constitutive expression of the inositol polyphosphate 5
TRANSCRIPT
Clemson University Clemson University
TigerPrints TigerPrints
All Theses Theses
August 2021
Constitutive Expression of the Inositol Polyphosphate 5- Constitutive Expression of the Inositol Polyphosphate 5-
Phosphatase Gene Alters Plant Development and Enhances Phosphatase Gene Alters Plant Development and Enhances
Abiotic Stress Tolerance in Creeping Bentgrass Abiotic Stress Tolerance in Creeping Bentgrass
Chen Chang Clemson University, [email protected]
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Recommended Citation Recommended Citation Chang, Chen, "Constitutive Expression of the Inositol Polyphosphate 5- Phosphatase Gene Alters Plant Development and Enhances Abiotic Stress Tolerance in Creeping Bentgrass" (2021). All Theses. 3587. https://tigerprints.clemson.edu/all_theses/3587
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CONSTITUTIVE EXPRESSION OF THE INOSITOL POLYPHOSPHATE 5- PHOSPHATASE GENE ALTERS PLANT DEVELOPMENT AND
ENHANCES ABIOTIC STRESS TOLERANCE IN CREEPING BENTGRASS
A Thesis Presented to
the Graduate School ofClemson University
In Partial Fulfillment of the Requirements for the Degree
Master of Science Biochemistry and Molecular Biology
by Chen Chang August 2021
Accepted by: Dr. Hong Luo, Committee Chair
Dr. Haiying Liang Dr. Guido Schnabel
ii
ABSTRACT
Inositol-1,4,5-triphosphate (IP3), a second messenger molecule and a very important
component of phosphoinositide (PI) signaling, participates in plant growth and response to
various abiotic stresses. Strict control of the IP3 balance is critical for normal plant
development. Type I Inositol polyphosphate 5-phosphatase (InsP 5-ptase) functions to
hydrolyze soluble inositol phosphates, such as IP3. It has previously been reported that
transgenic Arabidopsis, a dicotyledonous plant species overexpressing InsP 5-ptase exhibit
a sharply declined IP3 level, but enhanced tolerance to various environmental adversities,
indicating an important role the InsP 5-ptase plays in regulating phosphoinositide (PI)
signaling to mediate plant stress responses. To investigate how InsP 5-ptase is involved in
stress responses in monocots, we have generated transgenic creeping bentgrass (Agrostis
stolonifera L.), an important C3 cool-season turfgrass that constitutively expresses a
mammal type I InsP 5-ptase. Data obtained revealed that overexpression of InsP 5-ptase
gene alters plant development and leads to enhanced plant tolerance to drought, salt and
heat stresses associated with improved physiological parameters. Further characterization
of the InsP 5-ptase transgenic plants will allow a better understanding of InsP 5-ptase-
mediated plant stress response, providing information to develop novel biotechnology
approaches for crop genetic improvement.
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ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. Hong Luo to give me a chance to learn and
conduct my thesis research in his lab and to help me solve a lot of problems in research. I
thank my committee members, Dr. Guido Schnabel and Dr. Haiying Liang, for their
valuable suggestions. I also thank Qian Hu for teaching me plant tissue culture and I benefit
a lot from her experience. I thank Zihe Zhu, Rui Che, Yu Liu, and Xiaotong Chen for their
help during my thesis research.
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TABLE OF CONTENTS
Page
TITLE PAGE ....................................................................................................................... i
ABSTRACT ........................................................................................................................ ii
ACKNOWLEDGEMENTS ............................................................................................... iii
TABLE OF CONTENTS ................................................................................................... iv
LIST OF FIGURES .............................................................................................................v
LIST OF TABLES ............................................................................................................. vi
CHAPTER
1. LITERATURE REVIEW ............................................................................................... 1 Abiotic Stress ..................................................................................................... 1 The Response of Plants to Stresses ...................................................................... 3 Inositol-1,4,5-triphosphate (IP3) Signaling Pathways ........................................... 5 Inositol polyphosphate 5-phosphatase ................................................................. 8 Agrobacterium-mediated Plant Transformation ..................................................10
2. CONSTITUTIVE EXPRESSION OF THE INOSITAL POLYPHOSPHATE5-PHOSPHATASE GENE ALTERS PLANT DEVELOPMENT
AND ENHANCES ABIOTIC STRESS TOLERANCE IN CREEPING BENTGRASS ....................................................................13
Introduction .......................................................................................................13 Materials and Methods .......................................................................................16 Results ...............................................................................................................21 Discussion and Conclusion ................................................................................42
REFERENCE .....................................................................................................................49
v
LIST OF FIGURES
Figure Page
1. Molecular analysis of TG lines overexpressing InsP 5-ptase gene........................24 2. Development of wild-type (WT) and transgenic (TG) plants ................................27 3. Response of wild-type (WT) and transgenic (TG) plants to drought stress ..........34 4. Response of wild-type (WT) and transgenic (TG) plants to heat stress ................38 5. Response of wild-type (WT) and transgenic (TG) plants to salt stress .................41 6. A model of the IP3-mediated signaling pathway and the
InsP 5-ptase-regulated stomatal closure.............................................................45
vi
LIST OF TABLES
Table Page
1. Primer sequences were used in this study ..............................................................48 2. The mediums were used in this study ....................................................................48
1
CHAPTER ONE
LITERATURE REVIEW
Abiotic Stress
Although it is difficult to accurately estimate the impacts of abiotic stress on crop
production, it has become evident that the number of publications on the effects of abiotic
stress on plants has increased dramatically in recent years. In early 1982, Boyer foreboded
that the environmental factors may limit crop production by as much as 70% (Boyer et al.,
1982, Cramer et al., 2011). According to the 2007 FAO report, the global land area not
affected by environmental constraints only occupies 3-5%. With the reduction of arable
land, the decline of water resources, increased global warming due to the climate changes,
it is expected that the outputs of crops will dramatically decline in many areas in the future
(Colville et al., 2011; Cramer et al., 2011).
Abiotic stress is considered the negative effect of non-living factors on living things
in a specific environment (Ben-Ari et al., 2012). Most of the abiotic stresses for plants are
caused by the soil factors, such as the high concentration of salt; air pollution, such as acid
2
rain; and climate changes, which is considered the most critical factor, incurring various
stresses, such as drought and heat (Phil Riddel et al., 2003). Abiotic stresses, especially
salinity and drought, are considered the leading causes of global crop yield loss. Contrary
to the resistance of plants to biotic stresses (mainly depending on monogenic traits), the
genetically complex response to abiotic stresses is multigenic and more challenging to
identify and manipulate (Ben-Ari et al., 2012).
Plants need a lot of water and nutrients throughout their life cycle, and all aspects of
plant development will be affected by the reduction of water content in the soil (Sarker et
al., 2005). Drought can lead to nutrient deficiencies (even in the fertilized soil) due to the
decreased mobility and absorption of individual nutrients, leading to the reduced diffusion
rate of minerals from the soil matrix to the roots (Silva et al., 2001). Therefore, drought is
undoubtedly the most important stress factor that limits plant life. Drought can trigger a
variety of plant responses (Anjum et al., 2011). Plant growth changes, which are translated
into reduced leaf size, reduced leaves, less fruit yield, and changes in reproductive stages.
