Regulatory Functions of Phospholipase D and Phosphatidic Acid in Plant Growth, Development, and Stress Responses

PLD-catalyzed hydrolysis of phospholipids, downstream targets, and cellular functions. The activity of PLD produces PA and free-head group (X). PA target proteins have been identified in animals, plants, and yeast (see also Table I). Multiple cellular effects of PLD and PA have been documented or implicated. Note that in addition to PLD, signaling PA can be generated from DAG kinase coupled to the activation of PLC and potentially from acylation reactions. Domain structures of Arabidopsis PLDs. Plant PLDs consist of two distinctive subfamilies: the C2-PLDs and the PX/PH-PLDs. Individual PLDs can differ in key amino acid residues in regulatory motifs, such as C2, PIP2 binding, and DRY. The duplicated HKD motifs are involved in catalysis. Note that the 12 Arabidopsis PLDs have now been classified into six types instead of five types; the only modification is that PLDα4 has been reclassified to PLDε because this PLD is quite distant from all the other 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. PLDα1 possesses the common PLD activity (Wang, 2002). A gene knockout of PLDα1 abolishes the millimolar Ca2+-requiring PLD activity in Arabidopsis (Zhang et al., 2004), demonstrating that PLDα1 is the predominant one responsible for the common PLD activity in the plant. Arabidopsis deficient in PLDα1 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). A working model depicting the activation of PLDs and the role of PA in regulating kinase and phosphatase activities in cell's response to ABA, H2O2, or other stimuli. Potential mediators in the PLD activation include changes in G protein function and membrane conformations and/or increases in cytosolic Ca2+, phopshoinositides, and oleic acid. PLD-produced PA may stimulate protein kinase cascades and also inhibit protein phosphatases. PA may do so by directly affecting the enzymatic activities and/or by tethering the signaling enzymes to specific membranes or regions of a membrane. These interactions modulate the cell's ability to respond to stresses and inhibit cell death. Both PLDα1 and Gα are involved in ABA signaling. Gene suppression and overexpression results show that that PLDα1 plays a role in the ABA response (Sang et al., 2001). A recent study has unveiled a mechanism by which PLDα1 mediates such a response (Zhang et al., 2004). PLDα1-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 PLDα1 inhibits the function of the negative regulator ABI1, thus promoting ABA signaling (Fig. 3). PLDδ 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 PLDδ increases in severe dehydration, high salt, and during cold acclimation. Analyses of PLDδ-altered Arabidopsis suggest that PLDδ positively regulates plant tolerance to stresses such as freezing, oxidative assault, and ultra-violet irradiation (Zhang et al., 2003; Li et al., 2004). PLDδ-null Arabidopsis is less tolerant to freezing, whereas overexpression of PLDδ increases freezing tolerance (Li et al., 2004). Lipid profiling indicates that PLDδ activity produces selective PA species but does not result in substantial lipid hydrolysis during freezing stress. By comparison, antisense suppression of PLDα1 increases freezing tolerance, and lipid profiling shows that PLDα1 activity results in a large decrease in phosphatidylcholine (PC) and an increase in PA (Welti et al., 2002). These results indicate that PLDα1 and δ are involved in the response to freezing via different mechanisms and also demonstrate that manipulation of different PLDs can have different phenotypic alterations. In addition, the PLDδ alterations do not result in changes in the expression of the cold-regulated genes COR47 or COR78, nor in any change in cold-induced increases in the levels of compatible osmolytes, Pro, or soluble sugars, which are known to play a role in plant freezing tolerance. These results suggest that PLDs and associated membrane lipid hydrolysis present a novel signaling pathway involved in mediating plant freezing tolerance. One mechanism by which PLDδ positively mediates plant stress tolerance is through its role in signaling resistance to damages inflicted by reactive oxygen species (ROS; Li et al., 2004). PLDδ 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 PLDδ 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 PLDδ and its derived PA in these processes may present a mechanism by which PLDδ plays a positive role in stress response. PLDδ is activated by H2O2, but it is not involved in H2O2 production under the conditions tested (Zhang et al., 2003). By comparison, PLDα1 and PA are involved in the production of reactive oxygen species (Sang et al., 2001). These results indicate that specific PLDs function in different steps in plant oxidative stress pathways; whereas PLDα1 promotes the ROS