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Plant Physiology 139:566-573 (2005) © 2005 American Society of Plant Biologists Regulatory Functions of Phospholipase D and Phosphatidic Acid in Plant Growth, Development, and Stress Responses1Department of Biology, University of Missouri, St. Louis, Missouri 63121; and Donald Danforth Plant Science Center, St. Louis, Missouri 63132
Phospholipase D (PLD) hydrolyzes membrane lipids to generate phosphatidic acid (PA) and a free-head group (Fig. 1), and this activity is widespread in plants. Recent results indicate that PLD plays multiple regulatory roles in diverse plant processes, including abscisic acid (ABA) signaling, programmed cell death, root hair patterning, root growth, freezing tolerance, and other stress responses (Fig. 1). In some cases, direct molecular targets of PLD and PA have been identified, providing insights into the mechanism by which the phospholipase and lipid messenger mediate plant functions.
PLD is composed of a family of heterogeneous enzymes with distinguishable biochemical, regulatory, and structural properties. The PLD gene family in plants is more complex than that in other organisms: 12 PLD genes are in Arabidopsis (Arabidopsis thaliana), whereas two PLD genes are in mammals and one in baking yeast (Saccharomyces cerevisiae; Wang, 2002  , 2004, and refs. therein). The 12 Arabidopsis PLDs can be classified into six types, PLD (3),  (2),  (3),  ,  , and  (2) (Fig. 2). Based on the overall protein domain structures, PLDs can be divided into two subfamilies, C2-PLD and PX/PH-PLDs (Fig. 2). C2 is a Ca2+ and phospholipid-binding domain, and the PX and PH domains refer to two distinct phosphoinositide-interacting structural folds, phox homology and pleckstrin homology, respectively. Ten of the 12 Arabidopsis PLDs (, , , , and ) contain the C2 domain. The PLDs contain the PX and PH domains, and this domain structure is present in mammalian PLDs (Elias et al., 2002; Qin and Wang, 2002). The overall sequences of PLDs are more similar to mammalian PLDs than to other Arabidopsis PLDs.
In addition, new structural motifs have been identified in PLDs that interact with G proteins, Ca2+, and phosphoinositides (Fig. 2; Zheng et al., 2002 ; Pappan et al., 2004; Zhao and Wang, 2004). Individual PLDs differ in key amino acid residues in these regulatory motifs. These differences underlie a structural basis for the distinguishable biochemical and regulatory properties in different PLDs. The information on the molecular, structural, and biochemical heterogeneity of PLDs provides clues as to the regulation and diverse functions of PLDs in plants. This Update focuses on the recent developments toward understanding the function of specific PLDs and PA that they produce.
The most abundant plant PLD requires high millimolar concentrations of Ca2+ for activity in vitro. PLD 1 possesses the common PLD activity (Wang, 2002). A gene knockout of PLD1 abolishes the millimolar Ca2+-requiring PLD activity in Arabidopsis (Zhang et al., 2004), demonstrating that PLD1 is the predominant one responsible for the common PLD activity in the plant. Arabidopsis deficient in PLD1 displays alterations in various plant processes, such as reactive oxygen production, wound-induced accumulation of jasmonic acid, freezing tolerance, water loss, and abscisic acid signaling (for review, see Wang, 2002).
Recent results indicate that PLD
Both PLD 1 and G are involved in ABA signaling. Gene suppression and overexpression results show that that PLD1 plays a role in the ABA response (Sang et al., 2001). A recent study has unveiled a mechanism by which PLD1 mediates such a response (Zhang et al., 2004). PLD1-derived PA binds to ABI1 (ABA insensitive), a protein phosphatase 2C (PP2C) that is a negative regulator of ABA responses in Arabidopsis. The PA binding decreases PP2C activity and also appears to reduce its translocation to nuclei in response to ABA by tethering ABI1 to the plasma membrane. These results show that activation of PLD1 inhibits the function of the negative regulator ABI1, thus promoting ABA signaling (Fig. 3).
PLD exhibits several properties that distinguish it from other PLDs. It is activated by oleic acid and is tightly associated with the plasma membrane and microtubule cytoskeleton. The expression of PLD increases in severe dehydration, high salt, and during cold acclimation. Analyses of PLD-altered Arabidopsis suggest that PLD positively regulates plant tolerance to stresses such as freezing, oxidative assault, and ultra-violet irradiation (Zhang et al., 2003; Li et al., 2004). PLD-null Arabidopsis is less tolerant to freezing, whereas overexpression of PLD increases freezing tolerance (Li et al., 2004). Lipid profiling indicates that PLD activity produces selective PA species but does not result in substantial lipid hydrolysis during freezing stress.