At the same time, excessive salt concentrations have a significant impact on plants. It can
cause osmotic stress and ion imbalance due to the accumulation of toxic ions (such as Cl-
and Na+). Salt stress also hurts mineral homeostasis, especially Ca2+ and K+ (Isayenkov et
al., 2012). In addition, high temperatures can cause significant damage to plants. At very
high temperatures, plants may suffer from severe cellular damage and even cell death
3
within minutes (Schöffl et al., 1999). Injuries or death occur in plants after long-term
exposure to moderately high temperatures. Direct damages caused by high temperatures to
plants include protein denaturation and increased membrane lipids fluidity. Indirect
thermal damages include inhibition of protein synthesis, protein degradation, inactivation
of enzymes in chloroplasts and mitochondria, and damage of membrane integrity (Howarth,
2005). These damages ultimately lead to growth inhibition, reduced ion flux, and
production of toxic compounds (Schöffl et al., 1999, Howarth, 2005). Therefore, tackling
the impact of drought, salinity, and high temperatures in agriculture is essential for
achieving food security worldwide (Rizwan et al., 2015). In the long-term evolution, plants
have formed many molecular, cellular, and physiological mechanisms to deal with these
abiotic stresses.
The Response of Plants to Stresses
The first step in plant response to abiotic stress is the perception of stress. Once plant
cells sense stresses, the signal is transmitted by second messengers, such as calcium ions,
nitric oxide (NO), reactive oxygen species (ROS), and different protein kinases (Kudla et
al., 2018; Testerink et al., 2011). Stress-induced increase in cytosolic Ca2+ concentration
4
can be detected in Arabidopsis guard cells within 15 seconds after osmotic stress treatment
(Yuan et al., 2014). The Ca2+ can then be detected by calcium-binding proteins, which
usually transfer the signal to interacting protein kinases, such as calcium-dependent protein
kinases (CPKs). ROS in plants can be accumulated by various organelles, such as
chloroplasts, mitochondria, and peroxisomes (Zhang et al., 2020). The accumulated ROS
can stimulate specific calcium and electrical signals, and also mediate transductions of
systemic signals in response to stress immediately (Choi et al., 2016). Various abiotic
stresses also promote phosphatidic acid (PA) production, which plays a positive or negative
role under different stress conditions (Hong et al., 2016; Testerink et al., 2011). In addition,
plants accumulate many organic and inorganic compounds such as amino acids (proline),
normal sugars (sucrose), and organic acids (oxalic acid) to protect cellular proteins under
stress conditions (Valliyodan et al., 2006). These osmoprotectants protect plant cells under
stress without affecting the biochemistry of the cellular environment (Kaur et al., 2020).
Stress signals in plants also involve different kinase families, including kinase families in
the mitogen-activated protein kinase (MAPK) module (Zelicourt et al., 2016)). For
example, MPK3, MPK4, and MPK6 can be activated within 2 minutes after exposure to
drought and salt stresses (Zhang et al., 2020). It is obvious that signaling transductions are
crucial during the entire regulation process of plants response to stress.
5
There are many types of signaling pathways in plant response to stresses, and the ABA
signaling pathway is one of them. The stress-induced biosynthesis of ABA mainly occurs
in vascular tissues, but ABA exerts its impact in various cells (Kuromori et al., 2010).
Therefore, the ABA response needs to be transferred from ABA-producing cells via cell-
to-cell transport to allow distribution into adjacent tissues rapidly (Danquah et al., 2014).
Under osmotic stress conditions, ABA can regulate expression of many genes. The ABA
response element (ABRE) is the main cis-element for ABA response to many gene
expressions. ABRE binding protein/ABRE binding factor transcription factors
(AREB/ABF TFs) regulate ABRE-dependent gene expression. Other transcription factors
are also involved in ABA-responsive gene expression. The SNF1-related protein kinase 2
is a crucial regulator of ABA signaling. In addition, studies have shown that the main ABA
signaling pathway interacts with other signaling factors in plant response to stresses.
Controlling the expression of ABA signaling factors can improve the tolerance of plants to
environmental stresses (Nakashima et al., 2013).
Inositol-1,4,5-triphosphate (IP3, InsP3) Signaling Pathways
6
All organisms need to respond to environmental stresses to survive. In order to
respond to extracellular signals, many organisms regulate the inositol-1,4,5-triphosphate
(IP3) signaling pathway. This pathway uses membrane-bound receptors coupled to the
second messenger IP3 (Berridge, 1993). Many pieces of evidence indicate that this
signaling pathway is used by plants (Munnik et al., 1998), and calcium ions release in
response to signals from this pathway to trigger downstream biological pathways
regulating plant development and stress response (Trewavas et al., 1998). For example,
gravity elicits increased IP3 in corn pulvini (Perera et al., 1999). Red light stimulates
intracellular Ca2+ release in etiolated wheat protoplasts by microinjection of IP3 (Shacklock
et al., 1992). Other signals that can generate second messenger IP3 include plant hormones.
It has been shown that endogenous IP3 levels increase within 2 minutes after the addition
of abscisic acid to the stomata (Berdy et al., 2001), and stomatal closure occurs by
microinjection of IP3 into the stomata in abscisic acid stimulation (Gilroy et al., 1990). In
addition to the role of IP3 as a second messenger in reversible turgor-driven processes (such
as regulating cellular osmotic homeostasis and stomatal aperture), increasingly more
evidence shows that long-term changes of IP3 may be associated with guiding differential
plant growth (Stevenson et al., 2000). Studies on the gravity-responsive pulvinal cells of
cereal grasses (Perera et al., 1999) and tip-growing cells (such as pollen tubes) (Kost et al.,
7
1999) have shown that the long-term increased synthesis of IP3 and PIP2 is involved in the
regulation of cell elongation (Perera et al., 2002).
IP3 is a second messenger molecule produced due to phospholipase C (PI-PLC)-
mediated reactions in response to stress (Drøbak et al., 2000). Phosphatidylinositol 4,5-
bisphosphate (PtdInsP2, PIP2), a rare phospholipid, plays an important signaling role in the
phosphoinositide (PI) signaling pathway. Its activation can release 1, 2-diacylglycerol
(DAG), which activates the protein kinase C (PKC), and IP3, which leads to Ca2+
mobilization by binding to the Ca2+ channel on the endoplasmic reticulum membrane. The
IP3 from hydrolysis of a small part of PtdInsP2 will increase and subsequently induce Ca2+
signal in several minutes under adversities (DeWald et al., 2001). Furthermore, IP3 can
diffuse signals rapidly. The transient increase of IP3 happens in plants to respond to
environmental stimuli under extreme stresses. The Ca2+ will activate various proteins in
the cytoplasm since IP3 increases the Ca2+ flow from the endoplasmic reticulum to the
cytoplasm to improve cell response. Meanwhile, the increased IP3 will increase sugar and
organic phosphate consumption with an increased primary metabolism (Khodakovskaya et
al., 2010).
In general, IP3 will be provoked by the cell stimulations and cellular control processes,
such as cell division, metabolism, differentiation, and cell migration, which finally
contribute to cell death (Vanderheyden et al., 2009). Consequently, it is essential to strictly
8
control the durability of IP3 and reduce it for its appropriate function (Perera et al., 2002).
