundendogenous NADH oxidase activity. Similarly, the vector-onlycontrol extract showed no substantial activity abovebackground with GR24 or with any of the other substrates. Theresults indicate that SDG8i encodes a glucosyltransferase within vitro activity against a strigolactone-like compound.

The stimulation of Orobanche germination is reducedin Arabidopsis expressing SDG8i

Root exudates from Arabidopsis can stimulate thegermination of Phelipanche ramosa (Orobanchaceae) [29]. TheOrobanchaceae germination bioassay has been usedextensively to determine the level of strigolactone secretedfrom host plant roots [30]. To determine if SDG8i activity hasan effect on the root secretion of these stimulus signals that arerequired by parasitic plants to germinate, four SDG8itransgenic Arabidopsis lines were compared with wild-typeCol-0 Arabidopsis plants for the ability to stimulate Orobanchegermination in axenic culture (Figure 6C and Fig. S7). GR24,Sorghum bicolor and S. stapfianus were included as controls.Germination of 95% was achieved by treatment with GR24,while 11% germination was observed in the water control. Thelevels of Orobanche seed germination stimulated by sorghumand S. stapfianus was 71% and 52% respectively. The 60%germination of Orobanche seeds by wild-type Col-0 plants wassignificantly higher than that of the all the transgenic linestested (p<0.001) with SDG8i transgenic lines stimulating



42-47% germination. The transgenic line, F6bA, showed thehighest reduction in Orobanche germination (30%) comparedwith the wild-type Col-0 Arabidopsis control. These resultssuggest that SDG8i activity may be reducing the level ofstrigolactone germination stimulants secreted from the roots ofArabidopsis.

GR24 inhibition of shoot branching is reduced in SDG8itransgenic plants

The branching response of wild-type Col-0, SDG8itransgenic and the branching mutant max2 Arabidopsis plantswere tested by the application of GR24 to leaf axils and axillarybuds before and during bolting as described by Gomez-Roldanet al. [31] in order to see whether they are able to respond toSL-mediated shoot branching inhibition (Figure 6D). In wild-type Col-0 plants, shoot branching decreased by 55% inresponse to GR24 compared to control plants (p<0.001).However, in all SDG8i transgenic plants and max2 mutants,application of GR24 decreased shoot branching by only10-15% compared to untreated plants (p<0.001) with branching

remaining elevated over treated or untreated wild-type Col-0plants.

Discussion

The phenotypic changes mediated by overexpressionof SDG8i suggests the enzyme may affectstrigolactone-related processes

Several UGTs, capable of influencing the biological activity ofplant hormones via glycosylation, have been isolated [9-15].The ability of SDG8i recombinant protein to glycosylate thesynthetic strigolactone analogue GR24 in vitro, combined withthe phenotypic changes observed in SDG8i overexpressinglines, suggests that SDG8i glucosyltransferase activity may beeffecting the strigolactone signalling pathway in the transgenicplants. As well as mediating the interactions of host plants withsymbiotic fungi or parasitic plants, the well-conservedstrigolactone hormone signaling system contributes toenvironmental regulation of plant growth. Strigolactones havebeen shown to respond to environmental cues, such as low

Figure 5. Dark- and ABA-induced senescence in wild-type and SDG8i leaves. (A) Chlorophyll content and (B) photochemicalefficiency in dark-treated detached leaves of WT and SDG8i plants.(C) Chlorophyll content and (D) photochemical efficiency in detached leaves of WT and SDG8i plants under continuous light in theabsence or presence of 50 µM ABA.doi: 10.1371/journal.pone.0080035.g005



nutrient conditions (Pi, N), to interact with abscisic acid (ABA),and to be involved in growth-related auxin and ethylene activity[32-35]. Strigolactones are carotenoid-derived terpenoidlactones that are synthesized mainly in the roots and can betransported in the xylem sap to the shoots [29]. In some plantspecies, mutations in strigolactone production or perceptiongenes have been shown to delay flowering time, reducesenescence and decrease root mass [36,37]. Root exudatescontaining strigolactones can stimulate parasitic Striga andOrobanche plant germination and hyphal branching ofsymbiotic arbuscular mycorrhizal fungi [38]. Strigolactoneshave been shown to regulate root growth in response tophosphate and/or carbohydrate availability [39,40]. In theshoots, increased strigolactone levels inhibit tiller formation orlateral bud outgrowth [29,41] and mutations in the strigolactone

biosynthesis and signaling pathway are often associated with ahyperbranched, dwarfed phenotype [32,33].

Several characteristics of the SDG8i ectopic expression linesmimic those of the strigolactone MAX mutants, includinghyperbranching, reduced senescence and interactions withauxin and ABA [33,34,37]. However, the reduced stimulation bySDG8i plants of Orobanche germination, indicative ofdiminished strigolactone levels [30], is modest and the SDG8ienhanced shoot and root growth phenotypes are opposed tothose of SL-deficient mutants which are usually dwarfed. Whilethe moderate affinity of SDG8i for GR24 could be expectedgiven the artificial nature of the substrate and assay conditions,it is clear that the SDG8i enzyme in Arabidopsis is affecting thebioactivity of an endogenous growth-related compound/s.MAX2/ORE9, the F-box leucine-rich repeat signaling protein is

Figure 6. Analysis of the enzyme activity of SDG8i in vitro. (A) The glycosyltransferase activity of SDG8i recombinant proteinextract using the plant hormones gibberillin (GA3), salicylic acid, kinetin, auxin (IAA), methyl jasmonate, ABA([±]-cis, trans-abscisicacid and the synthetic strigolactone analogue GR24 as substrate (p < 0.05). The coupled enzyme assay [9] was conducted at pH7.4 using 25μg of protein extract with a final substrate concentration of 1mM. Rates were calculated per mg total protein extract.GR24 was obtained from Chiralix B.V. Nijmegen,The Netherlands. All other hormones were obtained from Sigma St. Louis, MO.(B) A Lineweaver-Burke plot of SDG8i activity with varying concentrations of GR24 indicating a Km of 0.349mM and a Vmax of 5.67μmole/min/mg. Rates were calculated per mg total protein extract.(C) Level of stimulation of germination of Orobanche seeds in response to root induction by wildtype Col-O and SDG8i transgenicArabidopsis, sorghum and S. stapfianus seedlings in vitro. The percent germination was calculated by counting the number of seedshaving an emerged radicle. Values are the means ± SE of 5 replicates.(D) Effect of GR24 (0 or 5 µM) on bud outgrowth of wild-type Col-0, SDG8i transgenic and max2 Arabidopsis plants. Plants weretreated with GR24 on the rosette shoot meristem, axillary buds and leaf axils every third day for 20 days and the number ofbranches was counted after 43 days. Data are means ± 4 replicates.doi: 10.1371/journal.pone.0080035.g006



expressed in the vasculature and plays multiple roles indifferent parts of the plant, not all of which are dependent onthe activity of the SL branching signal generated by MAX3 andMAX4 [42]. Several of the processes associated with MAX2activity appear to be negatively affected in SDG8i plants. Inaddition to auxin interactions and suppression of branching,MAX2 acts to promote leaf senescence [37] and represshypocotyl elongation in the light [43] and is also involved inoxidative stress and drought responses [44,45]. Hence thephenotype of SDG8i transgenic plants (which includeselongated hypocotyls) appears to be consistent with areduction in the activity of MAX2. The reduced branchinginhibition in SDG8i plants by GR24 application also supportsthis contention. This would suggest that crossing SDG8itransgenics with MAX3 or MAX4 mutants would be unlikely tolead to suppression of the mutant phenotypes, howevercrossing SDG8i transgenics with plants overexpressing MAX2may be informative in elucidating the mode of action of SDG8i.The genetic analyses with strigolactone mutants and anexamination of expression levels of MAX pathway genes areunderway, however, full comprehension of the mode of SDG8iaction requires identification and structural analysis of theendogenous target metabolite/s. Identification of themetabolite/s could substantially enhance our knowledge of howenvironmental influences regulate plant growth. Sinceglycosylation is widely accepted as having a role in bioactivity,transport and stability of many growth regulators [7], an effort toidentify an endogenous enzyme with similar SDG8i activity inArabidopsis and other plants may be worthwhile, althoughsubstrate specificity of UGTs is not necessarily reflected ingene phylogeny and in vitro substrate screening ofrecombinant UGTs is usually required to identify functionallysimilar UGTs between species [8].

SDG8i activity may impede the stress-related growthretardation response

Plant productivity, even when plants are growing under near-ideal conditions, can be limited by the innate response of plantsto seasonal influences or short-term stress events. Theseresponses often involve reduced growth and/or reallocation ofresources into less desirable growth patterns. The imposition ofstress below toxic levels can elicit apoptosis or growthredistribution within minutes to diminish stress exposure [46].The pathways integrating environmental stressors withinhibition of internal growth-related processes in plants is notwell understood [47] and the introduction of stress-relatedgenes to increase constitutive stress tolerance often correlateswith diminished growth. In contrast, SDG8i activity promotescell expansion and division in shoots and roots under bothstress and non-stress conditions. In the resurrection grassS. stapfianus, SDG8i gene activity, following transcriptaccumulation in desiccated tissue, may be associated with leafregeneration, which is more than twice as rapid following adehydration/rehydration cycle as in a well-watered plant [4].The enhanced growth exhibited by SDG8i transgenicArabidopsis plants under stress, suggests that SDG8i activitymay impede the growth retardation response which wouldnormally occur under these conditions. A reduction in the ability

of a plant to initiate morphogenic changes in response toenvironmental influences could be expected to compromise thesurvival of wild species over long-term stress events. However,substantial growth advantages may be conferred in the contextof crop cultivation with ample mineral nutrition and shorter-termstress events. The phenotype of the SDG8i transgenic plantsprovide an important example of the potential of bioengineeringto enhance shoot-root biomass and seed yield in plants, whilstsimultaneously conferring substantial improvements in salt,cold and drought resistance. The ability to utilize SDG8i activityto control stress-related and seasonal morphogenic responsesin crops could represent a substantial advance indomestication of food plants.

