stable transgenic arabidopsis thaliana

1
Stable transgenic Arabidopsis thaliana ? Floral organ ablation atp9 related Sequences * * Figure 2. Mitochondrial open reading frames associated with pollen collapse in S male-sterile maize Co-transcribed mitochondrial open reading frames, orf355 and orf77, are associated with S male-sterile maize (Zabala et al. Genetics 147:847). Post-transcriptional, C-to-T RNA editing of orf77 introduces a stop codon that creates both orf17 and orf26. Orf17 (Gallagher et al. Curr. Genet. 42:179). orf355 orf17 orf26 11 bp orf77 rearranged normal / unknown origin Figure 3. Similarity between the mitochondrial open reading frame associated with CMS-S pollen collapse and the ATP synthase subunit 9 (atp9) gene Orf17 predicts a peptide similar to the C-terminus of maize mitochondrial ATP9. The genome- encoded C nucleotides that are changed to U by plant mitochondrial RNA editing are underlined [Gallagher et al. Curr. Genet. 42:179], and the corresponding amino acid changes are indicated above the codons. orf17 M F A S F D S M M A F L I S>L F V F R>* ATG TTT GCA TCG TTT GAC TCA ATG ATG GCC TTT CTG ATC TC A TTC GTA TTC C GA atp9 (C terminus) A I A S>L F A P>L M M A F L I S>L F V F * GCT ATT GCA TC G TTT GCC CC A ATG ATG GCC TTT CTG ATC TC A TTC GTT TTC TAA Construct Promoter Target Signal GFP or Orf PCD-1 35S -- sGFP(S65T) PCD-2 35S atp9 DL sGFP(S65T) PCD-3 AP3 atp9 DL sGFP(S65T) PCD-4 AP3 atp9 DL orf 77 PCD-5 AP3 atp9 DL orf 17 PCD-6 AP3 atp9 DL orf 355 PCD-7 AP3 atp9 DL orf 26 CMS-S young collapsed microspore bi-cellular pollen bi- cellular pollen Acknowledgements This research was supported by USDA CSREES NRI award 00-35300-9409, and by National Science Foundation award IOS-0816782. Dr. Alice Barkan provided the PETD antibody. The ATP6 antibody was raised against synthetic peptides and affinity-purified by Bethyl Laboratories, Inc. The GFP antibody was purchased from Novus Biologicals, Littleton, CO. Carwayna McColley and Modupe Durojaiye contributed to the development of transgenic A. thaliana lines. Abstract: In maize, the S type of cytoplasmic male sterility (CMS-S) conditions pollen collapse at the early bi-cellular stage. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated nuclear and chromatin fragmentation in pre-collapse CMS-S pollen consistent with mitochondrial-signaled programmed cell death. The main objective of this research is to identify the mitochondrial open reading frame (orf) responsible for these events. Mitochondrial transcripts are associated with CMS-S pollen collapse, but post-transcriptional RNA editing creates different versions of the orfs suspected to be responsible for pollen cell death. The up-stream frame (orf355) is not edited, but the down-stream frame (orf77) is edited to create two shorter orfs (orf17 and orf26). Furthermore, partial editing within orf17 creates S14 and L14 coding variants. Thus far, plant mitochondrial genomes cannot be genetically transformed, so nuclear transformation approach was taken. Candidate orfs were fused to a mitochondrial targeting sequence, the ATP9 double leader (ATP9DL) from Neurospora crassa, and expressed behind the 35S or ap3 promoter. Fluorescence microscopy of stable Arabidopsis transformants demonstrated that ATP9DL targets GFP to mitochondria creating the potential to successfully target orf translation products. Floral phenotypes of Arabidopsis plants expressing ap3-driven, mitochondria- targeted, GFP or ORF17 support the hypothesis that orf17 is the reading frame responsible for S male sterility in maize. Expression of mitochondrial-targeted peptides linked to cell death signaling and pollen collapse in S male-sterile maize Kanchan Bhan 1 , Prestina Smith 3 , Karen Chamusco 2 , Jeffery Seib 1 , Cristina Moreira 3 , Maria Gallo 1 , Christine Chase 2 1 Agronomy Department, University of Florida, Gainesville, FL 32611 2 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611 3 Department of Biology, Bennett College for Women, Greensboro, NC 27401 Figure 5. In vivo assays for orf-associated cell death phenotypes Figure 4. P rogrammed C ell D eath (PCD) constructs designed to express candidate ORFs and GFP The pCAMBIA 1302 vector was used to transform Arabidopsis and Petunia. PCD-1 predicts GFP accumulation in the plant. PCD-2 predicted GFP targeted to the mitochondria by the Neurospora crassa ATP9 double leader (ATP9DL) sequence (Galanis et al. FEBS Lett. 282:425). PCD-3 predicts GFP expression, with mitochondrial targeting in petals and stamens. PCD-4 through 7 predict mitochondrial-targeted ORF77, ORF17, ORF355 and ORF26 respectively in petals and stamens. Figure 1. CMS-S maize pollen collapse