At present, the hydrolysis mechanisms of IP3 in plants are not very clear. There is no
biochemical evidence of InsP3 3-kinase in plants (Brearley et al., 2000). However, there
are many reports on InsP phosphatase, indicating that the degradation of IP3 in plants is
mainly via dephosphorylation. Early biochemical studies (Drøbak et al., 1991) have shown
that both inositol polyphosphate 1-phosphatase (InsP 1-ptase) and inositol polyphosphate
5-phosphatase (InsP 5-ptase) are involved in IP3 hydrolysis (Brearley et al., 1997).
Although IP3 changes may be an essential part of the PI signaling pathway, it has been
difficult to associate these changes with specific physiological responses. Pharmaceutical
preparations (such as the aminosteroid PLC inhibitor U73122) have effectively inhibited
the IP3 production, blocking downstream reactions in specific plant systems (Staxen et al.,
1999; Takahashi et al., 2001). But this method has its limitations, mainly due to problems
in the uptake of the compound into intact plant tissues (Cho et al., 1995). Molecular
methods to decrease IP3 will have broader applicability, and the InsP 5-ptase enzyme is
considered an obvious target for operation (Perera et al., 2002).
Inositol polyphosphate 5-phosphatase
9
The inositol polyphosphate 5-phosphatases (InsP 5-ptases) comprise a large protein
family that can hydrolyze several specific lipids and soluble inositol phosphates. For
instance, the type I InsP 5-ptase can hydrolyze soluble inositol phosphates only, such as
IP3, while the type II InsP 5-ptase can hydrolyze both soluble and lipid inositol phosphates.
In mammals, the InsP 5-ptase is an enzyme that can catalyze triphosphate and
tetraphosphate into biphosphate and trisphosphate, respectively. It is easy to decrease the
level of IP3 by phosphorylation of D-3 position on inositol ring to create inositol 1,3,4,5-
tetrakisphosphate (InsP4) by InP3 kinase (Perera et al., 2002) or by dephosphorylation of
D-5 position to create inositol bisphosphate (InsP2) by InsP 5-ptase to terminate the signal
(Tsujishita et al., 2001). The InsP3 will be hydrolyzed into InsP2 by type I InsP 5-ptase and
deduce Ca2+ signal (Majerus et al., 2000) because increased InsP3 will raise the release of
Ca2+ (Finch et al., 1991).
In type I InsP 5-ptase transgenic tobacco, the increased level of InsP2 from increased
hydrolysis of InsP3 indicated the importance of up-regulated phosphoinositide pathway and
the synthesis of InsP2 (Perera et al., 2002). In addition, the InsP 5-ptase either reduces or
delays the level of salicylic acid (SA) (Hung et al., 2014) (an essential fat-soluble organic
acid signaling molecule in abiotic stress response (Shah et al., 2003)) and SAR (a
progressive resistance from many uninfected organs when the plants' organs inoculated by
some pathogen (Ryals et al., 1996)). Moreover, the response
10
from Arabidopsis overexpressing InsP 5-ptase under drought stress was delayed, and the
level of abscisic acid (ABA) is lower than the wide-type (Perera et al., 2008). Additionally,
some defense genes, such as PR-1, PR-2, PR-5, also showed reduced or delayed levels in
plants overexpressing InsP 5-ptase when inoculated with pathogen compared with wide-
type (Hung et al., 2014). Transgenic tomatoes overexpressing InsP 5-ptase exhibited
declined InsP3, but increased drought tolerance, biomass, CO2 fixation, lycopene, and the
storage of hexoses and phosphate (Khodakovskaya et al., 2010).
Agrobacterium-mediated Plant Transformation
When manipulating gene expression in transgenic plants for trait improvement, two
major approaches have been used to transfer the target genes into plants: Agrobacterium-
mediated plant transformation and particle bombardment. The first method is the most
popular because it is easy to increase the transformation efficiency and can transfer large
fragments of DNA with defined ends (Komari et al., 1996). As a consequence, most
researchers use Agrobacterium-mediated plant transformation, which is better than other
ways. Agrobacterium-mediated plant transformation is the primary biological method for
the production of transgenic plants.
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Agrobacterium is a bacterium ubiquitous in the soil, infecting the injured parts of most
dicotyledonous plants. As the cells from injured areas are secreting lots of phenolic
compounds, Agrobacterium can move to these cells. The Agrobacterium
tumefaciens contains a Ti plasmid, in which there is a section of Transferring DNA (T-
DNA) (Gelvin et al., 2003). Ti plasmids can replicate in Agrobacterium and E. coli. A
binary vector consists of a T-DNA region (Transferring DNA that can move into plant
cells), vector backbone (with the replication origin of E. coli and Agrobacterium), and Vir
region (help T-DNA get into plant cells) (Komori et al., 2007). Upon Agrobacterium
tumefaciens infection, T-DNA enters plant cells and then can integrate into the plant
genomes. Then this gene can be stably passed on to the offspring through meiosis. This
feature makes the Agrobacterium-mediated plant transformation become the primary
method for researches in transgenic studies. Researchers insert the transgene into modified
T-DNA regions, then that gene integrates into plant cells by Agrobacterium plant cell
infection. Transgenic plants subsequently regenerate through plant tissue culture
techniques. In the beginning, Agrobacterium-mediated plant transformation was only used
in dicotyledonous plants, but, in recent years, Agrobacterium-mediated transformation has
also been widely used in many monocotyledonous plants (Gelvin et al., 2003).
How does the Agrobacterium tumefaciens transfer the gene into plant cells? Firstly,
damaged plant cells produce phenolic substances as a sign of Agrobacterium infection. The
12
induction of these chemical substances will pass through the cell membrane of
Agrobacterium, then activate VirA and VirG, and induce other genes on the Vir region. The
activated Vir region expresses VirD1 and VirD2. The VirD1 and VirD2 bind to both sides
of the T-DNA region to cut off the single-stranded T-DNA and deliver it into plants (Tzfira
et al., 2006).
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CHAPTER TWO
CONSTITUTIVE EXPRESSION OF THE INOSITAL POLYPHOSPHATE 5-
PHOSPHATASE GENE ALTERS PLANT DEVELOPMENT AND ENHANCES
ABIOTIC STRESS TOLERANCE INCREEPINGBENTGRASS
Introduction
Agriculture in the 21st century is facing daunting challenges. Abiotic stresses caused
by climate changes can significantly affect crop yields, for example flooding after high
temperatures and freezing damage after low temperatures. Upon exposure to environmental
stresses, plants show multiple impairments, including overproduction of ROS (such as
superoxide anion radicals (O2 ̄) and hydrogen peroxide (H2O2)), which leads to cell injury
(Wallace et al., 2016), decreased photosynthetic functions (Deeba et al., 2012), lipid
peroxidation, and increased frequency of programmed cell death processes (Gill et al.,
2010).
To adapt to environmental stresses, plants have evolved various acclimation
mechanisms. The perception of abiotic stress conditions induces a signaling cascade that
activates many downstream regulatory processes in plants, including antioxidant defense
14
systems and osmotic adjustments, (Fu et al., 2001; Khaleghi et al., 2019), ion channels,
kinase cascades, and the accumulation of plant hormones (such as SA, ethylene, and ABA).
