This document is a dissertation submitted by Satya Prakash on the topic of abscisic acid signaling in guard cell movement. It begins by acknowledging the author's dissertation supervisor, Dr. Girish Mishra, and thanking family for their support. The document then provides an introduction to ABA's role in regulating guard cell movement and stomatal closure during drought stress. It discusses several key molecules involved in ABA signaling, including hydrogen peroxide, phospholipase D, phosphatidic acid, reactive oxygen species, and ion channels. The role of these signaling molecules and various mechanisms of hydrogen peroxide generation in guard cells are described over multiple sections.

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Abscisic acid signaling in
guard cell movement
Paper 409: Dissertation
Supervisor:- Dr. Girish Mishra
Submitted by:- Satya Prakash
MSc Botany, Sem IV (2013)

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ACKNOWLEDGEMENTS
I would never have been able to finish my dissertation without the guidance of my
dissertation teacher, Dr. Girish Mishra , help from friends, and support from my
family .
Foremost, I would like to express my deepest gratitude to my well known
supervisor , Dr. Girish Mishra , for his excellent guidance, caring, patience, and
providing me help whenever I needed inspite of his very busy schedule. His
guidance helped me in all the time of study of dissertation topic and writing it.
Beside my supervisor , I would like to show my sincere gratitude towards my
father Mr. Raj Kumar Chaurasia and brother Mr. Sonu Chaurasia for their
motivation and financial help they provided me for my studies.

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ABA SIGNALING IN GUARD CELL MOVEMENT
CONTENTS: Pg. no.
1. Introduction 4
2. Changes necessary for stomatal closure. 6
3. Role of hydrogen per oxide in ABA induced stomatal movement. 7
3.1 Mechanisms of H2O2 generation in guard cells. 7
4. Role of phospholipase D-α 1 and phosphatic acid in ABA induced stomatal closure. 8
5. Role of PI3P AND PI4P in ROS generation. 13
6. Role of MAP kinases in ROS mediated ABA signaling. 14
7. Plasma Membrane Receptor Kinase GHR1 in ABA signaling. 14
8. Nitric Oxide and Abscisic Acid Cross Talk in Guard Cells. 17
9. Abscisic Acid Regulation of Guard Cell Anion Channels. 19
9.1 Channels in the plasmalemma. 20
9.2 Tonoplast ion channels. 21
10. Common techniques used to study stomatal functions and hidden physiology behind: 23
10.1 Patch clamp. 23
10.2 Infrared thermal imaging. 24
11. Summary 25

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1. Introdution
Guard cells can integrate and process multiple complex signals from the environment. It gives its
response by opening and closing stomata to its fluctuating environment. The plant hormone
abscisic acid (ABA) participates in diverse physiological processes, such as stomatal movement,
seed dormancy , germination, vegetative growth, and response to abiotic and biotic stresses
(Schroeder et al., 2001; Finkelstein et al., 2002; Assmann, 2003; Xiong and Zhu, 2003; Hirayama
and Shinozaki, 2007). ABA regulates guard cell movement in drought stress as endogenous anti-
transpirant. During drought its synthesis is increased and it is redistributed and accumulated in the
guard cells. Accumulation of ABA in guard cells results in efflux of ions followed by release of
water. As a result, there is a loss of turgour of guard cells causing stomatal closure. This regulation
requires the involvement of key regulatory elements such as PIP, PA , G-protiens as well as
several reactive oxygen species such as H2O2 and reactive nitrogen species, NO ( Neill at al.,
2002a). Several more key molecules are gradually being discovered. They are generated in
response to various abiotic and biotic stresses. Hydrogen per oxide and nitrogen oxide are
synthesized in parallel and act in tandem. They act either individually or in concert.
In this regulation, H2O2 plays an important role as a second messenger. Here, ABA induces H2O2
production that leads to an elevation of basal concentration of cytosolic H2O2. Although there
are multiple routes of H2O2 production. Its production in Arabidopsis has been shown to be
mediated by plasma membrane NAD(P)H oxidases. Nitrate Reductase has been identified as
source of NO in Arabidopsis. Enzyme catalase that degrades H2O2 counteracts the ABA.
Mutation in CAT genes that code for catalase potentiates the ABA induced stomatal closure.
Exogenous catalase reduced H2O2 and inhibited ABA induced stomatal closure.. Furthur,
treatment by AT(3-aminotriazole), catalase inhibitor, promotes ABA induced ROS production.
Thus , decrease in catalase activity potentiates the ABA induced stomatal closure to reduce
transpirational loss( Jannat et al., 2011) .