production, PLDδ mediates plant responses to ROS. The role of PA in ROS production is supported by another study: PA treatment of whole leaves and single cells increased the levels of ROS and promoted leaf cell death (Park et al., 2004). This study also suggests that PA promotes the ROS production via activating the Rho-related small G protein GTPase-mediated pathway. The PA-induced cell death appears contradictory to the role of PLDδ-derived PA in mitigating H2O2-induced cell death. However, the location and timing of PA production and appropriate cellular concentrations and molecular species of PA are important determinants of PA function. Unlike C2-containing PLDs, the PX/PH-containing PLDζ1 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 PLDζ1 (Ohashi et al., 2003). PLDζ1 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 PLDζ1 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 PLDζ1. However, loss of PLDζ1 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). PA binds to the 3′-phosphoinositide-dependent kinase-1 (AtPDK1) and stimulates the activity of AGC2-1 in an AtPDK1-dependent manner. AGC refers to a kinase superfamily comprising the prototypes of the cAMP-dependent protein kinases A (PKA), cGMP-dependent protein kinases G (PKG), and protein kinases C (PKC). AGC2-1 is a target protein kinase of AtPDK1, and the AGC2-1 knockout plants exhibit a decrease in root hair length. It remains to be determined whether the AtPDK1-binding PA results from the activation of PLDs because signaling PA can be derived from diacylglycerol (DAG) kinase coupled to the activation of phospholipase C (PLC; den Hartog et al., 2003; De Jong et al., 2004) and potentially from acylation reactions (Fig. 1). PLD and PA have been implicated in affecting both actin and tubulin cytoskeletons. Arabidopsis PLDβ1 binds to α-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 PLDβ1 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 PLDζs, 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). PA is found to bind a heterodimeric capping protein from Arabidopsis (AtCP; S. Huang and C.J. Staiger, personal communication). The PA-binding inhibits the activity of AtCP; in the presence of PA, AtCP is unable to cap the barbed or rapidly growing and shrinking end of actin filaments and fails to nucleate filament formation. The PA inhibition of capping-protein activity in plant cells results in stimulation of actin polymerization from a large pool of profilin-actin. Following the finding that PLDδ binds to tubulin, two studies explored the effect of PLD on microtubule cytoskeleton organization. In one study, seeds and young seedlings of Arabidopsis were incubated with 1-butanol, and this treatment disrupted the organization of interphase cortical microtubules and also inhibited seed germination and seedling growth (Gardiner et al., 2003). In another study using tobacco (Nicotiana tabacum) Bright Yellow-2 cells expressing the microtubule reporter green fluorescent protein-MAP4, 1-butanol treatment promoted a partial depolymerization of cortical microtubules and the release of them from the plasma membrane (Dhonukshe et al., 2003). It has been postulated that PLD links microtubules via the PLD catalytic intermediate phosphatidyl moiety. Results from both of the studies suggest that activation of PLD is critical to triggering microtubule reorganization. It is likely that, besides PLDδ, other PLDs are involved in microtubule reorganization because the PLDδ-knockout plants grow and develop normally under regular growth conditions. However, PLDδ-null plants are less tolerant to stresses (Zhang et al., 2003; Li et al., 2004), and their role in cytoskeletal reorganization may contribute to the weakening of plant stress responses. 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. Unique roles of different PLDs, multifaceted functions of a PLD, and multiple modes of action of PA. Unique functions have been shown for PLDα1 and PLDδ. Box 1 lists some of the available findings that support different functions of PLDs. Box 2 indicates that a given PLD can be involved in different cellular processes, depending upon the nature of biotic and abiotic cues, such as the severity of stresses. Box 3 lists several effects that PA has on cell regulation. Documented examples are given in parentheses, and those in bold denote PA target proteins identified in plants. The symbol and function of the target proteins are explained in Table I and text. FFA, Free fatty acid; DAG-PPi, DAG-pyrophosphate. 