Under stress conditions, soluble sugars and proline accumulate in various plants as
osmolytes to help stabilize membrane proteins and ultimately improve plant resistance to
stresses (Ashraf et al., 2007; Per et al., 2017). In addition, ROS scavenging enzymatic
antioxidants, such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)
can be activated to remove excessive ROS (Gill et al., 2010). These signals will eventually
induce the expression of a specific subset of defense genes, leading to the assembly of the
overall defense responses (Jaspers et al., 2010). Signaling pathways are considered the
most important part of the plant stress response mechanisms.
In plants, the phosphoinositide (PI) pathway is considered a particularly important
signaling pathway. Upon stresses, PtdInsP2 is hydrolyzed by PLC to produce IP3 and DAG
(Berridge, 1993). InsP3 can act as soluble second messengers to mediate the release of Ca2+
(Sanders et al., 1999). The rapid transient increase of InsP3 has been confirmed in various
plant tissues in response to environmental stimuli. For IP3 to function as second messengers,
the IP3 signal must be strictly regulated, and the signal rapidly degraded to impact temporal
and discrete response (Perera et al., 2002). As the most important component of the PI
signaling pathway, IP3 was reported to be reduced in transgenic tobacco overexpressing
InsP 5-ptase leading to enhanced stress resistance in plants (Perera et al., 2002).
15
Human type I InsP 5-ptase was found to have about two folds of magnitude higher
activity for hydrolysis of IP3 than the plant InsP 5-ptase enzyme (Perera et al., 2002). The
mammal type I InsP 5-ptase was also found to be able to induce the expression of a drought-
responsive gene, DREB2A, in transgenic Arabidopsis (Perera et al., 2008). Transgenic
tomato overexpressing a mammal type I InsP 5-ptase exhibited increased biomass (Mariya
et al., 2010). So far, research in different dicotyledonous plants overexpressing the
mammal type I InsP 5-ptase has demonstrated the important role the InsP 5-ptase plays in
plant stress response. Its biological function in monocots, especially important monocot
crop species remains unclear.
Creeping bentgrass (Agrostis stolonifera L.) is a cool-season lawn grass suitable for
golf courses. It can also be used in parks, factories, mines, institutions, schools, and urban
green spaces. It is a good material for soil protection and has a wide range of uses and
strong adaptability. The current research aims to study the feasibility of using InsP 5-ptase
as a new candidate gene to genetically engineer creeping bentgrass, an important monocot
grass species, for enhanced performance under adverse environmental conditions. We
show that overexpression of a human type I InsP 5-ptase gene in transgenic creeping
bentgrass leads to altered plant development and an enhanced tolerance to drought, salt,
and heat stress, indicating that InsP 5-ptase gene is a good candidate for use in genetically
engineer monocot species for improved performance under environmental adversities.
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Materials and Methods
Plasmid Construction
A binary vector pSB11 (Komari et al., 1996) was used to prepare the InsP 5-ptase
expression chimeric gene construct, p35S-bar/Ubi-InsP 5-ptase (pHL083) (Fig. 1a) for
turfgrass transformation. The chimeric gene construct contains the cauliflower mosaic
virus 35S (CaMV35S) promoter driving the selectable marker gene bar and corn ubiquitin
promoter driving the InsP 5-ptase gene. The plasmid was generated by cloning the InsP 5-
ptase gene expression cassette from PCR-Blunt-InsP 5-ptase into pSBbarUbiGUS (Hong
Luo, 12/97). The chimeric gene construct was mobilized into Agrobacterium tumefaciens
strain LBA4404 by electroporation for the subsequent plant transformation (Li et al., 2010).
Agrobacterium-mediated Plant Transformation
Creeping bentgrass cv. Penn A-4 (provided by HybriGene, Hubbard, Oregon, USA)
was used for the plant transformation in this study. The TG creeping bentgrass stably
17
expressing InsP 5-ptase were produced using Agrobacterium-mediated embryogenic callus
(produced by Surface-sterilized treatment) transformation from mature seeds (Li et al.,
2010).
Plant Propagation and Stress Treatments
Transgenic plants expressing InsP 5-ptase were transferred in mixture soil (Fafard 3-
B Mix, Fafard Inc., Anderson, SC, USA) or pure silica sand, and maintained in the
greenhouse under 16-hour photoperiod with supplemental lighting at 27 °C in the light and
25 °C in the dark (Li et al., 2010).
To produce lots of plant materials for use in assessing plant response to stresses, wild-
type (WT) controls and transgenic creeping bentgrass (TG) were clonally propagated from
stolons in the small cone-tainer (five individual stolons per cone-tainer) (Dillen Products,
Middlefield, OH, USA). They were grown in the greenhouse for 4 weeks under the
aforementioned conditions firstly. They were then transferred to a growth room for 10
weeks under a 14-hour photoperiod, during which the grass shoots were trimmed every
other week to achieve uniform plant growth. The temperature was maintained at 25°C in
the light and 17°C in the dark, and the relative humidity is 30 in the light and 60% in the
18
dark. Water the plants with 200 ppm of water-soluble fertilizer (20-10-20 Peat-Lite Special;
The Scotts Company, Marysville, OH, USA) every other day (Li et al., 2010).
The drought, heat, and salinity treatments were performed after ten weeks of
maintenance in the growth room. Experimental groups were started by watering every day
without fertilizer, while control groups were treated without water for drought treatment.
Experimental groups were started by watering every day with 10 ml (for cone-tainers) of
200 ppm 20-10-20 fertilizer supplemented with 150 mM NaCl, while control groups were
started by watering every day with 10 ml (for cone-tainers) of 200 ppm 20-10-20 fertilizer
without NaCl for salt treatment. Experimental groups were started by 42 °C growth
conditions, while control groups were treated at room temperature for heat treatment. They
were started by watering every day with 10 ml (for cone-tainers) of 200 ppm 20-10-20
fertilizer (Li et al., 2010).
Molecular Analysis
The cetyltrimethylammonium bromide (CTAB) method was used to isolate plant
genomic DNA (from 0.1 g of young leaves) (Luo et al., 1995). The insertion of the
transgene into TG plants host genomes was confirmed by PCR (BioRad MJ MiniThermal
19
Cycler (Bio-Rad Laboratories, Inc., CA)) amplification of 430 bp fragment of InsP 5-ptase
gene and a 440 bp fragment of the selectable marker gene bar. The two primers used for
InsP 5-ptase gene amplification were InsP5-PCR-F (5’-AAT CCC AGG AGC ACT TCA
CG-3’) and InsP5-PCR-R (5’-GGA ATC CAG CCG GAA GTT GA-3’). The design of the
bar gene (bar-F and bar-R) was described by Li (Li et al., 2010). The PCR production was
fractionated through a 0.8% (w/v) agarose gel.
