functional analysis of flrs and flr in wheat during
TRANSCRIPT
Functional analysis of FLRS and FLR in wheat during drought stress
By Sarah Alsaiari
A thesis Submitted to the Faculty of Graduate Studies and Research of Carleton University, in
partial fulfillment of the requirements for the degree of a Master of Science in Biology.
Department of Biology
Carleton University
Ottawa, ON, Canada
2012
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ACKNOWLEDGEMENTS
I would like to thank my supervisor, Dr. Tim Xing, for giving me the opportunity to
complete my Master degree at Carleton University. I am also grateful for the financial
support provided by Saudi Culture Bureau. I would also like to thank Dr. Ashkan Golshani
and Dr. Doug Johnson for being my advisory committee members and giving many helpful
comments and suggestions on this project. Final thanks must be given to my family, lab
members Kelly , Lubna El-Sarji and Kristina Sabou for their support throughout Carleton
University.
TABLE OF CONTENTS
Table of Contents i
List of Figures iv
List of Tables v
List of Abbreviations vi
Abstract 1
Chapter I:
Introduction 3
1.0 Plant and plant stress 3
1.1 Protein phosphorylation 4
1.2 MAPK in plant response pathways 5
1.3 Programmed cell death 12
1.4 My objectives 14
Chapter II:
Materials and Methods 18
2.1 Plant materials and growth conditions 18
2.2 Drought treatment 18
2.3 RNA extraction 19
2.4 RT-PCR 21
2.4.1 RT-PCR for FIR and FIRS 21
2.4.2 RT-PCR for Wtcbf2-2 21
i
2.5 Protein isolation and western blotting 22
2.6 Determination of protein concentration 23
2.7 One dimensional SDS-PAGE 26
2.8 Chlorophyll analysis 28
2.9 Trypan blue staining 29
2.10 Bioinformatics analysis 29
Chapter III:
Results 31
3.1 Transcriptional regulation of FLR and FLRS genes by drought stress 35
3.2 ERK-like MAPKs are post-translationally regulated during drought treatments 40
3.3 The effect of ERK inhibitor, general protein kinase inhibitor and drought on the expression of Wtcbf2-2 43
3.4 Drought stress induces programmed cell death 46
3.5 Chlorophyll analysis for wheat (Fielder) under drought stress 51
3.6 Bioinformatics analysis of expression profiles FLRS and FLR genes in wheat 53
3.6.1 Analysis of FLRS 55
3.6.2 Analysis of FLR 61
Chapter IV Discussion 67
4.1 FLR and FRLS genes do not regulate plant response to drought at the transcriptional level 69
4.2 FLR may regulate drought response at post-translational level 71
ii
4.3 Drought induces cell death inwheat 72
4.4 The effect of ERK inhibitor, general protein kinase inhibitor and drought on the expression of Wtcbf2-2 gene 73
4.5 Bioinformatics analysis 74
4.6 Conclusion 75
References 76
iii
LIST OF FIGURES
Figure 1.1: The mitogen-activated protein kinase cascades in plants 6
Figure 1. 2: The MAPK cascade consists of three different steps. MAPKKK 8
Figure 1.3: The three MAPK modules (ERKs, JNKs, p38), showing stimuli, the three tiers regulatory cascade within each module (MAPK, MAPKK and MAPKKK levels) 10
Figure 3.1: Expression of FLR and FLRS genes under different levels of drought exposure in wheat (Fielder) leaves using RT-PCR analysis 36
Figure 3.2: Expression of FLR and FLRS genes under different level of drought exposure in wheat (Fielder) leaves using RT-PCR analysis 38
Figure 3.3: ERK-type MAPK (including FLRS) is activated post-translationally during drought stress 41
Figure 3.4: The effect of ERK inhibitor, general kinases inhibitor and drought on the expression of Wtcbf2-2 44
Figure 3.5: Trypan blue staining of drought treated wheat (Triticum aestivum, CV Fielder) leaves afterexposure to different levels of drought 47
Figure 3.6: Macroscopic analysis of drought treated wheat (Triticum aestivum, CV Fielder) leaves afterexposure to different levels of drought 49
Figure 3.7: Level of chlorophyll content after drought treatment 52
Figure 3.8: Three steps of bioinformatics analysis 54
Figure 3.9: Overall expression of FLRS (Ta.23916.1.S2_2_at) during different stages of development from wheat (Triticum aestivum) 56
Figure 3.10.A: Effect of drought on FLRS (Ta.23916.1 .S1_at) expression in roots of wheat (Triticum aestivum) 57
Figure 3.10.B: Effect of drought on FLRS (Ta.23916.1.S1_at) expression in control roots of wheat (Triticum aestivum) 57
Figure 3.11 .A: Effect of drought on FLRS (Ta.23916.1 ,S1_at) expression in leaves of wheat (Triticum aestivum) 58
Figure 3.11.B: Effect of drought on FLRS (Ta.23916.1.S1_at) expression in control leaves of wheat (Triticum aestivum) 58
iv
Figure 3.12: FLRS (Ta.23916.1.S1_at) expression levels in different wheat cultivars exposed to drought stress 59
Figure 3.13: High stringency motif scan ouput for FLRS amino acid sequence indicating possible protein phosphorylation sites 60
Figure 3.14: Overall expression of FLR (Ta.12668.1_at) during different stages of plant development from wheat (Triticum aestivum) 62
Figure 3.15.A: Effect of drought on FLR (Ta.12668.1_at) expression in leaves of wheat (Triticum aestivum) 63
Figure 3.15.B: Effect of drought on FLR (Ta.12668.1_at) expression in control leaves of wheat (Triticum aestivum) 63
Figure 3.16.A: Effect of drought on FLR (Ta.12668.1_at) expression in roots of wheat (Triticum aestivum) 64
Figure 3.16.B: Effect of drought on FLR (Ta.12668.1_at) expression in control roots of wheat (Triticum aestivum) 64
Figure3.17: FLR (Ta.12668.1_at) expression levels in different wheat cultivars exposed to drought stress 65
Figure 3.18: High stringency motif scan ouput for FLR amino acid sequence indicating possible protein phosphorylation sites 66
LIST OF TABLE
Table 2-1: Dilutions of BSA solution used for strandard curve preparation 25
v
ABBREVIATIONS
cDNA: complementary DNA
DEPC: diethylpyrocarbonate
Kb: kilobase
kDa: kilo dalton
PCD: programmed cell death
MAPK: mitogen-activated protein kinase
mRNA: messenger RNA
NCBI: National Center for Biotechnology Information
PAGE: polyacrylamide gel electrophoresis
PCD: programmed cell death
PVDF: polyvinylidine fluoride
RT-PCR: reverse-transcriptase polymerase chain reaction
ROS: reactive oxygen species
SDS: sodium dodecyl sulphate
Abstract
The MAPK cascade plays an important role in intracellular signal transduction
processes involved in response to abiotic stress such as drought, high salinity, and low
temperature. Plants respond to abiotic stress through the activation and coordination of
different pathways. The regulation of some of these pathways requires the
phosphorylation of proteins by MAP kinase cascade. This protein kinase cascade is
composed of three classes of enzymes: MAPK, MAPK kinase (MAPKK), and MAPK
kinase kinase (MAPKKK). Our study has implicated that at the transcriptional level, the
level of FLR (a wheat MAPKKK) and FLRS (a wheat MAPK) gene transcripts do not
change during drought stress. This suggests that FLR and FLRS may not play a role in
wheat response to drought stress at the transcriptional level. However, ERK-type
MAPKs (including FLRS) may be involved in the signalling pathway at post-translational
level as seen by Western blot analysis with specific phospho-ERK antibody.
Programmed cell death (PCD) is associated with the accumulation of reactive oxygen
species (ROS) in wheat and the level of PCD increased during drought stress. The
transcription of Wcbf2-2 gene did not change during drought stress in the presence of
ERK inhibitor or general protein kinase inhibitor, suggesting Wtcbf2-2 did not respond to
drought stress in wheat. This may also indicate that Wtcbf2-2 might not be downstream
of ERK-type MAPK pathway in wheat. Bioinformatics analysis has indicated that there is
no significant difference in FLR and FLRS expression level between drought tolerant
and drought sensitive genotypes. Motif analysis has identified the possible
1
phosphorylation sites for FLR as T104, T149, and T958, and for FLRS as T365, T617,
T1028 and T1753. Work in this thesis shed light on the role of MAPK in plant response
to abiotic stress and more specifically drought stress.
2
Chapter I
Introduction
1.0 Plant and plants stress
Plants struggle to survive, though plants have evolved mechanisms to defend
themselves against different environmental stresses which include a rage of bacterial,
fungal, cold, drought, hyper osmotic conditions and even nematode and insect
pathogens with different lifestyles. In addition, environmental stresses attack plants in
different ways, some colonize the tissue within the plant, others settle on the surface of
the plant, and others may attack specific organs such as the roots, stems, and leaves
that cause problems like tissue death, browning, a decrease in fruiting, problems with
setting flowers, and may result in death of the host plant. For example, drought limits
plant growth due to a decrease in photosynthesis, osmotic stress-imposed constraints
on plant processes and interference with nutrient availability as the soil dries (Huang et
ai, 2011). Often healthy plants can control the attack and defend themselves by various
mechanisms. Most cellular defence responses are activated when receptor proteins
directly or indirectly detect environmental presence resulting in the triggering of ion
channel gating, oxidative burst, cellular redox changes, protein kinase cascades, or
other responses that directly activate cellular changes (such as cell wall reinforcement),
or activate changes in gene expression to defend the plant. The ability of plants to adapt
to environmental stress is dependent on the activation of a cascade of molecular
3
networks involved in stress perception, signal transduction, and the expression of
specific stress related genes and metabolites.