The SDG8i-mediated drought-tolerance improvementmay be associated with inhibition of the drought-related senescence program

Considerable research effort has been put into increasingcrop production utilizing the isopentenyltransferase (IPT) geneto increase cytokinin biosynthesis. Increased cytokinin levelscan delay drought-induced senescence and allow retention ofhigher chlorophyll levels under water-deficit [48-50]. Theproblems associated with lower yields due to altered sourcesink distribution appears to have been overcome by utilizing asenescence associated receptor kinase (SARK) promoter todrive the IPT gene [51-53]. Use of the SDG8i gene presents analternative approach to reducing drought-induced senescenceand potentially has the additional benefit of increasing yieldunder non-stress conditions. The two-fold improvement in thewater potential survived by the SDG8i transgenic lines,compared with the untransformed wild-type Arabidopsis plants,is considerable and is similar to the maximum ‘droughthardening’ reported for a crop plant droughted for 3 weeks [54].The reduced senescence displayed by transgenic SDG8iplants suggests that SDG8i activity may also be associatedwith inhibition of dehydration-induced senescence programs, aphenomenon that occurs in the younger leaves of thedesiccation-tolerant S. stapfianus plant. In non-resurrectionplants, leaf senescence is thought to be an efficient strategy forsurviving water-deficit by reducing canopy size andtranspiration [55]. Interestingly, SDG8i transgenic plantsshowed no sign of senescence after 13 days without wateringyet survived much better than wild-type plants. The droughtedwild-type plants ceased vegetative growth, the leavessenesced rapidly, and the plants produced an inflorescence of2-3 cm before dying. The transgenics on the other handremained green and healthy. Some transgenics formed one ormore extra rosette leaves and a reproductive meristem duringthe drying stage, but did not appear to accelerate reproductivedevelopment. The reduced senescence, in combination withthe larger leaves, may be related to the higher seed yield of theSDG8i transgenic plants.

The use of SDG8i to generate a more robust productive plantwith enhanced growth and stress resistance, combined with thebenefit of reduced stimulation of parasitic seed germination,could herald a novel approach for increasing food production inagriculturally important crop plants.



Supporting Information

Figure S1. RNA gel-blot analysis showing the presence ofSDG8i transcripts in SDG8i transgenic lines.(TIFF)

Figure S2. Flower morphology, seed development andgrowth characteristics, of wild-type Col-0 plants (WT) andSDG8i plants (T) growing at 21°C in LD conditions.(TIFF)

Figure S3. Growth characteristics of SDG8i plantsgrowing in short days.(TIFF)

Figure S4. Palisade cells in the second fully expandedrosette leaves of wild-type Col-0 and SDG8i transgeniclines.(TIFF)

Figure S5. Histochemical staining showing differentialGUS activity at various stages of development typical ofwild-type Col-0 plants and SDG8i transgenic (D5aA) plantscrossed with DR5::GUS Arabidopsis seedlings.

(TIFF)

Figure S6. Salt and freezing stress tests of wild-type Col-0(WT) and SDG8i transgenic (T) Arabidopsis seedlings invitro.(TIFF)

Figure S7. Stimulation of germination of Orobancheseeds.(TIFF)

Acknowledgements

We thank Dr John Matthews for assistance with Orobanche.

Author Contributions

Conceived and designed the experiments: ADN CKB JDHDFG. Performed the experiments: SI CAG TNL CKB ADN.Analyzed the data: ADN SI CAG CKB DFG. Contributedreagents/materials/analysis tools: ADN JDH. Wrote themanuscript: ADN SI CAG CKB TNL JDH.

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REVIEW ARTICLEpublished: 12 February 2014

doi: 10.3389/fpls.2014.00036

Drying without senescence in resurrection plantsCara A. Griffiths, Donald F. Gaff and Alan D. Neale*School of Biological Sciences, Monash University, Clayton, VIC, Australia

Edited by:John Moore, Stellenbosch University,South Africa

Reviewed by:John Chandler Cushman, Universityof Nevada, USAJill Margaret Farrant, University ofCape Town, South AfricaAndrew Wood, Southern IllinoisUniversity, USA

*Correspondence:Alan D. Neale, School of BiologicalSciences, Monash University,Wellington Road, Clayton, VIC 3800,Australiae-mail: [email protected]

Research into extreme drought tolerance in resurrection plants using species such asCraterostigma plantagineum, C. wilmsii, Xerophyta humilis, Tortula ruralis, and Sporobolusstapfianus has provided some insight into the desiccation tolerance mechanisms utilized bythese plants to allow them to persist under extremely adverse environmental conditions.Some of the mechanisms used to ensure cellular preservation during severe dehydrationappear to be peculiar to resurrection plants. Apart from the ability to preserve vital cellularcomponents during drying and rehydration, such mechanisms include the ability to down-regulate growth-related metabolism rapidly in response to changes in water availability,and the ability to inhibit dehydration-induced senescence programs enabling reconstitutionof photosynthetic capacity quickly following a rainfall event. Extensive research on themolecular mechanism of leaf senescence in non-resurrection plants has revealed a multi-layered regulatory network operates to control programed cell death pathways. However,very little is known about the molecular mechanisms that resurrection plants employ toavoid undergoing drought-related senescence during the desiccation process. To survivedesiccation, dehydration in the perennial resurrection grass S. stapfianus must proceedslowly over a period of 7 days or more. Leaves detached from the plant before 60%relative water content (RWC) is attained are desiccation-sensitive indicating that desiccationtolerance is conferred in vegetative tissue of S. stapfianus when the leaf RWC has declinedto 60%. Whilst some older leaves remaining attached to the plant during dehydration willsenesce, suggesting dehydration-induced senescence may be influenced by leaf age or therate of dehydration in individual leaves, the majority of leaves do not senesce. Rather theseleaves dehydrate to air-dryness and revive fully following rehydration. Hence it seems likelythat there are genes expressed in younger leaf tissues of resurrection plants that enablesuppression of drought-related senescence pathways. As very few studies have directlyaddressed this phenomenon, this review aims to discuss current literature surrounding theactivation and suppression of senescence pathways and how these pathways may differin resurrection plants.

Keywords: desiccation tolerance, senescence, drought, Sporobolus stapfianus, photosynthesis

INTRODUCTIONIn crop plants, drought-induced senescence can cause nutri-ent loss and limit the growth phase resulting in a substantialreduction in yield. A major goal of agricultural science is toincrease food production in a water-limited environment. Delay-ing drought-induced senescence to allow retention of higherchlorophyll levels has considerable potential to increase crop pro-duction under water-deficit (Gan and Amasino, 1997; Jordi et al.,2000; Sykorová et al., 2008). An understanding of the mechanismwhereby resurrection plants suppress drought-induced senescencemay enhance our knowledge of an important biological processand provide a novel means of increasing crop yields under adverseconditions.

Much resurrection plant research has focused on understand-ing how these plants survive desiccation and how desiccation-tolerance pathways are regulated. Approaches have involvedidentifying genes that are differentially regulated during dehy-dration and more recently, global analyses of the transcriptome(Oliver et al., 2004; Rodriguez et al., 2010), proteome (Oliver et al.,

2010) and metabolome (Oliver et al., 2011) in dehydrating andrehydrating tissue have been undertaken. This information, whencoupled with the detailed molecular genetic information avail-able on drought responses of non-resurrection plants, may allowa more targeted molecular approach to identifying those genesfunctioning in dehydration-related pathways which are thought tobe pivotal to the ability of resurrection plants to survive extremedehydration. Such genes could then be utilized to increase thestress-resilience of important food crops.

In many non-resurrection plants, surviving water-deficitinvolves the up-regulation of drought tolerance genes that alterthe osmotic potential of plant tissues to enable water retention(Zhang et al., 1999). When water loss becomes more severe, non-resurrection plants initiate leaf senescence, which is thought tobe an efficient strategy for surviving water-deficit by reducingcanopy size and transpiration, and allowing remobilization ofwater and nutrients to preserve organs crucial for the futuresurvival of the plant (Chaves et al., 2003; Munné-Bosch and Ale-gre, 2004). Although the perennial resurrection grass Sporobolus

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Griffiths et al. Drying without senescence

stapfianus initiates similar osmotic responses to slow down the rateof dehydration and allow the desiccation tolerance program to beestablished, the survival of S. stapfianus does not rely on the reten-tion of water but rather on the ability of the plant to withstandcomplete desiccation (Gaff and Ellis, 1974). Hence the initiationof leaf senescence, as a vital strategy to preserve water and cel-lular nutrients for the remaining plant tissues, does not occur inresurrection plants.

This leads to the question of how resurrection plants suppressthe drought-induced senescence response. It is also not clear if asimilar senescence-suppression pathway occurs during seed devel-opment. In most plants, dry mature seeds, and pollen tissues fora certain period of time, are composed of desiccation-toleranttissue capable of producing new life (Walters et al., 2005) and atleast some resurrection plants are thought to have evolved theirvegetative desiccation-tolerance pathways by ectopic expressionof desiccation-related pathways normally found in reproductivetissue (Oliver et al., 2000). This suggests that many plants may con-tain the required desiccation tolerance genes within the genome(Bartels, 2005). Many desiccation-tolerance-associated genes havehomologs in non-resurrection plants but they appear not to beactivated in vegetative tissue during dehydration.

The ability of resurrection plants to preserve healthy tissues andcompletely avoid senescence during dehydration is a key featureof desiccation tolerance. By attempting to synthesize the currentknowledge of the desiccation program in the resurrection grassS. stapfianus with that of age- and drought-related senescence innon-resurrection plants, we hope to shed some light on possi-ble mechanisms associated with dehydration-related senescenceavoidance in resurrection plants.

LEAF PROCESSES IN DEHYDRATING S. stapfianusThe desiccation of resurrection plants involves whole plant dehy-dration to an air-dried state with minimal cellular damage. Theleaves of the desiccation tolerant grass S. stapfianus can survivedrying down in equilibrium with air at 2% relative humidity,and remain in this anabiotic state for 2 years (Gaff and Ellis,1974). The leaf signal initiating the desiccation-tolerance pro-gram appears to originate from a decreased water supply inthe roots of S. stapfianus, as dehydration of plants with dis-turbed roots can lead to death of the plants (Gaff et al., 1997).Investigations into cellular structure during the dehydration ofS. stapfianus leaves have revealed that there is a higher inci-dence of closed stomata and epicuticular wax on the adaxial leafsurface. The closing of stomata reduces water loss and waterflux throughout the plant and reduces the transpiring leaf area(Tardieu, 2005). These features may aid in the deceleration ofcellular dehydration in order to protect thylakoid membranesand allow time for the induction of pathways that facilitate des-iccation tolerance in S. stapfianus (Quartacci et al., 1997). Inaddition, large bulliform cells in leaf tissue have a reduced rateof water loss, suggesting that they may act as an internal waterreservoir, again aiding in slowing the rate of tissue dehydration(Dalla Vecchia et al., 1998). It has been shown that a higherrate of photorespiratory electron transport occurs in desicca-tion sensitive leaves undergoing drought-induced senescence incomparison to desiccation-tolerant leaves. This suggests that the

protection of desiccation-tolerant leaves is not aided by the capac-ity of photorespiration to scavenge free electrons (Martinelli et al.,2007).

Sporobolus stapfianus retains around 40% of chlorophyll con-tent in dry leaves which reconstitutes quickly to around 90% onrehydration (Gaff and McGregor, 1979).