RNA was extracted from 0.1g of the young leaves of TG and WT plants with Trizol
reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I. The
expression of the transgene into TG plants host genomes was confirmed by RT-qPCR
(BioRad IQ5 Real-time PCR System (Bio-Rad Laboratories, Inc., CA)) amplification of
85 bp fragment of InsP 5-ptase gene (The two primers used for InsP 5-ptase gene
amplification were InsP5-qF and InsP5-qR) and a 100 bp fragment of the bar gene (The
two primers used for bar gene amplification were pKT-Bar-qF and pKT-Bar-qR). The
qPCR production was fractionated through a 2.0% (w/v) agarose gel.
Measurement of Physiological Parameters
Leaf Relative Water Content (RWC)
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Leaf RWC was evaluated using the following formula: RWC=[(FW-DW)/(TW-DW)]
× 100%, where FW is the fresh weight, TW is the turgid weight, and DW is the dry weight.
The leaves from WT and TG plants were harvested and immediately weighed (FW). TW
was weighed by cutting them into pieces and immersed in Millipore water at 4 °C for 16
hours. After measuring TW, the leaves were dried in the oven at 80°C for 24 hours and
weighed (DW) (Li et al., 2010).
Leaf Electrolyte Leakage (EL)
Leaf EL was measured using the following formula: EL%=(Ci/Cmax) * 100% to
evaluate cell membrane stability. For EL analysis, fresh leaf fragments (0.5 g) of each
sample were incubated in 20 ml of Millipore water at 4 °C for 16 hours. A conductance
meter (AB30, Fisher Scientific, Suwanee, GA, USA) was used to measure the incubation
solution’s initial conductance (Ci) to estimate the number of ions released from cells under
different conditions. The leaf tissue in the incubation solution was then heated in
autoclaving for 30 minutes. After autoclaving, the conductance (Cmax) of the incubation
solution was measured after 24 hours of incubation on a shaker (Li et al., 2010).
21
Proline content
Proline concentration was measured from a standard curve and used the following
formula: proline μmol g-1= [(proline μg/ml * extraction buffer ml)/115.5 μg μmol-1]/g
sample. Proline was extracted from 0.1 g of plant leaves of WT and TG plants by grinding
in 2 ml of 3% sulfosalicylic acid. 200 μl of the extract was reacted with 200 μl of acid
ninhydrin and 200 μl of glacial acetic acid at 100°C for 60 minutes. Terminate the reaction
with an ice bath. 1 ml of toluene has been used to extract the reaction mixture. Read the
absorbance of the toluene layer at 520 nm in Thermo Spectronic BioMate 3 (Thermo
Electron Corp., Waltham, MA, USA) (Li et al., 2010).
Chlorophyll Content
Scissors were used to cut 0.1 g of fresh leaf tissue into small pieces. Pigments were
extracted by grinding in 10 ml of 85% acetone in a mortar and pestle for 5 minutes. Transfer
the homogenate into 15 ml Falcon tube, then spun at 3000 g for 15 minutes. Then transfer
the supernatant into new 15 ml Falcon tube, then made up to volume with 85% acetone.
The absorbance of the extract was measured at 663 and 644 nm in Thermo Spectronic
22
BioMate 3. The concentration of chlorophyll a and b were calculated by the following
formula (Li et al., 2010):
Chlorophyll a mg/g FW=1.07*(OD663)-0.094(OD644)
Chlorophyll b mg/g FW=1.77*(OD644)-0.280(OD663)
Total chlorophyll mg/g FW= Chlorophyll a mg/g FW+ Chlorophyll b mg/g FW
23
Results
Production of Transgenic Creeping Bentgrass Overexpressing InsP 5-ptase Gene
To investigate the possible involvement of InsP 5-ptase in determining plant
adaptation to abiotic stresses in grass species, we prepared a chimeric gene construct in
which a mammal type I InsP 5-ptase gene was under the control of a constitutive corn
ubiquitin promoter linked to a CaMV35S promoter driving a herbicide resistance gene, bar
(Fig. 1a). This construct was introduced into creeping bentgrass “Penn A-4” by
Agrobacterium-mediated plant transformation. We obtained six independent transgenic
(TG) lines (all these transgenic lines were from different callus). We subsequently detected
the insertion of both bar gene and the InsP 5-ptase gene in all six TG lines using PCR on
genomic DNA (Fig. 1b, c). Overall, the six TG lines appeared to grow more erectly than
the creeping wild type (WT) controls (Fig. 2a),and could be divided into two distinct groups
based on other characteristics in morphology (Fig. 2), especially the chlorophyll content
(Fig. 2j). TG group 1 plants (TG 1, TG 4 and TG 6) had darker green leaves with a higher
chlorophyll content than TG group 2 (TG 2, TG 3 and TG 8) (TG group 2 exhibited a
significantly lower average chlorophyll content than TG group 1). RT-qPCR analysis of
24
bar gene revealed transgene expression in all TG lines, but not in WT control plants (Fig.
1d). Interestingly, TG lines in group 2 exhibited a higher transgene expression than those
in group 1. Further analysis of the InsP 5-ptase gene expression from representative plants
in both groups confirmed the observation (Fig. 1e). TG8 from group 2 has a significantly
higher InsP 5-ptase gene expression than TG1 and TG6 from group 1, whereas no
significant difference was observed between TG 1 and TG 6 (Fig. 1e).
25
Figure 1. Molecular analysis of transgenic (TG) lines overexpressing InsP 5-ptase gene.
(a) InsP 5-ptase chimeric gene construct, Ubi-InsP5-ptase/CaMV35S-bar. The human type
I InsP 5-ptase gene, controlled by the corn ubiquitin promoter, was linked to an herbicide
resistance gene, bar, driven by the cauliflower mosaic virus 35s promoter. (b) The gel
electrophoresis of PCR product of the bar gene (about 440 bp) amplified from genomic
DNA of both TG and wild type (WT) plants. Control was the chimeric plasmid DNA from
(a) as the template. (c) The gel electrophoresis of PCR product of the InsP 5-ptase gene
using the InsP5-PCR-F and InsP5-PCR-R primers (the product length is about 430 bp).
Control was the chimeric plasmid DNA from (a) as the template. (d) RT-qPCR analysis
from 50 ng cDNA of TG lines and WT plants using the pKT-Bar-qF and pKT-Bar-qR
primers (the product length is about 100 bp). (e) RT-qPCR analysis using the InsP5-qF and
InsP5-qR primers (the product length is about 85 bp). TG 1 (from group 1) was used as a
reference to compare the relative transgene expression levels in group 1 and group 2 TG
lines in (d), (e). The significant difference was between TG 1 and other TG lines. Data are
presented as means of three biological replicates (n = 3), and the error bars represent
STDEV. The asterisk indicates a significant difference between the TG group 1 and group
2 by Student’s t-test (* is P<0.05; ** is P<0.01, and *** is P<0.001).