1.1 Protein phosphorylation
Protein phosphorylation is one of the best studied post-translational processes in
plants and it plays an important role in activating pathways that respond to
environmental stress and pathogen attacks (Umezawa et al., 2004). In addition,
phosphorylation plays an important role in regulating and co-ordinating the interaction
among protein partners that must form complexes to function and this usually occurs on
serine, threonine, tyrosine and histidine residues in eukaryotic proteins (Pawson, 1995).
The phosphorylation and dephosphorylation of proteins not only regulates plant defence
response but also plays a role in the activation of developmental processes such as
metabolism, cell signalling, cell differentiation and membrane transportat (Hunter et al.,
1995; Xing et al., 2002). Recent studies have demonstrated the involvement of protein
phosphorylation in almost all signalling pathways in plants (Luan, 2003). As such the
phosphorylation of a protein can alter its behaviour in almost every conceivable way
including the modulation of intrinsic biological activity, subcellular location, half-life and
docking with other proteins and DNA (Xing et al., 2002). Previous studies showed that
plants contain many kinases and phosphatases (Zhang and Klessing, 2001; Xing et al.,
2002). In the Arabidopsis genome, there are about 1000 protein kinase genes and 200
phosphatase genes (Tena et al., 2001). The ability of protein kinases to amplify weak
signals is displayed when one active kinase is able to phosphorylate hundreds of target
proteins. This is done when specific protein kinases act in series giving a cascade.
4
Mitogen-activated protein kinase (MAPK) cascades are the best examples of the key
phosphorylation pathways.
1.2 MAPK in plant stress response pathways
Plants are able to develop mechanisms to rapidly sense a changing environment
or physical damage and have the ability to protect themselves from abiotic and biotic
stresses (Takahashi et al., 2007). Mitogen-activated protein kinase cascade is a
universal module of signal transduction, which plays important roles in plant growth,
development, and in plant response to environmental stress (Xing et al., 2002). MAPK
cascades relay and amplify signals by three types of reversibly phosphorylated kinases,
due to the phosphorylation of the substrate proteins the cascade activity leads to altered
activities of many genes (Maria et al., 2010). The MAPK cascade is presented in Figure
1.1 and the cascade consists of at least three enzymes: the MAP kinase kinase kinase
(MKKK or MAP3K), MAP kinase kinase (MKK or MAP2K), and MAP kinase (MAPK or
MAPI K). These enzymes are activated in series by phosphorylation from an upstream
kinase that belongs to the class of MAPKKKs to downstream targets (Marshall, 1994).
The Arabidopsis genome contains 23 MAPKs, 10 MAPKKs, and 60 MAPKKKs
(Ichimura et al., 2002). At the same time, numerous studies have shown that multiple
MAPK pathways play a role under biotic and abiotic stress such as drought, wounding,
pathogen infection and temperature stress (Kyra et al., 2003; Wrzaczek and Hirt, 2001).
5
I
t
MAPKKK
t MAPKK
• MAPK
i
\ ©
0 d
'N* I
corwegenoe fcWPKK
it EL
m
8 £ X I
1
J I
Cascade acSvafion by diferent sayalsiindiKling R-pinMnedHteti pathways
Mufipfelod regulation and de-enrmatkm ofmagrifcLide and duration of ihe adr-otion
TSaSfStiiPSsatSiHaa
Figure 1.1: The mitogen-activated protein kinase cascades in plants (From Xing et al., 2002). PR= pathogenesis-related; HR= hypersensitive response; SAR= systemic acquired resistance.
6
MAPKKs are activated by MAPKKKs through phosphorylation on serine and
threonine residues in a conserved S/T-X3-5-S/T motif. MAPKKs are dual-specificity
kinases that phosphorylate MAPKs on threonine and tyrosine residues in the T-X-Y
motif (Figure 1.2). MAPKs are serine and threonine kinases that phosphorylate a variety
of substrates including transcription factors, protein kinases and cytoskeleton-
associated proteins. After activation, the MAPK module is either translocated into the
nucleus or stays in the cytoplasm to initiate the cellular response through
phosphorylation of downstream proteins (Pedley and Martin, 2005; Nadarajah and
Sidels, 2010). The MAP kinase pathways are intracellular signal modules that mediate
signal transduction from the cell surface to the nucleus. Studies have also shown that
the pathways are down-regulated by phosphatases through dephosphorylation at
tyrosine or serine or threonine residues (Luan et al., 2003; Sun et al., 2003). MsERKI
in alfalfa (Duerr et al., 1993), D5 kinase in pea (Stafstrom et al., 1993), a few MAPKs in
Arabidopsis thaliana (Mizoguchi et al., 1993), and tobacco (Wilson et al., 1993) are
among the first reported plant MAPKs. Also, in wheat, PKABA1 was among the first
plant protein kinase genes that were found to be up-regulated by drought, low
temperature and NaCI, as well as by ABA (Holappa and Walker-Simmons, 1995).
7
MAPK Pathway
Diverse Group of Stimuli MAPK highly conserved >
Cascade of at least three kinases
Ser-Thr Kinases
S/T-X3-5-S/T Dual specificity Kinases
MAPKK
~I TXY
I MAPK
* To the Nucleus
Transcription Factors
Physiological Response
Figure 1.2: The MAPK cascade consists of three different steps.
MAPKKK
Researchers showed that the MAPK superfamily is made up of three main and
distinct signalling pathways which include the extracellular-signal-regulated protein
kinases (ERKs), the c-Jun N-terminal kinases or stress-activated protein kinases
(JNK/SAPK), and the p38 family of kinases (Cvetkouska et al., 2005; Katuo et al., 2005;
Qi and Elion 2005) (Figure 1.3). The plant MAP kinases that have been identified to this
date mainly belong to the ERK group, with a few belonging to the p38 subfamily and
none to the JNK subfamily (Morris, 2001; Nakagami et al., 2005). The ERK subfamily is
activated in response to growth factors to regulate cell proliferation and cell
differentiation (Wang et al., 2002). The activation of ERK is also required for the
execution of apoptosis (Wang et al., 2002). Moreover, the JNK and P38 sub-families are
also required for cell proliferation and differentiation which are activated by stress such
as ultraviolet light and heat shock (Cano and Mahadeven, 1995; Kyriakis and Avruch,
1996; Ono and Han, 2000; Xia etai, 1995).
9
Stimulus: Growth facte Cytokines/Stress Cytokiiies/Stress
MAPKKK:
y
Raf
w
MEKKMASK^MIK MEKK,MLK,ASK1
MEK1/2 MKK4/7
ERK1/2
Cellularresponse: Proliferation Development Differentiation Cellularsurvival
JNKl/2,3
i Apoptosis
Inflammation Tumourigenesis
P38
Cell motility Apoptosis
Chromatin remodeling
Osmoregulation
Figure 1.3: The three MAPK modules (ERKs, JNKs, p38). It shows stimuli, the three tier regulatory cascade within each module (MAPK, MAPKK and MAPKKK levels) and the various cellular responses elicited by MAPK control (Kyra et a!.,2003).
10
The MAPK cascade can be activated by a variety of biotic and abiotic stress
factors such as pathogen infection, wounding, temperature, and reactive oxygen
species involving upstream receptors and downstream targets (Takahashi et al., 2007).
MAPK pathways can bring together transduction pathways initiated by different types of
signals and deliver messages to diversified downstream components (Xing et al., 2002).
The cascade components are regulated at the transcriptional, translational and post-
translational levels (Xing et al., 2002). Components at different tiers in a module are
associated with each other or bound together by scaffold proteins, which make the
cascade efficient and able to respond to a stimulus within minutes.
A cascade conveys information to effectors and coordinates incoming information
from other signalling pathways then amplifies signals and allows for a variety of
response patterns. For example, MAPK signalling acts as a positive regulator for
environmental stress-induced stomata closure but as a negative regulator for stomata
development (Bergmann et al., 2004; Wang, 2007; Neil, 2008). Many genes encoding
these protein kinases are induced by drought, high salt and low temperature, and they
have been identified in Arabidopsis (Qiang et al., 2000; Bohnert et al., 2001). The
MAPK4 and MAPK6 are such examples since they are activated by cold, salt and
drought (Droillard et al., 2004). In addition, a role for the MAPK module consisting of
MAP3K1-MAP2K1/MAP2K2-MAPK4/MAPK6 has been confirmed in cold and salt stress
by yeast two hybrid analysis (Teige et al., 2000). Moreover, many studies have shown
that the MAPK's, WIPK (wound-induced protein kinase) and SIPK (salicylic acid
11
induced protein kinase) in tobacco, alfalfa and tomato are activated by a wide range of
biotic and abiotic stressors such as wounding, osmotic shock, high salinity, drought, UV
irradiation, ozone exposure, extreme temperature, oxidative stress and pathogen
infection (Asai et al., 2002; Del Pozo et al., 2004; Holley et al., 2003; Ichimura et al.,
2000; MAPK group 2002; Kroj et al., 2003; Seo et al., 1999; Tena et al., 2001; Zhang
and Klessig, 2001). The rice OsMPK5a and OsMPK6 MAPKs are also activated by a
variety of stressors such as wounding, drought, high salinity, cold and pathogen
infection (Agrawal et al., 2003; Kurusu et al., 2005; Lieberherr et al., 2005; Xiong and
Yang, 2003). A more comprehensive data analysis has indicated that MAPK cascade is
associated with the activation of several processes such as redox chain (Grant et al.,
2000), systemic acquired resistance (SAR), protective genes (Tena et al., 2001),
hypersensitive response (HR)-like cell death (Yang et al., 2001), and pathogenesis-
related (PR) genes in downstream defence responses (Hirt, 1997).
