Phospholipase D and phosphatidic acid ... - Wiley Online Library

Plant, Cell and Environment (2010) 33, 627–635

doi: 10.1111/j.1365-3040.2009.02087.x

Phospholipase D and phosphatidic acid signalling in plant response to drought and salinity pce_2087

627..635

YUEYUN HONG1, WENHUA ZHANG2 & XUEMIN WANG3 1 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China; 2College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China and 3Department of Biology, University of Missouri, St. Louis, MO 63121, USA; Donald Danforth Plant Science Center, St. Louis, MO 63132, USA

ABSTRACT

INTRODUCTION

The activity of phospholipase D (PLD) in plants increases under different hyperosmotic stresses, such as dehydration, drought, and salinity. Recent results begin to shed light onto the involvement of PLD in response to water deficits and salinity. Different PLDs have unique and overlapping functions in these responses. PLDa1 promotes stomatal closure and reduces water loss. PLDa1 and PLDd are involved in seedling tolerance to salt stress. PLDa3 and PLDe enhance plant growth and hyperosmotic tolerance. The different PLDs regulate the production of phosphatidic acid (PA) that is a key class of lipid mediators in plant response to environmental stresses. Further studies on the upstream regulators that activate different PLDs and the downstream effectors of PLDs and PA have the potential to unveil the linkage between the stimulus perception at the cell membrane to intracellular responses to drought and salinity stresses.

Drought and soil salinity are major environmental stresses that adversely affect plant growth and development. Studies in recent years have provided valuable insights into the molecular and cellular mechanisms by which plants respond to and tolerate salinity and drought stresses. These include the identification and characterization of transporters and channels such as the Na+/H+ transporter, K+ inward and outward channels, signalling components in transducing salt and drought challenges, and epigenetic regulators including histone variants, histone post-translational modifications and DNA methylation (Apse et al. 1999; Zhu 2002; Chinnusamy & Zhu 2009). In addition, increasing evidence indicates that lipid signalling is an integral part of the complex regulatory network in plant response to salinity and drought (Wang et al. 2006; Hong et al. 2008a, 2009; Li, Hong & Wang 2009; Munnik & Testerink 2009). Modifications of membrane lipids produce different classes of signalling messengers, such as phosphatidic acid (PA), diacylglycerol (DAG), DAG-pyrophosphate (DAGPP), lysophospholipids, free fatty acids (FFAs), oxylipins, phosphoinositides and inositol polyphosphates (Wang 2004; Bargmann & Munnik 2006; Boss, Lynch & Wang 2008). The production of these mediators is regulated by different families of enzymes, particularly phospholipases (Fig. 1), lipid kinases and/or phosphatases. Phospholipase D (PLD), which hydrolyses membrane phospholipids to PA and a free head group, is a major family of phospholipases in plants (Fig. 1). Recent studies indicate that PLD and PA play important and complex roles in plant drought and salt stress tolerance (Wang 2002, 2004, 2005; Bargmann & Munnik 2006; Wang et al. 2006; Hong et al. 2008a, 2009; Bargmann et al. 2009). PLD activity has been implicated in signalling and/or catabolic functions (Hong, Zheng & Wang 2008b). Here we will focus on the signalling and regulatory functions of PLDs and PA in Arabidopsis response to drought and salinity.

Key-words: Arabidopsis; hyperosmotic stress; phospholipid signalling. Abbreviations: ABA, abscisic acid; C2, Ca2+-dependent phospholipid-binding; C2-PLD, PLD containing the C2 domain; DAG, diacylglycerol; DAG-PP, diacylglycerol pyrophosphate; DGK, diacylglycerol kinase; G protein, heterotrimeric GTP-binding protein; KO, knockout; mTOR, mammalian target of rapamycin; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; PA, phosphatidic acid; PDK1, phosphoinositide-dependent protein kinase1; PI(4)P, phosphatidylinositol 4 phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PH, pleckstrin homology; PI-PLC, phosphoinositide (4,5) bisphosphate (PIP2)-hydrolysing PLC; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PP2C, protein phosphatase 2C; PX, phox homology; PX/PH-PLDs, PLD containing PX and PH domains; ROS, reactive oxygen species; S6K, ribosomal S6 kinase.