26
Transgenic Plants Overexpressing InsP 5-ptase Gene Exhibit Altered Growth and
Development
In order to study whether overexpression of InsP 5-ptase in creeping bentgrass affects
the overall plant growth and development, we conducted experiments to compare the two
groups of TG lines and WT control plants (Fig. 2). To this end, we chose one representative
from each TG group (TG 2 and TG 6) for further analysis (Figs. 2b-i). The results showed
that both in the early (8-week-old) or late plant growth stage (10-week-old), TG plants had
significantly fewer tiller numbers than WT controls, whereas no significant difference
between the two TG groups was observed (Fig. 2k). Interestingly, TG group 1 appeared to
have a more extended shoot growth although insignificantly, whereas group 2 exhibited a
shorter shoot than WT controls (Fig. 2b-f, l). No significant difference in internode length
was observed between WT and TG group 1 plants, but the group 2 TG plants exhibited a
significantly shorter internode length than WT and group 1 TG plants (Fig. 2g, m). Further
analysis revealed a pronounced difference in the leaf width between TG plants and WT
controls at both the early (4-week-growth) and late stage (8-week-growth) (Fig. 2h-i, o).
This difference decreased at the latter stage, but remained significant (Fig.2o). Although
there was no significant difference in root length (Fig. 2p), the root numbers of the TG
plants in their early stage (4-week-old), especially those of the TG group 2 plants, far
27
exceeded that of the WT controls (Fig. 2q). However, no significant difference in root and
shoot biomass was observed between TG and WT control plants (Fig. 2r). On the contrary,
a significant difference in leaf clipping was observed between TG and WT control plants
(Fig. 2s). The leaf clipping of the TG plants from both groups was significantly lower than
that of the WT controls (Fig. 2s).
28
29
Figure 2. Development of wild-type (WT) and transgenic (TG) plants. (a) Ten-week-old
WT and TG lines initiated from five tillers and grown under normal conditions. (b) and (c)
Eight-week-old WT and TG plants initiated from a single tiller in soil under normal growth
conditions. (d) and (e) Plants from (b) and (c), respectively, upon removal from soil and
washing with tap water. (f) The most extended shoot of WT and TG lines cut from (b) and
(c). (g) All the internodes of a representative tiller from the 8-week-old WT and TG plants
sliced from top to bottom and displayed from left to right. (h) and (i) Representative fully
developed top leaves taken from the representative tillers of 8-week-old WT and TG plants.
(j) Statistical analysis of the total chlorophyll content between WT and TG plants under
normal growth conditions. (k) Tiller numbers in WT and TG plants 5 and 10 weeks after
initiation from a single tiller. (l) The most extended shoots from WT and TG plants 4 and
8 weeks after initiation from a single tiller. (m) Statistical analysis of the average internode
length of a representative tiller between WT and TG plants 5 and 10 weeks after initiation
from a single tiller. (n) and (o) leaf blade length and width of the top representative leaf
from the most extended tiller in WT and TG plants. (p) The length of the most extended
30
root from WT and TG plants 4 and 8 weeks after initiation from a single tiller. (q) Root
numbers of the WT and TG plants 4 weeks after initiation from one tiller. (r) Statistical
analysis of root and shoot biomass between WT and TG plants. Fresh weight (FW) of all
shoot and root was determined with 8-week-old plants developed from a single tiller. Dry
weight (DW) was measured by incubating plant materials in an oven at 80 ℃ for 24 h. (s)
Ten-week-old WT and TG lines initiated from five tillers were mowed to the same height
every two weeks. The clippings were collected to measure the fresh and dry weight. Data
are presented as means of three or four biological replicates (n = 3 or 4), and the error bars
represent STDEV. The asterisk indicates a significant difference between the wild-type and
TG lines by Student’s t-test (* is P<0.05; ** is P<0.01, and *** is P<0.001).
Overexpression of InsP 5-ptase Results in Enhanced Drought Resistance in Transgenic
Plants
To test how TG creeping bentgrass overexpressing InsP 5-ptase responds to water
deficiency, we analyzed both TG and WT control plants subjected to drought stress. As
shown in Fig. 3a and b, three days after water withholding, the WT control plants were
31
seriously damaged and became withered, whereas the TG lines remained fresh and green,
hardly displaying any dehydration symptoms (Fig. 3a, b).
Relative water content (RWC), an indicator of plant water states (Mullan et al., 2012),
is a parameter reflecting how plants resist stress. The normal range of plant RWC is 85-98%
in fresh leaves and 30-40% in withered leaves (Barrs et al., 1962). A low RWC in plants
indicates a state of water shortage. Analysis of RWC in both WT and TG plants revealed
no significant difference before drought treatment and both WT and TG plants had a RWC
of about 80% (Fig. 3c). Upon three days of water withholding, the RWC of WT dropped
significantly to less than 10%, whereas that of TG plants remained as high as 50-70% (Fig.
3c), suggesting an enhanced water retention capacity in TG lines.
Electrolyte leakage (EL) reflects the degree of damage to the cell membranes (Cottee
et al., 2007). An increase in cell EL indicates an increase in cell membrane permeability
and decreased resistance to environmental stresses (Cottee et al., 2007). At the same time,
stress may change the biofilm's chemical composition and physical structure (Lauriano et
al., 2000, SENARATNA et al., 1987), which directly affects cell EL (Knowles et al., 2001).
For this reason, EL in plant leaves can be used to evaluate the adaptability of plants to
environmental stresses (Wilson et al., 2004). Under normal growth conditions, we
observed that InsP 5-ptase TG plants had significantly lower EL than WT controls, only
about 50% of that in WT (Fig. 3d). After drought treatment, the cell EL of WT increased
32
sharply from 15% to 60%, about 73% higher than that under the normal conditions,
whereas the EL increase in TG plants was only about 24%-35% (Fig. 3d).
When subjected to stress conditions, plants accumulate substances such as proline to
decrease the osmotic potential to protect themselves. Their value reflects the osmotic
adjustment function of plants under stress (Zhu et al., 2009). Generally speaking, plants
that can resist stresses will have more proline under normal conditions, and can accumulate
proline faster under stress conditions (Xin. 2020). We have measured protein content in
both TG and WT plants before and after water withholding and observed a considerable
accumulation of proline in WT control plants after drought treatment, whereas no apparent
proline accumulation occurred in TG plants upon drought stress compared to normal
growth conditions (Fig. 3e). This observation, contrary to the previous assumption, is quite
interesting since highly drought resistant TG plants exhibited a lower proline accumulation,
while WT plants highly susceptible to drought showed a significantly higher proline
accumulation.
It has previously been shown that drought stress affects the chlorophyll content of
leaves (Yi et al., 1995, Kozlowski et al., 1968). Meanwhile, the photosynthesis decrease
caused by non-stomatal inhibition is an essential physiological manifestation of plants
under drought stress (Yang et al., 1993). For example, both the photosynthetic intensity
and chlorophyll content decreased in Hippophae leaves under drought stress (Lin et al.,
33
1996). This suggests that the more the plant chlorophyll content declines, the less resistant
to drought the plants would become. We have therefore measured chlorophyll content in
both TG and WT control plants before and after water withholding and found that upon
drought stress, the chlorophyll content in WT plants dropped sharply, reducing by 25%
compared to the normal conditions (Fig. 3f). However, only a slight change in chlorophyll
content was observed in TG plants compared to the normal growth conditions. Especially,
the chlorophyll contents of TG 1 and TG 8 were reduced by 7%, while that of TG 6 was
only reduced by 4% (Fig. 3f).