In wheat very few MAPK cascade components have been identified and
characterized. My project will focus on identifying MAPK pathway components in wheat
followed by studying their possible functions in drought stress response.
1.3 Programmed cell death
Programmed cell death (PCD) plays a number of important roles in plant
developmental pathways, including xylogenesis (formation of woody tissues in trees and
perennials), leaf abscission, self-incompatibility, as well as defence responses to a wide
variety of pathogen and environmental stresses (Nancy, 2006). A molecular mechanism
for eliminating developmentally unwanted cells is essential for successful development 12
and growth of complex multicellular organisms. To regulate the rate of cell division,
multicellular organisms such as plants have developed a biochemical pathway to control
cell death (Chen and Dickman, 2004; Hoeberichts and Woltering, 2003). The role of cell
death is not limited to development and is used in a number of other processes such as
the control of cell populations and in the defence against invading microbes (Ellis and
Horvitz, 1986; Raff, 1992; Greenberg, 1996; Jones and Dangl, 1996; Mittler and Lam,
1996). The Hypersensitive response (HR) has been characterized extensively in the
response to pathogen attack in plants (Pennell and Lamb, 1997; Lam et al., 2001).
Several studies have indicated that the MAPK cascade is involved in the regulation of
plant HR as well as in other defence responses during incompatible interactions
between plants and their pathogens. In plants, PCD is characterized by the generation
of reaction oxygen species (ROS) and fragmentation of DNA, which eventually leading
to HR (Lam et al., 2001). ROS are usually generated when plants are exposed to biotic
or abiotic stress conditions (Xiong and Zhu, 2002). In plants, ROS generation involves
MAPK pathway consisting of the MAPK kinase kinase MEKK1 and the MAPK, MPK4
(Pitzschke, 2008). The hallmark of PCD was visible after 1 hour under saline (NaCI)
stress (Affenzeller et al., 2009). Other studies have suggested that at least 4 hours of
salinity treatment was needed for PCD to develop (Li et al., 2007). Drought inhibits
photosynthesis which in turn leads to the accumulation of ROS (Cohen, 2009). Several
studies have also shown that diverse environmental stressors such as salt stress,
drought and nutrient starvation are able to induce PCD in plant roots (Huh et al., 2002).
PCD is also triggered by high or low temperature, water stress and UV irradiation
(Beers and McDowell, 2001). For example, over 90% of Arabidopsis cell cultures are
dead by 24 hours after a heat treatment of 55°C for 10 minutes (Watanabe and Lam,
2006). The dying cells exhibit changes indicative of PCD such as cytoplasmic
condensation and cleavage of genomic DNA by endonucleases (McCabe and Leaver,
2000).
1.4 My objectives
Global warming is expected to increase yield loss in crop plants due to increasing
temperatures and decreasing water availability during the next two decades (Cattivelli et
al., 2008; Lobell et al., 2008). The major cereal crops such as rice, wheat and maize
are important components of human life (Cattivelli et al., 2008; Guy et al., 2006; Vasil,
2007). Drought stress affects plant growth and development and several responses are
often observed or detected due to abiotic stress. When water content of a plant
decreases, its cells shrink and the cell walls relax. This decrease in cell volume results
in lower turgor pressure and thus turgor-dependent activities such as leaf expansion
and root elongation are the most sensitive to water deficits. Inhibition of cell expansion
results in a slowing of leaf expansion early in the development of water deficits. The
smaller leaf area effectively reduces the loss of water through transpiration. If plants
become water stressed after a substantial leaf area has developed, leaves will senesce
and eventually fall off. Such a leaf area adjustment is an important long-term change
(Biosic et al., 2007). Roots preferentially grow into the soil zones that remain moist.
Deeper root growth into wet soil zones can be considered a second line of defence
against drought. When the onset of stress is more rapid or the plant has reached its full
leaf area before initiation of stress, other responses protect the plant against immediate
desiccation. Stomata closure reduces evaporation from the existing leaf area (Neslihan,
2009). The photosynthetic rate of the leaf is seldom as responsive to mild water stress
as leaf expansion. A common developmental response to water stress is the production
of a thicker cuticle that reduces water loss from the epidermis. Studies on gene
expression under water stress is "complexed" by the following factors: (i) when water is
limiting, plants can become more susceptible to other stresses; (ii) when in light, plants
dissipate heat mainly through transpiration, a process inhibited by drought and salinity.
Accordingly, heat stress often accompanies water deficit. As such, some gene products
induced by water deficit may function primarily to minimize other stresses, and several
appear to have multiple stress-related roles (Lee, 2009; Li et al., 2003; Zhu, 2002).
Osmotic stress responsive genes can be regulated by ABA-dependent and ABA-
independent processes. Some genes induced by water stress are responsive to ABA.
Not all water deficit responsive genes are induced by ABA (Jain et al., 2002; Lee et al.,
2009; Munns, 2002; Ruiz-Sanches et al., 1993). Although considerable progress has
been achieved in this field over the last two decades, there is clearly much yet to be
learned about the molecular and cellular mechanisms of plant response to drought
stress. Plants have well-developed survival and reproduction strategies under drought
conditions through changes at the molecular, cellular, and physiological levels
depending on factors such as species genotype, and severity of water loss (Ruize-
Sanchez et al., 1993). Drought is one of the most common abiotic stresses and some of
the side effects limits the productivity and growth of plants (Pradreep et al., 2006).
Securing supplies and achieving food safety worldwide is essential for the future with
the growing world population.
A large array of genes are found to be induced by drought (Manuela et al., 2003).
Many drought-inducible genes with various functions have been identified by molecular
and genomic analysis. For example, OsCDPK7 in rice is one of the hundreds of genes
that regulate plant response to drought and high salinity stress (Saijo et al., 2000). In
Arabidopsis and other plants there is a large group of transcription factors that regulate
the expression of stress-inducible genes. In addition, several MAPKs identified in plants
that are subjected to water stress are shown to be involved in relaying the dehydration
signal at the plasma membrane to the nucleus (Ramanjulu and Bartels, 2002). One of
the best studied cases on linking MAPKs to abiotic stress signalling is Arabidopsis
MAPK4 and MAPK6 genes, which are activated by cold, salt, drought, wounding and
touch (Ichimura et al., 2000,). In this project, I focus on two MAPK cascade components
in wheat and look at their possible role in wheat response to drought stress.
FLR (Fusarium and Leaf Rust Responsive) and FLRS (Fusarium and Leaf Rust
Sensitive) genes have been identified as components of MAPK cascades in our lab
(Gao, 2010; Gao et al., 2011). FLRS (accession#: AY173962) is identified as a Triticum
aestivum MAPK, while FLR (accession#: AY173961) is identified as a Triticum aestivum
MAPKKK. FLRS is further identified as an ERK-type MAPK (Gao et al., 2011). Both
kinases are involved in wheat response to pathogen attack (Gao, 2010; Gao et al.,
2011). The objectives of my project are:
16
1. Analyze the gene expression level of FLR and FLRS under drought stress by RT-
PCR;
2. Study the post-translational activation of ERK type MAPKs under drought stress by
Western blotting;
3. Confirm the involvement of ERK-type MAPKs in the activation of drought-induced
gene expression using ERK-specific inhibitor;
4. Study PCD under drought stress;
5. Use bioinformatics tools to analyze the role of FLR and FLRS under drought stress in
different genotypes.
17
Chapter II
Materials and Methods
2.1 Plant materials and growth conditions
Wheat (Triticum aestivum, CV Fielder) seeds were sterilized for 2 minutes in 70%
ethanol and then soaked for 8 minutes in a sterilization solution (25% bleach v/v and
0.01% Triton X-100 v/v). Seeds were then rinsed 10 times with autoclaved water to
remove any residue of the sterilization solution. The seeds were then sowed directly into
autoclaved soil and were grown in long day conditions (16 h of light at 17°C and 8 h of
dark at 15°C) in growth chambers (ENCONAIR technologies INC, Winnipeg, Manitoba).
2.2 Drought treatment
The drought treatment was administered using two different approaches. The
first drought treatment involved cutting 2 cm leaf segments from 3-week-old plants and
placing them on a paper towel for 0, 1,2, 3, 4, 6, 7, 8, 9, and 10 h to dry (Jing et a!.,
2006). The second treatment was carried out over a period of days (0, 1, 2 and 3 days)
instead of hours. The last watering was counted as day 0. The plants in the second
treatment were divided into two groups; experimental group (no watering) and control
group (watering every the other day). The leaf segments were collected in individual
18
falcon tubes and sharp-frozen in liquid nitrogen and then stored at -80°C for RNA
extraction.
2.3 RNA extraction
Total RNA was extracted from the leaves after exposure to drought. TRIzol
Reagent kit (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction according to
the manufacturer's protocol. Leaf tissue (100 mg) was homogenized in 1 mL of TRIzol.
The homogenized sample was incubated for 5 min at room temperature (RT) to permit
the complete dissociation of nucleoprotein complexes. An aliquot of 200 pL of
chloroform was then added to the sample, and mixed vigorously by hand for 15
seconds, and then incubated for 2-3 min at (RT). The sample was centrifuged for 15
min at 12,000 x g at 4°C. Following centrifugation, the mixture separated into a lower,
phenol-chloroform phase, an interphase, and a colourless upper aqueous phase. RNA
remains exclusively in the aqueous phase. The volume of the aqueous phase was
about 60% of the volume of TRIzol reagent used for homogenization. The aqueous
phase was transferred into a fresh tube and 500 pi of isopropyl alcohol was added. The
sample was incubated for 10 min at (RT) and centrifuged for 10 min at 12,000 x g at
4°C. The RNA precipitate formed a gel-like pellet at the bottom side of the tube. The
supernatant was removed and the RNA pellet was washed once with 1pL of 75 % v/v
ethanol (made with DEPC water). The sample was mixed by vortexing and then
centrifuged for 5 minutes at 7,500 x g at 4°C. The RNA pellet was air-dried for 5
minutes. The pellet was dissolved in 50 pL of RNase-free water and stored at -80°C.
