plant phospholipasesarquivo.ufv.br/.../phospholipasesreview.pdf · abstract phospholipases are a...

P1: FXY

April 11, 2001 17:43 Annual Reviews AR129-08

Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:211–31Copyright c© 2001 by Annual Reviews. All rights reserved

PLANT PHOSPHOLIPASES

Xuemin WangDepartment of Biochemistry, Kansas State University, Willard Hall, Manhattan,Kansas 66506; e-mail: [email protected]

Key Words lipid messengers, signal transduction, lipid hydrolysis, membranes,stress response

■ Abstract Phospholipases are a diverse series of enzymes that hydrolyze phos-pholipids. Multiple forms of phospholipases D, C, and A have been characterized inplants. These enzymes are involved in a broad range of functions in cellular regulation,lipid metabolism, and membrane remodeling. In recent years, increasing attention hasbeen paid to the many roles of phospholipases in signal transduction. This review high-lights recent developments in the understanding of biochemical, molecular biological,and functional aspects of various phospholipases in plants.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211PHOSPHOLIPASE D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

Subfamilies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213Catalysis and Substrate Specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213Regulation and Activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214Cellular Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

PHOSPHOLIPASE C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216Subfamilies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216Catalysis and Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217Cellular Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

PHOSPHOLIPASE A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220Subfamilies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220Identification and Catalytic Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221Cellular Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

PHOSPHOLIPASES A1 AND B; LYSOPHOSPHOLIPASES. . . . . . . . . . . . . . . . . 222NETWORK OF PHOSPHOLIPASES IN CELLULAR FUNCTION. . . . . . . . . . . . 224CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

INTRODUCTION

Phospholipases constitute a diverse series of enzymes that can be classified intophospholipases D (PLD), C (PLC), A2 (PLA2), A1 (PLA1), and B (PLB) according

1040-2519/01/0601-0211$14.00 211

P1: FXY


212 WANG

Figure 1 Hydrolysis of PtdCho by PLD, PLC, PLA2, PLA1, PLB, and lysoPLA and therespective reaction products. Note that the arrow lines for PLD, PLC, and PLA2 indicatetheir site of hydrolysis, but those for PLB, lysoPLA, and PLA1 do not. PLA1 hydrolyzes thesn-1 acylester bond, whereas lysoPLA removes the last fatty acid from lysophospholipidsthat can be produced by PLA2 and PLA1, as marked by the curved arrows. PLB sequentiallyremoves two fatty acids from phospholipids, and its final reaction products are the same asthose of lysoPLA. Cho, choline; P-Cho, phosphocholine; FA, fatty acid.

to their sites of hydrolysis on phospholipids (Figure 1). Each class is dividedfurther into subfamilies based on sequences, biochemical properties, or a combi-nation of both. A closely related class of enzymes comprises lysophospholipases(lysoPLAs), which hydrolyze the products of PLAs (Figure 1); some phospholi-pases also exhibit this activity. Phospholipids provide the backbone for biomem-branes, serve as rich sources of signaling messengers, and occupy important junc-tions in lipid metabolism. The activities of phospholipases not only affect thestructure and stability of cellular membranes, but they also regulate many cel-lular functions. The past two decades have brought rapid growth in knowledgeabout the role of phospholipases in cell regulation and signaling processes, par-ticularly in animal systems (2, 42, 64, 98). Activation of phospholipases often isan initial step in generating lipid and lipid-derived second messengers. In thepast several years, significant advances have been made toward understanding

P1: FXY


PLANT PHOSPHOLIPASES 213

PLD, PLC, and PLA2 in plants. Several reviews covering various aspects of plantphospholipases and phospholipids in cellular regulation have appeared recently(5, 11, 49, 82, 94, 95). This article is not intended to cover exhaustively all litera-ture in the field, but to highlight recent developments in the knowledge of molecularbiology, biochemistry, and cellular functions of plant phospholipases.

PHOSPHOLIPASE D

Subfamilies

PLD cleaves the terminal phosphodiesteric bond of phospholipids to phosphatidicacid (PtdOH) and water-soluble free head groups (Figure 1). Based on the re-quirements for Ca2+ and lipids of in vitro assays, PLDs can be grouped into threeclasses: (a) the conventional PLD that is most active at millimolar levels of Ca2+

(20 to 100 mM), (b) the polyphosphoinositide (PI)-dependent PLD that is most ac-tive at micromolar levels of Ca2+, and (c) the phosphatidylinositol (PtdIn)-specificPLD that is Ca2+-independent (reviewed in 95). The conventional PLD is the mostprevalent and best studied class in plants and has been purified to apparent homo-geneity from several plant sources (95 and references therein). The PI-dependentPLD was characterized recently inArabidopsis(58). The PtdIn-specific PLD wasidentified in suspension cells ofCatharanthus roseus(99).