Resurrection plants have evolved strategies to reduce themechanical stress of cell wall stiffening during water loss.Arabinose-rich polymers have been implicated in maintenanceof cell wall flexibility in several resurrection plants (Moore et al.,2013). Various resurrection plants may also employ drought-inducible cell wall modifications including calcium ion deposition,xyloglucan remodeling, and elevated cell wall expansins whichcan act to increase cell wall flexibility and allow cells to con-tract and fold without collapsing (Quartacci et al., 1997; Wu et al.,2001; Jones and McQueen-Mason, 2004; Vicré et al., 2004). InS. stapfianus the increase in poly-unsaturated phospholipids whichaccumulates during dehydration is thought to increase cellularmembrane fluidity (Quartacci et al., 1997). Maintenance of cellu-lar volume during dehydration, via water replacement in vacuoleswith substances such as sugars, proline, polyphenols, and glyc-erol can also occur (Vander Willigen et al., 2004; Moore et al.,2007; Farrant et al., 2009). In some cells of resurrection plants,the controlled fragmentation of the vacuole into multiple smallervacuoles may act to facilitate mechanical stabilization (Quartacciet al., 1997; Vander Willigen et al., 2004).

The drought-induced growth retardation in S. stapfianusis thought to be rapid compared to plants that utilize well-developed drought avoidance mechanisms (Puliga et al., 1996).The accumulation of amino acids (Gaff and McGregor, 1979)and sugars, in particular sucrose (Ghasempour et al., 1998) isalso apparent during dehydration in S. stapfianus leaves. Theco-ordination of carbon partitioning between these competingpathways alters throughout dehydration. In the initial stagesof dehydration both cellular starches and photosynthesis directequal energy into both these pathways (Whittaker et al., 2007).In the later stages of dehydration, after photosynthesis ceasesand the starch stores are exhausted, carbon flux is directed tosucrose and amino acid biosynthesis. However, this increase inamino acid may also be attributable to insoluble protein break-down (Whittaker et al., 2007). Amino acid accumulation has beenassociated with the stabilization of cytoplasmic constituents, ionsequestration and water retention (reviewed in Chen and Murata,2002). The increase in sucrose is associated with a process knownas vitrification, where the formation of biological glasses inthe drying cell protects organelles from damage. The interac-tion between accumulating sugars and dehydration-induced lateembryogenesis abundant (LEA) proteins (Ingram and Bartels,1996), which are commonly associated with embryo develop-ment at the later stages of seed maturation (Dure, 1993), isthought to be important for the protection of cellular com-ponents. Recently, sucrose and glucose has been shown toaccumulate in all viable desiccation tolerant tissue (Martinelli,2008). In the rehydrating resurrection plant S. stapfianus, theinitial energy requirement for revival appears to be generatedpreferentially from catabolism of the accumulated amino acids(Whittaker et al., 2004).

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GENE EXPRESSION IN DEHYDRATING S. stapfianusChanges in leaf gene expression during dehydration in S. stapfi-anus was first examined by Gaff et al. (1997) where mRNA wasisolated from air dry, dehydrating and fully hydrated leaf tis-sue, and translated in vitro. This process revealed novel genesbeing expressed during dehydration. A comparison of in vivoprotein extracts with the in vitro translated proteins suggestedthat some dehydration-related transcripts may not be trans-lated until the rehydration stage. These products are presumablyrequired to protect against cellular damage during the rapid rehy-dration which generally occurs within 24 h following rainfall(Gaff et al., 1997) or to allow for the very rapid growth thatoccurs during the short wet periods when conditions are favorable(Blomstedt et al., 2010).

Early work on isolating genes up-regulated during the ini-tial stages of dehydration of S. stapfianus leaf tissue identified anumber of genes encoding dehydrins, group 3 LEA, glyoxalase I,thiol proteases, an eukaryotic initiation factor 1A (eIF1A) proteintranslation initiation factor, glycine- and proline-rich proteins,a tonoplast intrinsic protein, and an early light inducible protein(Blomstedt et al., 1998a,b; Neale et al., 2000; Le et al., 2007). A moreextensive list of S. stapfianus dehydration-responsive genes is avail-able in Gaff et al. (2009). Many of these genes have high levels ofhomology to genes associated with protection from dehydration inmany non-resurrection plants or are expressed in desiccated seedsof higher plants. Some of these proteins have been suggested to beinvolved in the rapid translation of proteins during dehydration,stabilization of membranes during desiccation, solute flow duringdehydration/rehydration, and reducing photosynthetic appara-tus damage (Neale et al., 2000). Interestingly these genes wereidentified as being differentially expressed in desiccation-toleranttissue and not in tissues dehydrated following excision from thehydrated plant, or in the related S. pyramidalis, both of whichare desiccation-sensitive (Gaff et al., 1997). This indicates that anelevated and/or persistent drought response occurs in desiccation-tolerant tissues. In some cases the desiccation-tolerance-relatedgenes were specific sequences from multi-gene families. The sig-nificance of this is not well understood. In severely dehydrated ordesiccated resurrection grass tissue, the profile of the transcriptpool is somewhat different compared to that of early dehydrationstages. As might be expected, several of the transcripts presentin high levels in desiccated resurrection plant tissues encodegrowth-related protein products for utilization upon rehydration(Blomstedt et al., 2010; Islam et al., 2013).

While this research has uncovered some of the key genes beingup-regulated during dehydration of S. stapfianus and has set upthe framework for understanding the molecular basis of desicca-tion tolerance, we are some distance from identifying the pivotalmolecular mechanisms responsible for the successful negotiationof a complete desiccation and rehydration cycle. By necessity oneof these mechanisms must involve suppression of the dehydration-induced cell death response which is implemented by many higherplants.

LEAF SENESCENCELeaf senescence is a complex, degenerative, developmentally reg-ulated programed cell death (PCD) process that is associated with

the final stages of leaf development. Both multiple developmentaland environmental signals (drought, detachment, and darkness)can affect this process (Lim et al., 2003). Leaf senescence is thoughtto have evolved to allow remobilization of nutrients and othermolecules to younger, actively growing areas of the plant (Nam,1997). Generally, plants will exhibit two types of age-related senes-cence, one being replicative (mitotic) senescence (associated withthe inability of the cell to continue cell division upon aging) andpost-mitotic senescence (associated with the degenerative processoccurring after cellular maturation, for example, leaf organ matu-ration; Lim et al., 2003). Stress-related senescence, most similar topost-mitotic senescence, is degenerative involving the dismantlingof cellular organelles, degradation of protein, nucleic acid, lipid,and chlorophyll, and the remobilization of nutrients and nitrogencompounds (Smart, 1994). During the senescence process, cellularrespiration is maintained and transcriptional machinery remainsactive, being required to transcribe senescence-associated genes(SAGs).

AGE-DEPENDENT SENESCENCEAge-related senescence in perennial plants occurs from the old-est to the youngest leaves (Munné-Bosch, 2008). In differentdesiccation-tolerant angiosperm plant species, the interactionbetween the developmental stage of individual leaves, drying andsenescence varies widely. In S. stapfianus, the leaves that havemore recently attained full length will survive desiccation entirely,whereas in older leaves, the tissue at the tip of the leaves dies. Asthe leaves age, more of the length of the leaf from the tip diesduring dehydration (Gaff and Giess, 1986; Gaff, 1989; Martinelliet al., 2007). Two extremes of the interaction are found inter alia inspecies of Borya (a genus formerly in the Liliaceae). In Borya scor-pioides Lindley only the basal 4 mm of the youngest three leavesrecover from desiccation, whereas 20 successive leaves on a shootof Borya mirabilis Churchill survive desiccation (Gaff and Oliver,2013).

Rehydration of young in vitro-cultured S. stapfianus seedlingsthat have been allowed to desiccate, results in the revival of theentire plant. Senescence of the oldest healthy leaves in more maturesoil-grown S. stapfianus plants (Martinelli et al., 2007) suggeststhat desiccation tolerance of leaf tissue has an age-specific com-ponent and genes associated with repression of drought-inducedsenescence are not expressed in older leaves. Regulation of replica-tive leaf senescence may be responsible for the senescence of olderleaves in S. stapfianus during desiccation. As the senescence ofolder leaves in plants has been shown to aid in the remobiliza-tion of nutrients to younger tissues (Munné-Bosch and Peñuelas,2003) it is possible that senescence of older tissues in dehydratingS. stapfianus provides resources in the younger leaves for survivalof the desiccation/rehydration cycle.

Telomere length, a suggested notification of cellular agehas been shown to control replicative senescence in mammals(reviewed by Campisi, 1997). However, the effect of changes intelomeres as a result of cell replication in plants is not well stud-ied. The length of telomeres in Arabidopsis thaliana at differentdevelopmental stages has not been shown to change; suggestingage-related senescence is not triggered by a reduction in telom-ere length in plants. However, plant telomeric DNA is involved in





a DNA–protein complex with a telomere binding protein ATBP1(A. thaliana binding protein 1) which is bound to the telomericDNA in all stages of development. During the onset of senes-cence, a protein–protein interaction between ATBP1 (bound tothe telomeric DNA) and ATBP2 (A. thaliana binding protein 2)occurs. This binding of ATBP2 during the onset of replicativesenescence suggests that it may be disturbing the DNA–ATBP1complex, thereby inducing senescence (Zentgraf et al., 2000). Theprogression of the age-dependent senescence process may alsobe facilitated by lipid degrading enzymes that can provide pre-cursors for the synthesis of the senescence-promoting hormonejasmonic acid (JA; He et al., 2002). In animals, a high metabolicrate associated with elevated oxidative stress may be a deter-mining factor in aging (Guarente, 1997). As yet there is scantevidence for this phenomenon in plants, although the delayedsenescence, which appears to be specifically age-related in theArabidopsis ore4-1 mutant, may be attributable to a partiallyimpaired chloroplast function providing a reduced metabolic rate(Woo et al., 2002).

Live air-dry leaves are easily distinguished from dead air-dryleaves (senesced) by color differences on drying S. stapfianusplants and during analyses of gene expression associated withdesiccation-tolerance the senescing tissue would normally beexcluded. A comparison of gene expression occurring in live andsenescing leaf tissue from the same dehydrating plant may beuseful for distinguishing drought-induced senescence genes fromdrought-induced protective genes and could provide evidence thatATBP2 or other as yet unidentified genes are up-regulated bydrought stress specifically to induce senescence.

