CHAPTER 8

PHOSPHOLIPID SIGNALING IN PLANTRESPONSE TO DROUGHT AND SALT STRESS

XUEMIN WANG1, WENHAU ZHANG2, WEIQI LI3,AND GIRISH MISHRA1

1Department of Biology, University of Missouri, St. Louis, MO 63121 and Donald Danforth PlantScience Center, St. Louis, MO 63132, USA2College of Life Science, State Key Laboratory of Crop Genetics and Germplasm Enhancement,Nanjing Agricultural University, Nanijing 210095, P.R. China3Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204 and Department ofBiology, Honghe University, Mengzi, Yunnan 661100, China

Abstract: Many stresses trigger transient increases in minor phospholipids, such as phosphatidicacid (PA) and phosphoinositides (PIs), in plants. Such changes are early events insignaling plant stress response. Lipid mediators affect cellular functions through directinteraction with proteins and/or structural effects on cell membranes. The identifiedlipid targets in plants include protein phosphatases, kinases, and proteins involved inmembrane trafficking and cytoskeleton. The effect of lipids on signaling, intracellulartrafficking, and cytoskeletal organization plays important roles in plant coping withdrought and salinity

Keywords: lipid signaling; phospholipases; phosphoinositides; drought; salt; stress response;osmotic stress; abscisic acid; phosphatidic acid

1. INTRODUCTION

As the physical barrier that separates the interior of a cell from its surroundings,cell membranes play a pivotal role in plant responses to environment stresses.Membranes are the initial site of cellular perception of stress cues. Many subsequentsteps in signaling cascades, such as activation of effector proteins, generation ofsecond messengers, and alteration in cellular metabolism, are often associated withmembranes. While proteins have been the focus of most research on membrane-associated signaling events, recent advances have made it evident that membranelipids and their derivatives are important players in the signaling network of plantresponses to stress, including drought and salinity.

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Membrane lipids give rise to various signaling messengers, such as phosphatidicacid (PA), diacylglycerol (DAG), DAG-pyrophosphate (DAG-PP), lysophospho-lipids, free fatty acids (FFAs), oxylipins, phosphoinositides, and inositol polyphos-phates. The production of these mediators is regulated by different families ofenzymes, particularly phospholipases, lipid kinases, and/or phosphatases (Wang,2004). Great strides have been made recently to understand the role of lipid signalingin different plant processes. Several recent reviews have dealt with signaling aspectsof various lipids and enzymes in plants (Chapman, 2004; Ryu, 2004; Testerink andMunnik, 2005; van Schooten et al, 2006; Wang, 2004; Wang et al., 2006; Zhanget al., 2005). Here, we will focus on the involvement of phospholipid-mediatedsignaling in plant response to drought and salt stress.

2. PHOSPHATIDIC ACID AS A MESSENGERIN OSMOTIC STRESS

PA has been identified as a new class of lipid mediators regulating numerous cellularprocesses, including signal transduction, cytoskeletal rearrangement, secretion,endo/exocytosis,and oxidative burst. PA is a minor membrane lipid, constitutingless than 1% of total phospholipids in Arabidopsis leaves (Zhang et al., 2004;Wang et al., 2006). Cellular levels of PA in plants change rapidly under variousconditions, including abiotic and biotic stresses as well as during plant growth anddevelopment (Testerink and Munnik, 2005; Wang et al., 2006). In particular, PAis produced under different forms of osmotic stress, such as dehydration, drought,salinity, freezing, and treatment with the stress hormone abscisic acid (ABA).