PA AS LIPID MESSENGERS IN MEDIATING HYPEROSMOTIC STRESS Correspondence: Y. Hong. e-mail: [email protected]; X. Wang. Fax: +3145871519; e-mail: [email protected] © 2010 Blackwell Publishing Ltd

Genetic and pharmacological manipulations have shown that PA produced by PLDs plays a role in stomatal closure, 627

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Cellular and physiological effects: Stomatal movement ABA response ROS production ROS response Root hair growth Root elongation Na+/H+ exchange Anti-cell death M b Membrane ttrafficking ffi ki Cytoskeletal changes

Figure 1. The sites of phospholipid hydrolysis by phospholipase D, C, A and the targets of PA identified in plants that are potentially involved in hyperosmotic stress responses. PLD hydrolyses the terminal phosphodiester bond of glycerophospholipids to generate PA and a free head group. Head group can be choline, ethanolamine, serine, and/or glycerol. In the presence of primary alcohols, such as 1-butanol or ethanol, PLD also has a unique ability to transfer phosphatidyl group to a primary alcohol to form phosphatidylalcohol at the expense of PA. PLC hydrolyses the first phosphodiester bond to generate a phosphorylated head group and DAG. DAG can be phosphorylated to PA by DGK. PA molecular species refer to PAs with different fatty acid chains in the sn-1 and sn-2 positions. Several PA protein targets have been identified in plants and the PA-protein interactions are involved in plant response to hyperosmotic stresses.

root growth, and plant tolerance to salinity and water deficits. The production of the lipid mediator PA is a key mode of action by which PLDs modulate plant functions. PA is a minor class of membrane lipids, constituting less than 1% of total phospholipids in most plant tissues. Cellular levels of PA change rapidly under various conditions, and the magnitude of PA change varies, depending on the treatment and tissue. PA is comprised of various molecular species because the two fatty acyl chains vary in the numbers of carbons and double bonds (Devaiah et al. 2006). It has been reported that the hydrogen bond at the phosphate group of PA enhances the negative charge to recruit positively charged proteins (Kooijman et al. 2007). PA may affect cellular processes via different modes of action, such as direct interaction with proteins and biophysical effects on membrane structures (Wang et al. 2006).

PLD-produced PAs and its molecular targets in hyperosmotic stress responses Alterations of different PLDs have resulted in the change in PA production and cellular responses, indicating that different PLDs and their PAs have unique functions. PLDs have been shown to be regulated differently by several cellular effectors (see further discussion; Li et al. 2009), some display different subcellular association and substrate preferences (Fig. 2), and some show different expression patterns in response to hyperosmotic challenges. It is conceivable that the activity of different PLDs regulate the location, timing, and the molecular species (i.e. PAs with different fatty acyl chains) of PA production. For example, the major molecular species of PAs in Arabidopsis are 34:2 (16:0–18:2), 34:3 (16:0–18:3), 36:4 (18:2–18:2), 36:5

(18:2–18:3) and 36:6 (18:3–18:3) (Devaiah et al. 2006; Zhang et al. 2009). The PA species 34:1, 34:2, 34:3, 36:3, and 36:6 markedly increase in response to abscisic acid (ABA) application. In comparison, in PLDa1 knock-out mutant the PA species induced by ABA were primarily 34:2, 34:3, 36:4, and 36:5 PAs. In addition, the magnitude of increase in 34:2 PA at 10 min was lower in the mutant than in wild-type leaves. After a 10-min ABA treatment, the contents of 34:2, 34:3, 36:3, 36:4, 36:5 and 36:6 PAs in wild-type leaves were significantly higher than those in plda1 mutant leaves (Zhang et al. 2009). The results show that abrogation of PLDa1 leads to changes in the amount, the magnitude and molecular species of PA in response to ABA. However, in freezing response, both wild-type and PLDd knock-out mutant increase about four fold of PA, but the total amounts of PA accumulate in the PLDd mutant is only 20% less than that in wild type. In particular, 36:5 and 36:6 PAs in the mutant leaves are significantly lower than those in wild type (Li et al. 2004). Therefore, in response to various stimuli, the substrate specificity of different PLDs probably generates the PAs with certain fatty acids. Different PAs generated from specific PLDs may have their own specific targets. PA has been shown to interact with a number of proteins in plants, animals and yeast (Fig. 1). Different PA species show different affinity to specific proteins, suggesting that different PA species have different functions (Zhang et al. 2004, 2009). One documented PA target in plants is ABI1 that is involved in ABA signalling in stomata movement (Zhang et al. 2004). Site-specific mutation showed that arginine 73 in ABI1 is critical for PA-ABI1 binding in vitro and in vivo (Zhang et al. 2004; Mishra et al. 2006). The PA-ABI1 interaction promotes closure of open stomata, possibly via tethering ABI1 to the