SENESCENCE-ASSOCIATED GENESResearch into senescence in several species such as rice, Ara-bidopsis, tomato, and maize has identified a large number ofSAGs responsible for the execution of the senescence process (Heand Gan, 2002). Up to 15% of Arabidopsis genes may be differ-entially regulated during senescence (Buchanan-Wollaston et al.,2003; Guo et al., 2004; Zentgraf et al., 2004). The expression pat-terns of SAGs up-regulated during leaf aging and the roles SAGsplay during senescence have been reviewed in detail by Buchanan-Wollaston (2008). Basal expression of SAGs is observed acrossmany stages of leaf development, indicating that the genes playa role in non-senescent tissue, and increase in expression as theleaf ages (Weaver et al., 1998). In Brassica napus genes identifiedas being associated with senescence encode two types of protease,glutamine synthase, ATP sulfurylase, two types of metallothionein,ferritin, catalase, and an antifungal protein (Buchanan-Wollastonand Ainsworth, 1997) as well as chitinase, and PR1 (pathogenesis-related 1; Guerrero et al., 1990). The action of proteases duringsenescence has been suggested to be involved in senescence-relatedprotein degradation (Lohman et al., 1994) which is one of thefirst cellular responses to senescence induction. The remobiliza-tion of nutrients may be attributed to the action of glutaminesynthase and metallothioneins, although metallothioneins mayalso provide a protective role by scavenging free ions. Furtherprotection may be offered by catalase, which detoxifies oxygen rad-icals (Buchanan-Wollaston and Ainsworth, 1997). The roles of theantifungal and pathogenesis-related proteins during senescence

are not understood, although chitinase may have a role in cellwall disruption and cell signaling affecting hormone homeostasis(Kasprzewska, 2003). The pathogenesis-related protein, PR10, hasrecently been shown to interact with leucine-rich repeat protein 1to initiate cell death-mediated defense (Choi et al., 2003) suggest-ing that the function of some pathogenesis-related proteins maybe associated with signaling induction of the cell death pathway.In Arabidopsis, a number of YLS (yellow leaf specific) genes werefound to be up-regulated during natural senescence (Yoshida et al.,2001). Of these genes, a lipid transfer protein (YLS3) was shownto up-regulated in the early stages of leaf yellowing, and genes up-regulated during the later stages of senescence were homologousto β-glucosidase (YLS1), strictosidine synthase (YLS2), aspartateaminotransferase (YLS4), protease I (YLS5), cytochrome P450(YLS6). Some of the YLS genes could play similar roles to theBrassica napus SAGs discussed previously. YLS4 is thought to beinvolved in nutrient remobilization. The hypothesized role forYLS1 is to supply glucose for respiration whilst YLS3 may beinvolved in lipid transfer activity. The function of several of theseYLS genes (YLS2, -5, -6, -7, -8, -9) remains unknown (Yoshidaet al., 2001). Interestingly, in artificially induced senescence [byapplication of darkness, ethylene, or abscisic acid (ABA)] notall of these YLS genes were responsive. A subtractive hybridiza-tion technique identified a suite of Arabidopsis SAGs that weregrouped in several categories involved in degradation and remo-bilization of metabolites, the production of antioxidant- anddefense-related compounds, and secondary metabolite biosynthe-sis (Gepstein et al., 2003). Included were several genes suspected ofbeing associated with regulation of the initiation and progressionof senescence. More recently, transcriptome analyses have revealeddifferential expression of around 800 SAGs associated with thedramatic change in physiology accompanying the PCD process(Buchanan-Wollaston et al., 2005; van der Graaff et al., 2006).

INDUCTION OF SENESCENCEThe regulation of PCD is vital to plant survival and requiresthe activity of several plant hormones to integrate a large num-ber of external environmental influences in concert with thedevelopmental stage of the plant (Figure 1). The actions ofhormones can either facilitate or inhibit the senescence of leaftissues. As yet, the network of regulatory mechanisms control-ling SAG expression, and the precise molecular functions of manyof these genes during senescence remains to be fully elucidated.The control and regulation of leaf senescence has been recentlyreviewed by Lim et al. (2007) and summarizes some of the genesthought to be involved in this process. In the early stages of leafsenescence, the activation of the SENESCENCE-ASSOCIATEDRECEPTOR KINASE (SARK) may initiate the senescence pro-gram by functioning as a regulatory factor that perceives andtransduces leaf senescence signals (Hajouj et al., 2000). Severalother proteins involved in signal perception and transduction arealso thought to play an early role in the induction of senescenceand include the transcription factors WRKY53, a MYB proteinand a zinc finger protein (Buchanan-Wollaston et al., 2003), aswell as AtSIRK (senescence-induced receptor-like kinase) which isstrongly expressed during leaf senescence (Robatzek and Somssich,2002). Several WRKY genes have been found to regulate gene





FIGURE 1 | Outline of hormonal influences on leaf senescence andchlorophyll degradation processes in non-resurrection plants. SAGs,senescence-associated genes; SAUR, small auxin up RNA gene; SARK,senescence-associated receptor-like kinase; SAUL, senescence-associated E3ubiquitin ligase (see text for details). Arrows indicate senescence promoting

pathways. Lines terminated by bars indicate inhibitory pathways. Dotted linesindicate the mechanisms may be indirect. Asterisks indicate hormones thatinduce desiccation tolerance* or protoplasmic drought tolerance** in someresurrection plant species. For details on the genetic components associatedwith these pathways, see Fischer (2012) and Khan et al. (2013).

expression during pathogen defense and senescence, includingsome SAGs and AtSIRK, suggesting that the control or progressionof senescence may rely on the action of these genes (Robatzek andSomssich, 2002; Zhou et al., 2011). Examples include WRKY70which acts as a negative regulator of senescence during salicylicacid (SA), JA, or ethylene-mediated defense responses (Ülker et al.,2007). WRKY22 accelerates senescence but only under dark treat-ment (Zhou et al., 2011). WRKY53 also acts as a positive regulatorduring early senescence. Increasing levels of JA during senes-cence may activate the EPITHIOSPECIFYING SENESCENCEREGULATOR (ESR) protein which can bind to and inhibit thesenescence-promoting activities of WRKY53 (Miao and Zentgraf,2007). Hence WRKY transcription factors, which can form homo-and heterocomplexes (Xu et al., 2006) appear to act in a regu-latory network integrating positive and negative influences onsenescence (Miao and Zentgraf, 2007). Several members of thelarge family of NAC transcription factors found in plants alsoshow differential expression during both age-related and dark-induced senescence and mutations in NACs have been shownto delay senescence (Lim et al., 2007). The E3 ubiquitin ligase,SENESCENCE-ASSOCIATED UBIQUITIN LIGASE 1 (SAUL1)acts as a negative regulator of senescence under low-light condi-tions via the targeting of senescence-promoting WRKY and NACtranscription factors. Arabidopsis saul1 mutants placed in low-light conditions accumulate SA and exhibit chlorophyll loss andcell death (Raab et al., 2009; Vogelmann et al., 2012).

The hormone auxin (IAA) may have both positive and nega-tive influences on senescence. Bioactive auxin increases twofoldin senescing leaves and may play a positive role in senescence

induction (Lim et al., 2007). Auxin induces the expression off theSMALL AUXIN UP RNA gene, SAUR36, as well as SARK, both ofwhich promote senescence (Xu et al., 2011; Hou et al., 2013). Alter-natively, overexpression of YUCCA6 in Arabidopsis increases auxinlevels and represses the senescence gene SAG12. Transgenic plantsover-expressing YUCCA6 are delayed in dark-induced senescence(Kim et al., 2011).

The induction of expression of several SAGs has been shownto be different during hormone and stress treatments. ABA andethylene treatments increase the expression of many SAGs, butnot necessarily to the same levels as do standard darkness-inducedsenescence assays. Hence, whilst there is overlap between natural-and stress-related senescence, a complex regulatory network oper-ates to integrate the senescence process (Lim et al., 2003). Theregulatory network has been reviewed by Fischer (2012) and Khanet al. (2013). Cytokinin, a noted inhibitor of senescence, reducesthe expression of many SAGs (Weaver et al., 1998). Interestingly,dehydration produced a quick response, with half of the SAGstested being up-regulated after 3 h of dehydration (Weaver et al.,1998). The results of this study indicates that the expression ofsome SAGs are associated with a general response to stress inducedby both darkness and drought (Weaver et al., 1998) and providedsome of the first data linking age-related SAG expression withdrought.

Additional support for a common pathway shared betweenhormone-induced senescence and age-related senescence has beendemonstrated by the effect of the mutation of the ORE9/MAX2protein on senescence in Arabidopsis (Woo et al., 2001). ORE9,an F-box leucine-rich repeat signaling protein is expressed in





the vasculature and plays multiple roles in different parts of theplant. ORE9/MAX2 mediates strigolactone signals produced inthe roots in response to nutrient stress to suppress shoot branch-ing (Stirnberg et al., 2007). Plants with defects in the strigolactonepathway exhibit delayed senescence (Snowden et al., 2005). Thisdelayed senescence is thought to be mediated by reduced activityof ORE9/MAX2 which acts to promote leaf senescence and represshypocotyl elongation in the light and is also involved in oxidativestress and drought responses (Woo et al., 2001, 2004; Stirnberget al., 2002; Tang et al., 2005). Hormonally induced senescenceutilizing ABA or ethylene or methyl jasmonate (MeJA), as well asage-induced senescence was delayed in Arabidopsis ORE9 mutants.This study suggests that ORE9 limits leaf longevity by remov-ing proteins that delay leaf senescence via ubiquitin-dependentproteolysis (Woo et al., 2001).

Cytokinin has long been known to inhibit senescence (Rich-mond and Lang, 1957) and the delay of leaf senescence has beenachieved by engineering the increased production of cytokinin insenescing tissues (Smart et al., 1991; Gan and Amasino, 1997).Increased levels of cytokinin during the initial stages of senescenceinhibit degradation of chlorophyll and photosynthetic tissues(Smart et al., 1991). The protective effect of cytokinin on photo-synthetic protein complexes appear to be mediated by repressionof drought-induced ABA responses and by the upregulation ofbrassinosteroid synthesis and signaling pathways (Rivero et al.,2010). Xia et al. (2009) have shown that exogenous brassinosteroidcan protect photosystem II (PSII) and promote photosynthesis.The activity of photosynthetic enzymes is also negatively regulatedby accumulating sugar which can block the effects of cytokinin(Wingler et al., 1998). Correspondingly, the cytokinin-mediateddelay of senescence is inhibited when extracellular invertase, theenzyme involved in sucrose cleavage and the phloem unload-ing pathway, is inhibited (Balibrea Lara et al., 2004). Althoughcytokinin has been shown to play a pivotal role in the progressionof senescence, cytokinin deficit does not appear to trigger senes-cence onset. This was shown in a study by Werner et al. (2003),where cytokinin-deficient Arabidopsis did not display acceleratedsenescence. Similarly, an examination of the response of the onsetof leaf death 1 (old1) Arabidopsis mutant to ethylene exposure indi-cates that ethylene accelerates age-related senescence, but is notsufficient to initiate leaf senescence in younger plants (Jing et al.,2005). However, a number of genetic studies implicate JA and SAas well as ethylene in promoting senescence. Exogenous JA acceler-ates senescence in the leaves of Arabidopsis but is not effective in theJA-insensitive coronatine insensitive 1 mutant (He et al., 2002). Ara-bidopsis plants defective in SA signaling exhibited delayed yellow-ing and reduced necrosis (Morris et al., 2000). However senescenceappears to proceed fairly normally in plants deficient in JA, SA, andethylene indicating that these hormones are not essential for ini-tiating senescence (Grbic and Bleecker, 1995; Morris et al., 2000;Stintzi and Browse, 2000).