Stress conditions can affect the stomatal closure. Under normal circumstances, the
stomata of plants are in open states because a certain amount of CO2 intake must be ensured
to maintain photosynthesis. Nevertheless, plants will quickly respond when subjected to
drought stress: increased release of ABA leads to the closure of the stomata (Chen et al.,
1999). That is, a decrease in stomatal conductance in plants indicates a drought stress
condition. It was further revealed that the sensitivity of stomata to the increased ABA
would increase under lower water potential, which means that the more severe the water
loss of the leaf, the more significant the decrease of stomatal conductance (Chen et al.,
1999). Meanwhile, photosynthesis will decrease with the closure of stomata. Under mild
drought stress, the main reason for photosynthesis decline is the closure of stomata. Under
severe drought stress, chloroplast decomposition strengthens with the protein
34
decomposition and chlorophyll content and photosynthesis reduction (Yang et al., 1993).
In our results, we found that the stomatal conductance of WT plants decreased sharply with
the decrease of leaf RWC (Fig. 3c, g). However, the stomatal conductance of TG plants did
not show significant change after three days of water withholding, remaining the same as
in the normal conditions. Furthermore, TG plants also maintained a significantly higher
photosynthesis rate than WT controls under drought stress (Fig. 3h) most likely due to the
suppressed stomatal closure and stable chlorophyll content (Fig. 3f, g). Therefore, the
suppression of stomatal closure under drought stress leads to an enhanced drought
resistance in TG creeping bentgrass overexpressing InsP 5-ptase.
35
Figure 3. Response of wild-type (WT) and transgenic (TG) plants to drought stress. (a)
Fourteen-week-old WT and TG lines developed from five tillers in sand before water
withholding. (b) WT and TG lines 3 days after water withholding. The back row shows
control plants grown under normal conditions. (c) Statistical analysis of RWC between WT
and TG plants before and 3 days after water withholding. (d) Statistical analysis of cell EL
between WT and TG plants before and 3 days after water withholding. (e) Statistical
analysis of proline content between WT and TG plants before and 3 days after water
withholding. (f) Statistical analysis of total chlorophyll content between WT and TG plants
before and 3 days after water withholding. (g) Statistical analysis of stomatal conductance
between WT and TG plants before and 3 days after water withholding. (h) Statistical
analysis of photosynthetic rate between WT and TG plants before and 3 days after water
36
withholding. Data are presented as means of three or four biological replicates (n = 3 or 4),
and the error bars represent STDEV. The asterisk indicates the significant difference
between the WT and TG lines by Student’s t-test (* is P<0.05; ** is P<0.01, and *** is
P<0.001).
Overexpression of InsP 5-ptase Gene Leads to Enhanced Heat Tolerance in Transgenic
Plants
It has previously been shown that tomatoes overexpressing InsP 5-ptase had enhanced
drought resistance (Khodakovskaya et al., 2010). However, it remains unclear how InsP 5-
ptase would impact plant response to other stresses. We therefore further investigated the
difference between WT and InsP 5-ptase TG plants under high temperature conditions. As
shown in Fig. 4, six days of heat stress at 42ºC caused severe damage in WT plants, which
were unable to recover, whereas TG plants barely showed any significant heat-elicited
symptoms and were all recovered (Fig. 4b, c).
Analysis of the relative water content (RWC) in WT and TG lines revealed that under
heat stress, the TG lines had an RWC of over 70%, more than twice as much as WT controls,
whereas there was no difference between them under normal growth conditions (Fig. 4d).
37
Usually, the RWC in a normal plant is about 85-95% (Xin, 2020), just like that of the TG
plants under high-temperature conditions (Fig. 4d). However, when the RWC is less than
60%, it indicates that the plant leaves are almost withered (Xin, 2020), just like the RWC
of WT plants under high-temperature conditions (Fig. 4d).
The electrolyte leakage (EL) of plant leaves will show an increase under heat stress
(Xin. 2020) because high temperatures destroy the integrity of the cell membrane. As
shown in Fig. 4e, the TG lines had a significantly lower cell EL than WT controls under
normal growth conditions. The cell EL increased sharply in both TG and WT plants when
subjected to heat stress. However, the EL of the TG lines was still lower than WT controls
although the difference was insignificant (Fig. 4e).
As discussed above, the more proline plants accumulate under stress, the more
resistant to stress the plants are. Under normal conditions, no significant difference in
proline content was observed between TG lines and WT controls (except TG 2 and TG 3).
Proline accumulation was significantly elevated in both WT and TG plants under heat
stress. However, the elevation in proline accumulation was more pronounced in TG plants
than in WT controls (Fig. 4f), indicating the enhanced heat tolerance in TG plants was
associated with elevated proline accumulation.
High-temperature stress will affect the chlorophyll content in plants and inhibit plant
photosynthesis (Xin, 2020). As shown in Fig. 4g, TG plants had a significantly lower total
38
chlorophyll content than WT controls under normal growth conditions. This difference was
decreased when plants were subjected to heat stress as the chlorophyll content of the WT
controls decreased by 46%, whereas that of the TG lines decreased only by 22% (Fig. 4g).
39
Figure 4. Response of wild-type (WT) and transgenic (TG) plants to heat stress. (a)
Fourteen-week-old WT and TG lines Developed from five tillers in sand before heat stress.
(b) Development of WT and TG lines 6 days after heat stress. (c) Development of WT and
TG lines 20 days after recovery from 6-day heat stress. The back row in (b) shows control
plants grown under normal conditions. (d) Statistical analysis of RWC between WT and
TG plants before and 6 days after heat stress. (e) Statistical analysis of cell EL between WT
and TG plants before and 6 days after heat. (f) Statistical analysis of proline between WT
and TG plants before and 6 days after heat stress. (g) Statistical analysis of total chlorophyll
between WT and TG plants before and 6 days after heat stress. Data are presented as means
of three or four biological replicates (n = 3 or 4), and the error bars represent STDEV. The
asterisk indicats a significant difference between WT and TG lines by Students’s t-test (*
is P<0.05; ** is P<0.01, and *** is P<0.001).
Transgenic Creeping Bentgrass Overexpressing InsP 5-ptase exhibits Enhanced Salt
Tolerance
TG creeping bentgrass overexpressing InsP 5-ptase exhibited enhanced resistance to
drought stress and high temperatures. To investigate if overexpression of InsP 5-ptase
40
would also impact plant response to salt stress, we applied 150 mM NaCl to both TG and
WT plants for 18 days and evaluate plant performance. As shown in Fig. 5b, although TG
and WT plants were both impacted by the stress displaying wilted and yellowing leaves,
TG plants exhibited less salt-elicited tissue damage, and the symptoms appeared later than
WT controls. Examination of RWC revealed no significant difference between TG and WT
control plants before treatment, but a significant reduction in WT controls after salt stress
(Fig. 5c). The RWC of the WT controls under salt stress was less than 60% (only about
48%), 40% lower than that under normal growth conditions, indicating that the leaves were
suffering from severe water shortage. On the contrary, the RWC of the TG plants remained
almost unchanged before and after salt stress (Fig. 5c). We also measured cell EL to check
plant cell membrane integrity and found that the cell EL of the TG plants was significantly
lower than that of the WT controls under both normal and salt stress conditions even though
both of their cell EL increased sharply upon stress (Fig.5d). These results indicated that
overexpression of InsP 5-ptase led to enhanced salt tolerance in TG creeping bentgrass
associated with maintained plant RWC and sustained cell membrane integrity.