19
After TRIzol extraction, the Deoxyribonuclease I kit (amplification grade, Invitrogen,
Carlsbad, CA, USA) was used to eliminate genomic DNA contamination in the sample.
The following was added to an RNase-free 0.5 mL micro-centrifuge tube on ice: 1-5 |jg
RNA sample, 1 \iL 10X DNase I reaction buffer (100 mM Tris- HCL (pH 7.5), 25 mM
MgCb, and 5 mM CaCh), 1 pL of 1U/ pL DNase I, and DEPC treated water to 10 pL.
The sample was then incubated for 15 minutes at room temperature. DNase I was
inactivated by the addition of 1 jjL of 25 mM EDTA solution to the reaction mixture. The
sample was then heated for 10 minutes at 65°C. Next, the cloned AMV First-Strand
cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) was used for cDNA synthesis
according to the manufacturer's protocol. A master mixture of 4 pL of 5x cDNA
synthesis buffer (250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM
magnesium acetate, stabilizer, 20 pg/mL BSA), 1 pL of 0.1 M DDT, 1 pL of RNaseOUT
(40 U/ pL), 1.5 mL of DEPC-treated water and 0.5 pL of cloned AMV RT (15 U/mL) was
prepared and kept on ice. In a 0.5 mL tube, the following components were mixed: 1pL
of 50 pM oligo (DT) 20, 1-5 pg RNA sample, 2 pL of 10 mM dNTP mix and DEPC-
treated water to 12 pL. The mixture was incubated for 5 minutes at 65°C. 8 pL of the
master mixture was added to the above 0.5 mL reaction tube. The reaction tube was
transferred to a thermal cycle and heated for 48 minutes at 48°C, and then for 5 minutes
at 85 °C.
20
2.4 RT-PCR
2.4.1 RT-PCR for FLR and FLRS
PCR was performed with Taq polymerase to semi-quantify the amount of mRNA.
The cDNA quantity was measured using a Nanodrop ND-1000 spectrophotometer
(ThermoFisher Scientific, Wilmington, USA). The primers used for RT-PCR were 5'-
GCCATTCGAGGAACATCTA-3' and 5' -TTGTCGTCATTATCGGCGTA-3' for FLR, and
5'-CAGGTGGCCAT CAAG AAG AT -3' and 5-GCAGT GCT CCT CCGAT AAAG-3* for
FLRS, and 5-GCCACACT GTT CCAAT CT AT GA-3' and 5'
TGATGGAATTGTATGTCGCTTC- 3' for the wheat actin gene (GenBank accession
number AB181991). PCR was carried out under the following conditions: 2 minute at 94
°C, 1 minute at 61 °C, and 1 minute at 72°C for 25 cycles; followed by 10 minutes at
72°C. The expected size of FLR and FLRS PCR products are 218 base pairs and 237
base pairs respectively, and for wheat actin the size is 369 base pairs.
2.4.2 RT-PCR for Wtcbf2-2
Three-week-old wheat leaves were treated by distilled water, with 250M ERK docking
domain inhibitor (3-(2-Aminoethy1 )-5-((4-ethoxypheny1) methylene)-2, 4-
thiazolidinedione hydrochloride, Sigma, USA), or 0.4 jjM general kinase protein inhibitor
(K252b solution, Sigma, USA). Total RNA was extracted from wheat leaves after
21
treatment for 0, 4, and 10 hours. TRIzol Reagent Kit was used for RNA extraction
according to manufacturer's protocol. The cDNA was obtained according to the protocol
described in section 3.2. The primers used for RT-PCR were 5'- TAC CGA GGG AGT A
GT TTC CAA A -3' and the 5'- TTT GGA AAC TAC TCC CTC GGT A -3' primer. RT-PCR
was carried out under the following conditions: 94 °C for 1 minute; 94 °C for 1 minute,
60 °C for 1 minute, and 1 minute for 75 °C for 25 cycles; and then 10 minutes at 72 °C.
2.5 Protein isolation and western blotting
Using a porcelain mortar and pestle approximately 100 mg of frozen leaf tissue
was homogenized in 1 mL of TRIzol Reagent. The homogenate was then transferred
into a sterile tube and after 5 min of incubation at (RT), 0.2 mL of chloroform was
added. This mixture was shaken vigorously for about 15 seconds and then incubated for
3 min at (RT). After incubation the sample was centrifuged using a bench-top centrifuge
at 12000 x g for 15 min at 4°C. Following centrifugation the top aqueous layer was
disposed of and DNA was precipitated from the remaining solution by the addition of 0.3
mL of 100% ethanol. To sediment the precipitated DNA the content of the tube was
centrifuged at 2000 x g for 5 min at 4°C. Once the tubes were removed from the
centrifuge the phenol-ethanol supernatant was carefully removed using a pipette and
transferred into clean tubes to which 1.5 mL isopropyl alcohol was added. Samples
were stored for 10 min at 15 to 30°C (RT), and the protein precipitate was sedimented
at 12,000 x g for 10 min at 2 to 8°C. The supernatant was removed and the protein
pellet was washed 3 times in 2 mL of solution containing 0.3 M guanidine hydrochloride
in 59% ethanol. During each wash cycle, the protein pellet was stored in the wash
solution for 20 minutes at 15 to 30°C and centrifuged at 7,500 x g for 5 minutes at 2 to 8
°C. After the final wash, the protein pellet was vortexed in 2 mL of 100% ethanol. The
protein was stored in ethanol for 20 minutes at 15 to 30°C and centrifuged at 7,500 x g
for 5 minutes at 2 to 8 °C. The protein pellet was vacuum-dried for 5 to 10 minutes, and
dissolved in 1% SDS by pipetting. Complete dissolution of the protein pellet requires
incubating the sample at 50 °C for 10 minutes. Then the insoluble material was
sedimented by centrifugation at 10,000 x g for 10 minutes at 4°C. The supernatant was
saved and transferred to a fresh tube. The protein sample was stored at -20 °C until
time of use.
2.6 Determining protein concentration
Protein concentration was determined using the Coomassie blue dye binding
method with the Bradford reagent and bovine serum albumin (BSA) as the standard
(Bradford, 1976). 200 mL 85% phosphoric acid and distilled water was diluted to 200
mL. The Bradford reagent was filtered just before use. A 10 mg/mL solution of BSA
(mixed with 1 % SDS) was diluted to make a BSA standard curve as Table 2.1.
23
20 jjL of each dilution and 1 mL Bradford dye reagent were placed in a 1.5 mL
tube and vortexed. After 5 minutes, 1 mL of each dilution was transferred to a UV
spectrometer cuvette. Absorbance at 595 nm was measured for each dilution. The BSA
standard curve was generated by plotting Abs 595 against BSA concentration. The
protein sample with Bradford dye reagent was placed in cuvette in the same way
described above and absorbance at 595 nm was read. The concentration of the protein
samples was determined from the BSA standard curve.
24
Table 2.1: Dilutions of BSA solution used for standard curve preparation.
Final BSA concentration
(mg /mL)
10mg/mL BSA
volume (pL)
1% SDS(uL)
0.25 2.5 97.5
0.50 5.0 95.0
0.75 7.5 92.5
1.00 10.0 90.0
1.25 12.5 87.5
25
2.7 One dimensional SDS-PAGE
An SDS-PAGE gel usually has two parts, the separating gel and the stacking
gel. For separating gel, 2.5 mL of separating solution was added to 4.16 mL of dH20
and 3.3 mL of 30% acrylamide/bis-acrylamide stock solution. This solution was allowed
to settle for 15 minutes. While the solution was settling the plates were assembled. The
glass plates were pressed together and then loaded into the gel casting apparatus.
Once the plate was ready, 0.03 mL of 10% ammonium persulfate and 6.6 mL of TEMED
were added to the settled solution. The final solution was then mixed by gentle swirling.
Using a 1000 mL pipette the finished solution was added into the space between the
glass plates until the solution was about 1.5 cm from the top of the plates. The gel was
then overlaid with 10mL of isobutyl alcohol and allowed to polymerize for one hour
during which the stacking gel will be generated. To create the stacking gel, 0.65 mL of
30% acrylamide/bisacylamide, 1.25 mL of stacking solution, and 3.05 mL of water were
mixed together and allowed to settle. After the separating gel solidified, 25 mL of
ammonium persulfate and 5 mL of TEMED was added to the separating solution and
swirled gently to mix. The isobutyl alcohol was removed from the top of the separating
gel and the stacking gel solution was added and allowed to solidify for one hour. The gel
was stored at 4°C until needed. The concentration of protein samples was adjusted to
0.8 mg/mL with 2x SDS-PAGE sample buffer containing 100 mM Tris (pH 6.8), 4% w/v
lauryl sulphate (SDS), 20% v/v glycerol, 10% v/v p-mercaptoethanol and 0.2% w/v
bromophenol blue, and the sample was then heated for 10 minutes at 90°C. Equal
26
amounts of proteins (8 mg) from water control, and samples exposed to different levels
of drought (0, 1, 2, and 3 days and 0, 1,2, 3, 4, 6, 7, 8, 9, and 10 hours) were then
loaded into each well of 10% SDS polyacrylamide gels. Pre-stained SDS-PAGE
standards (Bio-Rad, USA) were also loaded in one well to estimate the size and
positions of sample proteins. Proteins were separated using the discontinuous buffer
system of Laemmli (1970). Electrophoretic separation was generally carried out in 1x
running buffer (3.03 g Tris base, 14.4 g glycine and 1 g SDS per litre, pH-8.3) at 175V
for about 50 minutes at room temperature using the Bio-Rad Mini-PROTEAN 3 system.