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 PLDβ1 (PLDβcat) and other PLDβ1 domain constructs, a recent study has shown that Ca2+ also interacts with the catalytic regions of PLD (Pappan et al., 2004). The Ca2+-PLDβcat 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 Gα, actin, and tubulin; (3) altering membrane lipid composition; and (4) membrane degradation. The role in changes of membrane lipid composition has been documented for PLDα1 in response to freezing (Welti et al., 2002). PLDα1 selectively hydrolyzes PC and alters the ratios of membrane lipids and contributes to membrane lipid degradation. Historically, PLD activity was associated with lipid degradation and membrane deterioration. This is perhaps best reflected by or attributed to a large increase of PA under conditions such as wounding, tissue handling, or uncontrolled lipid extraction. Such high PLD activity is likely to result from deregulation of PLD, such as disruption of cellular compartments. This deregulation may occur in plant tissues under severe stress conditions such as freezing, aging, or storage and thus is physiologically relevant. However, under normal plant growth, PLD activity is tightly regulated; for example, it is activated in response to specific stimuli, such as ABA and H2O2. These results indicate multifaceted functions of a PLD. 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 (Wang, 2002, 2004; and Munnik, It be that signaling PA can be produced by another (Fig. the activation of the kinase pathway can occur under stress conditions, such as hyperosmotic stress and Jong et al., 2004; and Munnik, has been in understanding how PA functions as a signaling In direct molecular of PA have been identified in two specific plant In ABA PLDα1-derived PA has been shown to bind to ABI1 PP2C (Zhang et al., 2004). The amino acid required for interaction has been identified and in the region of The binding of PA to ABI1 decreases the PP2C activity and also ABI1 to the plasma membrane. These changes reduce the translocation of ABI1 to the and thus inhibit the ABI1 function (Zhang et al., 2004). In mediating root growth and initiation, PA binds to and stimulates the activity of a downstream kinase AGC2-1 (Anthony et al., 2004). In addition, a protein kinase and the regulatory are potential PA targets, as they were identified through et al., 2004; Table I of PA-binding proteins identified in plants, animals, and yeast other proteins were also found to be associated with of PA-binding proteins identified in plants, animals, and yeast other proteins were also found to be associated with The PA in plants protein and proteins were associated with a these include and et al., 2004; Table I). The binding to has been shown to be specific to PA. PA target proteins have been identified in other I). In the yeast S. PA on the directly to the soluble to it as the et al., 2004). A decrease in PA to lipid from the its translocation and of target These results show that PA functions as both an intermediate and a signaling In PLD-derived PA regulates mammalian target of which to the of phosphatidylinositol kinases and regulates initiation in and and cytoskeletal in yeast 2004). PA directly interacts with the domain in the and this binding is critical for ability to downstream et al., 2001). It is important to that PA may mediate cellular processes via different of action (Fig. 4). as a signaling PA can bind to its proteins, and this binding may or inhibit the function of its target proteins, as described as a lipid in PA can its proteins to and the tethering may to direct proteins to a specific or region of a membrane. PA may function via its structural effect on membranes to promote membrane and The of head group is than that of its and this PA a negative and lipid et al., PLD-derived PA is required for cell formation during and in yeast et al., and is a key of membrane in different mammalian cells et al., In addition, PA as a substrate and/or an for enzymes that promote the formation of other lipid such as fatty and (Wang 2002, 2004; and Munnik, has been toward understanding the regulation and function of PLDs. These present new to the signaling mechanisms in plants. The finding of the novel signaling interaction between PLDα1 and Gα several PLDs function as proteins or How the interaction the interactions of Gα with and with be the function and of the protein interaction in the The that PA interacts with protein and kinases PLD and PA may play an important role in the of protein by regulation of the two groups of signaling enzymes with biochemical functions in a specific signaling response (Fig. 3). In the of the it is that that PLD and PA may also the functions of other because Arabidopsis A combination of the multiple of PA production, different PA diverse protein targets, and modes of action PA mediators in cell regulation. of the structural determinants that are required for interaction to determine the of the interaction and to how that interaction and function in the A understanding of the regulation and function of PLDs and their derived messengers not only the understanding of the phospholipase but also the of cell signaling and signaling in plant growth and stress responses.