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 627–635

Phospholipase D and phosphatidic acid signalling

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Figure 2. PLD family involved in Enhanced root growth

Less water loss Drought and salt tolerance

plasma membrane to prevent ABI1 translocation from the cytosol to the nucleus (Zhang et al. 2004). Recently, PA is found to bind to AtrbohD and AtrbohF NADPH oxidases, and the PA binding motifs in AtrbohD and AtrbohF have been identified near the N terminus (Zhang et al. 2009). The mutated AtrbohD, which lacks PA binding, failed to mediate PA-induced ROS elevation and stomatal closure and was compromised in ABA-promoted ROS production and stomatal closure. In addition, the ABA-induced production of nitric oxide (NO) was impaired in plda1 guard cells. Disruption of PA binding to the ABI1 protein phosphatase 2C (PP2C) did not affect the ABA-induced production of ROS or NO, but inhibited stomatal closure induced by ABA, H2O2 or NO. In addition, PA acts as an activator of other proteins, such as H+-ATPase, protein kinase C (PKC), and mitogen-activated protein (MAP) kinases (Zhang et al. 2003). Suppression of PLD-catalysed PA production abolished salt-induced increases in H+-ATPase activity, but addition of PA stimulates H+-ATPase activity. The PLD-produced PA has been proposed to activate H+-ATPase and Na+/H+ exchange to enhance salt tolerance (Zhang et al. 2006). In mammalian cells, PLD1-derived PA was shown to activate mammalian target of rapamycin (mTOR) signalling to promote protein synthesis and generate survival signals under stress conditions (Fang et al. 2001; Sun & Chen 2008). Sequence analysis suggests that the domains that are important for TOR function are conserved in the AtTOR protein (Menand et al. 2002). Plant TOR has been implicated in embryo development, meristem-driven cell growth and hyperosmotic stress response (Menand et al. 2002; Mahfouz et al. 2006). AtTOR regulates cellular activities by activating downstream kinases. One characterized TOR target is ribosomal S6 kinase (S6K) that phosphorylates ribosomal

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salt and drought stresses in different pathways. Different PLDs exhibit distinguishable reaction conditions, substrate preferences and subcellular localization. Four members of PLD family are found to be involved in drought and salt responses through different pathways.

proteins and promotes translation in both animal and plant (Fang et al. 2001; Mahfouz et al. 2006). In particular, S6K has been implicated in hyperosmotic stress response in Arabidopsis (Mahfouz et al. 2006). In addition, S6K is a substrate of the phosphoinositide-dependent protein kinase 1 (PDK1), and PA has been shown to bind to PDK1 from Arabidopsis. The PA–PDK1 interaction activates the PDK1 and AGC2-1 kinases to promote root hair growth in Arabidopsis (Anthony et al. 2004) (Fig. 1). PLDe-produced PA promotes root hair formation under nitrogen deprivation and salt stress in Arabidopsis (Hong et al. 2009). PLDa3 KO reduces PA formation and have lower levels of TOR and AGC2-1 expression and less phosphorylated S6K protein under hyperosmotic stress, suggesting that PLDa3 and PA affect the expression of the players in TOR pathway (Hong et al. 2008a). Enhanced growth and biomass by PLDa3 and PLDe under hyperosmotic stress may indicate PLDe or PLDa3 produced PA is involved in mTOR pathway. It would be of interest to determine whether the targets of PLDe and PLDa3 derived PA are PDK1, TOR or both or directly to S6K.