To prevent non-specific binding, PVDF membranes were blocked with 5% non-fat milk
in TBST (20M Tris, pH7.5, 150 mM, 0.05% Tween-20) for 30 minutes at room
temperature. The blot was washed with TBST (5 minutes x 3) and then incubated with
10 mL TBST buffer containing primary antibody against the protein of interest on a
shaking platform at 4°C overnight. The primary antibody used was Phospho-p44/42
MAP kinase (Erk 1/2) (Thr-202/Tyr-204) antibody at 1:1000 (v/v) (Cell Signalling
Technology, USA). After the overnight incubation with the primary antibody, the blot was
washed with TBST (5 minutes x 3) and then incubated at room temperature for 1 hour
with 1:2000 (v:v) dilution of the secondary antibody (anti-rabbit IgG, HRP-linked) (Cell
Signalling Technology, USA). The target protein on the PVDF membrane was detected
using an enhanced chemiluminescence (ECL) system containing 1x LumiGLO Reagent
and 1x peroxide (Cell Signalling Technology, USA). The membrane was scanned using
FluorChem Q imaging system (Alpha Innotech Cooperation, USA) and the resulting
image was analyzed with the FluorChem Q Band Analysis tools. To confirm equal
loading in each lane of the blot, total protein on the PVDF membrane was also
visualized by staining for 10 minutes with Coomassie blue solution (25% w/v Coomassie
Brilliant blue R, 50% v/v methanol and 7.5% v/v acetic acid) followed by destaining for
30 minutes with destain solution (20% distilled water, 20% acetic acid, 60% methanol).
2.8 Chlorophyll analysis
Wheat plants (Fielder) were grown under long day conditions (16 hours of light at
17°C and 8 hours of dark at 15°C) in a growth chamber. Three week-old leaves were
exposed to drought for 0, 4, 10, 24, 48, 72 or 96 hours. Fresh tissues (50 mg) from the
samples were collected after exposure to drought. One mL of 95% EtOH was added to
tissues and incubated at 65 °C for 2 hours. The OD649 and OD665 were measured.
The micro-molar concentration of total chlorophyll per gram of fresh weight of tissue
was calculated using the following equation:
Total micromoles [chlorophyll a + chlorophyll b] = 6.1 OD665 + 20.04 OD649
28
2.9 Trypan blue staining
Cells of wheat leaves undergoing cell death were photographed using an
Axioplan 2 microscope (Carl Zeiss). Methods used are described in Tang et al., (1999)
and Stone et al., (2000) with slight modifications. Falcon tubes containing the leaves
were boiled at 95°C for 4 minutes and kept at room temperature for 20 minutes instead
of 30 minutes for the detection of auto-fluorescent material. The leaves were then
stained with trypan blue. Wheat leaves were immersed in 10 mL of ethanol-lactophenol
(2 volumes of ethanol and 1 volume of phenol-glycerol-lactic acid-water (1:1:1:1)) that
contained 0.05% trypan blue in 15 mL Falcon tubes and were then covered. The
samples were incubated at 95°C for 4 minutes and then kept at room temperature for 20
minutes. The staining solution was removed and 1.5 mL chloral hydrate destaining
solution (2.5 g/mL in nano pure water) was added to each tube. The leaves were
cleared for 2 days by replacing the destaining solution twice. After destaining, leaves
were suspended in 50% glycerol and examined under an Axioplan 2 microscope (Cark
Zeiss) with white light.
2.10 Bioinformatics analysis
Genevestigator was used for microarray data mining. The data was manually
recorded and then plotted using Microsoft Office Excel 2007. Motif analysis was
29
performed using the ScanSite analysis tool (http://scansite.mit.eduA while protein-
protein interactions were predicted using Atted-ll 6.0 (http://atted.ioA and STRING 9.0
(http://string-db.oraA databases.
30
CHAPTER III
RESULTS
As mentioned in Chapter I, abiotic stress affects plant growth and development
by acting on specific pathways such as the mitogen-activated protein kinase (MAPK)
pathway which plays an important role in plant responses to stress. The MAPK cascade
includes three proteins: MAPK, MAPKK, and MAPKKK which are activated by a series
of phosphorylation reactions. Several studies using a variety of plant species have
shown that the MAPK cascade is involved in signalling pathways activated by abiotic
stress such as cold, drought, UV, osmotic shock, salt, touch, wounding, heat, and heavy
metals (Sinha et al., 2010). Drought can be a major environmental factor limiting plant
productivity (Boyer, 1982). Many studies have shown that some MAPKs are activated at
the transcriptional level while others are activated post-translationally when exposed to
drought stress (Ichimura et al., 2000; Jonak et al., 1996). This chapter will describe how
plants respond to abiotic stress by looking at how two MAPK pathways genes are
regulated when leaves from wheat are exposed to drought stress. Recently, studies
have identified the FLR and FLRS genes as components of the MAPK cascade. The
FLR and FLRS genes were not shown to be regulated at the transcriptional level in
response to salicylic acid (SA) and fumonisin B1 (FB1), however, they were regulated at
the post-translational level by phosphorylation (Gao, 2010). The FLR and FLRS genes
will be monitored at the transcriptional level by examining the gene transcript level using
31
reverse transcriptase PCR (RT-PCR). MAPKs are components of signalling pathways
that transduce diverse extracellular stimuli to multiple intracellular response (Seo et al.,
1995). Some studies suggest that stress induced transcription of MAPK genes evolved
early in plants, and might play an important role in plant defense response (Zhang and
Klessing et al., 2001). Some of the early evidence have shown that plant MAPKs are
involved in stress signalling where the WIPK gene is transcriptionally activated by
wounding (Seo et al., 1995). Further studies have showed that the WIPK transcript was
induced by various elicitors and pathogens (Zhang et al., 1998; Romeis et al., 1999).
Other members in the same subfamily are induced likewise at the mRNA level such as
MMK4 in alfalfa (later called SAMK), MPK3 in Arabidopsis and ERMK in parsley
(Petroselinum crispum) (Jonak et al., 1995; Takezawa et al., 1999). In wheat, the
TaWCKI gene is activated transcriptionally by a fungal elicitor (Takezawa et al., 1999).
These results suggest that stress induced transcription of MAPK genes play an
important role in plant defence response.
Post-translational modifications of proteins play a very important role in the
regulation of protein functions under abiotic stress signal transduction (Xiong et al.,
2003). Phosphorylation is one of the cascade mechanisms which can regulate MAPK
activities and we will use Western blot analysis to detennine if the ERK-like MAPKs are
post-translationally regulated under drought stress. The phospho p44/MMK4 was
indicated to be post-translationally activated after drought and cold treatment (Jonak et
al,. 1996). The MAPKs can be categorised into three broad subfamilies which are C-
jun2, P38 MAPK and ERKs (Schramek, 2002). The P38 MAPK family are primarily
activated at dual phosphorylation sites in their Thr-Gly-Tyr motif, the JNKs are activated
at dual phosphorylation at their Thr-Pro-Tyr motif and the ERKs are activated by dual
phosphorylation of a Thr-Glu-Tyr motif (Schramek, 2002).
In this study, we focus on the FLRS gene which was identified as an ERK-type
MAPK, which contains a Thr-Glu-Tyr motif (Gao et al., 2011). We used the available
anti-active MAPK antibody to prove that the extracellular signal-regulated kinases play a
role in drought stress response.
Programmed cell death will also be examined using microscopy and chlorophyll
analysis. PCD is a primary process in plant growth and development which can be
triggered by several environmental stresses (Zuppini et al., 2012). High salt-induced
PCD in rice root tip cells and in cultured tobacco cells occurred with characteristics of
the well described morphological and biochemical PCD hallmarks such as ROS
accumulation, nuclear alteration and degradation, chromatin condensation,
mitochondrial involvement, endonuclease activity and DNA fragmentation (Lin et al.,
2006, Jiang et al., 2008, Banu et al., 2009). ROS are produced during the cell's
response to abiotic stress and are recognized as important signals in the activation of
plant PCD (Apel et al., 2004; Zuippini et al., 2012). A key role for ROS has been
documented in PCD induced by salt stress (Lin et al., 2006, Affenzeller et al., 2009,
Affenzeller etal., 2009, Chen etal., 2009).
Bioinformatics data will be used and both sensitive (TTD.22) and tolerant
(TR39477) genotype expression will be analysed under drought stress. The level of
expression in different genotypes under drought and the overall expression of FLR and
FLRS at different development stages will be studied.
The involvement of the ERK-type MAPKs in the activation of drought-induced
gene expression will be examined using an ERK-specific inhibitor and the K252b
inhibitor. Wtcbf2-2 is a marker for drought in wheat (Kume et al., 2005) and will be used
as a marker for drought genes downstream of ERK-type MAPK in wheat. RT-PCR will
be used to evaluate the transcription levels of Wtcbf2-2 gene in the presence of both the
ERK docking domain inhibitor and general kinase inhibitor.
34
3.1 Transcriptional regulation of FLR and FLRS genes by
drought stress
The MAPK cascade is activated by several gene-mediated pathways and
regulated by abiotic stresses pathogen attack. This suggests that the MAPK pathway
genes FLR and FLRS might be involved in the plant response to stress. Given that the
MAPK cascade mediates the plant innate immune response, it is expected that
signalling events in leaves may also represent responses to drought stress (Asai et al.,
2001).