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ABA-induced stomatal closure involves a net increase in guard cell cytoplasmic Ca2+
concentrations. Furthermore, CADPR, ryanodine receptors, and phospholipases C and D have
been also involved in this signaling pathway (MacRobbie, 1998; Jacob et al., 1999; Sanders et
al., 1999; Schroeder et al., 2001b). Perception of ABA by receptors in guard cells activates a
complex web of signaling pathways. In guard cells, the early events in ABA signal transduction
after receptor activation involve ion channel regulation, cytosolic Ca2+
changes, and intracellular
coupling mechanisms. A plasma membrane receptor GHR1 has been identified in Arabidopsis
guard cells. ghr1 mutants were defective ABA and H2O2 induction of stomatal closure( Hua et
al., 2012). Ca2+
, Protein Kinases and cyclic GMP act downstream to ROS and RNS( Reactive
Nitrogen Species). Exposure of vicia faba guard cells with exogenous H2O2 elevated the
cytosolic concentration of calcium ions which resulted into stomatal closure. H2O2 is essential
for ABA induced stomatal closure in various species.
In addition to the above signaling molecules lipid mediators such as PA and S1P generated by
phospholipase D (PLD), phospholipase C (PLC), and sphingosine kinase respectively have been
identified as integral parts ofthe complex signaling cascades in the ABA response (Fan et al.,
2004; Zhang et al., 2005; Wang et al., 2006). Stomatal closure requires the activation of ion
channels and the synthesis of calcium mobilizing molecules such as cyclic ADP ribose and
inositol trisphosphate(IP3) thereby elevating cytosolic Ca2+
ion levels. Abscisic acid (ABA)-
induced stomatal closing is mediated by a reduction in the turgour pressure of guard cells, which
requires an efflux of potassium and anions, sucrose removal and the conversion of malate to
osmotically inactive starch. Slow anion channels have been proposed to play a rate-limiting role
in ABA-induced stomatal closing. ABA strongly activates slow anion channels in wild-type
guard cell ( Pie et al.,1997).
Patch-clamp studies have led to the identification of a number of ion channel types in the plasma
membrane and vacuolar membrane of guard cells that can function in unisonto inhibit stomatal
opening and mediate stomatal closing (Schroeder and Hedrich, 1989; MacRobbie, 1992;
Assmann, 1993; Ward et al., 1995). When slow (S-type) anion channels are activated, the
resulting sustained efflux of anions from guard cells would produce long-term depolarization
(Schroeder and Keller, 1992). At the plasmalemma, loss of K+
requires depolarization of the
membrane potential into the range at which the outward K+
channel is open. Inward K+
channels

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are known to be inhibited by the direct application of ROS to guard cells. ABA-induced
activation of a non-specific cation channel, permeable to Ca2+
, may contribute to the necessary
depolarization, together with ABA-induced activa- tion of S-type anion channels in the
plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are
activated by Ca2+
and by phosphorylation, but the precise mechanism of their activation by ABA
is not yet clear (Mori et al., 2006).
2. Changes necessary for stomatal closure.
Loss of K+
and anions is necessary from the guard cell so that stomata can close. This is a kind
of event which requires a signal to happen. Here, ABA has been found to be acting as a closing
signal. There are two membranes across which the transfer of potassium ions and anions takes
place. These membranes are plasmalemma and the tonoplast. K+
-permeable and anion permeable
channels of the plasmalemma and tonoplast need to be activated. For channels which are
voltage-dependent, voltage across the membrane concerned is adjusted within the range for
opening of that channel. Down-regulation of the inward fluxes of K+
and anions is not essential
for stomatal closure, but will speed the process driven by stimulation of the efflux
processes.There are now clear evidences that all of these changes do follow the application of
ABA to guard cells, that for both anions and cations there is stimulation of efflux at the
plasmalemma, and of the flux from the vacuole to the cytoplasm.
Figure 1. Diagrammatic representation of voltage-gated K+
channel protein.
Hydropathy analysis & topology studies predicted the presence of 6 transmembrane α -helices in
the voltage-gated K+
channel protein. The core of the channel consists of helices 5 & 6 & the