at the early bi-cellular stage accompanied by chromatin and nuclear fragmentation Left: CMS-S (a-c) and normal (d-f) pollen development visualized with DAPI stain. g) enlarged nuclei from panel b, showing marginalized, fragmented chromatin (red arrows). Microspore mother cells and tetrads are not shown. Right: Terminal dUTP nick end labeling (TUNEL) assay of CMS-S (a-c) and normal (d-f) pollen development. b) TUNEL positive staining (red arrows) is observed in the nuclei of young, bi- cellular CMS pollen. c) The cytoplasm of collapsed, CMS-S pollen (red brace) was condensed and disorganized with punctuate TUNEL-positive bodies indicating “packets” of nicked DNA, possibly remnants of a degraded nucleus. Bars=50µm a) b) e) d) c) f) g) CMS-S young collapsed microspore bi-cellular pollen bi-cellular pollen microspore bi-cellular pollen tri-cellular Normal pollen e) d ) f) b) c) a) microspore bi-cellular pollen tri-cellular Normal pollen Conclusions In CMS-S maize, expression of a mitochondrial orf initiates a cell-death pathway characterized by chromatin and nuclear fragmentation and culminating in pollen collapse. The N. crassa ATP9DL targeted GFP to the mitochondria in A. thaliana sepals & petals and in petals of petunia. Candidate CMS orf17, expressed from the A. thaliana AP3 promoter, was associated with ablation of floral petals and stamens. The AP3-driven GFP construct conditioned homeotic floral phenotypes consistent with silencing of the endogenous AP3 promoter. Future prospects Subsequent floral dips are underway in Arabidopsis plants for each of the constructs to verify previous results. In the recent observation it was discovered that the edit responsible for the creation of orf 17 also creates orf 26 down stream of orf 17. Future work will include determining of expression of these proteins and whether or not they work in concert to cause PCD in the microspores of the CMS-S maize system. Work is in progress to transform normal maize pants with mitochondria targeted candidate orfs to confirm the cause of the collapsed pollen phenotype. Figure 6. The N. crassa ATP9DL targets GFP to the mitochondria in transgenic Arabidopsis kDa ATP6 GFP PETD Crude Extract 1 ,500 g pellet 1 ,500 g sup 15,000 g mit ochondrial pelle t 1 5,000 g po s t- mitoc hondrial s u p 22 40 30 27 25 17 Left: Fluorescent imaging of sepals from a PCD-2 transformed Arabisopsis plant. Red auto- fluorescent chlorophyll indicates the location of chloroplasts in the sepals. Green punctate staining ,non-concordant with the chloroplasts , consistent with mitochondrial targeting of the GFP. Right: Western blots of cell fractions from PCD-2 transformed Arabidopsis. plants. ATP6, GFP and PETD antibodies were used to determine the co-fractionation of these proteins by differential centrifugation. A mature, 25 kDa form of GFP co-fractionated with mitochondrial ATP6. A 40 kDa form corresponds to the predicted size of the primary translation product. Intermediate forms of 30 and 27 kDa were enriched in the post- mitochondrial supernatant. PETD was enriched in the 1,500 g pellet, which contained chloroplasts. PETD in the mitochondrial fraction might be attributed to broken thylakoids. Figure 7. The N. crassa ATP9DL targets GFP to the mitochondria in a petunia petal transient expression assay Petunia petals (Easy Wave) were inoculated at the base with Agrobacterium tumefaciens strain GV3101 (O.D. 0.05) carrying the PCD-3 construct. a - c) Time-lapse pictures of the same cell to illustrate the movement of the GFP-labeled mitochondria. a) and b) are 14 seconds apart and c) is 7 seconds after b). The red signal is autofluorescence from the chloroplasts. Bar=50µm a ) b) c) a) Normal Arabidopsis flower stamen, pistil, sepals and petals b) Abnormal floral structures of Arabidopsis transformed with PCD-3, consistent with silencing of the endogenous AP3 promoter. Sepals and pistil were normal. Stamens were converted to pistil-like structures and petals had sepal-like features. c) Abnormal floral structures of Arabidopsis transformed with PCD-5 were consistent with the ablation of petals and stamens. Sepals and pistil-like structures were observed. Figure 8. Floral abnormalities observed in Arabidopsis plants expressing PCD-3 and PCD-5 Sepal Petal Pisti l Stamen Sepal Pisti l Sepaloid petal Stigmatic tissue Ovule s Pisti l Sepal Pistilloid stamen a) b) c) Table1. A. thaliana (Lansberg) flowers after transformation with programmed cell death constructs 3 and 5 (PCD3 & 5) Wild Type PCD-3 PCD-5 T 0 Plants Flower Phenotype PCD3 1-1, 1-3, 1-4, 3-1, 3-2, 7-2, 7-4,8-1 and 9-1 Normal PCD3 1-2,1-5 and 4-1 Silenced PCD-5 7-1,7-2,8-1 Normal PCD-5 5-1, Ablated