Cellular PA may be produced by multiple enzymes (Wang et al., 2006; Figure 1).Available data indicate that signaling PA is generated by two principal routesin plants. One is the phospholipase D (PLD)-catalyzed hydrolysis of commonmembrane phospholipids to produce directly PA. Another is phospholipase C(PLC) hydrolysis of phosphatidylinositol (4, 5) bisphosphate PI(4, 5)P2 followedby phosphorylation of DAG by DAG kinase (DGK). PLD, PLC, and DGK eachconsist of multiple enzymes in plants. For example, Arabidopsis has 12 PLD genesthat are grouped into six types, PLD�, �, �, �, �, and �. Different type of PLDsdisplay distinguishable properties, such as their requirements for Ca2+, phospho-inositides (PI), and/or free fatty acids, their lipid vesicle composition, substratepreferences, subcellular location, and patterns of gene expression. These differencesplay an important role in regulating the spatial and temporal production of PA andalso indicate distinguishable functions among different PLDs. It has been shownthat Arabidopsis PLD�1 is responsible for ABA-induced PA (Zhang et al., 2004),whereas PLD� is involved in the dehydration-induced PA formation (Katagiri et al.2001). PLD and PA have been suggested to affect osmotic-stress induced productionof proline (Thiery et al., 2004). 1-Butanol, an inhibitor of PA production by PLD,reduces NaCl- induced H+-ATPase activation, whereas applied PA stimulated H+-ATPase activity (Zhang et al., 2006). These results point to a role of PLD and PAin salt stress response.

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PA DAG

PLD NS -PLC

LPP

PC, PE, PG, PS PIP2 PC, PE, PG, PS

PAKDAG-PPi

PI-PLC

LPP

DGK

Acyl transferase

LysoPA

PA-binding protein Protein function Effect of PA binding

ABI1 PP2C and a negative regulator of ABA response Inhibit ABI1 function and promote stomatal closure

AtPDK1 3-Phopshoinositide-dependent kinase 1 Activate AGC kinase and promote root growth

AtCP Heterodimeric actin capping protein Inhibit capping and stimulate actin polymerization

mTOR Mammalian target of rapamycin Activate TOR signaling and promote cell survival

Opi1p Yeast soluble transcriptional repressor Regulate Opi1p location between ER and nucleus and the hemeostasis of glycerolipid synthesis

Figure 1. PA production and selected PA effects (Wang et al., 2006). Enzymatic reactions leading tothe PA production (upper) and removal (lower). DGK, diacylglycerol kinases; DAG-PPi, diacylglycerolpyrophosphate; LPP, lipid phosphate phosphatase;LysoPA, lysophosphatidic acid; PAK, phosphatidicacid kinase; PI-PLC, phosphoinostide-specific PLC; NS-PLC, non-specific PLC. PC, phosphatidyl-choline; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine. PA binds todifferent types of proteins and the specific examples including proteins from plants, animals, and yeast

Arabidopsis has muliple PI-PLCs that hydrolyze PI(4, 5)P2 to generates DAGand inositol 1, 4, 5-trisphosphate (IP3). DAG serves as a potent activator of proteinkinase C in animal cells, but its target is unclear in plants. Under salt and hyper-osmotic conditions, PLC-produced DAG is phosphorylated to PA by DGK. Inseveral cases, it has been found that the PLD and PLC/DGK reactions are activateddifferentially in response to different stimuli (den Hartog et al., 2003; Zonia andMunnik, 2004).

The increase in PA can impact cell function in different ways:i) PA can act asa messenger by its interaction with specific target proteins. PA has been foundto tether target proteins to membranes and/or modulate the catalytic activity ofits targets (Fan et al., 2001; Anthony et al., 2004; Zhang et al., 2004; Huanget al., 2006). ii) PA may alter membrane structure, promoting membrane fusionand interaction of certain soluble proteins with membranes (Kooijman et al., 2005;Wang et al., 2006). iii) PA may be converted to other signaling molecules, suchas DAG, lysoPA, DAG-PPi, and free fatty acids, or may be involved in membranelipid metabolism (Testerink and Munnik, 2005; Wang et al., 2006; Figure 1).A number of PA-binding proteins have been identified in plants, animals, and yeast,which include protein kinases (Anthony et al., 2004; Fang et al., 2001), protein

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phosphatases (Zhang et al., 2004), transcriptional factors (Loewen et al., 2004), andproteins involved in vesicular trafficking and cytoskeletal dynamics (Huang et al.,2006; Testerink et al., 2004).