CHLOROPHYLL DEGRADATION, PHOTOSYNTHESIS, ANDSENESCENCEThe breakdown of the chloroplast occurs early during senescence.The accumulation of sugars and changes in cytokinin/ABA lev-els during drought stress results in degradation of chlorophyll,

and a halt to cellular respiration (Richmond and Lang, 1957;Krapp et al., 1991; Smart, 1994). Chlorophyll breakdown duringsenescence is conducted by chlorophyll catabolic enzymes (CCEs)and begins with the removal of phytol and the Mg atom fromchlorophyll, catalyzed by chlorophyllase and a metal chelatingsubstance. The porphyrin cycle is then opened and pheophorbidea is removed by pheophorbide a oxygenase and red chlorophyllcatabolite reductase. This releases a fluorescent catabolite whichis exported from the plastid. The subsequent steps of chloro-phyll breakdown involve converting the phytotoxic fluorescentbreakdown products into non-fluorescent compounds, which aresequestered in the vacuole in a manner similar to widely occurringdetoxification processes (Kreuz et al., 1996).

The regulation of chlorophyll degradation has been associatedwith the stay-green protein which binds to the light-harvestingcomplex II (LHCII) in the thylakoid membrane (Park et al., 2007).Recently LHCII has been shown to interact both directly and indi-rectly, with the five CCEs. Stay-green protein has been associatedwith the recruitment of CCEs in chloroplasts undergoing senes-cence leading to the breakdown of chlorophyll (Sakuraba et al.,2012). Staygreen mutants are defective in chlorophyll breakdown,however, the mechanisms that lead to this defect are not pre-cisely known. The Staygreen mutants have the ability to retainchlorophyll during leaf senescence and are divided into types A–E.Briefly, type A delays the induction of senescence, but the rateof chlorophyll degradation is the same as the wild-type. Type Bshows no delay in the onset of senescence, however, the degra-dation of chlorophyll and photosynthetic activity is much slower.Type C retains chlorophyll during leaf senescence, however, pho-tosynthetic capabilities decrease. Type D results in the death ofleaf tissue during drying or freezing and type E maintains a chloro-phyll level higher than the wild-type throughout leaf development,however, the photosynthetic capacity of the plant does not increase(Thomas and Howarth, 2000).

Analysis of a type C Staygreen mutant has shown that stay-greenprotein acts upstream of pheophorbide a oxygenase in the chloro-phyll breakdown pathway (Aubry et al., 2008). This Staygreenmutant has also been shown to regulate this process at a transcrip-tional level and that the ability of the plant to retain chlorophyll isassociated with the failure to destabilize light-harvesting chloro-phyll binding complexes in the thylakoid membrane, a prerequisitefor chlorophyll degradation.

CHLOROPHYLL DEGRADATION IN RESURRECTION PLANTSChlorophyll reduction/degradation is typically displayed in non-resurrection plants during prolonged dehydration as well as duringsenescence. Unlike many other resurrection plants, S. stapfianusonly experiences partial chlorophyll loss during dehydration andthylakoid membranes are retained. Thus S. stapfianus is not con-sidered either a poikilochlorophyllous (total chlorophyll loss andthylakoid degradation) or a homoiochlorophyllous (chlorophylland thylakoid retention) resurrection plant (Gaff and McGre-gor, 1979; Quartacci et al., 1997). In S. stapfianus photosyntheticactivity seems to be tightly coupled to water availability. Photo-synthesis decreases gradually as the water content declines anddoes not cease till around 45% relative water content (RWC;Di Blasi et al., 1998). Following rehydration from the desiccated





state, CO2 assimilation begins at 45% RWC and the chloro-phyll content is rapidly restored. Hence S. stapfianus has amechanism for preventing total chlorophyll degradation dur-ing desiccation. The ability of S. stapfianus to retain chloro-phyll and reduce photosynthetic activity during dehydrationhas some similarities to the Staygreen type C mutant phe-notype and an examination of the activity and regulation ofpheophorbide a oxygenase in dehydrating S. stapfianus may beinformative.

The LHCII/CAB transcripts in dehydrating S. stapfianus showa substantial increase at 60% RWC, before declining follow-ing further dehydration. Despite this reduction, the level ofLHCII transcripts in dehydrating desiccation-tolerant leaf tis-sue remained higher than in the equivalent desiccation-sensitiveleaf tissue (Blomstedt et al., 1998a). This is consistent with themaintenance of some photosynthetic activity in the dehydrat-ing resurrection plant. Interestingly, Oliver et al. (2010) observedan increase in the abundance of type II light-harvesting LHCIIduring desiccation of S. stapfianus. The observation that LHCIIin S. stapfianus increases during dehydration, despite the reduc-tion in photosynthetic activity, suggests that the LHCII proteinsmay be stored for use in regaining photosynthetic capacity fol-lowing rehydration. Alternatively, the increased abundance ofLHCII may attenuate CCEs activity or recruitment by the stay-green protein and result in a reduction in the rate of chlorophyllbreakdown during desiccation. An increase in the abundanceof PSII stability/assembly factor during desiccation (Oliver et al.,2010), may also account for the protection of PSII. This factoris located in the lumen of stromal thylakoids and is essentialfor the formation of the PSII complex (Meurer et al., 1998).One or more of these mechanisms may operate to enable theplant to retain some photosynthetic activity during dehydra-tion and to stabilize photosynthetic reaction centers in the airdried state so photosynthetic activity is regained rapidly afterrehydration.

Some monocot and dicot species in Western Australia presentan interesting case where foliage may become yellow duringdrought in summer or autumn and yet survive to regreen (indays or weeks) after winter rain (George, 2002). In most ofsuch “diallagous” species (99 species, 59 genera, 24 families)there is no evidence that the foliage becomes air-dry; these maythen represent an intermediate situation where drought resultsin the loss of chlorophyll, a process usually associated withsenescence but here (as in most diallagous species) yellowingoccurs without subsequent senescence and without inductionof desiccation tolerance – an interesting situation that deservesinvestigation of the regulatory molecular mechanisms associatedwith it.

ROLE OF CYSTEINE PROTEASE INHIBITORS (PHYTOSTATINS)Cysteine proteases function in nitrogen remobilization duringsenescence and PCD (Grudkowska and Zagdanska, 2004) aswell as modulating auxin physiology (Chen et al., 2007). Cys-teine proteases may also be involved in degradation of LHCII(Forsberg et al., 2005). Transcripts encoding cysteine proteaseinhibitors (phytostatins) are up-regulated in dehydrating S. stap-fianus (Blomstedt et al., 2010). The activity of cysteine protease

inhibitors during dehydration may aid in the protection of chloro-plast components, resource allocation, or may be deceleratingdrought-related senescence by inhibiting the breakdown of LHCIIby cysteine proteases.

CARBON FIXATIONA decrease in carbon fixation is associated with water deficits(Kramer, 1983). The primary enzyme involved in carbon fixationis ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).The synthesis of Rubisco is elevated during leaf expansion, whereasthe rate of Rubisco synthesis declines during senescence andRubisco is degraded (Suzuki et al., 2001). Between 15 and 30%of total nitrogen content in leaves is present in Rubisco. Dur-ing senescence the Rubisco degradation products are used as asource of nitrogen in developing tissues (Makino et al., 1985).S. stapfianus displays a reduction in the abundance of Rubiscolarge subunit, as well as Rubisco small subunit binding proteinsduring desiccation (Oliver et al., 2010) similar to that displayedby senescing leaf tissues in desiccation-sensitive plants. In nat-ural senescence, autophagy has been shown to be responsiblefor the degradation of Rubisco (Ono et al., 2013). Autophagicprocesses can be responsible for enveloping and remobilizationof nutrients and maintenance of cellular viability under nutri-ent limited conditions. Furthermore, senescence is acceleratedwhen autophagy is disrupted in Arabidopsis (Hanaoka et al., 2002).In rehydrating S. stapfianus, carbon fixation resumes at 40–45%RWC; initially rising rapidly and then more gradually to 75%of the level found in the initial hydrated plant (Di Blasi et al.,1998). S. stapfianus is able to resume normal metabolism 48 hafter rehydration (Gaff and Ellis, 1974) suggesting the need fora pool of stored nutrients to achieving this state so rapidly afterrehydration. Autophagy during dehydration in S. stapfianus maybe responsible for the recruitment and relocation and storageof nutrients from older desiccation-sensitive leaves to youngerdesiccation-tolerant leaf tissues to enable rapid synthesis of car-bon fixation enzymes and other proteins following rehydration.Interestingly, while the abundance of Rubisco declines, severalother enzymes involved in carbon fixation increase in abun-dance during the dehydration of S. stapfianus. These enzymesinclude chloroplastic phosphoglycerate kinase, sedoheptulose1,7-bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase,chloroplastic aldolase, and phosphoribulose kinase. The accu-mulation of these enzymes suggests that a partial Calvin cyclemay be required for the establishment of desiccation tolerance(Oliver et al., 2010). The increase in abundance of some of theseenzymes which also have a role in glycolysis, may indicate ashift between autotrophy and heterotrophy during desiccation,and the degradation of Rubisco may supply nitrogen and othernutrients to the desiccating tissues to provide a nutrient sourcefor the large increase in amino acids and sugars during des-iccation (Whittaker et al., 2007). The application of exogenoussugar to mature spinach leaves results in a decrease in Rubiscoand chlorophyll content, leading to a decrease in photosynthesis,whilst cellular respiration is stimulated (Krapp et al., 1991). Thisobservation suggests some of the changes observed in desiccat-ing S. stapfianus tissues may be driven by the accumulating sugarlevels.