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e). d). f). Expression of mitochondrial-targeted peptides linked to cell death signaling and pollen collapse in S male-sterile maize. Kanchan Bhan 1 , Prestina Smith 3 , Karen Chamusco 2 , Jeffery Seib 1 , Cristina Moreira 3 , Maria Gallo 1 , Christine Chase 2 - PowerPoint PPT Presentation

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Page 1: Stable transgenic  Arabidopsis thaliana

Stable transgenic Arabidopsis thaliana ? Floral organ ablation

atp9 related Sequences * *

Figure 2. Mitochondrial open reading frames associated with pollen collapse in S male-sterile maize

Co-transcribed mitochondrial open reading frames, orf355 and orf77, are associated with S male-sterile maize (Zabala et al. Genetics 147:847). Post-transcriptional, C-to-T RNA editing of orf77 introduces a stop codon that creates both orf17 and orf26. Orf17 (Gallagher et al. Curr. Genet. 42:179).

orf355 orf17 orf2611 bp

orf77

rearranged normal / unknown origin

Figure 3. Similarity between the mitochondrial open reading frame associated with CMS-S pollen collapse and the ATP synthase subunit 9 (atp9) gene

Orf17 predicts a peptide similar to the C-terminus of maize mitochondrial ATP9. The genome-encoded C nucleotides that are changed to U by plant mitochondrial RNA editing are underlined [Gallagher et al. Curr. Genet. 42:179], and the corresponding amino acid changes are indicated above the codons.

orf17 M F A S F D S M M A F L I S>L F V F R>*ATG TTT GCA TCG TTT GAC TCA ATG ATG GCC TTT CTG ATC TCA TTC GTA TTC CGA

atp9 (C terminus) A I A S>L F A P>L M M A F L I S>L F V F *GCT ATT GCA TCG TTT GCC CCA ATG ATG GCC TTT CTG ATC TCA TTC GTT TTC TAA

Construct Promoter Target Signal GFP or Orf

PCD-1 35S -- sGFP(S65T)

PCD-2 35S atp9 DL sGFP(S65T)

PCD-3 AP3 atp9 DL sGFP(S65T)PCD-4 AP3 atp9 DL orf 77PCD-5 AP3 atp9 DL orf 17PCD-6 AP3 atp9 DL orf 355PCD-7 AP3 atp9 DL orf 26

CMS-S young collapsed microspore bi-cellular pollen bi-cellular pollen

Acknowledgements This research was supported by USDA CSREES NRI award 00-35300-9409, and by National Science Foundation award IOS-0816782. Dr. Alice Barkan provided the PETD antibody. The ATP6 antibody was raised against synthetic peptides and affinity-purified by Bethyl Laboratories, Inc. The GFP antibody was purchased from Novus Biologicals, Littleton, CO. Carwayna McColley and Modupe Durojaiye contributed to the development of transgenic A. thaliana lines.