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intervening H5 segment of each of the 4 copies of the protein. Helices 1-4 function as a voltage-
sensing domain, with helix #4 having a special role in voltage sensing. This domain is absent in
K+ channels that are not voltage-sensitive. (E. A. C.MacRobbie, 1998)
3. Role of H2O2 in ABA induced stomatal movement.
In guard cells, ROS generated by ABA play an important role as signal mediators for the
activation of multiple downstream events that are important for signal-induced stomatal
movements, including the opening of Ca2+
channels (Pei et al., 2000), intracellular alkalization
(Zhang et al., 2001b), and closure of inward potassium channels (Zhang et al., 2001a).
Treatment of guard cells with exogenous ABA causes rapid H2O2 generation in Vicia faba (Miao
et al., 2000) and Arabidopsis (Pei et al., 2000). Zhang and colleagues conducted a detailed study
of the generation of H2O2 in response to exogenous application of ABA to the guard cells of V.
faba (Zhang et al., 2001c). The results of these experiments performed using the fluorescent
probe dichlorofluorescein showed that generation of H2O2 was dependent on ABA
Concentration.
3.1 Mechanisms of H2O2 generation in guard cell.
There are two potential mechanisms by which H2O2 might be generated in guard cells. First
proposed mechanism for generating H2O2 involves guard cell chloroplasts as principle source for
ROS generation in plant cells (Foyer & Harbinson, 1994). Under normal photosynthesis,
chloroplasts generate about 150–250 μmol of H2O2 mg−1
chlorophyll h−1
.
A second mechanism for generating H2O2 involves NADPH oxidase which is located in the cell
membrane (Pei et al., 2000; Zhang et al., 2001c). Enzymatic sources of H2O2 also result from
those reactions that are catalysed by cell wall peroxidases, amine oxidases and other flavin-
containing enzymes (Neill et al., 2002b, 2002c). Allan & Fluhr (1997) suggested that H2O2 was
generated via intracellular flavincontaining enzymes, apoplastic peroxidases and amine
oxidasetype enzymes in guard cells and epidermal cells of tobacco in response to elicitor
challenge. A pH-dependent cell wall peroxidase can also generate H2O2 (Peng & Kuc, 1992).

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ABA-induced stomatal closure was inhibited by diphenylene iodinium (DPI) in Arabidopsis
(Cross & Jones, 1986). Hydrogen per oxide-induced calcium channel activation is dependent on
NADPH . This finding suggested a role of NADPH oxidase-like enzyme mediating H2O2
formation in response to ABA in Arabidopsis guard cells (Murata et al., 2001). Analysys of the
ABA insensitive1 (abi1) and ABA insensitive2 (abi2) point mutant plants with strongly reduced
phosphatase activities showed that ABA was unable to generate ROS in the abi1 mutant plants,
but ABA could still induce ROS production in the abi2 mutant plants (Murata et al., 2001).
These data suggested that the abi2-1 mutation impairs ABA signalling downstream of ROS
production (Murata et al., 2001).
4. Role of phospholipase D-α 1 and phosphatic acid in ABA induced stomatal
closure.
Recent studies show that PLD and its lipid product phosphatidic acid (PA) interact with a G
protein and protein phosphatase to mediate the ABA response in guard cells. PA produced by
PLD binds to the ABI1 protein phosphatase 2C, a negative regulator of ABA action. This
inhibits the function of ABI1(Zhang et al., 2009). The depletion of PLD-α1 decreases ROS
production in leaves and addition of PA results in recovery of ROS production in pldα1 mutants .

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Figure 2. Enzymatic reactions lead to PA production and degradation. .(Sang et al., 2001)
(A) Sites of hydrolysis by four types of phospholipases. X denotes the head group that defines
different head classes of phospholipids. (B) The enzymatic reactions leading to the PA
production (upper) and removal (lower). DGK, diacylglycerol kinases; DAG-PPi, diacylglycerol
pyrophosphate; LPP, lipid phosphate phosphatase; LysoPA, lysophosphatidic acid; PAK,
phosphatidic acid kinase; PE, phosphatidylethanolamine; PS, phosphatidylserine.
PLD α mediates the ABA effects on stomata through interaction with a protein phosphatase 2C
(PP2C) and a heterotrimeric GTP-binding protein (G protein) in Arabidopsis (Mishra et al.,
2006). Phospholipase D-α 1( PLD- α 1) and phosphatic acid are involved in ABA induced ROS
production in Arabidopsis guard cells. PLD α1 positively regulates ABA-induced stomatal
closure (Zhang et al., 2004) and the inhibition of stomatal opening by ABA (Mishra et al., 2006).
Here the phosphatic acid is the lipid product of phospholipase D-α 1. PLD hydrolyzes membrane

10. 10
lipids to produce phosphatic acid. PA binds to the ABI1 which is a negative regulator of ABA
response (Gosti et al.,1999 ; Schroeder et al., 2001). The pldα1 mutant failed to produce ROS in
guard cells in response to ABA. But , a pld α1 loss-of-function mutation alone did not inhibit
ABA induced stomata closure (Siegel et al., 2009), which suggests that other PLDs are involved
in ABA signaling in guard cells.
Figure 3. The main domain structures of the Arabidopsis and mammalian PLD families. ( Siegel
et al., 2009)
Plant PLDs consist of two distinctive groups: the C2-PLDs and the PX/PH-PLDs. Individual
PLDs can differ in key amino acid residues in these regulatory motifs such as C2, PIP2-binding,
and DRY. Note that the Twelve Arabidopsis PLDs have now been classified into six types,
instead of five types; the only modification is that PLDa4 has been reclassified to PLDe because
this PLD is quite distantly related to all the other PLDs. C2, Ca2+
/phospholipids binding domain;
PH, Pleckstrin homology domain; PX, phox homology domain. The duplicated HKD motifs are
involved in catalysis.