To investigate the expression pattern of FLR and FLRS genes in response to
drought stress, RT-PCR was conducted using total RNA isolated from 3-week-old wheat
(Fielder) seedlings. RT-PCR analysis indicated that the expression level of the FLR and
FLRS genes did not change when exposed to increasing time intervals of drought stress
(Figure 3.1 and Figure 3.2).
35
Figure 3.1: Expression of FLR and FLRS genes under different levels of drought
exposure in wheat (Fielder) leaves using RT-PCR analysis.
a) FLR gene expression in wheat leaves exposed to drought from 0 to 4 hours.
b) FLRS gene expression in wheat leaves exposed to drought from 0 to 4 hours.
c) FLR gene expression in wheat leaves exposed to drought from 0 to 10 hours.
d) FLRS gene expression in wheat leaves exposed to drought from 0 to 10 hours.
36
a)
c)
Drought (hour)
0 6 7 8 9 1 0 d)
Drought (hour)
0 6 7 8 9 1 0
37
Figure 3.2: Expression of FLR and FLRS genes under different levels of drought
exposure in wheat (Fielder) leaves using RT-PCR analysis.
a) FLR gene expression in wheat leaves treated with water for 0 to 3 days.
b) FLRS gene expression in wheat leaves treated with water for 0 to 3 days.
c) FLR gene expression in wheat leaves treated without water for 0 to 3 days.
d) FLRS gene expression in wheat leaves treated without water for 0 to 3 days.
38
aj Control (with water)
Time (day)
1 2
Actin
Drought Time (day)
0 1 2
FLR
Actin
I)] Control (with water)
Time (day)
0 1 2 3
Drought Time (day)
d ) 0 1 2 3
FLRS I
Actin
39
3.2 ERK-like MAPKs are post-translationally regulated during
drought treatments
Protein phosphorylation plays an important role in activating pathways in plants
during their response to environmental stresses and pathogen attacks (Umezawa et al.,
2004). Since a change in the expression pattern of FLR and FLRS genes was not
visible at the transcriptional level, Western blot analysis with the anti-pTEYp antibody
was used to determine the phosphorylation levels. To study the activation of
phosphorylation of FLRS during drought stress, protein extracts from drought treated
leaves from three-week-old wheat plants were subjected to Western blot analysis using
phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (anti-ERK-TEY). This
antibody specifically detects the dual phosphorylation of Thr and Tyr residues which is
an essential feature of the post-translational activation of ERK-like-MAPKs (including
FLRS) (Gao et al., 2011). Western blot analysis indicated that the phosphorylation level
of ERK-type MAPKs increased with increasing time of treatment in hours (0, 1, 2, 3, 4,
6, 7, 8, 9, and 10 hours) and in days (0, 1, 2, 3 days) (Figure 3.3).
40
Figure 3.3: ERK-type MAPK (including FLRS) is activated post-translationally
during drought stress
a) Phosphorylation levels of ERK-type MAPKs in wheat leaves exposed to drought
(without water) from 0 to 4 hours.
b) Phosphorylation levels of ERK-type MAPKs in wheat leaves exposed to drought
(without water) from 0 to 10 hours.
c) Phosphorylation levels of ERK-type MAPKs in wheat leaves exposed to no drought
(with water) from 0 to 3 days.
d) Phosphorylation levels of ERK-type MAPKs in wheat leaves exposed to drought
(without water) from 0 to 3 days.
41
a) Drought Time (hour)
Drought b) Time (hour)
0 1 2 3 4 0 6 7 8 9 10
Protein level 44kDa Protein level 44kDa
Anti-ERK-TEY 44kDa P—PI Anti-ERK-TEY 44kDa
c) Control (with water) Time (day)
0 1 2 3
d) Drought Time (day)
0 1 2 3
Protein level 44kDa
Anti-ERK-TEY 44kDa
Protein level 44kDa
Anti-ERK-TEY 44kDa
42
3.3 The effect of ERK inhibitor, general protein kinase
inhibitor and drought on the expression of Wtcbf2-2
Wtcbf2-2 was used in this study as a downstream drought-responsive gene
(Kume et a!., 2005). The expression pattern of Wtcbf2-2 did not change at the
transcription level, when an ERK inhibitor or a general protein kinase inhibitor (K252b)
were applied (Figure 3.4). This experiment was performed once.
43
Figure 3.4: The effect of ERK inhibitor, general kinases inhibitor and drought on
the expression of Wtcbf2-2.
a) The Wtcbf2-2 gene expression level in wheat leaves under drought in the
presence of the ERK inhibitor from 0,4 and 10 hours.
b) The Wtcbf2-2 gene expression level in wheat leaves exposed to drought from 0,
4 and 10 hours.
c) The Wtcbf2-2 gene expression level in wheat leaves under drought in the
presence of ERK inhibitor and general protein kinases inhibitor K252b from 0, 4
and 10 hours.
44
a) ERK inhibitor and drought
Oh 4h ldh
WtCBF2-2
c) K252b and drought
Oh 4h lOh
WtCBF2-2
Actin
bj drought Oh 4h lOh
WtCBF2-2
Actin
45
3.4 Drought stress induces programmed cell death.
Programmed cell death (PCD) is recognized as an essential physiological and
genetic process during plant development (Beers and McDowell, 2001). PCD can be
used as an indicator of physical changes in plants due to abiotic stress such as drought.
In this project, wheat was exposed to drought stress and the level of PCD was
determined. Leaves were collected after exposure to drought conditions for 0, 4, 10, 24,
48, 72 and 96 hours, after which an image was captured under a microscope. Trypan
blue was used to stain the leaves since it selectively stains dead tissues or cells blue.
Dead cells appeared to be much darker compared to living cells and cell death in the
leaves was constrained to a limited area in the leaf. Physical analysis of the leaves
showed that the first signal of cell death started to appear after 24 hours of exposure to
drought stress and as exposure time increased the amount of cell death detected
increased (Figure 3.5). It is important to note that there was no cell death detected at 0,
4, and 10 hours of exposure to drought (Figure 3.5). At the same time, macroscopic
analysis by using a digital camera has shown that at 0, 4, 10 hours no physical changes
in leaves but after 24, 48, 72, and 96 hours of exposure to drought cell death became
visible in leaves (Figure 3.6).
46
Figure 3.5: Trypan blue staining of wheat (Fielder) leaves after exposure to
different levels of drought.
Leaves were treated for 0-96h. The circle indicates areas of dead cells.
47
Time (hour)
Oh
48h
4h
lOh
72h
96h
48
Figure 3.6: Macroscopic analysis of drought treated wheat (Fielder) leaves after
exposure to different levels of drought. Leaf segments were dried on a filter paper
for 0-96h.
49
Time (hour)
Oh
48h
96h
3.5 Chlorophyll analysis for wheat (Fielder) under drought
stress
Microscopic analysis has shown changes in leaves after drought stress. Thus,
we examined the response of wheat leaves by testing the chlorophyll content. Wheat
was grown in a chamber under long day conditions (16h light / 8h dark). A decrease in
the level of chlorophyll was observed after exposure to drought stress for 4h, 10h, 24h,
48h, 72h, and 96h. The highest level of decrease was at 96 h (Figure 3.7).
51
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Oh 4h lOh 24h 48h 72h 96h
Expousre to drought (Hours)
Figure 3.7: leaf chlorophyll content after drought treatment. The change of chlorophyll content in detached leaves was recorded at 0, 4, 10, 24, 48, 72, and 96 hours exposure to drought conditions.
52
3.6 Bioinformatics analysis of expression profiles of FLRS
and FLR genes in wheat
Bioinformatics is the science of information as applied to biological research and
the management of biological information. Bioinformatics allows researcher to generate
information about gene function, protein structure, and molecular evolution. When
looking at a gene of interest, information such as expression level in different tissue
types, expression at different stages of development, and expression due to response
to a variety of factors can be determined using bioinformatics databases (Figure 3.8).
In this part, we will focus on bioinformatics analysis of expression profiles of
FLRS and FLR genes. We will describe the expression of FLRS and FLR by using
online bioinformatics tools.
53
Bioinformatics
r~rt
r*±j
m •^^^Biological
Databases -y
Modrliuf, analysis Biotofjol rvperimHit* Si ttypothtti? jwirtartoi* Bioto(i«l data oifanbatf an vfanalteatiott, prediction
Life Sclent** liifoi mutiou Sdmtexj Uf* Sd*ite*s «& InfotmaUott Sciences
Figure 3.8: Three steps of bioinformatics analysis, a) The first step is life sciences, which include biological experiments and hypothesis generation, b) The second step is information sciences, which include biological data organization, c) The third step is life sciences and information sciences, which include modelling, analysis, visualization and prediction (From Google images).
54
3.6.1 Analysis of FLRS
Overall expression level of FLRS at different stages of plant development was
plotted. There is no significant change in FLRS gene expression at different
developmental stages including germination, seedling growth, tillering stage, stem
elongation, booting stage, inflorescence emergence stage, anthesis, milk development,
dough development and ripening (Figure 3.9).
In the roots, FLRS expression in the drought sensitive genotype (TTD.22) and
the tolerant genotype (TR39477) showed no significant change under drought treatment
compared to the control (Figure 3.10 A, B). Similarly, the expression level in the control
was the same with exposure in both genotypes (TTD.22 and TR39477) in the leaves
(Figure 3.11 A, B). However, FLRS expression was down-regulated after drought stress
in almost all the different genotypes (TR39477 (root and leaf), TTD.22 (root and leaf),
Chinese spring, Cresoand Cs-5AL-10) after drought stress treatment while in two other
genotypes ((Y12-3) flag leaf and (A24-39) flag leaf) FLRS was up-regulated (Figure
3.12). Two probe sets were analysed in the above work with similar results. Only one
data set is presented here.