Abstract: In maize, the S type of cytoplasmic male sterility (CMS-S) conditions pollen collapse at the early bi-cellular stage. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated nuclear and chromatin fragmentation in pre-collapse CMS-S pollen consistent with mitochondrial-signaled programmed cell death. The main objective of this research is to identify the mitochondrial open reading frame (orf) responsible for these events. Mitochondrial transcripts are associated with CMS-S pollen collapse, but post-transcriptional RNA editing creates different versions of the orfs suspected to be responsible for pollen cell death. The up-stream frame (orf355) is not edited, but the down-stream frame (orf77) is edited to create two shorter orfs (orf17 and orf26). Furthermore, partial editing within orf17 creates S14 and L14 coding variants. Thus far, plant mitochondrial genomes cannot be genetically transformed, so nuclear transformation approach was taken. Candidate orfs were fused to a mitochondrial targeting sequence, the ATP9 double leader (ATP9DL) from Neurospora crassa, and expressed behind the 35S or ap3 promoter. Fluorescence microscopy of stable Arabidopsis transformants demonstrated that ATP9DL targets GFP to mitochondria creating the potential to successfully target orf translation products. Floral phenotypes of Arabidopsis plants expressing ap3-driven, mitochondria- targeted, GFP or ORF17 support the hypothesis that orf17 is the reading frame responsible for S male sterility in maize.

Expression of mitochondrial-targeted peptides linked to cell death signaling and pollen collapse in S male-sterile maize

Kanchan Bhan1, Prestina Smith3, Karen Chamusco2, Jeffery Seib1, Cristina Moreira3, Maria Gallo1 , Christine Chase2

1Agronomy Department, University of Florida, Gainesville, FL 326112 Horticultural Sciences Department, University of Florida, Gainesville, FL 326113Department of Biology, Bennett College for Women, Greensboro, NC 27401

Figure 5. In vivo assays for orf-associated cell death phenotypes

Figure 4. Programmed Cell Death (PCD) constructs designed to express candidate ORFs and GFP

The pCAMBIA 1302 vector was used to transform Arabidopsis and Petunia. PCD-1 predicts GFP accumulation in the plant. PCD-2 predicted GFP targeted to the mitochondria by the Neurospora crassa ATP9 double leader (ATP9DL) sequence (Galanis et al. FEBS Lett. 282:425). PCD-3 predicts GFP expression, with mitochondrial targeting in petals and stamens. PCD-4 through 7 predict mitochondrial-targeted ORF77, ORF17, ORF355 and ORF26 respectively in petals and stamens.

Figure 1. CMS-S maize pollen collapse at the early bi-cellular stage accompanied by chromatin and nuclear fragmentation

Left: CMS-S (a-c) and normal (d-f) pollen development visualized with DAPI stain. g) enlarged nuclei from panel b, showing marginalized, fragmented chromatin (red arrows). Microspore mother cells and tetrads are not shown.

Right: Terminal dUTP nick end labeling (TUNEL) assay of CMS-S (a-c) and normal (d-f) pollen development. b) TUNEL positive staining (red arrows) is observed in the nuclei of young, bi-cellular CMS pollen. c) The cytoplasm of collapsed, CMS-S pollen (red brace) was condensed and disorganized with punctuate TUNEL-positive bodies indicating “packets” of nicked DNA, possibly remnants of a degraded nucleus. Bars=50µm

a) b)

e)d)

c)

f)

g)

CMS-S young collapsedmicrospore bi-cellular pollen bi-cellular pollen

microspore bi-cellular pollen tri-cellular Normal pollen

e)d) f)

b) c)a)

microspore bi-cellular pollen tri-cellularNormal pollen

Conclusions

• In CMS-S maize, expression of a mitochondrial orf initiates a cell-death pathway characterized by chromatin and nuclear fragmentation and culminating in pollen collapse.