PA produced by other reactions In addition to PLD, PA can also be produced in other reactions such as phosphorylation of DAG by DAG kinases (DGKs) (Fig. 1), dephosphorylation of DAGpyrophosphate by lipid phosphatase, and de novo synthesis (Munnik et al. 2000; Wang et al. 2006). The Arabidopsis genome contains multiple DGK genes, and the role of DGK in plants is yet to be identified. Phospholipase C (PLC)-mediated hydrolysis of phospholipids is one source of DAG used for PA formation by DGK (Testerink & Munnik 2005) (Fig. 1). Phosphoinositide (4,5)


630 Y. Hong et al. bisphosphate (PIP2)-hydrolysing PLC (PI-PLC) has been implicated in plant response to hyperosmotic stress such as salinity (Hirayama et al. 1995; Dewald et al. 2001), drought (Zonia & Munnik 2004; Das et al. 2005), and ABA response (Lee et al. 1996; Burnette, Gunesekera & Gillaspy 2003; Hunt et al. 2003). Transgenic maize with enhanced expression of ZmPLC1 improved plant drought tolerance (Wang et al. 2008). In animal systems, the PI–PLC reaction can produce two potent second messengers, inositol 1,4,5trisphosphate (IP3), a Ca2+ mobilizer and DAG that activate PKC. The target of DAG in plants is unknown, but PLCproduced DAG can be rapidly phosphorylated to PA (Bargmann et al. 2009). In addition, plants have another family of PLCs, named non-specific PLC (NPC) for their hydrolysis of common membrane phospholipids, such as phosphatidylcholine and phosphatidyethanolamine. Specific NPCs are shown to be involved in plant adaptation of phosphate deficiency (Nakamura et al. 2005; Gaude et al. 2008) and they could potentially be involved in providing DAG for PA formation in response to hyperosmotic stresses. In rice detached leaves, the NaCl-induced PA formation was suggested to come primarily from a DGK reaction under the assay condition (Darwish et al. 2009). This result differs from that of Arabidopsis whole plants grown under NaCl stress conditions under which multiple PLDs have been found to contribute to the salt-induced PA production (Hong et al. 2008a; Bargmann et al. 2009). It is unclear whether these differences result from the different assay conditions and/or species differences. In addition, the function of the PA produced by the PLC/DGK reaction in plant tolerance to hyperosmotic stress remains to be determined.

INVOLVEMENTS OF DIFFERENT PLDS IN DROUGHT AND SALT STRESSES The activity of PLD increases under various hyperosmotic stresses, such as high salinity (Testerink & Munnik 2005; Bargmann et al. 2009), dehydration (Munnik et al. 2000; Katagiri, Takahashi & Shinozaki 2001), freezing (Welti et al. 2002; Li et al. 2004), as well as ABA, a phytohormone that regulates plant water homeostasis (Zhang et al. 2004). Recent analyses of mutant plants deficient or overexpressing specific PLDs have provided insights into the function and the cellular mechanism by which the PLD family is involved in plant hyperosmotic responses. The PLD family in higher plants is composed of multiple enzymes with distinguishable biochemical, structural, and molecular properties (Elias et al. 2002; Qin & Wang 2002; Li, Lin & Xue 2007; Dippe & Ulbrich-Hofmann 2009). Different PLDs have unique and overlapping functions in mediating plant growth and development under prolonged drought, rapid dehydration and different levels of salinity. Recent results also indicate that different PLDs mediate plant stress response through different molecular and cellular events.