ROLE OF SUGAR ACCUMULATION IN SENESCENCECellular stability and vitrification within desiccating tissuesof S. stapfianus has been attributed to sucrose accumulation(Ghasempour et al., 1998). The accumulation of sugars alsooccurs in drought-stressed tissues of non-resurrection plants andis thought to provide cellular protection during water-deficit(Wingler and Roitsch, 2008). Accumulation of glucose, fructose,and sucrose occur at the beginning of desiccation in S. stapfianus,and in the later stages of dehydration the glucose and fructosedisappear and sucrose forms the leaf sugar pool (Ghasempouret al., 1998; Hoekstra et al., 2001). This large increase in the leafsugar pool may not only protect the cellular constituents fromdamage but may also act as a signal to slow cellular metabolismonce the cell is stabilized to withstand extreme dehydration. Thistheory is supported by the observation that an increase in sugaraids in the inhibition of photosynthesis (Krapp et al., 1991).In non-resurrection plants the presence of sugars in tissues islinked to the regulation of senescence during drought. In lettuce,increased cytokinin production during senescence results in anabnormally high accumulation of sugars in the upper leaves. Thissugar accumulation causes premature senescence (McCabe et al.,2001). The causal relationship between sugar accumulation andthe reduction of photosynthesis and initiation of senescence iscomplex. Research has shown that the senescence associate pro-moter PSAG12 is repressed by sugars, however, above a thresholdlevel of sugar accumulation the expression of SAGs is induced(McCabe et al., 2001). High level sugar accumulation duringdehydration is a common feature of resurrection plants and sug-gests that these plants have a mechanism that allows sugar toaccumulate to high levels without triggering the induction ofsenescence.

HORMONAL INVOLVEMENT IN DROUGHT-RELATEDSENESCENCE AND ITS EVASIONThe induction of senescence in the leaves of non-resurrectionplants during adverse conditions has been the topic of several stud-ies (Weaver et al., 1998; reviewed by Munné-Bosch and Alegre,2004). Drought, darkness, pathogen attack, and oxidative stresscan elicit senescence, remobilization of resources, and changes ingrowth patterns to diminish stress exposure (Potters et al., 2007).While there are differences in the genetic responses between stress-induced senescence and age-related senescence, there are alsocommon elements that are mediated by cross-talk between hor-mones that trigger or repress senescence (Park et al., 1998; Weaveret al., 1998).

The relationship between senescence and cytokinin has longbeen recognized (Richmond and Lang, 1957) where increasingthe endogenous cytokinin content can inhibit leaf senescence(McCabe et al., 2001; Rivero et al., 2010). It has been suggestedthat cytokinin mediates the source–sink relationship during age-related senescence (Roitsch and Ehness, 2000) with the sourcebeing the older leaves and the sink being the younger leaves. Inter-estingly, the production of cytokinins during senescence has alsobeen associated with enhanced drought tolerance (Rivero et al.,2007). When cytokinin synthesis was enhanced using isopentenyl-transferase (IPT) under the control of the SARK promoter in bothtobacco and rice, drought-related senescence was reduced (Rivero

et al., 2010; Peleg et al., 2011). The grain yield of the transgenic riceunder drought stress was greater than the wild-type (Peleg et al.,2011). Hence, the focus of agricultural research into increasingcrop yields through the inhibition of drought-induced senescencehas for the most part been directed at increasing production ofcytokinin via transgenic means. This increase in yield caused bythe increase in cytokinin synthesis was also associated with theinduction of brassinosteroid-associated gene expression, and areduction in jasmonate-associated gene expression during waterstress (Peleg et al., 2011). These studies demonstrate the complex-ity of hormone interaction associated with the drought-relatedsenescence process.

CYTOKININ ACTIVITY IN RESURRECTION PLANTSCytokinin levels have not been examined during dehydration ofS. stapfianus, however, in the resurrection plant Craterostigmawilmsii, cytokinin concentrations during the initial stages of des-iccation are low (Vicré et al., 2004). This low cytokinin contentduring the early stages of desiccation is similar to the situationthat occurs during senescence in tobacco which is accompaniedby a decrease in leaf cytokinin content (Balibrea Lara et al., 2004).However, a substantial increase in cytokinin occurs when C. wilm-sii plants dehydrate below 20% RWC. During rehydration from thedesiccated state, the cytokinin levels progressively decrease againand are reduced to initial low levels at 70% RWC (Vicré et al.,2004). A non-resurrection plant experiencing 20% RWC would beseverely stressed and it seems unlikely that the increase in cytokinincontent in C. wilmsii at 20% RWC and below would be a mech-anism associated with halting senescence. It is not clear whetherincreased cytokinin is essential to allow the leaf tissue to survive thedesiccated state or is associated with resumption of metabolism orregrowth after rehydration.

The cytokinin-mediated delay of senescence has been shownto involve extracellular invertase activity in tobacco (Balibrea Laraet al., 2004). Extracellular invertase contributes to the ability ofplant cells to import sugars and undertake heterotrophic growth(Roitsch et al., 2003). In Corallium rubrum, the induction of extra-cellular invertase by cytokinin also induces a hexose transporter,which doubles sucrose accumulation (Ehness and Roitsch, 1997).If this is mirrored in resurrection plants, this could be a mech-anism of accumulating sucrose in desiccating tissues for cellularprotection and potential energy supply. Extracellular invertase andhexose transporter induction by cytokinin has not been studiedin S. stapfianus. However, hexokinase activity, which regulatesthe entry of hexose sugars for primary metabolism and sucrosestorage is associated with desiccation of both S. stapfianus andX. viscosa resurrection plants. Hexokinase activity peaks at 30%RWC, coinciding with the decline in cellular glucose and fruc-tose content and rapid accumulation of sucrose beginning tooccur at 50% RWC (Whittaker et al., 2001). Peak sucrose contentin S. stapfianus occurs at 30% RWC and remains at a simi-lar level below 30% RWC when the plant is considered air-dry(Whittaker et al., 2001). In non-resurrection plants a decrease incytokinin levels may be required to allow senescence to proceed.The presence of cytokinin can block some cellular responses tosugar accumulation, which includes necrosis and chlorosis (Janget al., 1997) as well as inhibiting chlorophyll and photosynthetic





tissue degradation (Richmond and Lang, 1957). Sucrose accu-mulation in S. stapfianus continues essentially through the entiredrying process and reaches levels around 10 times those foundin hydrated plants (Gaff et al., 2009). This level of sugar accu-mulation is thought to be vital for surviving the final stages ofdesiccation where dehydration is extreme. As well as inhibitingphotosynthesis, and thereby preventing potential damage gener-ated by reactive oxygen species (ROS) and other toxic by-productsarising from photosynthesis, the vitrification of cellular com-ponents by accumulated sugar allows this state to be sustaineduntil rehydration without decomposition. Cytokinin increases,as seen in the severely dehydrated resurrection plant C. wilm-sii, may aid the sugar accumulation process in the final stages ofdesiccation.

ABA INTERACTIONS WITH CYTOKININ AND SUGARACCUMULATIONAbscisic acid has been implicated in the senescence of tissues dur-ing drought stress. In rice plants, ABA was shown to aid in carbonremobilization from senescing tissue into grain production (Yanget al., 2003). ABA has traditionally been thought of as mediat-ing drought responses in many plants. An increase in endogenousABA levels in tissues, leads to an increased transcription of stress-responsive genes which aids the plant in surviving drought stress(Sharp, 2002). In tomato, increasing endogenous levels of ABAwithin tissues has been demonstrated to enhance drought toler-ance (Thompson et al., 2007). Hence ABA appears to have bothcellular protection activities and senescence promoting activities.The precise nature of the interaction between ABA and cytokininduring drought stress is still under examination (Yamaguchi-Shinozaki and Shinozaki, 2006). It has been shown that increasedcytokinin synthesis under the control of the SARK promoter intobacco, prevented the activation of some drought-related ABAresponses, including preventing the degradation of protein com-plexes involved in photosynthesis. Despite these reduced ABAresponses, the increased cytokinin synthesis during drought stressproduced a plant that is more drought tolerant than wild-typeplants (Rivero et al., 2010). The transgenic plants survived droughtfor 15 days and retained higher photosynthetic activity and watercontent in comparison to the wild-type plants which did notsurvive under these conditions (Rivero et al., 2007). This demon-strates the antagonistic nature of plant responses to cytokinin andABA and could be related to the opposing effects these two hor-mones have on growth signaling. Interestingly, the effect of ABAon growth signaling is observed in Arabidopsis mutants deficientin ABA synthesis or ABA signaling. These plants show no growtharrest when exposed to high sugar concentrations (reviewed byRolland et al., 2006).

Whilst sugar accumulation during drought stress is thoughtto have a protective effect on cellular components and to delaywater loss, sugar accumulation, along with ABA, may also act as agrowth retardation signal via regulation of the growth inhibit-ing activities of Snf1-related protein kinase 1 (SnRK1; Jossieret al., 2009). SnRK1 has been strongly implicated in the con-trol of metabolic enzymes and along with SnRK2 and SnRK3acts in a network to link metabolic and stress signaling in plants(Halford and Hey, 2009). The activities of the tobacco SnRK1

complex can lead to reallocation of carbon resources (Schwachtjeet al., 2006) and interestingly, in animal systems the “upstream”kinase (LKB1) of the SnRK1-related protein (AMPK) can trig-ger apoptosis in response to energy stress (Shaw et al., 2004).Sugar accumulation has also been suggested to be involved inredox signaling (Hare et al., 1998), as has SnRK1 (Halford andHey, 2009). The environmental signals that affect metabolismvia the SnRK family, including drought, cold, salt, and nutri-ent stress all impose sink-limiting growth conditions and maylead to sugar accumulation and ultimately cause senescence.Sink-limiting conditions refers to situations where the growth oftissues is impeded by adverse environmental conditions despitethe ample supply of sugar. Nunes et al. (2013) have shown thattrehalose-6-phosphate (T6P) accumulation correlates closely withsucrose accumulation under sink-limited conditions imposed bycold temperatures and N-limitation. This finding, along with theobservation that T6P inhibits SnRK1 in growing tissues of plants(Zhang et al., 2009; Debast et al., 2011) has led to the proposalthat whilst SnRK1 inhibits growth under conditions when sugarand energy is scarce, under sink-limiting conditions, T6P accu-mulates to inhibit the growth-retarding activities of SnRK1. Thisprepares the plant for rapid growth recovery in the presence ofhigh sucrose following alleviation of the environmental stress(Nunes et al., 2013). In these experiments, alleviation of a cold-treatment produced a threefold higher growth rate than that ofunstressed plants, which was dependent on T6P levels. A simi-lar accumulation of growth-related transcripts and a growth rateincrease is seen in S. stapfianus that has gone through a dehydra-tion/rehydration cycle compared with plants kept fully hydrated(Blomstedt et al., 2010). T6P levels have not been measured indehydrating S. stapfianus but trehalose increases up to 10-fold inS. stapfianus dehydrated below 40% RWC. However, the accumu-lated amount is too small to provide substantial protection alone(Gaff et al., 2009) and may be more consistent with a role in sig-naling. It is not clear that there is a direct link between trehaloseaccumulation and senescence inhibition, although senescence hasbeen shown to be delayed in cut Gladiolus spikes by the immersionof the spike in trehalose, which increased the vase-life by 2 days(Otsubo and Iwaya-Inoue, 2000).

