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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

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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.

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(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

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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.

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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.

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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

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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 Ca

2+-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+, Ca

2+, and Mg

2+, 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

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

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

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

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

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

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