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PLD and PA actually regulate NADPH- oxidase activity. NADPH oxidases are the source of
ROS produced in ABA response and other processes, including pathogen recognition and root
hair growth (Torres et al., 2002; Foreman et al., 2003; Kwak et al., 2003; Torres and Dangl,
2005). Plant NADPH oxidases, termed respiratory burst oxidase homologs (Rbohs), are
homologs of the mammalian NADPH oxidase catalytic subunit gp91phox. Different Rboh genes
have been isolated from rice (Oryza sativa), Arabidopsis, tomato (Solanum lycopersicum),
tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) (Torres and Dangl, 2005).
Arabidopsis RbohD are located in the plasma membrane (Torres and Dangl, 2005) and are
expressed in Arabidopsis guard cells (Kwak et al., 2003). ABA stimulated NADPH oxidase
activity in wild-type guard cells but not in plda1 cells, whereas PA stimulated NADPH oxidase
activity in both genotypes. PA stimulates ROS production in Arabidopsis leaves (Sang et al.,
2001a). PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst
oxidase homolog D) and RbohF. People identified PA binding motifs. Mutation of the Arg
residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA
activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was weak in ABA-
mediated ROS production and stomatal closure. plda1 and rbohD mutants show similar
phenotypes; they are both insensitive to ABA-induced stomatal closure (Kwak et al., 2003;
Zhang et al., 2004).

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Figure 4. A bifurcating model for interaction among PLDa1, PA, ABI1, and GPA1 (Ga) in
mediating ABA effects on stomatal closure and opening. ( source:- Mishra et al., 2006)
PLDα1-produced PA binds to ABI1, and this binding removes ABI1 inhibition of ABA
promotion of stomatal closure. On ABA inhibition of stomatal opening, PLDα1- produced PA
acts upstream of GTP-bound Gα (Gα-GTP) to inhibit stomatal opening, whereas GDP-bound Gα
(Gα-GDP) binds to PLDα1 to suppress PLD activity. This model is not comprehensive.

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5. Role of PI3P AND PI4P in ROS generation.
Guard cells contain PI3P (Phosphatidylinositol 3-Phosphate) activity. An inhibitor of PI3P also
inhibited ABA induced ROS generation and stomatal closure (Park et al., 2003). PI3P is a
product of phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates the D-3 position of
phosphoinositides. There are 3 different types of PI3K have been reported in animals but in
plants only type III PI3K have been reported which makes PI3P from phosphoinositides. Broad
bean (Vicia faba) guard cells have type III PI3-kinase activity, and PI3P is necessary in ABA-
induced stomatal closing (Jung et al., 2002). Guard cells overexpressing PI3P-binding protein
showed decreased stomatal closing in response to ABA, and the same effects were observed in
guard cells treated with the PI3K inhibitors wortmannin (WM) and LY294002 (LY);( Jung et al.,
2002). These inhibiters also suppressed ca2+
oscillations indicating that PI3K may be acting
upstream to ca2+
signaling. Hydrogen peroxide (H2O2) is also involved upstream of Ca2+
signaling (Pei et al., 2000).
Phosphatidylinositol 4-Phosphate (PI4P) is the product of PI4K activity and plays a central role
in signaling pathway. Both wortmannin (WM) and LY294002 inhibited PI3K and PI4K activities
in guard cells, they promoted stomatal opening induced by white light and inhibited stomatal
closing induced by abscisic acid (ABA) (Jung et al., 2002). Overexpression of a protein in guard
cell which binds to PI3P or PI4P partialy inhibited closure induced by ABA. Also, WM and
LY294002 inhibited ABA induced cytosolic Ca2+
increases in guard cells. These results suggest
that PI3P and PI4P play an important role in the modulation of stomatal closing and that
reductions in the levels of functional PI3P and PI4P enhance stomatal opening.
Figure 5. Brght field images of guard cell. ( Jung et al., 2002)
(2)(1)

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1) A guard cell(transformed one) expressing GFP:EBD( protein that binds to PI) treated
with 50 micro molar ABA for 1 h. stomatal closure is inhibited.
2) Untransformed guard cell showing more closed stoma.
6. Role of MAP kinases in ROS mediated ABA signaling
Among the identified molecular elements working in ABA signaling are protein kinases and
phosphatases that play a central role in regulating the signaling network. Two MAPK genes,
MPK9 and MPK12 are preferentially and highly expressed in guard cells.The two genes are
functionally redundant as mutation in any one of them does not produce an altered phenotype. If
the both MPK9 and MPK12 transcripts are silenced the ABA induced stomatal closure is
impaired. Furthermore, ABA and calcium failed to activate anion channels in guard cells of
mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA
signaling. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H2O2
treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic
evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA
signaling positively. (Jammes et al .,2009)
7. Role of Plasma Membrane Receptor Kinase GHR1 in ABA signaling.
Both plant and animal cells perceive and process extracellular signals through plasma membrane
receptors. In animals, the main cell surface receptors, called receptor tyrosine kinases (RTKs),
are key regulators of many signaling events (Lemmon and Schlessinger, 2010). In plants, the
largest group of membrane receptors is the receptor-like kinases (RLKs), and there are more than
600 different RLKs in Arabidopsis thaliana and more than 1,100 in rice (Oryza sativa) (Morillo
and Tax, 2006).
Hua et al., 2012, isolated GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1),
which encodes a receptor-like kinase localized on the plasma membrane in Arabidopsis thaliana.