Potentially relevant to drought and salt stress is the PA binding to ABI1 proteinphosphatase 2C (PP2C) (Zhang et al., 2004), to 3-phosphoinositide-dependentkinase 1 (AtPDK1) (Anthony et al., 2004), and to the mammalian target ofrapamycin (mTOR), a phosphatidylinositol-3 kinase like protein kinase (Fang et al.,2001). The PA-ABI1 interaction is required for ABA promotion of stomatal closure(Mishra et al., 2006), whereas the PA-AtPDK1 binding promotes root growth(Anthony et al., 2004). The TOR kinase pathway mediates translational regulationof cell growth and proliferation in animal cells. The TOR pathway in plants,animals, and yeast are affected by various adverse conditions including osmoticstress (Mahfouz et al., 2006). The TOR target ribosomal S6 kinase 1 (S6K1) isalso regulated by AtPDK1. Thus, it would be of interest to test whether the TORsignaling pathway is a PA target in plant osmotic stress response.

3. PHOSPHATIDIC ACID AND PHOSPHOLIPASED IN STOMATAL CLOSURE AND WATER LOSS

One documented function of PA is to mediate stomatal response to ABA. ABAis a phytohormone involved in diverse plant processes, including stomatal closure.ABA causes stomatal closure by affecting two separable processes:it promotes theclosing of opened stomata and inhibits the opening of closed stomata. Recent resultsshow that stomatal responses to ABA are regulated by a bifurcating pathway thatincludes PLD�1, PA, ABI1, and G� (Mishra et al., 2006; Figure 2). To promoteclosure of open stomata, PLD�1 produces PA that binds to the ABI1 PP2C (Zhanget al., 2004). ABI1 is a negative regulator of ABA response, and the PA-ABI1interaction is necessary to remove the ABI1 inhibition of the ABA promotion ofstomatal closure. PA regulates ABI1 function by inhibiting its phosphatase activityand by sequestering it in the plasma membrane. The membrane tethering by PAdecreases ABI1’s translocation from the cytosol to the nucleus and promotes ABAsignaling (Zhang et al., 2004).

To inhibit opening of closed stomata, PLD�1 modulates the function of G� (onlycanonical G� subunit of a heterotrimeric G protein in Arabidopsis) through multipleinteractions (Figure 2). PLD�1 activates the intrinsic GTPase activity that convertsactive G�-GTP to inactive G�-GDP (Zhao and Wang 2004). In turn, G�-GDP binds toPLD�1 and decreases its activity. Weakening the G�-GDP and PLD�1 interactionrenders Arabidopsis plants hypersensitive to ABA because both the G� and PLD�1functions are less inhibited by the subdued interaction between PLD�1 and G�(Mishra et al., 2006). On the other hand, both PLD�1 and G� are positive regulatorsin ABA inhibition of stomatal opening. The positive role of G� may result fromthe exchange of GTP with GDP; the binding of GTP to G� (G�-GTP) dissociatesG� from PLD�1, thus removing the inhibition of PLD�1 activity. PA resulting

PHOSPHOLIPID SIGNALING IN PLANT RESPONSE 187

PLDα1

ABI1-PP2C ABI1-PP2C PA

Promotesclosure

Inhibitsclosure

Gα-GTP Gα-GDP

Inhibitsopening

Promotesopening

More closed stomata

More open stomata

PA

PLDα1

DroughtABA

Figure 2. A bifurcating pathway by which PLD and PA in signaling ABA response in guard cells(Mishra et al., 2006). PLD�1-derived PA binds to ABI1, and this interaction tethers ABI1 to the plasmamembrane and also decreases the PP2C activity. Thus, PA promotes ABA response by suppressing thenegative effect of ABI1. In addition, PLD�1 binds to G�, the � subunit of heterotrimeric G proteins,and this interaction regulates reciprocally the activity of PLD�1 and G�, and thus the production ofPA. Note that this model is not comprehensive and only includes some of the signaling componentsimplicated in the ABA signaling cascades

from PLD�1 activity promotes inhibition of stomatal opening (Figure 3). Thus, thePLD�1- G� interaction regulates mutually the activity of both proteins.