By comparison, antisense suppression of PLD
One mechanism by which PLD positively mediates plant stress tolerance is through its role in signaling resistance to damages inflicted by reactive oxygen species (ROS; Li et al., 2004). PLD is activated by hydrogen peroxide (H2O2), and the resulting PA functions to decrease H2O2-promoted programmed cell death (Zhang et al., 2003). Examining mitogen-activated protein (MAP) kinase activity suggests that PLD and PA play a role in H2O2-induced activation of MAP kinase cascades (Zhang et al., 2003). The ROS H2O2 is an important cellular mediator, and its concentration increases under various stress conditions, including freezing. Activation of MAP kinase cascades is involved in plant response to H2O2 and various stress responses. The involvement of PLD and its derived PA in these processes may present a mechanism by which PLD plays a positive role in stress response.
PLD
Unlike C2-containing PLDs, the PX/PH-containing PLD 1 is Ca2+ independent and PC specific (Qin and Wang, 2002). A study of the molecular target of the homeobox gene GLABRA2 (GL2) has provided insights into one physiological function of PLD1 (Ohashi et al., 2003). PLD1 is a direct target of GL2, a key component of a regulatory circuit composed of transcription factor genes that regulate the root hair pattern of Arabidopsis. GL2 is thought to be a negative regulator of root hair development. Inducible expression of PLD1 promoted ectopic root hair initiation, whereas inducible suppression inhibits root hair initiation (Ohashi et al., 2003). The results suggest that GL2 regulates root hair development through modulation of PLD1. However, loss of PLD1 does not cause obvious changes in root hair patterning (M. Li, C. Qin, and X. Wang, unpublished data). These results may indicate that more than one PLD could be involved in the root hair growth and development.
How PLD regulates the root hair patterning and initiation is not known. A recent study indicates that PA regulates a protein kinase pathway involved in root growth and initiation (Anthony et al., 2004
PLD and PA have been implicated in affecting both actin and tubulin cytoskeletons. Arabidopsis PLD 1 binds to -actin (Kusner et al., 2003). The effects of actin on the activities of the PLD are polymerization dependent; monomeric G-actin inhibits PLD activity, whereas polymerized F-actin augments PLD activity. Actin modulation of PLD1 has kinetic characteristics, efficacies, and potencies similar to those of human PLD1 (Kusner et al., 2003). This conservation of PLD-actin interaction between PLDs from plants and mammals indicates that other plant PLDs, such as PLDs, may also bind to actin because of their close similarity to mammalian PLDs.
PA is reported to promote actin polymerization in soybean (Glycine max) cells (Lee et al., 2003
Following the finding that PLD
It is worth noting that butanol treatment takes advantage of the unique, transphosphatidylation activity of PLD, which uses primary alcohols as substrates to form phosphatidylalcohol at the expense of PA. Because there is no specific, effective inhibitor for PLD, this approach has been useful in helping to determine the cellular functions of the PLD activity. However, primary alcohols stimulate rather than inhibit PLD activity. Also, the diversion of PA formation by alcohol is incomplete. Thus, an effect associated with an alcohol treatment needs to be interpreted with caution because it may result from other alcoholic effects, such as increases in lipid hydrolysis, changes in lipid composition, and/or release of lipid head groups that have regulatory functions (Chapman, 2004
Tobacco pollen germination and tube growth are stopped in the presence 0.5% 1-butanol, and this inhibition could be overcome by addition of exogenous PA, suggesting that PLD-derived PA plays a role in stimulating pollen germination and tube elongation (Potocky et al., 2003 ). In another study, the expansion of the pollen tube apical region is associated with a severalfold increase in PA, which is generated by PLD (Zonia and Munnik, 2004). Different phospholipid signals are differentially stimulated or attenuated in pollen tube volume changes in response to the osmotic perturbations. Hypo-osmotic stress induces rapid increases in PA, but hyperosmotic stress and cell shrinkage inhibit PLD activity and induce increases in the levels of phosphatidylinositides.
In other cells or tissues, however, PLD is activated under hyperosmotic stresses, such as high salinity and dehydration (Testerink and Munnik, 2005
All C2-PLDs require Ca2+ for activity, but how Ca2+ affects PLD activity is not well understood. It was previously reported that Ca2+ binds to the regulatory C2 domain that occurs in the N terminus of PLDs (Zheng et al., 2000 ). Using a C2-deleted PLD1 (PLDcat) and other PLD1 domain constructs, a recent study has shown that Ca2+ also interacts with the catalytic regions of PLD (Pappan et al., 2004). The Ca2+-PLDcat interaction increases the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate (PIP2), but not for the substrate, PC. This is in contrast to the effect of Ca2+ binding to the C2 domain, which stimulates PC binding but inhibits PIP2 binding of the domain. The PIP2-bound catalytic domain increases the enzyme's affinity for its substrate PC. These results demonstrate the contrasting and complementary effects of the Ca2+- and lipid-binding properties of the C2 and catalytic domains of plant PLD, and provide insight into the mechanism by which Ca2+ regulates PLD activity. A membrane-scooting model has been proposed to explain the regulation of PLD activity by changing cellular Ca2+ concentrations. These findings also account in part for why Ca2+ is required for the activity of the C2-PLDs but not the PX/PH-PLDs (Pappan et al., 2004).