• The N. crassa ATP9DL targeted GFP to the mitochondria in A. thaliana sepals & petals and in petals of petunia.

• Candidate CMS orf17, expressed from the A. thaliana AP3 promoter, was associated with ablation of floral petals and stamens.

• The AP3-driven GFP construct conditioned homeotic floral phenotypes consistent with silencing of the endogenous AP3 promoter.

Future prospects

• Subsequent floral dips are underway in Arabidopsis plants for each of the constructs to verify previous results.

• In the recent observation it was discovered that the edit responsible for the creation of orf 17 also creates orf 26 down stream of orf 17. Future work will include determining of expression of these proteins and whether or not they work in concert to cause PCD in the microspores of the CMS-S maize system.

• Work is in progress to transform normal maize pants with mitochondria targeted candidate orfs to confirm the cause of the collapsed pollen phenotype.

Figure 6. The N. crassa ATP9DL targets GFP to the mitochondria in transgenic Arabidopsis

kDaATP6

GFP PETD

Crud

e Ex

tract

1,50

0 g

pelle

t1,

500

g su

p 15,0

00 g

m

itoch

ondr

ial p

elle

t

15,0

00 g

pos

t-

mito

chon

dria

l sup

22

40302725

17

Left: Fluorescent imaging of sepals from a PCD-2 transformed Arabisopsis plant. Red auto-fluorescent chlorophyll indicates the location of chloroplasts in the sepals. Green punctate staining ,non-concordant with the chloroplasts , consistent with mitochondrial targeting of the GFP.

Right: Western blots of cell fractions from PCD-2 transformed Arabidopsis. plants. ATP6, GFP and PETD antibodies were used to determine the co-fractionation of these proteins by differential centrifugation. A mature, 25 kDa form of GFP co-fractionated with mitochondrial ATP6. A 40 kDa form corresponds to the predicted size of the primary translation product. Intermediate forms of 30 and 27 kDa were enriched in the post-mitochondrial supernatant. PETD was enriched in the 1,500 g pellet, which contained chloroplasts. PETD in the mitochondrial fraction might be attributed to broken thylakoids.

Figure 7. The N. crassa ATP9DL targets GFP to the mitochondria in a petunia petal transient expression assay

Petunia petals (Easy Wave) were inoculated at the base with Agrobacterium tumefaciens strain GV3101 (O.D. 0.05) carrying the PCD-3 construct. a - c) Time-lapse pictures of the same cell to illustrate the movement of the GFP-labeled mitochondria. a) and b) are 14 seconds apart and c) is 7 seconds after b). The red signal is autofluorescence from the chloroplasts. Bar=50µm

a) b) c)

a) Normal Arabidopsis flower stamen, pistil, sepals and petalsb) Abnormal floral structures of Arabidopsis transformed with PCD-3, consistent with silencing of the endogenous AP3 promoter. Sepals and pistil were normal. Stamens were converted to pistil-like structures and petals had sepal-like features. c) Abnormal floral structures of Arabidopsis transformed with PCD-5 were consistent with the ablation of petals and stamens. Sepals and pistil-like structures were observed.

Figure 8. Floral abnormalities observed in Arabidopsis plants expressing PCD-3 and PCD-5

Sepal

Petal

Pistil

Stamen Sepal

Pistil

Sepaloid petal

Stigmatic tissue

Ovules

Pistil

Sepal Pistilloid stamen

a) b) c)

Table1. A. thaliana (Lansberg) flowers after transformation with programmed cell death constructs 3 and 5 (PCD3 & 5)

Wild Type PCD-3 PCD-5

T0 Plants Flower PhenotypePCD3

1-1, 1-3, 1-4, 3-1, 3-2, 7-2, 7-4,8-1 and 9-1 Normal

PCD31-2,1-5 and 4-1

Silenced

PCD-57-1,7-2,8-1

Normal

PCD-55-1,

Ablated