The PA/PLD regulation of stomatal closure affects plant water loss. Terrestrialplants lose water primarily via stomata. During drought, ABA levels in plantsincrease and promotes stomatal closure. This change is crucial to maintaining ahydration status in leaves and permitting plant survival. In Arabidopsis, the stomataof PLD�1-deficient plants fail to close in response to ABA (Sang et al., 2001;Mishra et al., 2006). PLD�1-deficient plants exhibit a higher rate of transpirationalwater loss than wild-type plants, whereas overexpression of PLD�1 reduces transpi-rational water loss in tobacco by rendering the plants more sensitive to ABA (Sanget al., 2001). These insights into the pathways regulating stomatal function may beused to produce plants with enhanced water-usage efficiency and drought tolerance.

4. PHOSPHOINOSITIDES IN OSMOTIC STRESS

Phosphoinositides (PIs) are phosphorylated phosphatidylinositols. They include threemonophosphorylated PI3P, PI4P, and PI5P; three bisphosphorylated PI(4, 5)P2,PI(3, 4)P2, and PI(3, 5)P2; and one trisphosphorylated PI(3, 4, 5)P3 (Figure 3). Exceptfor PI(3, 4, 5)P3, the occurrence of all other PIs have been reported in plants.Several PIs, such as PI5P, PI(3, 5)P2, and PI(4, 5)P2, are elevated in plants underhyperosmotic stress (Meijer and Munnik 2003; Zonia and Munnik, 2004).

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PI 5P

PI3P

PI4P

PI (3,5)P2

PI(3,4)P2

PI(4,5)P2

PI(3,4,5)P3

DAG I(1,4,5)P3

PA [Ca 2+]

PI

Bind proteins with specific domains:

PH, PX, FYVE, and other motifs

PLC

DGK

Figure 3. Phosphoinositide metabolism and selected functions. The phosphorylation and dephosphory-lation of PIs are catalyzed by specific PI kinases and phosphatases. The arrow points to known directionof PI metabolism, but some of the reactions are yet to be demonstrated in plants. One function of PIs isto interact with proteins with specific domains such as PH, PX, and FYVE. In addition, PI(4, 5)P2 is asubstrate of PLC that produces IP3 and DAG

A study on Ssh1p, a soybean Sec14-like, phosphatidylinositol transfer proteins(PITP), has shed light on the early events of osmosensory signaling and PI synthesisin plants (Monks et al., 2001). Under hyperosmotic stress, Ssh1p kinases, SPK1and/or SPK2, are activated and rapidly phosphorylate Ssh1p. This modificationdecreases membrane association of Ssh1p. Ssh1p enhances the activities of plant PI3-kinase and PI 4-kinase, suggesting that Ssh1p’s function in cellular signaling isto alter the plant’s capacity to synthesize PIs during hyperosmotic stress (Figure 4).

Hyperosmotic stress

Osmosensor Membrane

SPK1/2

Activation

Sship

Sship

Increase PI synthesis

PI effects onsignaling, vesicular trafficking,

cytoskeletal rearrangement, ion channels

Osmotic response

p

Figure 4. A hypothetic model of hyperosmotic stress-induced production of phosphoinositides andfunctions (Monks et al., 2001). Hyperosmotic stress triggers activation of the kinases SPK 1 and 2 thatphosphorylate the Sec14-like Ssh1p. Activated Ssh1p stimulates PI 3-kinase– and PI 4-kinase to increasethe production of PIs. PIs modulate cellular processes and participate in osmotic stress responses

PHOSPHOLIPID SIGNALING IN PLANT RESPONSE 189

Recently, dysfunctions of specific PITPs have been linked to plant root hairgrowth and stress response (Bohme et al., 2004; Vincent et al., 2005). TheArabidopsis Sec14p-nodulin domain proteins AtSfh1p regulate intracellular andplasma membrane PI polarity directing membrane trafficking, Ca2+ signaling, andcytoskeleton functions to the growing root hair apex (Vincent et al., 2005). It hasbeen proposed that Sec14p-nodulin domain proteins represent a family of regulatorsof polarized membrane growth in plants (Vincent et al., 2005). In yeast, PLDactivity is required for suppression of PITP defect (Xie et al., 1998), indicating thatcrosstalk exists among PITPs, PLDs, PIs, and PA in cell regulation.