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GHR1 is a leucine-rich repeat (LRR) RLK. They analyzed ghr1 mutants. These mutants were
defective ABA and H2O2 induction of stomatal closure. Genetic analysis indicates that GHR1 is
a critical early component in ABA signaling. The ghr1 mutation impaired ABA- and H2O2-
regulated activation of S-type anion currents in guard cells. Furthermore, GHR1 physically
interacted with, phosphorylated, and activated the S-type anion channel SLOW ANION
CHANNEL ASSOCIATED1(SLAC1) when coexpressed in Xenopus laevis oocytes, and this
activation was inhibited by ABA-INSENSITIVE2 (ABI2) but not ABI1. The interaction between
GHR1 and SLAC1 provides a simple model for the regulation of downstream targets by an RLK
in plants.Their study identified a critical component in ABA and H2O2 signaling that is involved
in stomatal movement and resolves a long-standing mystery about the differential functions of
ABI1 and ABI2 in this process.
They randomly isolated an Arabidopsis mutant that lost more water and wilted earlier than the
wild type when growing in a pot with soil. They named this mutant ghr1. They determined that
its stomata were resistant to changes in H2O2 . The detached leaves of ghr1 lost water more
readly than the wild type (figure.A), although the number of stomata were similar (figure.B).
Figure 6. Comparison of water loss and guard cell number between WT and ghr1. (Hua et al.,
2012)
(A) Water loss of detached leaves of the wild type (WT) and the ghr1 mutant. Values are means
6 SE of three replicates (40 leaves from one pot per replicate) for one experiment (**P < 0.01
from the fourth time point), and three experiments were performed with similar results.

16. 16
(B) Number of guard cells in the leaf abaxial epidermis of the wild type (WT) and the ghr1
mutant.
They used solated epidermal peels to test the stomatal responses to ABA: the ghr1 mutation
impaired ABA-induced stomatal closure and ABA-mediated inhibition of light induced stomatal
opening.
Figure 7. Model showing the roles of constitutive- and ABA-inducible cytosolic H2O2
accumulation in ABA signaling in guard cells. Arrows are indicating the positive regulation.
Open blockes indicate negative regulation. Mitochondria and chloroplast give constitutive H2O2
when there is absence of ABA and this is independent from stomatal movement. In a wild type
guard cell, CAT3 decomposes the constitutive H2O2 to O2. In the presence of ABA, ABA
activates NAD(P)H oxidases in the plasma membrane that produces H2O2 from superoxide anion
(•O2−
). This H2O2 migrates into cytosolic space across the plasma membrane, raises cytosolic

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H2O2 concentration (ABA-inducible H2O2) and functions for following stomatal movement
signaling in the cell. ABA-inducible H2O2 is also decomposed into O2 by CAT3 for
downregulating the signal. Thus, disruption of CAT3 causes a slight increase of ABA sensitivity
of stomata.
8. Nitric Oxide and Abscisic Acid Cross Talk in Guard Cells.
Nitric oxide( NO) is a short life bioactive molecule first described as a toxic compound. But now
we recognized it as an important signal and effector molecule both in animal and plant cell
physiology. Even though NO research in plants is not as advanced as in animals, in the last
decade NO was proved to participate in many key physiological processes such as growth,
pathogen defense reaction, development, programmed cell death, and stress tolerance (Foissner
et al., 2000; Pedroso et al., 2000; Beligni and Lamattina, 2001a). In plants, as in animals, NO
was proved to interact with other signaling elements such as cADPR, lipids, cGMP, ion
channels, Ca2-
, and others. In addition, much evidence is coming about cross talk between NO
and some plant hormones during adaptive responses to adverse conditions (Hausladen and
Stamler, 1998; Durner and Klessig, 1999; Jacob et al., 1999; Beligni and Lamattina, 2001b;
Wendehenne et al., 2001).
Treatment of V. faba epidermal stripes with increasing concentrations of ABA in the presence of
increasing concentrations of NO releaser sodium nitroprusside (SNP). As expected, both ABA
and SNP induced stomatal closure in a dose-dependent manner. Thus, small and rapid changes in
both ABA and NO concentrations can determine variations in percentages of stomatal closure
and probably explain the spatial and temporal heterogeneity in stomatal behavior, as has been
already described (Mott and Buckley, 2000).
Application of the specific NO scavenger 2-(4- carboxyphenyl)-4,4,5,5-tetramethylimidazoline-
1-oxyl- 3-oxide (c-PTIO) on the ABA-induced stomatal closure showed that in the presence of
PTIO the percentage of open stomata and remained constant through all the tested ABA
concentrations, showing that the guard cells were not responding to ABA treatment(Carlos
Garcia-Mata and Lorenzo Lamattina., 2002). In addition, when 200 micro molar SNP was added