PLDa1 in promoting ROS production, ABA effect on stomatal closure and salt tolerance PLDa1, the most predominant PLD found in plants, is activated by ABA to produce PA. Terrestrial plants lose water primarily through the stomata. In response to drought and salt stimuli, the cellular level of ABA increases, promoting stomatal closure in order to decrease water loss. PLDa1deficient Arabidopsis plants exhibited a higher rate of transpirational water loss than wild-type plants, whereas the overexpression of PLDa1 reduced transpirational water loss in tobacco by rendering the plants more sensitive to ABA (Sang et al. 2001a). The stomata in epidermal peels from PLDa1-deficient Arabidpsis plants fail to close in response to ABA, whereas external supply of PA, the lipid product of PLD, promoted the stomatal closure in wild-type and PLDa1-deficient Arabidopsis plants (Sang et al. 2001a; Mishra et al. 2006). The PLD-mediated production of PA has also been implicated in promoting stomatal closure in Vicia faba (Jacob et al. 1999). These results suggest that PLDa1 and PA play a positive role in ABA effects on preventing water loss. Further studies show that PLDa1 and PA mediate ABA signalling and stomatal movement via a bifurcating pathway (Mishra et al. 2006). PLDa1produced PA binds to ABI1, a PP2C, which functions as a negative regulator in ABA signalling in stomata closure (Zhang et al. 2004). On the other hand, PLDa1 also interacts with Ga protein, a heterotrimeric Ga protein in Arabidopsis to prevent closed stomata from opening (Zhao & Wang 2004; Mishra et al. 2006). Ga and two G protein-coupled receptor-like proteins GTG1 and GTG2 are involved in mediating ABA response in Arabidopsis (Pandey & Assmann 2004; Pandey, Nelson & Assmann 2009). More recently, two ABI1 interactors were identified as ABA receptors that bind and inhibit PP2C activity (Ma et al. 2009; Park et al. 2009). Therefore, the interactions of PLD/PA with ABI1 and the G protein place PLDa1 and PA in the initial events in the ABA sensing and signalling. The proposition mentioned earlier is consistent with other findings. Suppression of PLDa1 results in changes in the expression of many genes involved in transcription, signal transduction, lipid signalling, hormone response and metabolism under progressive drought stress (Mane et al. 2007). PLDa1 and PA regulate NADPH oxidase activity and the production of reactive oxygen species (ROS) in ABA-mediated stomatal closure (Zhang et al. 2009). Ablation of PLDa1, by either antisense suppression or gene knockout (KO), suppressed ROS production whereas PA stimulated NADPH oxidase activity and ROS production in wild-type and PLDa1-deficient cells (Sang, Cui & Wang 2001b; Zhang et al. 2009). These results indicate that PA-NADPH oxidase interaction is essential to ABAmediated ROS generation and stomatal closure. They also suggest that although the PA-ABI1 interaction is not required for the ABA-induced production of ROS and NO, it is important in mediating the ROS effect on stomatal closure. These findings further demonstrate that PLD and PA are central lipid messengers that link cellular regulators


Phospholipase D and phosphatidic acid signalling in the ABA signalling network. In addition, PLDa1 is also found to be involved in response to salt stress. KO of Arabidopsis PLDa1 rendered plants less tolerance to salt stress (Bargmann et al. 2009).

PLDd in plant response to ROS, dehydration, and salt stresses PLDd differs from PLDa1 in subcellular association, gene expression pattern, activity requirements and substrate preference (Wang & Wang 2001; Qin & Wang 2002; Wang et al. 2006) (Fig. 2). PLDd is activated in response to high salinity and rapid dehydration (Katagiri et al. 2001; Bargmann et al. 2009). PLDd mRNA is induced by dehydration and high salinity (Katagiri et al. 2001). PLDd-antisense plants did not display overt changes in phenotype, but PLDd-KO Arabidopsis plants were more susceptible to salt stress. PLDa1/PLDd double mutants accumulated only 30% of the PA in wild type, and the plants were more susceptible to salt stress than were PLDa1 and PLDd single mutants (Bargmann et al. 2009). The results indicate that PLDa1 and PLDd have distinctively different as well as overlapping functions in salt stress. Salt and dehydration stress cause the production of ROS such as H2O2. It has been shown that Arabidopsis PLDd is activated by H2O2 (Zhang et al. 2003). The H2O2-induced PA level in PLDd -KO cells was approximately 50% of that in wild-type cells, and the PLDd -KO cells exhibited a higher rate of H2O2-induced cell death than wild type. (Zhang et al. 2003). This suggests that PLDd plays an important role in protecting cells from damage by ROS. However, unlike PLDa1-KO plants, PLDd-KO plants are still able to produce the normal levels of ROS in response to stresses, such as ABA and freezing. Compared with wild-type plants, PLDd-KO plants exhibit less tolerance to freezing injuries whereas OE plants exhibit more tolerance (Li et al. 2004). Freezing stress also causes hyperosmotic stress in cells (Wolfe & Steponkus 1983). The involvement of PLDd in salt and freezing stresses suggests that PLDd plays a positive role in hyperosmotic response possibly through its role in mediating cellular response to ROS.