PIs are important signaling molecules that regulate actin organization, membranetrafficking, endo/exocytosis, and ion channels. For example, PIs are required foractivating plant shaker-type K+ channels (Liu et al., 2005) and for normal stomatalmovement (Jung et al., 2002). PIs affect the location and function of proteins withspecific PI-binding domains, such as pleckstrin homology (PH), phox homology(PX), FYVE finger, and other motifs. In plants, several proteins have shown tointeract with PIs, and these include certain PLDs (Pappan et al., 1997; Zheng et al.,2002) and Patellin1, a novel Sec14-like protein, localized to the cell plate (Petermanet al., 2004). PI(4, 5)P2 is a required activator of PLD�, �, and �, and also stimulatesPLD�1 activity (Pappan et al., 2004; Qin and Wang 2002). PLD�s contain twoPI-binding domains, PH and PX, whereas of PLD� has been shown to bind to PIsthrough a polybasic residue-motif (Zheng et al., 2002). The role of PLD in osmoticstress-induced production of PA and ABA signaling is described earlier. Thus, it ispossible that increases in PIs under hyperosmotic stress contribute to the activationof PLD and PA production in the stress response.

In addition to be a mediator on its own, PI(4, 5)P2 is the substrate of PLCthat produces DAG and IP3. While osmotic stress-induced DAG has been shownto be rapidly converted to PA, IP3 accumulates in salt, cold, and osmoticallystressed plants (Smolenska and Kacperska 1996; Takahashi et al., 2001; Xiong et al.,2001). IP3 promotes an increase in Ca2+ in guard cells and stomatal closure, andsuppression of a PLC reduces ABA-promoted closure of stomata (Hunt et al., 2003).

The signaling process of PIs and the derivative inositol polyphosphates isterminated through the action of PI phosphatases and inositol polyphosphatephosphatases. Perturbation of these phosphatases by either overexpression orablation affects the expression of stress-responsive genes under salt, drought,and ABA treatment and alters plants stress tolerance. At5PTase1 is up-regulatedin response to ABA and has bee suggested to act as a signal terminator ofABA signaling (Burnette et al., 2003). FRY1 encodes an inositol polyphosphate1-phosphatase, and fry1 mutant plants had elevated levels of IP3 after ABAtreatment. The mutant plants are compromised in tolerance to freezing, drought,and salt stresses (Xiong et al., 2001). Knockout of the suppressor of actin mutation(SAC) domain phosphatase results in elevated levels of PI(4, 5)P2 and IP3 ascompared to wild-type plants under unstressed conditions (Williams et al., 2005).The sac9 mutants display constitutive stress response, including closed stomata,anthocyanin accumulation, overexpressing stress-induced genes, and accumulating

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reactive-oxygen species. The results indicate that cellular levels of PIs and inositolpolyphosphates are tightly regulated to achieve optimal plant performance understress.

5. PERSPECTIVES

Drought and salinity are the two crucial environmental factors that limit plantgrowth, productivity, and geographic distribution. Lipid-mediated signaling playsimportant roles in plant responses to these stresses. Information is growing rapidlyon the production of potential lipid mediators under different growth conditions.However, the current knowledge is still rather limited on the exact role of specificlipid signaling reactions in plant adaptation to drought and salt stress.

Genetic manipulation of enzymes that produce lipid mediators provides valuableinsights into the function of specific lipid signaling reaction. The distinguishablephenotypes resulting from the loss of different PLDs indicate that some PLDs haveunique functions in plant response to different forms of osmotic stresses. Suchdistinctions may be related to the location and timing of PA production regulatedby different PLDs. Spatial and temporal regulation is important to all signalingevents, but it is particularly critical to intracellular lipid messengers because oftheir limited mobility in the cell. Similarly, distinguishable functions are expectedto occur for other phospholipid-signaling enzyme families, such as PLCs, DGKs,PI kinases, and phosphatases. Therefore, it is important to identify specific geneand enzymes involved when the roles of given type of lipid signaling enzymesin a specific physiological response are addressed. Such information will also benecessary for potential biotechnological manipulation of lipid signaling pathwaysto improve plant stress tolerance.

ACKNOWLEDGMENTS

We thank Eric Weldon for critical reading of the manuscript. Work fromauthors’ laboratory was supported by grants from the National Science Foundation(IBN-0454866) and the U.S. Department of Agriculture (2005-35318).

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