The distinguishable phenotypes resulting from loss of different PLDs indicate that the loss of one PLD is not compensated for, at least completely, by the other 11 PLDs in Arabidopsis (Zhang et al., 2003 , 2004; Li et al., 2004). Unique functions of different PLDs may occur via one or a combination of the following (Fig. 4): (1) Individual PLDs are regulated and activated differently in the cell; (2) they have different substrate preferences and have the potential to generate different PAs or other derivatives; (3) they are associated with different membranes; and (4) they have different temporal and spatial patterns of expression. The differences in regulation, activation, substrate preference, intracellular association, and expression patterns have been demonstrated for several PLDs (Wang 2002, 2004, and refs. therein). These differences ultimately regulate the location and timing of the PLD activities and PA produced by the enzymes. Spatial and temporal regulation is important to all signaling events, but it is particularly critical to intracellular lipid messengers because of their limited mobility.
In addition, one PLD can mediate cell function via different modes of action, depending upon the nature of stimuli and severity of stresses (Fig. 4). These include (1) production of lipid mediators, such as PA, N-acyletanolamine, and other lipid derivatives; (2) direct interaction with other proteins, such as G
PLD is activated under various growth conditions, and PA is the direct lipid product of such activation (Welti et al., 2002 ; den Hartog et al., 2003; Potocky et al., 2003; Zhang et al., 2003, 2004; Li et al., 2004; Zonia and Munnik, 2004). Genetic and pharmacological manipulations of PLD-mediated PA formation suggest that PA mediates various signaling processes, as described here and reviewed earlier (Wang, 2002, 2004; Testerink and Munnik, 2005). It should be noted that signaling PA can be produced by another route (Fig. 1); the activation of the PLC/DAG kinase pathway can occur under certain stress conditions, such as hyperosmotic stress and pathogen elicitation (De Jong et al., 2004; Testerink and Munnik, 2005).
Major progress has been made recently in understanding how PA functions as a signaling messenger. In particular, direct molecular targets of PA have been identified in two specific plant processes. In ABA responses, PLD
The PA targets in plants go beyond protein phophatases and kinases. Several additional proteins were associated with a PA-affinity column; these include phosphoenolpyruvate carboxylase, Hsp90, and 14-3-3 (Testerink et al., 2004
It is important to note that PA may mediate cellular processes via different ways of action (Fig. 4). First, as a signaling messenger, PA can bind to its effector proteins, and this binding may activate or inhibit the function of its target proteins, as described above. Second, as a lipid in membranes, PA can tether its effector proteins to membranes, and the tethering may help to direct proteins to a specific type or region of a membrane. Third, PA may function via its structural effect on membranes to promote membrane fusion and trafficking. The space of PA's head group is smaller than that of its acyl chains, and this geometry renders PA a negative curvature and fusigenic lipid (Kooijman et al., 2005
Substantial progress has been made recently toward understanding the regulation and function of PLDs. These advances present new opportunities to investigate the signaling mechanisms in plants. The finding of the novel signaling interaction between PLD 1 and G raises several intriguing questions. For instance, would PLDs function as GTPase-activating proteins or guanine nucleotide exchange factors? How would the PLD-G interaction affect the interactions of G with G subunits and with G-protein receptors? What would be the function and specificity of the PLD-G protein interaction in the cell? The observations that PA interacts with protein phosphatases and kinases warrant further investigation. PLD and PA may play an important role in the homeostasis of protein phosphorylation by concerted regulation of the two groups of signaling enzymes with opposite biochemical functions in a specific signaling response (Fig. 3). In the case of the PA-ABI1 interaction, it is conceivable that that PLD and PA may also affect the functions of other PP2Cs because Arabidopsis contains 69 PP2C-like genes. A combination of the multiple routes of PA production, different PA species, diverse protein targets, and varied modes of action makes PA versatile mediators in cell regulation. Elucidation of the structural determinants that are required for PA-protein interaction will help to determine the specificity of the lipid-protein interaction and to understand how that interaction modulates structure, dynamics, and function in the ensuing lipid-protein complex. A better understanding of the regulation and function of PLDs and their derived messengers will advance not only the understanding of the major phospholipase family, but also the field of cell signaling and signaling networks in plant growth and stress responses. Received July 25, 2005; returned for revision August 7, 2005; accepted August 12, 2005.
1 This work was supported by grants from the National Science Foundation and the U.S. Department of Agriculture.  www.plantphysiol.org/cgi/doi/10.1104/pp.105.068809. * E-mail wangxue{at}umsl.edu; fax 314–587–1519.
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