High stringency motif scan analysis of FLRS using Scan Site analysis tool
predicted a possible phosphorylation sites at T104, T149, and T958 (Figure 3.13).
Protein-protein interaction data for FLRS (AY173961.1) when analyzed using Atted-ll
(http://atted.jp) and STRING 9.0 (http:string-db.org) were not available.
55
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Stages of development for FLRS
Figure 3.9: Overall expression of FLRS (Ta.23916.1.S2_2_at) during different stages of development from wheat (Triticum aestivum). Chart was generated from pre-existing microarray data obtained from Genevestigator.
56
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TR39477 (4h) TR39477 (8h) TTD.22 (4h) TTD.22 (8h)
Tolerant (TR39477) and sensitive (TTD.22) genotypes
Figure 3.10.A: Effect of drought on FLRS (Ta.23916.1.S1_at) expression in roots of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single repeat experiment; no error value provided.
14
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Tolerant (TR39477)and sensitive (TTD.22) genotypes
Figure 3.10.B: Effect of drought on FLRS (Ta.23916.1.S1_at) expression in control roots of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single repeat experiment; no error value provided.
57
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Tolerant (TR39477) and sensitive (TTD.22) genotypes
Figure 3.11.A: Effect of drought on FLRS (Ta.23916.1.S1_at) expression in leaves of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single repeat experiment; no error value provided.
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Tolerant (TR39477)and sensitive (TTD.22) genotypes
Figure 3.11.B: Effect of drought on FLRS (Ta.23916.1.S1_at) expression in control leaves of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single repeat experiment; no error value provided.
58
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Different wheat cultivars
Figure 3.12: FLRS (Ta.23916.1.S1_at) expression levels in different wheat cultivars exposed to drought stress. Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single experiment; no error value provided.
59
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PKC delta €S@m Site Score Percentile Sequence
PKC delta
Site Score Percentile Sequence
SA
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SA
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Figure 3.13: High stringency motif scan ouput for FLRS amino acid sequence indicating possible protein phosphorylation sites.
60
3.6.2 Analysis of FLR
The overall expression level of FLR at different stages of the plant development
displays a double bottom chart pattern. There is no obvious change at different
developmental stages (Figure 3.14). For the level of FLR expression in leaves there is
no significant difference between the sensitive genotype (TTD.22) and the tolerant
genotype (TR39477) in the control and in the drought treatment (Figure 3.15 A,B). In
addition, the expression level in the control roots was very similar in the control and
drought-treated TTD.22 and TR39477 genotypes (Figure 3.16 A, B).
The FLR gene was up-regulated for almost all of different genotypes (TR39477
(leaf) TTD.22 (leaf), Chinses spring, Creso, Cs-5AL-10, (Y12-3) flag leaf and (A24-39)
flag leaf) after drought stress, expect for one genotype (TR39477 in (root) and (leaf))
which was down-regulated (Figure 3.17). High stringency motif scan analysis of FLR
using the Scan Site analysis tool predicted possible phosphorylation sites at T365,
T617, T1028, and T1775 (Figure 3.18). Protein-protein interaction for FLR
(AY173962.1), when analyzed using Atted-ll (http://atted.jp) and STRING 9.0
(http:string-db.org) were not available.
61
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Figure 3.14: Overall expression of FLR (Ta.12668.1_at) during different stages of plant development from wheat (Triticum aestivum). Chart was generated using preexisting microarray data obtained from Genevestigator.
62
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Figure 3.15.A: Effect of drought on FLR (Ta.12668.1_at) expression in leaves of wheat (Triticum aestivum). Chart was generated using pre-existing 'microarray data obtained from Genevestigator database. Single repeat experiment; no error value provided.
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Tolerant (TR39477) and sensitive (TTD.22) genotypes
Figure 3.15.B: Effect of drought on FLR (Ta.12668.1_at) expression in control leaves of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single experiment; no error value provided.
63
16
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Tolerant (TR39477) and sensitive (TTD.22) genotypes
Figure 3.16.A: Effect of drought on FLR (Ta.12668.1_at) expression in roots of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single repeat experiment; no error value provided.
14 -|
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Tolerant (TR39477) and sensitive (TTD.22) genotypes
Figure 3.16.B: Effect of drought on FLR (Ta.12668.1_at) expression in control roots of wheat (Triticum aestivum). Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single experiment; no error value provided.
64
-1.5
Different wheat cultivars
Figure3.17: FLR (Ta.12668.1_at) expression levels in different wheat cultivars exposed to drought stress. Chart was generated using pre-existing microarray data obtained from Genevestigator database. Single experiment; no error value provided.
65
ill!
1
Site Score Percentile Sequence SA T365 0.3542 0.043 % AACTTCATCACCGAO 0.052
MXMsma ̂ •1 Site T617
Score Percentile 0.3764 0.085 %
Sequence CACAGCGTCATCGGC
SA 0.137
HHI 1^— HHI Site T1028
Score Percentile 0.3848 0.113%
Sequence ATGACCATCACCGGC
SA 0.052
•1 Site Score Percentile Sequence SA T1775 0.3921 0.141 % AACACCGTCGGCGCG 0.092
Figure 3.18: High stringency motif scan ouput for FLR amino acid sequence indicating possible protein phosphorylation sites
66
Chapter IV
Discussion
Plants have evolved mechanisms to defend themselves against abiotic and biotic
stress by activating and coordinating various pathways. The regulation of some of these
pathways requires the phosphorylation of proteins by kinases (Thurston et al., 2005;
Xing et ai, 2009). The mitogen-activated protein kinase (MAPK) pathway is one of the
key phosphorylation pathways, as it has been shown to play an essential role in plant
signal transduction (Hirt et ai, 1997; Pitzchke et al., 2009). In eukaryotes, MAPKs can
be divided into three interlinked protein kinases: MAPKK kinases (MAP3Ks or
MAPKKK), MAPK kinases (MAPKKs or MAP2Ks) and MAPK (Nakagami et al., 2005).
Studies have indicated that plants rapidly activate MAPKs when exposed to abiotic
stress stimuli (Sinha etai, 2010). In Arabidopsis, MAPK cascades have been shown to
be involved in pathways activated by abiotic stress such as cold, salt, touch, wounding,
heat, UV, osmotic shock, and heavy metals (Sinha et al., 2010). The MAPK signal plays
an important role in molecular response to dehydration in plants, and as a result, plants
have adapted the ability to survive under different levels of drought (Bartels and Souar,
2004). Evidence has been shown in alfalfa, where the stress-activated MAPKKK kinase
(MKK4) was found to be activated by drought and cold (Jonale etai, 1996). The role of
the MAPK pathway during plant adaptation to drought conditions has also been
indicated for the Arabidopsis AtMEKKI and AtMPK3 genes, the rice OsMSRMK2 and
OsMAPK5 genes, the malus MaMAPK gene, and the maize ZmMPK3 gene, where the
expression of these MAPK genes were induced by drought (Agrawal et al., 2002; Pang
et al., 2006; Mizoguchietalc et al., 1996; Wang et al., 2010; Xiong et al., 2003). The
components of the MAPK pathway can be regulated at multiple levels such as
transcriptional, translational and post-translational levels (Xing etai., 2002; Zhang etai.,
2000). The NtWIPK gene from tobacco displayed post-translational and transcriptional
regulation during the defence response against tobacco mosaic virus (TMV) infection
(Wilson et al., 1997; Zhang et al., 1998). In maize, the transcription of ZmMPK3
accumulated markedly after the plant was subjected to various abiotic stimuli (Wang et
al., 2010). This was also seen in rice where the transcription of OsMAPKKK
accumulated significantly after roots and leaves were exposed to drought stress (Lee et
al., 2011; Ning et al., 2010). The expression patterns of MEKK1, AtMAPK3 and
AtMAPK4 were changed at the transcriptional level and up-regulated in response to
drought, touch, cold, mechanical stress, salt stress, low humidity and hyper-osmolality
in Arabidopsis (Covicef et al., 1999; Ichimura, 2000; Mizoguchi etai., 1996).
Research in our laboratory identified the wheat FLR and FLRS genes, which are
MAPKKK and MAPK genes respectively (Gao, 2010). Previous experiments performed
in our lab have indicated that the FLR and FLRS genes may not play a role in wheat
response to treatment with FB1 at transcriptional level, but the ERK-type MAPKs
(including FLRS) were found to be involved in wheat response to FB1 at the post-
translational level (Gao, 2010). Programmed cell death (PCD), which is associated with
concurrent accumulation of reactive oxygen species (ROS), was also apparent in the
leaves after FB1 treatment (Gao, 2010). In this project, we have focused on FLR and
FLRS genes and their possible function in the regulation of the plant response to 68
drought at the transcriptional level and at the post-translational level. Physiological
changes due to abiotic stress such as PCD and level of chlorophyll was also studied in
the examination of effects of drought on wheat.
4.1 FLR and FLRS genes do not regulate plant response to drought at
the transcriptional level.
Wheat is one of the most important crops in the world and a better understanding
of stress response mechanisms would undoubtedly have a considerable effect in the
development of wheat varieties that are immune to such stressors. Global warming is
expected to increase yield loss in plants due to the increasing temperatures and
decreasing water availability on earth in the next two decades (Lobell et al., 2008).
Water stress influences vital activities of plants in several ways including disrupting cell
walls, and misbalancing endogenous hormone metabolism (Jing et al., 2006). Drought
may cause serious sterilization of more than 50% of all arable lands by the year 2050.