PLDa3 in salt and mild drought responses PLDa3 and PLDa1 have both overlapping and distinguishable biochemical and regulatory properties. PLDa3 is more distant to PLDa1 than is PLDa2 in terms of sequence similarity and gene expression pattern. Genetic KO of PLDa3 rendered plants more sensitive to hyperosmotic stress including salt and water deficiency, whereas OE plants are more tolerant. The leaves of PLDa3-altered plants exhibited no significant difference from that of wild type in water loss through the stomata, suggesting that PLDa3 is involved in hyperosmotic stress in a different mechanism than that of PLDa1. Under salt stress and water deficiency, PLDa3-KO mutants reduce root growth, whereas PLDa3-OE enhances root growth, including root

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length and lateral root number. Enhanced root growth makes plants more accessible to water, resulting in better performance in hyperosmotic stress (Hong et al. 2008a). Moreover, under mild drought conditions, PLDa3-KO mutants exhibited later flowering, whereas OE plants flowered earlier than wild-type plants (Hong et al. 2008a). Earlier flowering enables plants to complete their life cycle early in order to escape adverse environments. The analysis of ABA response showed that PLDa3-KO plants accumulated a similar level of ABA as wild type, but displayed higher levels of ABA response gene expression. The root growth of PLDa3 -KO seedlings was also more inhibited by ABA treatment than wild type (Hong et al. 2008a). A robust root system enables plants to access water and maintains water homeostasis, thus delaying ABA-responsive gene expression. This result suggests that PLDa3 plays a positive role in salt and drought stress through regulating root growth and development (Hong et al. 2008a) (Fig. 2). Although the expression level of PLDa3 is much lower than that of PLDa1, KO of PLDa3 suppresses the level of PA under hyperosmotic stress; PLDa3-KO plants contained 60% of the PA in wild-type plants, suggesting that PLDa3 contributes to PA formation in plants (Hong et al. 2008a). Salt induced PA formation in PLDa1/PLDd double mutants showed that PA does not exclusively come from these two PLDs under salt stress (Bargmann et al. 2009), which is consistent with the involvement of PLDa3 in salt stress response.

PLDe in N signalling and plant growth under salt stress and water deficiency Genetic alterations of PLDe affect plant root architecture and biomass production under salt stress and water deficits. The PA content in PLDe-KO plants was approximately 50% of that in wild type. PLDe-KO plants had fewer, shorter lateral roots, whereas OE plants had higher PA level and exhibited more, longer lateral roots than wildtype plants (Hong et al. 2009). The conclusion that growth enhancement is mediated by PLDe-generated PA is supported by the observation that application of PA inhibitor. Suppression of PLD-catalysed PA formation by 1-butanol abolished the difference between PLDe-altered and wildtype plants. In addition, OE seedlings have more and longer root hairs, whereas KO seedlings have less and shorter root hairs than that of wild type under salt stress conditions (Hong et al. 2009; Hong unpublished data). The growth alterations also occurred with PLDe-KO and OE plants in response to nitrogen availability (Hong et al. 2009). OE and KO of PLDe had the opposite effect on lateral root elongation in response to nitrogen. Increased expression of PLDe also promoted root hair elongation and primary root growth at severe N deprivation. The results suggest that PLDe and PA promote organismal growth and play a role in N signalling. Under natural conditions, hyperosmotic stress is usually accompanied by inhibition of nutrient uptake and transport (Alfieri & Petronini 2007). It is possible that enhanced nitrogen sensing and increased root surface by


632 Y. Hong et al. PLDe enables plants to uptake and utilize water more efficiently during hyperosmotic stress (Fig. 2). Analyses of the biochemical properties and expression of PLDe provide some insights into the distinctive effects of PLDe on plant growth (Hong et al. 2009). PLDe is the most permissive of all the characterized PLDs in terms of reaction requirements; it is active under PLDa1, b and d reaction conditions (Hong et al. 2009). However, the level of PLDe expression in vegetative tissue is much lower than that of PLDa1 and PLDd. PLDe is associated with the plasma membrane, and this association would allow overexpressed PLDe ready access to membrane substrates, resulting in production of PA that mediates plant response to stimuli. PLDe-KO plants contain lower PA level and have less biomass and seed yield, whereas PLDe-OE plants have higher PA level and perform better than wild-type plants. The differences in whole plant growth among KO, OE and wild-type plants were abolished by the application of 1-butanol, which suppresses PLD-mediated PA production, into growth media. The results suggest that the enhanced growth by PLDe is mediated via generation of the lipid messenger PA (Hong et al. 2009).