18. 18
after the ABA + c-PTIO treatment, the stomatal closure was induced again, showing that the c-
PTIO-mediated inhibition of ABA-induced stomatal closure is reversible. All together, these data
suggest that NO might be acting downstream of the ABA-induced signaling cascade.
Figure 8. Schematic representation of ABA, H2O2 and NO signaling cross-talk in stomatal guard
cells. Solid lines represent those signaling pathways for which experimental evidence is
available; broken lines indicate predicted pathways. ( Adopted from Desikan et al.,2004)
Above figure shows that membrane bound ABA recepters are first activated in response to hike
in ABA concentration. This outer signal is then carried by several kinases and phosphatases.
Thus a chain of phosphorylation and dephosphorylation takes place. This results in the
production of reactive oxygen and nitrogen species like ROS and NOS. signal is then forwarded
in calcium dependent as well as independent manner.

19. 19
9. Abscisic Acid Regulation of Guard Cell Anion Channels.
When guard cells perceive increased ABA levels, their turgor and volume are reduced by efflux
of anions and potassium ions and by gluconeogenic conversion of malate into starch, causing
stomatal closure (MacRobbie et al., 1998). ABA triggers cytosolic [Ca2+]
cyt increases and
enhances [Ca2+]
cyt sensitivity (Siegel et al., 2009). It activates two different types of anion
channels, slow-activating sustained (S-type) and rapid-transient (R-type) anion channels (Linder
et al.,1992; Hedrichet al., 1990). Whereas S type anion channels generate slow and sustained
anion efflux, R-type anion channels are activated transiently within 50 ms, suggesting that two
different types of anion channels provide distinctive mechanisms for anion effluxe (Schroeder et
al., 1992). Anion efflux via anion channels causes membrane depolarization, which subsequently
drives K+
efflux from guard cells through outwardrectifying K+
out channels (Schroeder et
al.,1984; Hosy et al., 2003). H+
-ATPases induces K+
uptake through inward-rectifying K+
in
channels (Kwak et al.,2001; Lebaudy et al.,2007). ABA inhibits stomatal opening through
downregulation of K+
in channels and H+
-ATPase (Kinoshita et al.,1995).
Early patch clamp, cell signaling, and genetic studies suggested that S-type anion channels play a
key role in stimulus-induced stomatal closure (Grabov et al.,1997;Keller et al.,1989; Pei et
al.,1997). In guard cells, the early events in ABA signal transduction after receptor activation
involve ion channel regulation and cytosolic Ca2+
changes. There are a number of ion channel
types in the plasma membrane and vacuolar membrane of guard cells that can function in unison
to inhibit stomatal opening and mediate stomatal closing (Schroeder and Hedrich, 1989). During
drought stress ABA activates guard cell anion channels in a calcium-dependent as well as-
independent manner. Activation of slow anion channels in guard cells can function as a rate
limiting step in stomatal closing (Schmidt et al., 1995). When slow (S-type) anion channels are
activated, the resulting sustained efflux of anions from guard cells takes place. This causes long
term depolarization. It results in the activation of outward-rectifying K+
channel currents, which
mediate K+
efflux. Efflux of K+
and anions lowers the turgor and the volume of guard cells,
resulting in closure of stomatal pores.