As such, it is important to understand the regulators of plant response to drought (Wang
et al., 2005). Plants have a developed a set of defensive mechanisms to adapt to
environmental stress including recognition of stress signals, gene expression and
protein modification, and metabolism shift (Jing etai., 2006).
When wheat plants are exposed to drought stress the expression of FLR and
FLRS was not induced at the mRNA level since a change in expression was not
observed (Figures 3.1 and 3.2). These results indicate that transcriptional regulation of
FLR and FLRS may not play a role in wheat response to drought stress. However,
69
studies have shown that transcriptional regulation of MAPKs in plants is not always
correlated with a change at the translation level (Bogre et al., 1997; Seo et al., 1999).
Rapid and transient activation of the Arabidopsis MAPKs, AtMAPK4 and AtMAPK6, is
induced when plants are exposed to abiotic stress such as low temperature, touch,
hyper-osmolality and wounding (Ichimura et al., 2000). The activation of these proteins
was associated with tyrosine phosphorylation but not with the amount of mRNA
(transcriptional level) indicating that the level of gene transcript does not necessarily
reflect the amount of functional proteins in the plant (Buchanan-Wollaston et al., 2005;
Pierrat et al., 2007; Xing et al., 2002). Work in our lab showed that FLRS expression
was not induced at the transcriptional level but the kinase protein was more
phosphorylated under FB1 treatment in wheat (Gao et al., 2011). Regulation that was
seen at the post-translational level but not at transcriptional level may reflect that the
MAPK cascade components are regulated at multiple levels, which include
transcriptional, translational and post-translational levels (Zhang etai., 2000; Xing et al.,
2002). Many studies have shown that the expression level of genes does not
necessarily correlate with the protein level in cells (Gygi et al., 1999).
Our results have indicated that FLR and FLRS do not regulate plant response to
drought stress at transcriptional level since a significant change was not observed when
wheat plants were exposed to drought for up to 3 days.
70
4.2 FLRS may regulate drought response at post-translational level.
Protein phosphorylation and de-phosphorylation play a significant role in many
signal transduction pathways. Protein phosphorylation is one of the main mechanisms
of post-translational modification that regulate protein stability, biological activity and
cellular location (Shiniozaki and Yamaguchi, 2007). In yeast and higher plants, MAPK
activation is mediated by post-translational phosphorylation (Agrawal et al., 2003 and
Morris et al., 1997, and Widmann et al., 1999). In turn, post-translational modification
can activate an array of defence mechanisms in minutes (Thurston et al., 2005; Xing et
al., 2002). In Arabidopsis, AtMAPK4 and AtMAPK6 were activated by phosphorylation
within 5 minutes when exposed to abiotic stresses such as dehydration, cold, wound
and touch (Ichimura et al., 2000). The MAPK and MMK4 were also activated at the
post-translational level under drought and low-temperature stress in alfalfa (Jonek et al.,
1996). Our gene expression study has shown that the FLRS gene could be activated at
the post-translational level by dual phosphorylation of threonine and tyrosine residues
during drought treatment (Figure 3.3). Similar results were obtained when wheat was
exposed to FB1 (Fumonisin B1) toxin and SA (salicylic acid) (Gao et al., 2011). FLRS
was shown to be activated post-translationally by dual phosphorylation of threonine and
tyrosine residues in wheat (Gao et al., 2011). Evidence of this was also obtained in
Arabidopsis where the MAP kinases ATMAPK4 and ATMAPK6 were activated under
abiotic stress such as low temperature, low humidity, hyper-osmolality, touch and
wounding by the phosphorylation at tyrosine residue (Ichimura et al., 2000). During our
gene expression study, the ERK-type MAPKs (including FLRS) are post-translationally
activated by dual phosphorylation of threonine and tyrosine residues during drought
stress in wheat, indicating a role for the ERK-type MAPK in plant defence against
drought stress.
4.3 Drought induces cell death in wheat.
Programmed cell death (PCD) is the active process of cell death that happens
during development and in response to environmental stresses (Danon et al., 2000;
Greenberg, 1996; Lam et al., 2001). Abiotic stresses such as low or high temperature
have been shown to induce PCD in tomato, tobacco, Arabidopsis, cucumber, and wheat
(Balk etai., 1999; Fan and Xing, 2004; Koukalova et al., 1997; Swidzinski et al., 2002).
Reactive oxygen species (ROS) are produced during the cell's response to abiotic
stress and are recognized as important signals in the activation of plant PCD (Zuppini et
al., 2010). ROS plays a role in programmed cell death in plants along with the MAPK
cascades (Pitzschke et ai, 2009). MAPK cascades are key players in ROS signalling
(Pitzschke et al., 2009). Several studies have shown that MAPK signalling pathways are
not only induced by ROS but can also regulate ROS production (Pitzschke ef al., 2009).
Our results have shown that more cell death occurred in wheat leaves with
increasing exposure times as indicated by trypan blue staining (Figure 3.5 and Figure
3.7). Trypan blue is commonly used to selectively stain dead tissue or cells blue. When
exposing the wheat leaves to different levels of drought, the dead cells appeared to be
much darker compared to living cells. These blue dead cells scattered on leaves as
clusters without defined margins and the cell death did not seem to occur in the entire
leaf. As the time of drought increased, the amount of PCD increased.
4.4 The effect of ERK inhibitor, general protein kinase inhibitor and
drought on the expression of Wtcbf2-2 gene.
In Arabidopsis, several pathways respond independently to abiotic stress, and
Wtcbf2 plays a key role during drought stress (Fowler and Thomashow, 2002).
Recently, studies have identified that the Wtcbf2 transcript level decreased in the
Chinese Spring (CS) and "Mironovskaya 808" wheat cultivars after 4 to 9 weeks during
low temperature stress (Kume et al., 2005). In addition, the Wtcbf2-2 transcript
increased after 15 to 30 minutes of drought stress in the Chinese Spring (CS) and
'Mironovskaya 808' (M808) cultivars at transcriptional level responsive. The Wtcbf2-2
was used in this study as a marker for drought responsive genes and we would like to
see if it is downstream of ERK-type MAPKs in wheat. In this experiment Wtcbf2-2
transcript level was examined after drought treatment in the presence of ERK docking
domain inhibitor as well as a general protein kinase inhibitor (Figure 3.4). Our result
indicate that the transcript level of Wtcbf2-2 did not change during in the presence of
drought treatment, ERK inhibitor or general protein kinase inhibitor, suggesting Wtcbf2-
2 did not respond to drought stress in wheat (Figure 3.4). At the same time, Wtcbf2-2
may not be downstream of ERK-type MAPKs pathway.
73
4.5 Bioinformatics analysis
A tolerant (TR39477) and a sensitive (TTD.22) genotype of wheat were selected
based on their physiological and morphological response to drought stress for transcript
analysis by using Affymetrix Gene Chip Wheat Genome Array (Genevestigator). Along
with the analysis of gene expression profiles in these two genotypes, we also studied
the expression patterns of FLR and FLRS genes through data mining using
Genevestigator.
Bioinformatics analysis confirmed our results where the expression of FLR and
FLRS was not changed at the transcriptional level in drought stress and this indicates
that they may not play a role in drought stress at the transcriptional level. Bioinformatics
data have shown that both FLR and FLRS genes do not play a role in wheat response
during drought stresses in leaves and roots in both tolerant (TR39477) and sensitive
(TTD.22) genotypes (Figure 3.10 A, B; Figure 3.11 A, B; Figure 3.15 A, B and Figure
3.16 A, B).
Bioinformatics analysis has shown that FLRS is down-regulated after drought
stress in all the different genotypes (TR39477, TTD.22, Chinese spring, Creso and Cs-
5AL-10). FLR on the other hand is up-regulated in all genotypes (TR39477, TTD.22,
Chinese spring, Creso, Cs-5AL-10, Y12-3 flag leaf and A24-39 flag lea) (Figure 3.12
and Figure 3.17). Our previous data however indicates that FLRS and FLR were up-
regulated at transcriptional level when wheat was challenged by wheat leaf rust or
Fusarium head blight (Gao etai., 2011; Xing, unpublished).
74
4.6 Conclusion and future work
In conclusion, this study focused on the role that FLR (a MAPKK kinase) and
FLRS (a MAP kinase) genes play when wheat plants are exposed to drought stress.
There was no change at the transcriptional level for both FLR and FLRS genes during
drought stress, indicating that transcriptional regulation of FLR and FLRS may not play
a role in wheat response to drought stress. However, ERK-type MAPKs including the
FLRS protein have been shown to be activated at the post-translational level,
suggesting that ERK-type MAPKs may play a role against drought stress in wheat. The
level of PCD increased with the increasing time of drought treatment.
Bioinformatics analysis has indicated that there is no significant difference in FLR
and FLRS gene expression levels between drought tolerant and drought sensitive
genotypes. The bioinformatics analysis has also indicated that FLRS transcription is
down-regulated in TR39477, TTD.22, Chinese spring, Creso and Cs-5AL-10 genotypes
after exposure to drought stress. The FLR gene expression was shown to be up-
regulated in TR39477, TTD.22, Chinese spring, Creso, Cs-5AL-10, Y12-3 flag leaf and
A24-39 flag leaf genotypes. The possible phosphorylation site for FLR is at T104, T149
and T958 and for FLRS is at T365, T617, T1028 and T1775.
Quantitative PCR should be applied to confirm that the FLR and FLRS are not
induced at the transcriptional level under drought stress. It would be helpful if FLRS
protein is used in a protein-protein interaction analysis. Identification of the interactive
protein may lead to further understanding of the wheat MAPK signalling pathway.
75
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