REACTIVE OXYGEN SPECIESInjurious ROS such as superoxide, peroxide, and free hydroxylions has been shown to increase during drought stress (Alscheret al., 2002) and the accumulation of ROS has been implicatedin PCD (Del Río et al., 1998). Resurrection plants may expe-rience high levels of irradiation when dehydrating in the hotsun, with the potential to incur photo-oxidative damage fromthe formation of ROS. Some desiccation-tolerant plants reducephoto-oxidative stress by losing all chlorophyll during drying(Tuba et al., 1993; Gaff et al., 2009). While S. stapfianus leavesretain most of its chlorophyll, the plant has the ability to produceprotective pigments and decrease photosynthetic activity in syn-chrony with decreasing water availability. During drought stress,plants produce enzymes such as peroxidase (POD) and superoxidedismutase (SOD) to protect against damage to cellular compo-nents by ROS. These two enzymes are part of the first line ofdefense against ROS. In S. stapfianus, the Cu–Zn SOD which





provides cellular protection in chloroplasts and cytosol decreasesin abundance during dehydration. This is presumably due to thereduction of activity of SOD required, due to stabilization ofcellular structures by vitrification (Veljovic-Jovanovic et al., 2006;Oliver et al., 2010).

Catalase transcripts in S. stapfianus increase initially duringdehydration and peak during the mid-stages of drying (59–40%RWC), then drop to levels below those observed in fully hydratedplants as the tissue dries further (Blomstedt et al., 1998a). Upreg-ulation of catalase activity, along with the activities of otherantioxidant enzymes, has been demonstrated in several other res-urrection plants during the onset of dehydration. Interestingly,these antioxidant enzymes were found to be substantially moreresistant to dehydration-related damage than those from the non-resurrection plants examined (Farrant et al., 2007). The changes incatalase transcripts observed during dehydration in S. stapfianus,may be associated with the maintenance of photosynthetic activityduring the early stages of drying, as catalase is responsible for thebreakdown of hydrogen peroxide and provides protection againstoxidative damage during photosynthesis (Kaiser, 1976; Forti andGerola, 1977).

In one of the few studies undertaken on senescence in res-urrection plants, Veljovic-Jovanovic et al. (2006) found that theisoforms of POD produced by the resurrection plant Ramondaserbica during senescence and dehydration are different. It wassuggested that these isoforms may have alternate physiologicalroles, with a cationic isoform of POD being expressed duringdehydration of R. serbica, to protect cellular constituents, andan anionic isoform involved in lignifying cell walls during senes-cence, presumably to impede pathogens transgressing the cell wallbarrier.

PROTEIN SYNTHESISDuring the dehydration-induced instigation of desiccation-tolerance pathways in resurrection plants it is important thatcellular machinery remains active. Similarly, at least during theinitial stages of drought-induced senescence, the activity of thetranscriptional and translational machinery is maintained as thecell produces the required SAG products. However, desiccation-sensitive plant species cease protein synthesis at mild levels ofdrought stress; while desiccation tolerant species have the ability tocontinue protein synthesis until leaves are almost air-dry (Bartelset al., 1990; Gaff et al., 1997). Protein turnover during drying inresurrection plants is also essential for redirecting protein synthe-sis into production of protectant proteins. Increases in transcriptsencoding proteases and protein translation factors during dehy-dration in S. stapfianus and may contribute to continuing proteinturnover (Blomstedt et al., 1998b).

The content of soluble protein doubles in S. stapfianus leavesdrying on intact plants as they became desiccation-tolerant, butnot in desiccation-sensitive leaves dried detached (Whittaker et al.,2004). During rehydration, the soluble protein content in thedesiccation-tolerant leaves reduces again to control levels. Thissuggests that the activity of the translation machinery is elevatedduring the drying down of resurrection plants.

There is some evidence that changes in expression levels, orproduction of particular isoforms, of protein turnover enzymes

and translation machinery components may be important duringdehydration of resurrection plants. Eukaryotic translation initia-tion factor 5A (eIF5A) has been shown to be implicated in plantgrowth and development (Chou et al., 2004; Thompson et al.,2004). eIF5A is involved in facilitating protein synthesis, however,the precise cellular function is not understood. Recently it has beenshown that the abiotic stress responsive WRKY and RAV proteins,involved in the ABA signaling pathway, can bind to the pro-moter and drive expression of eIF5A. Yeast and poplar expressingeIF5A display elevated protein content, and an improved toler-ance to abiotic stresses (Wang et al., 2012). Furthermore, separateisoforms of eIF5A facilitate the translation of mRNAs encodingproteins involved in cell division and cell death, suggesting a roleof eIF5A in senescence (Thompson et al., 2004). Although expres-sion of eIF5A has not been studied in S. stapfianus, an increasein the levels of eIF1A has been observed in dehydrating tissues(Neale et al., 2000). The role of eIF1A has been shown to beimportant in the tolerance to salt stress (Rausell et al., 2003), how-ever, its role in dehydration stress and senescence has not beenstudied.

Sporobolus stapfianus may regulate the expression of eIF1A toallow the plant to facilitate dehydration-related protein synthesisduring desiccation (Neale et al., 2000). In addition to translationinitiation factors, S. stapfianus also has increased expression ofelongation factors during dehydration. In S. stapfianus, eEF1A(eukaryotic elongation factor 1A) is expressed in dehydrating tis-sues with high levels of expression appearing in fully hydrated leaftissue, at the initiation of the desiccation tolerance pathway at 60%RWC, and again at 20% RWC (Blomstedt et al., 2010). This elonga-tion factor catalyses the binding of aminoacyl-tRNA to the A-siteof the ribosome (Ejiri, 2002) demonstrating its ability to facili-tate protein synthesis. Like eIF5A, eEF1A transcripts accumulateduring abiotic stresses (Dunn et al., 1993). The function of eEF1Ais not well understood, but it may allow for the rapid synthesisof proteins involved in avoiding the senescence program whenthe desiccation-tolerance program is induced (∼60% RWC) andwhere sugar has accumulated to very high levels (∼20% RWC).

ENDOPLASMIC RETICULUM STRESSEndoplasmic reticulum (ER) stress has been linked with osmoticstress-induced cell death (Alves et al., 2011) where integrated sig-nals from both ER stress and osmotic stress are required to activatecell death (Figure 2). In all eukaryote cells, including plants,ER stress triggers the unfolded protein response (UPR). The ERlocated molecular chaperone binding protein BiP assists in fold-ing newly synthesized proteins in the ER lumen and also acts asa sensor of ER stress and stress-related signal generation (Reiset al., 2011). Environmental stress can lead to an accumulationof unfolded proteins in the ER. It is thought that a high level ofunfolded proteins sequesters BiP, releasing and activating severalER-associated receptors such as PERK (protein kinase RNA-like ERkinase), IRE1 (inositol-requiring protein-1), and ATF6 (activatingtranscription factor-6). Activation of these receptors triggers theUPR, leading to a down-regulation of translation and an increasein ER protein folding and process components including BiP (Reisand Fontes, 2012). Several key components of the plant UPRhave been identified although the precise roles of some of these





FIGURE 2 | Integrated cell death pathway model linking drought stressand ER stress. Model showing pathways thought to be associated withactivating cell death. Dotted lines represent proposed pathways. The trianglesindicate possible drought-induced senescence inhibition points. The bentarrow indicates an antagonistic relationship. The lightning bolt indicatesprotein degradation. The model is based on the results of Alves et al. (2011)from Glycine max and also incorporates information from Arabidopsisstudies. The ABA attenuation loop is based on AtERD15 (Kariola et al., 2006)which does not appear to activate NRP-B expression. The involvement of

GmERD15, which contains a PAM2 domain, in the regulation of ABA signalingis yet to be demonstrated. AGB1, a heterotrimeric G protein thought to have arole in triggering the cell death signal; ATF6, activating transcription factor 6related homologs (e.g., bZIP28 and 60); BiP, ER chaperone binding protein;Ire1p, inositol-requiring protein-1 homolog; NL, nuclear localization; NRP,N-rich proteins; PERK, protein kinase RNA-like ER kinase; SA, salicylic acid;eIF2α, eukaryotic initiation factor 2α (related references: Tang et al., 2005;Costa et al., 2008; Liu and Howell, 2010; Deng et al., 2011; Moreno andOrellana, 2011).

components remain to be clarified (Fanata et al., 2013). Whilstno homolog of PERK has been identified in plants, GCN2 mayplay a similar role in phosphorylating eIF2α to inhibit proteinsynthesis (Zhang et al., 2003). bZIP60 acts as a plant homologof ATF6 and up-regulates the BiP chaperone as well as SKP1, acomponent of the SCF-type E3 ubiquitin ligase complex, thatdegrades misfolded proteins via the 26S proteasome (Ye et al.,2011). When the stress is severe enough to prevent restorationof ER homeostasis, a signal is activated that involves activationof asparagine-rich protein (NRP) genes (Irsigler et al., 2007) andother downstream components that result in cell death (Costaet al., 2008; Wang et al., 2008). NRP expression causes chloro-phyll loss, ethylene production, and senescence (Reis and Fontes,2012). The transcription factor early responsive to dehydration15 (ERD15; Kiyosue et al., 1994) has been identified in severalplant species. ERD15 in soybean binds to the promoter and drivesexpression of the NRP-B gene and is proposed to link osmoticstress and ER stress to cell death (Alves et al., 2011). There maybe differences in the roles of ERD15 from Arabidopsis and soy-bean. Both the Arabidopsis and soybean ERD15 protein containPAM2 domains. PAM2 domains bind PABP and may be involvedin regulating mRNA translation. ERD15 has been proposed toattenuate ABA signaling in A. thaliana lines, with silenced ERD15expression showing hypersensitivity to the exogenous applicationof ABA during seed germination, and the over-expression of

ERD15 resulting in tolerance to exogenous ABA (Kariola et al.,2006). In Arabidopsis, ERD15 does not appear to directly driveNRP-B expression but may act through attenuation of the ABAantagonistic effect on SA signaling, since the SA hypersensi-tive response appears to induce NRP expression (Ludwig andTenhaken, 2001).

SENESCENCE ATTENUATION MECHANISMS INRESURRECTION PLANTSBy necessity resurrection plants must contain a mechanism forpreventing activation of the UPR cell death response during severedehydration. As resurrection plants can undergo age-related senes-cence, it would seem likely that the mechanisms for inhibitingdrought-related senescence would operate upstream of the termi-nal stages of the PCD process (see Figure 2). This may involveattenuation of the activity of ERD15. An examination of theactivity of ERD15 and potential interacting proteins during dehy-dration in resurrection plants may reveal if the UPR and celldeath pathway is modified in these plants. As ERD15 is a focalpoint for integration of these two pathways, attenuation of ERD15activity would allow the plant to withstand increased endogenousABA levels and would also modulate ER stress signaling duringdehydration to prevent induction of senescence in the youngerdesiccation-tolerant leaves.