41
Figure 5. Response of wild-type (WT) and transgenic (TG) plants to salt stress. (a)
Fourteen-week-old WT and TG plants developed from five tillers in sand before salt stress.
(b) Development of WT and TG lines 18 days after salt stress. The back row shows control
plants grown under normal conditions. (c) Statistical analysis of RWC between WT and
TG plants before and 18 days after salt stress. (d) Statistical analysis of cell EL between
WT and TG plants before and 18 days after salt stress. Data are presented as means of three
or four biological replicates (n = 3 or 4), and the error bars represent STDEV. The asterisk
indicates a significant difference between WT and TG lines by Student’s t-test (* is P<0.05;
** is P<0.01, and *** is P<0.001).
42
Discussion and Conclusion
Altered Plant Development in Creeping Bentgrass Overexpressing InsP 5-ptase May be
Associated with Modified ABA and Chlorophyll biosynthesis
Our results showed that TG creeping bentgrass overexpressing InsP 5-ptase exhibited
altered plant development with reduced leaf clippings and less chlorophyll production (Fig.
2s, j). This is different from previous studies in transgenic Arabidopsis (Perera et al., 2008).
Overexpression of InsP 5-ptase did not adversely affect plant growth. TG Arabidopsis had
less ABA accumulation than WT controls (Perera et al., 2008). ABA is an important plant
hormone that can inhibit plant growth (Nambara et al., 2017). Interestingly, the reduced
ABA accumulation in TG Arabidopsis did not show impaired plant growth. On the contrary,
InsP 5-ptase TG creeping bentgrass had reduced leaf clippings and decreased internode
length, especially in the group 2 TG plants (Fig. 2s, m). Most likely, the overexpression of
the InsP 5-ptase gene may have led to modified IP3 level in TG plants, altering ABA
biosynthesis, which negatively impacts plant growth. The fact that group 2 TG creeping
bentgrass exhibited more severely impacted internode growth than group 1 TG plants
indirectly supports this hypothesis. Further analysis of ABA level in TG plants compared
43
to WT controls would provide information to better understand impacted plant growth by
IP3-ABA module.
In this study, we also observed that TG plants exhibited a reduced chlorophyll content,
especially in group 2 TG plants, displaying a pale green leaf color (Fig. 2). It has previously
been shown that TG tomato plants with increased InsP3 hydrolysis in the cytosol exhibited
increased net CO2-fixation in source leaves (Khodakovskaya et al., 2010). Interestingly,
the rate of CO2-fixation in soybean was found to be four times faster in pale green plants
than in dark green plants (Koller et al., 1974). We speculate that a higher InsP 5-ptase
expression level in group 2 TG creeping bentgrass may cause more IP3 hydrolysis, leading
to impaired chlorophyll biosynthesis and therefore pale-green leaf color. It might also result
in more and faster net CO2-fixation. Further analysis of TG plant IP3 level and CO2-fixation
rate would provide evidence validating this hypothesis.
Enhanced Drought and Heat Resistance in TG Creeping Bentgrass Overexpressing InsP
5-ptase Is Likely Associated with Up-regulated DREB2A Expression
In the present study, TG creeping bentgrass overexpressing InsP 5-ptase exhibited
significantly enhanced drought (Fig. 3) and heat tolerance (Fig. 4). This is consistent with
44
a previous observation in TG Arabidopsis overexpressing InsP 5-ptase, which also
exhibited enhanced drought resistance (Perera et al., 2008). The enhanced drought
resistance was found to be associated with an up-regulated expression of DREB2A, a
dehydration-responsive element-binding protein 2A transcription factor gene (DREB2A)
(Perera et al., 2008). DREB2A was found to be highly expressed in drought and salt
treatment in an ABA-independent pathway (Liu et al., 1998). The intact DREB2A protein
cannot activate downstream genes under normal conditions. It needs posttranslational
modification to remove the negative regulatory region (NRD) for activation (Sakuma et al.,
2006). Similarly, we speculate that the enhanced drought and heat resistance in TG
creeping bentgrass overexpressing InsP 5-ptase was likely caused by IP3-mediated up-
regulation of the DREB2A gene, triggering downstream drought and heat resistance gene
expression.
In addition, InsP 5-ptase TG creeping bentgrass showed an enhanced drought
tolerance associated with a lower proline accumulation and a non-suppressed stomatal
conductance. This was probably because the three-day water withholding was perceived as
normal condition to TG plants, so the mechanisms regulating proline accumulation and
stomatal conductance change did not need to be activated to protect themselves from
drought stress. In fact, the increased IP3 hydrolysis in TG plants would cause a decreased
Ca2+ signaling and lead to non-suppression of H+-ATPases and inward-rectifying K+
45
channels, and therefore causing the suppression of stomatal closure (Fig. 6) (Blatt et al.,
1990; Lemtiri-Chlieh et al., 1994; Kim et al., 2010).
Figure 6. A model of the IP3-mediated signaling pathway and the InsP 5-ptase-regulated
stomatal closure. The stress-stimulated ABA signals, the first messenger, are received by
G protein, which then activates phospholipase C (PLC) to hydrolyze PIP2 into IP3 and DAG
(secondary messengers), triggering the transfer of more Ca2+ ions into the cytoplasm to
cause plant cell response to stresses. Meanwhile, the Ca2+ signal will inhibit the inward-
46
rectifying K+ channel to induce stomatal closure, while the InsP 5-ptase induces the
inhibition of stomatal closure by reduced IP3. The blue arrows are the regular regulations
of WT under drought stress, while the red arrows are the plants overexpressing InsP 5-
ptase.
Enhanced Salt Tolerance in the TG Creeping Bentgrass Overexpressing InsP 5-ptase May
be Associated with Altered ROS Production and Salt-responsive Gene Expression
Our results also showed that TG creeping bentgrass overexpressing InsP 5-ptase
exhibited enhanced salt tolerance (Fig. 5). It has previously been reported that the T-DNA
insertion mutant of Arabidopsis thaliana Inositol Polyphosphate 5-Phosphatase7
(At5PTase7) gene increased salt sensitivity, whereas overexpression of At5PTase7 in TG
plants increased salt tolerance (Kaye et al., 2011). Ten to fifteen minutes after salt treatment,
the At5PTase7 mutant Arabidopsis plants exhibited reduced production of ROS in roots.
In addition, the expression of salt-responsive genes (such as RD29A and RD22) was not as
highly induced in the mutants as in the wild type under salt stress (Golani et al., 2013).
This suggests the important role InsP 5-ptase gene play in regulating ROS accumulation in
plants and the expression of stress-related genes, such as RD29A and RD22. Most likely,
47
Overexpression of InsP 5-ptase gene in TG creeping bentgrass impacted plant ROS balance
and the expression of RD29A and RD22 or other stress-related gene, leading to improved
salt tolerance. Further analysis of ROS accumulation and different stress-related gene
expression in TG creeping bentgrass compared with WT controls would provide
information to better understand the molecular mechanisms underlying IP3-mediated plant
salt tolerance.
48
Table 1. Primer sequences were used in this study.
Table 2. The mediums were used in this study.
49
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