REGULATION OF PLDS IN HYPEROSMOTIC STRESSES Increases in PLD activity and PA production under drought and salinity have been reported in various plant species in addition to Arabidopsis (Jacob et al. 1999; Frank et al. 2000; Munnik et al. 2000). It has been long known that physical perturbation of plant tissues, such as wounding and grinding, can lead to massive lipid hydrolysis by PLD. This increase in PLD hydrolysis likely results from deregulation at least in part due to decompartmentalization of the enzymes and cofactors. On the other hand, significantly increasing PLDa1 protein by PLD overexpression in tobacco does not alter plant lipid composition under normal growth conditions, suggesting that PLD activity is highly regulated post-translationally. In addition, the mRNA level of Arabidopsis PLDd increased under high salinity and that of PLDa increased in tomato cell suspension cultures exposed to salt (Katagiri et al. 2001; Bargmann et al. 2009). These results suggest that transcriptional regulation also plays a role in modulating PLD levels under hyperosmotic responses. The precise mechanism by which a specific PLD is activated remains to be defined under salinity or water deficit conditions. Available results suggest that the regulation of the PLD family is complex, involving multiple effectors, including Ca2+, PIP2, G proteins, and positive and negative feedback loops.

Cofactors affecting PLD activity The activities of PLDs require several cofactors, including Ca2+, phosphoinositides and oleic acid, but the requirements differ among specific PLDs (Qin, Pappan & Wang 1997; Wang & Wang 2001; Qin & Wang 2002; Hong et al. 2009) (Fig. 2). Ca2+ is a key regulator for PLD activity in

hyperosmotic stress.All characterized PLDs involved in salt and drought stress belong to C2-PLD (Qin & Wang 2002; Zhang et al. 2004; Hong et al. 2008a, 2009; Bargmann et al. 2009). The C2-PLDs need Ca2+ for activity whereas PX/PHPLDs are not (Qin & Wang 2002). PLDa1 and PLDb have been shown to bind to Ca2+ with different binding affinities (Zheng et al. 2000). Whereas PLDa1 is most active under millimolar Ca2+, it is also active at micromolar Ca2+ under acidic pH conditions (Pappan & Wang 1999). The interactions and differential affinities are consistent with the domain structures of the plant PLD family. PLDa(3), b(2), g(3), d, and e contain the Ca2+-dependent phospholipid binding C2 domains, whereas two PLDzs have N-terminal phox homology (PX) and pleckstrin homology (PH) domains (Wang 2004;Wang et al. 2006). In addition to direct binding, Ca2+ may alter membrane microdomains to facilitate PLD interaction with the phospholipid surface and activate PLD activity (Kuppe et al. 2008). Increases in and oscillations of the cytosolic free Ca2+ are key messengers in plant response to hyperosmotic stresses. Phosphoinositides, particularly PIP2, is another key regulator of PLD activation. PLDb, PLDg, PLDd, and PLDz require PIP2 for activity (Qin & Wang 2002). The activity of other PLDs is also stimulated by PIP2. The binding region of PIP2 on PLDb1 has been mapped and the PLD-PIP2 binding enhances PLD interactions with membrane lipids and thus substrates (Qin & Wang 2002; Zheng et al. 2002). PIP2 is a class of important regulators in cell response to hyperosmotic stresses, and its activation of PLD constitutes one of the mechanisms by which PIP2 mediates cell functions. On the other hand, in animal systems, phosphatidyl 4-phosphate 5-kinase (PIP5K) that synthesizes PIP2 is a downstream target of PLD signalling pathways (Morris 2007). In plants, PIP5K was induced by drought (Mikami et al. 1998), and PA interacts with the enzyme phosphatidylinositol 4-kinase that produces the substrate for PIP5K (Stevenson, Perera & Boss 1998). Thus, it appears to be a positive loop in which PIP2 activates PLD to produce PA whereas PA activates PIP5K to produce more PIP2.