Exogenous ABA induces full desiccation tolerance in hydratedleaves of the monocot Borya constricta (Gaff and Loveys, 1984).In the resurrection plant C. plantagineum, the induction of thedesiccation tolerance pathway is also ABA-dependent (Bartelsand Salamini, 2001). As Borya constricta is poikilochlorophyllous(Gaff,1989) and C. plantagineum is homiochlorophyllous (Farrantet al., 2007) the importance of ABA in conferring desiccation tol-erance cannot be inferred from the degree of chlorophyll exhibitedby resurrection plants during dehydration. A non-ABA pathwayappears to regulate desiccation tolerance in S. stapfianus. ABA indehydrating S. stapfianus does not elevate substantially until thelatter stages of dehydration, reaching a peak between 40 and 15%RWC. This is well after the initiation of the desiccation toleranceprogram which occurs around 60% RWC (Gaff and Loveys, 1993).While ABA accumulation in non-resurrection plants inducesmechanisms that allow the survival of mild water-deficit, it canalso induce chlorophyll degradation and senescence (Back andRichmond, 1971; Lim et al., 2003). In many desiccation-tolerantpoikilochlorophyllous monocots, the link between dehydration-dependent chlorophyll degradation and leaf senescence has beenuncoupled. Whereas in ABA-dependent homiochlorophyllous res-urrection plants, such as C. plantagineum, it appears that amechanism for attenuating both ABA-induced chlorophyll degra-dation and senescence as it dehydrates is present. An interestingfunction associated with ABA signaling has been proposed forthe lysine-rich LEA-like protein CDeT11-24 from C. plantagineumbased on its ability to bind phosphatidic acid (PA) in vitro (Petersenet al., 2012). LEA proteins, which are highly expressed in theleaves of resurrection plants during dehydration, have no appar-ent inherent catalytic activity but their predicted functions includethe formation of stabilizing filaments within the cytoplasm andacting as molecular chaperones by retaining the hydration shellfor proteins during desiccation (Wise and Tunnacliffe, 2004; Bar-tels, 2005). The C. plantagineum LEA-like protein, CDeT11-24binds specifically to PA. PA is produced in plants in responseto several stresses including drought and acts as a stress sig-nal (Testerink and Munnik, 2005). A signaling function of PAinvolves the disruption of binding of 2C protein phosphatases(PP2Cs) to the ABA receptor (Park et al., 2009). PP2Cs, suchas ABI1, act as negative regulators of ABA signaling and thisdisruption of receptor binding allows transduction of the ABAsignal. Petersen et al. (2012) have hypothesized that the drought-induced membrane-binding of this lysine-rich LEA protein mayinterfere with the PA–ABI1 interaction and attenuate ABA sig-naling. Several LEA proteins that are differentially expressed indesiccation-tolerant tissue of S. stapfianus during dehydrationhave been identified (Blomstedt et al., 1998a,b; Le et al., 2007).Interestingly, one of the S. stapfianus lysine-rich LEA proteins,when ectopically expressed in Arabidopsis, locates to the chloro-plast and appears to reduce ABA-related stress responses of thetransgenic plants and to affect ABA-induced stomatal closure(Ling, 2011).

The activity of stress-related hormones, such as ABA, respon-sible for driving drought responses would normally be consideredadvantageous in the tissues of a dehydrating plant (Thompsonet al., 2007). However, the rate of water-loss and growth retarda-tion of resurrection plants under drought is rapid, when compared

to many other plant species. This suggests that the primarysurvival response of resurrection plants like S. stapfianus expe-riencing drought is not to initiate measures to restrict water-lossor maintain normal metabolism, but to instigate the desiccationtolerance pathway. Hence, S. stapfianus may have several mecha-nisms to eliminate traditional stress-related hormonal responsesto water-deficit during establishment of the desiccation toleranceprogram.

Apart from ethylene, the bioactivity of most knownplant hormones can be modulated by the action of UDP-glycosyltransferases (UGTs; Lim and Bowles, 2004). TheSporobolus drought-responsive gene 8i, SDG8i, gene encodes a UGTwhose transcript levels increase substantially under severe waterdeficit (Le et al., 2007). Whilst the endogenous substrate of thisUGT is unknown, the enzyme has in vitro activity against thesynthetic strigolactone analog GR24, suggesting that it may affectstrigolactone signaling in the resurrection plant (Islam et al., 2013).Strigolactones are produced in the roots of plants in response tonutrient stress and translocate to the shoot to reduce shoot growth(Gomez-Roldan et al., 2008; Umehara et al., 2010). Strigolactoneshave pleiotropic effects on plant growth. They can regulate rootgrowth in response to nutrient availability, affect flowering timeas well as regulating shoot branching (Stirnberg et al., 2002; Dunet al., 2009; Leyser, 2009; Umehara et al., 2010; Ruyter-Spira et al.,2011). Mutations in the strigolactone synthesis and signalingpathways can also inhibit senescence (Woo et al., 2001; Snowdenet al., 2005). When the SDG8i UGT is constitutively expressedin Arabidopsis it enhances the growth rate of the plant consider-ably, under both favorable and stress conditions. Auxin synthesisalso appears to be upregulated in these plants. The constitutiveexpression of SDG8i UGT also delays senescence substantially(Islam et al., 2013). The SDG8i transgenic plants have a pheno-type consistent with altered ORE9/MAX2 activity which acts asa signaling protein in the strigolactone pathway and promotessenescence (Woo et al., 2001; Stirnberg et al., 2007). While theeffect on MeJA- and ethylene-induced senescence was not tested,SDG8i UGT activity was demonstrated to inhibit both dark-induced senescence and ABA-induced senescence (Islam et al.,2013). Low levels of the UGT transcript are present in hydratedleaf tissue in S. stapfianus. UGT transcripts increase substan-tially during dehydration and persist in desiccated tissue (Le et al.,2007). These results raise the possibility that the drought-inducedexpression of this UGT in S. stapfianus inhibits drought-relatedABA-induced senescence and also promotes rapid plant growthfollowing rehydration.

Enhanced disease resistance I (EDR1) encodes a protein kinasethat acts as a negative regulator of ethylene- and SA-relatedsenescence pathways in Arabidopsis. The ethylene pathway thatEDR1 inhibits also appears to act through the reduced activityof ORE9/MAX2. Arabidopsis edr1 mutants display spontaneousnecrosis under drought suggesting that EDR1 acts to preventdrought-induced senescence (Tang et al., 2005). Assuming anortholog of EDR1 can be identified in resurrection plants, ananalysis of its expression and function during dehydration maybe informative.

The interactions between cytokinin and ABA during waterstress have revealed that cytokinin can inhibit the accumulation





of ABA during water stress, and ABA can increase cytokinin accu-mulation during water stress (Pospíšilová et al., 2005). However,the reason for the accumulation of high levels of ABA and/orcytokinin in resurrection plants at the later stages of the desicca-tion process remains obscure. Studies have shown that high levelsof growth-related gene transcripts accumulate at hydration lev-els well below 20% RWC (Le et al., 2007). This gene activity mayoccur in preparation for rapid growth following a rainfall event(Blomstedt et al., 2010).

CONCLUSIONThe ability to prevent initiation of drought-related senescenceis a major component of the desiccation-tolerance program ofresurrection plants. Mutational analysis in a number of non-resurrection plant species has revealed genetic mechanisms associ-ated with promoting or progressing senescence. Current researchhas indicated that senescence can be activated in non-resurrectionplants by a complex network of signal pathways that integratemetabolic signals with age-related information and environmentalstress. As many defects in basic metabolic processes could poten-tially lead to premature cell death, identifying genes specificallyinvolved in preventing senescence through mutational analysis ismore problematic (Lim et al., 2007). In younger leaf tissues, res-urrection plants can block the senescence signaling pathways andpotentially provide a valuable resource for identifying these block-ing mechanisms. Research in resurrection plants has indicated thatdesiccation-tolerance has not been conferred by the acquisitionof genes unique to resurrection plants, but rather by alterationsin the regulatory control of genes that are likely to be presentin the genomes of most plants. Since drought-related leaf senes-cence is a survival strategy utilized by non-resurrection plants, theidea that these senescence blocking mechanisms currently operatein the some cells of reproductive or root tissues of resurrectionplants may be worth exploring. As the input signaling into senes-cence is very diverse it seems likely that the leaves of resurrectionplants harbor multiple mechanisms to repress drought-relatedsenescence or alternatively may act at pivotal points of conver-gence. Recent senescence research indicates that ERD15, whichintegrates drought stress and ER stress signals to activate the NRPcell death pathway, represents one such convergence point andinvestigation of this pathway in resurrection plants may provefruitful. ABA signaling may also feed into senescence via ERD15.Currently, the evidence that lysine-rich LEA proteins localized invarious cellular compartments can attenuate ABA is not conclu-sive and warrants further investigation. Based on the precedenceof the regulatory mechanisms controlling cell growth, inductionof a particular cell death pathway may require both a positiveactivating signal and inactivation of a negative inhibitory sig-nal. Recent results from Islam et al. (2013) suggests drought-,ABA-, and dark-induced senescence can be blocked by the activityof the S. stapfianus dehydration-responsive UGT SDG8i. SDG8iprobably mediates this effect via repression of ORE9-mediatedubiquitination and degradation of senescence-inhibitory proteins.Further research into the key anti-senescence mechanisms associ-ated with the desiccation-tolerance program may uncover valuableinformation on how these remarkable plants survive prolongeddrought.

ACKNOWLEDGMENTThe authors would like to thank Dr. Cecilia K. Blomstedt for thecritical review of this manuscript.

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Conflict of Interest Statement: The authors declare that the research was conductedin the absence of any commercial or financial relationships that could be construedas a potential conflict of interest.

Received: 16 October 2013; accepted: 27 January 2014; published online: 12 February2014.Citation: Griffiths CA, Gaff DF and Neale AD (2014) Drying without senescence inresurrection plants. Front. Plant Sci. 5:36. doi: 10.3389/fpls.2014.00036This article was submitted to Plant Physiology, a section of the journal Frontiers inPlant Science.Copyright © 2014 Griffiths, Gaff and Neale. This is an open-access article distributedunder the terms of the Creative Commons Attribution License (CC BY). The use, dis-tribution or reproduction in other forums is permitted, provided the original author(s)or licensor are credited and that the original publication in this journal is cited, inaccordance with accepted academic practice. No use, distribution or reproduction ispermitted which does not comply with these terms.


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