20. 20
Slow anion channels are strongly activated by elevation in cytosolic Ca2+
and by phosphorylation
events (Schmidt et al., 1995). it is now clear that phosphorylation and dephosphorylation are
important componants of ABA signaling. Protein kinase Open stomata 1( OST1) and protein
phosphatase ABA insensitive 1( ABI1) are two key componenets of ABA signaling pathway.
The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in
this signaling pathway. OST1 is an interaction partner of SLAC1 and ABI1. Using protein–
protein interaction assays protein kinase OST1 and the protein phosphatase ABI1 were identified
as regulators of SLAC1 within the ABA transduction Pathway. Upon coexpression of SLAC1
with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those
observed in guard cells. But , Integration of ABI1 into the SLAC1/OST1 complex prevented
SLAC1 activation. Studies have demonstrated that SLAC1 represents the slow, deactivating,
weak voltage-dependent anion channel of guard cells controlled by
phosphorylation/dephosphorylation. Protein phosphatases ABI1 and ABI2 were identified as
elements of ABA signaling pathway on the basis of the ABA-insensitive abi1–1 and abi2–1
dominant mutations. Guard-cell activity is impaired in these ABA-insensitive Mutants and the
stomata remains constitutively open even during drought stress.
ABA-activation of guard cell anion channels in leaves of intact plants takes place under two
different scenarios: one is Ca2+-
independent( Levchenko et al., 2005 and Marten et al.,2007);
whereas the other is associated with changes in cytosolic Ca2+
levels (Hetherington et al., 2003
and 2004).CDPK protein kinases are able to control the activation state of the slow guard cell
anion channel in response to different Ca2+
concentrations as well in a Ca2+
-independent
manner(Geiger et al., 2009).
9.1 Channels in the plasmalemma:
There is clear evidence for activation of slow anion channels by ABA, in Arabidopsis (Pei et al.
1997) and in tobacco (Grabov et al. 1997). Protein kinases and protein phosphatases, of types 1-
2A and 2C, can be involved in the signalling chains by which anion channels are regulated. One

21. 21
aspect of the results in Arabidopsis, which is important, is that the anion channels were activated
by ABA in conditions where the rise in cytoplasmic Ca2+
was buffered out by Ca2+
chelator.
Thus, even if the anion channels are Ca2+
-activated, the ABA-induced activation of anion eflux
by activation of the S-type anion channels is, similar to the activation of K+
eflux through the
outward K+
channels, a Ca2+
-independent process. Whether this is also true of the anion channel
activation in Vicia, Commelina and tobacco remains to be seen. It remains to be established
whether anion channel activation can be achieved by alternative mechanisms, one of which is
Ca2+
-dependent and the other Ca2+
-independent, in the same cell.
9.2 Tonoplast ion channels:
A total of two K+
-permeable channels have been identified using patch-clamping technique of
isolated guard cell vacuoles. These are the ubiquitous SV channels, first observed in sugar beet
vacuoles by Hedrich & Neher (1987). The SV channel is Ca2+
-activated. It is voltage dependent
and opens at positive potentials. This channel is permeable to K+
, Ca2+
, and Mg2+
, with relative
permeabilities dependent on ionic conditions, but not permeable to anions.
SV channel carries the K+
efflux from the vacuole in response to ABA-induced increase in
cytoplasmic Ca2
. The necessary positive shift is by the operation ofthe second K+
-permeable
channel identified in the tonoplast, the VK channel (Ward & Schroeder 1994). This is K+
-
selective but insensitive to voltage. Like SV channel it is also activated by calcium
concentration. But the level of Ca2+
required is much less than the concentration that is required
by SV channel. The current hypothesis is that an ABA-induced increase in cytoplasmic Ca2+
will
first activate the VK channel, and the resultant K+
flux will drive the cytoplasm sufficiently
positive to activate the SV channel (whose activation voltage has also been shifted negative by
the increase in Ca2+
), allowing further eflux of K+.
Pottosin et al. (1997) found that the open probability of the SVchannel depended on the elec-
trochemical gradient for Ca2+
across the tonoplast.

22. 22
Figure 8. Proposed signalling pathways linking ABA to changes in specific ion channels in
guard cells, and to the changes which contribute to stomatal closure. There is evidence for each
of the links shown, but of variable weight, and the scheme must be regarded as a working
hypothesis. (Adopted from MacRobbie et al.,1998)
Above figure is an attempt to summarize potential signaling chains where the global changes and
protein phosphorylation may be linked to the flux changes in the plasmalemma and tonoplast
necessary for ABA-induced stomatal closure.

23. 23
10. Common techniques used to study stomatal functions and hidden
physiology behind:
10.1 Patch clamp
Patch clamping is used to measure ion currents across biological membranes. For example,
measurement of currents through K+
- channel and anion channels in guard cells. Membranes are
isolated using enzymes that digests away the cell wall. These naked cells, called protoplasts, are
kept in a solution (bath solution) with appropriate osmolarity and various ions depending on
which ion channels we are interested in and experimental design. A glass pipette is filled with a
pipette solution and an Ag/AgCl wire connected to an electrical device called the patch clamp
(amplifier). The patch clamp is connected to a computer so we can control experimental
parameters and analyze the acquired patch clamp data. The patch pipette is moved to the surface
of the protoplast and mild suction is applied to obtain a gigaohm seal between the pipette and the
plasma membrane. From there various approaches can be used.
Ion currents are converted into electrical currents and vice versa at the Ag/AgCl wire, so that ion
currents going across the membranes can be read as electrical currents by the patch clamp. We
use a technique called voltage clamping where the membrane potential of the cell is held
constant (clamped) while we measure ion currents across the membrane.
Bath dish with ground and pipette electrode mounted on the microscope:
Vicia faba guard cell protoplast being approached by the patch pipette:

24. 24
10.2 Infra-red thermal imaging:
The electronic detection and display of long wave or far-red radiation is known as thermal
imaging. Using a sensitive camera together with the appropriate image analysis software the
technique allows the surface temperature of objects to be both displayed visually and quantified
with a resolution of less than 0.07 ° C. Although, the main applications of this form of imaging
are in the defence or manufacturing industries the technique has also been used in botanical
research. Successful applications include, investigating the ascent of sap (Anfodillo et al 1993 Pl
Cell Env 16:997), flowering in aroid species (Skubatz et al 1990 Planta 182:432 and
Bermadinger-Stabentheiner and Stabentheiner 1995 New Phyt 131:41) and ice nucleation
(Wisniewski et al 1997 Plant Physiol 113:327).
It can also be used to identify mutants that display aberrant stomatal behaviour. People have used
it identify mutants in Arabidopsis. Raskin and Ladyman (1988 Planta 173:73) used thermal
imaging to isolate the ABA insensitive cool mutant of barley. Infra-red thermal imaging can be
used to visualise evapo-transpirational cooling of the leaf surface. As the loss of water vapour
during transpiration occurs through the stomata, thermal imaging can be applied to the problem
of identifying plants that display aberrant stomatal behaviour. For example, Arabidopsis plants
carrying the ABI1-1 mutation display stomata that are insensitive to the plant hormone abscisic
acid (ABA). Application of ABA to wild type Arabidopsis results in a reduction in stomatal
aperture, which is manifested in an increase in leaf temperature (as a result of a reduction in
evapo-transpirational cooling). In centrast stomata of the abi1-1 mutant are insensitive to ABA
and gape wide open. Comparison of abi1-1 to wild type reveals that the leaves of the mutant are
consistently cooler.

25. 25
Thermogram showing Wild Type (left) and abi 1-1 mutant (right) Arabidopsis thaliana plants
11. Summary
Guard cells are a unique signal transduction research tool that provide an elegant system to
dissect an intricate network of signalling pathways. Both H2O2 and NO play a central role in the
guard cell ABA signaling network. These molecules are synthesized in response to ABA and
both control a single Response the reduction in stomatal aperture. NADPH oxidase is a source
of H2O2 biosynthesis in guard cells, although other sources could also exist. Ca2+
functions as a
second messenger in guard cell signaling and stomatal movements, as was originally described
over 20 years ago (DeSilva et al., 1985; Schwartz, 1985; Schroeder and Hagiwara, 1989;
McAinsh et al., 1990). Early patch clamp, cell signaling, and genetic studies suggested that S-
type anion channels play a key role in stimulus-induced stomatal closure (Grabov et
al.,1997;Keller et al.,1989; Pei et al.,1997). In guard cells, events after receptor activation
involve ion channel regulation and cytosolic Ca2+
changes. There are a number of ion channel
types in the plasma membrane and vacuolar membrane of guard cells that can function in unison
to inhibit stomatal opening and mediate stomatal closing (Schroeder and Hedrich, 1989). Protein
kinase Open stomata 1( OST1) and protein phosphatase ABA insensitive 1( ABI1) are two key

26. 26
componenets of ABA signaling pathway. The recently identified guard cell anion channel
SLAC1 appeared to be the key ion channel in this signaling pathway. OST1 is an interaction
partner of SLAC1 and ABI1. Using protein–protein interaction assays protein kinase OST1 and
the protein phosphatase.
ABI1 were identified as regulators of SLAC1 within the ABA transduction Pathway. PLD α
mediates the ABA effects on stomata through interaction with a protein phosphatase 2C (PP2C)
and a heterotrimeric GTP-binding protein (G protein) in Arabidopsis (Mishra et al., 2006). PLD
and PA actually regulate NADPH- oxidase activity. NADPH oxidases are the source of ROS
produced in ABA response and other processes, including pathogen recognition and root hair
growth (Torres et al., 2002; Foreman et al., 2003; Kwak et al., 2003; Torres and Dangl, 2005).
Guard cells contain PI3P activity. An inhibitor of PI3P also inhibited ABA induced ROS
generation and stomatal closure (Park et al., 2003). Among the identified molecular elements
working in ABA signaling are protein kinases and phosphatases that play a central role in
regulating the signaling network. Two MAPK genes, MPK9 and MPK12 are preferentially and
highly expressed in guard cells. Hua et al., 2012, isolated GUARD CELL HYDROGEN
PEROXIDE-RESISTANT1 (GHR1), which encodes a receptor-like kinase localized on the
plasma membrane in Arabidopsis thaliana. GHR1 is a leucine-rich repeat (LRR) RLK. They
analyzed ghr1 mutants. These mutants were defective ABA and H2O2 induction of stomatal
closure.

27. 27
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