PLD-interacting proteins potentially involved in hyperosmotic responses Proteins that interact with PLDs are potentially important regulators for PLD activation in response to salt and water stresses. PLDa interacts with G protein to modulate stomatal closure and water loss. A PLDa-like activity is associated with G protein in the plasma membrane to mediate ABA signalling in barley aleurone cells (Ritchie & Gilroy 2000). Both PLDa1 and Ga play positive roles in ABA signalling. The PLDa1-G protein interaction affects reciprocally the activity of both proteins to inhibit the opening of closed stomata (Zhao & Wang 2004; Mishra et al. 2006). A recent study found that PLDb and g bind to 14-3-3 proteins that are involved in regulation of cell signalling and metabolism in plants (Chang et al. 2009). Further studies are needed to determine the role of the PLD-14-3-3 interaction in PLD function and stress responses.


Phospholipase D and phosphatidic acid signalling Cellular cytoskeletons undergo extensive rearrangement under hyperosmotic stress response. Plant PLDs interact with both actin and tubulin. Arabidopsis PLDb1 binds to a-actin, and monomeric G-actin inhibits PLD activity, whereas polymerized F-actin augments PLD activity (Kusner et al. 2003). Meanwhile, PA is reported to promote actin polymerization in soybean cells (Lee, Park & Lee 2003) and is found to bind a heterodimeric capping protein from Arabidopsis (AtCP) (Huang et al. 2006). The PA inhibition of capping protein activity in plant cells results in stimulation of actin polymerization from a large pool of profilin-actin. Thus, the PLD interaction with actin filaments may act as a positive loop to promote active polymerization. The actin binding motif is conserved in most PLDs, indicating the interaction is not only limited to PLDb. In addition, PLDd interacts with microtubule cytoskeleton and the activation of PLD is implicated in triggering microtubule reorganization (Gardiner et al. 2001; Dhonukshe et al. 2003). PLDd-null plants are less tolerant to stresses, and their role in cytoskeletal reorganization may contribute to the weakening of plant stress responses (Zhang et al. 2003). In addition, PA produced by PLD is suggested to act upstream of the accumulation of a MAP kinase to regulate hyperosmotically induced microtubule formation in plasmolysed turgidum root cells (Komis et al. 2006). Therefore, the PLD-cytoskeleton interaction regulates both actin and microtubule cytoskeleton reorganization in plant response to hyperosmotic stresses.

PERSPECTIVES Cell membranes are an initial and focal point of signal perception and transduction involved in various biological processes. Increasing results indicate that PLDs play important roles in plant response to water deficits and salinity.The function of different PLDs can be unique and overlapping, depending on the specific stress and severity and stages of the stress. The molecular heterogeneities of multiple PLDs play important roles in the diverse cellular functions of PLDs. PLD and PA may provide key linkages among cellular regulators, such as phytohormones, oxidative stress, G proteins, protein phosphatase and kinases in plant growth and stress responses. Further investigations are needed to understand the complex interplays and functions of specific PLDs in the cell and their mode of actions. Specifically, how are different PLDs activated by specific types of hypersomotic stresses? What are the molecular targets of PA and how the PLD and PA interactions with the effectors affect cell functions? In addition, what are the functions of lipid head groups released by PLD? What and how would the change in lipid composition and membrane structure resulting from PLD activation influence plant response and tolerance to hyperosmotic stresses? The plant response to and tolerance of drought and salinity involves complex molecular, cellular, and physiological changes. A better understanding of PLD and PA-based membrane lipid signalling has the potential to connect the stimulus perception at the

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cell membrane to intracellular actions and physiological responses to drought and salinity.

ACKNOWLEDGMENTS The work for the authors’ laboratories was supported by grants from the National Science Foundation (IOS0818740) and United States Department of Agriculture (2007-35318-18393) to X. Wang, the National Science Foundation of China (30871303) to Y. Hong, and the Chinese National Key Basic Research Project (2006CB100100) and the grant from National Science Foundation of China (30625027, 90817014) and Ministry of Education of China (20060307019)) to W. Zhang.

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