A cDNA for the conventional PLD was isolated initially from castor bean (97);this cloning revealed the first primary structure of an intracellular PLD. PLD hassubsequently been cloned from a number of plants (95), animals (21), and fungi(68) and found to constitute a supergene family (42). Taking into account thesimilarities of deduced amino acid sequence, gene architecture, and biochemicalproperties, PLDs inArabidopsisare divided into five groups, PLDα, β, γ , δ, andε. Three PLDγ s (γ1, γ2, andγ3) are located close together on chromosomeIV (95). Most of the PLDs cloned from other plant species belong to the PLDα

group, and multiple PLDαs have been cloned from cabbage (34, 54),Craterostigaplantagineum(16), and rice (47). The PLDα gene product is responsible for theconventional PLD activity (97), and PLDβ andγ possess the newly identifiedPI-dependent PLD activity (62). PLDβ-like genes also have been cloned fromcotton (10) and rice (D McGee & J Leach, personal communication). None of thecloned PLDs exhibits the PtdIn-specific PLD activity.

Catalysis and Substrate Specificity

The PLDs cloned from eukaryotes all contain two HxKxxxD motifs, which consti-tute two active-site regions necessary for PLD activity (101). The motif also wasobserved in some phospholipid-synthesizing enzymes, bacterial phosphatidylse-rine (PtdSer) synthase and cardiolipin synthase, endonucleases, and other proteinsof unknown functions in pathogenic viruses and bacteria (61). The presence ofthe HxKxxxD motif is used to define the PLD superfamily. Recently, crystal

P1: FXY


214 WANG

structures have been determined for a 16-kDa endonuclease member of the PLDsuperfamily (84) and a 54-kDa bacterial PLD (41). Such structural informationprovides valuable insights into the mode of action of PLD catalysis. The enzymesof the PLD superfamily use the conserved histidine for nucleophilic attack on thesubstrate phosphorus. PLD hydrolyzes phospholipids at the P-O rather than theC-O bond via a two-step Ping-Pong reaction mechanism involving a phosphatidy-lated enzyme intermediate (84).

Most plant PLDs have broad substrate specificity (1, 55), but different groupsof PLDs exhibit varied abilities to hydrolyze different phospholipids (55). PLDα,β, and γ all utilize phosphatidylcholine (PtdCho), phosphatidylethanolamine(PtdEtn), and phosphatidylglycerol (PtdGro) as substrates, but the substrate pre-sentation and Ca2+ levels required for PLDβ andγ are strikingly different fromthose for PLDα (55). In addition, PLDβ andγ use PtdSer andN-acylphosphatidy-lethanolamine (NAPtdEtn) as substrates. Although PLDβ andγ hydrolyze thesame substrates, PLDγ , but not PLDβ, prefers ethanolamine-containing lipids,PtdEtn and NAPtdEtn, to other lipids. None of these cloned PLDs uses phos-phatidylinositol (PtdIn), phosphatidylinositol 4,5-bisphosphate (PtdInP2), or car-diolipin as a substrate. In contrast, the Ca2+-independent PLD fromC. roseushydrolyzes PtdIn, but not PtdCho, PtdEtn, or PtdGro (99). This PLD also wasreported to lack the transphosphatidylation activity characteristic for all otherPLDs (55, 95). The varied substrate specificities and preferences suggest thatactivation of different PLDs may result in selective hydrolysis of membranephospholipids.

Regulation and Activation

The activities of PLD are affected by a number of factors, including Ca2+ (95, 104),PIs (58, 62), substrate lipid composition (55), pH changes (56), and mastoparan,a tetradecapeptide G-protein activator (16, 48, 90). PLDs bind Ca2+ and phos-phatidylinositol 4,5-bisphosphate (PtdInP2) (58, 104); significant progress hasbeen made toward understanding how Ca2+ regulates PLD. Sequence analysisindicates that plant PLDs contain a Ca2+/phospholipid-binding fold, called theC2 domain, at the N terminus (57). The C2 domains of PLDα andβ have beendemonstrated to bind Ca2+, and this binding causes conformational changes ofthe proteins (104). They also display distinctive thermodynamics of binding, withthe PLDβ C2 having a higher affinity for Ca2+. In addition, the Ca2+ require-ment of PLDα is influenced strongly by pH and substrate lipid composition (56).PLDα is active at near-physiological, micromolar Ca2+ concentrations at an acidicpH of 4.5–5.0 in the presence of mixed lipid vesicles. In contrast, PLDβ andαare most active around neutral pH, and their Ca2+ requirements are independentof pH.

Intracellular translocation between cytosol and membranes has been pro-posed as one important mechanism of PLD activation (95). Indeed, PLD asso-ciated with microsomal membranes are correlated with stress-induced activation

P1: FXY



of PLD-mediated hydrolysis (72, 93), and membrane-associated PLD fromC. plantagineumwas activated within minutes during dehydration (16). The rel-ative distribution of PLD between the soluble and membrane fractions changesduring development and in response to stress (72, 93). Study of the Ca2+ and C2domain interaction showed that Ca2+ binding increases the affinity of the C2 do-mains for membrane phospholipids (104). This indicates that the C2 domain inPLDs is responsible for mediating a Ca2+-dependent intracellular translocationbetween cytosol and membranes. The increased association with membranes ofpreexisting PLD in the cell may represent a rapid and early step in PLD activationin stress responses (93). In addition, gene expression also plays a role in regulatingthe cellular levels of some PLD isoforms as described below.

Cellular Functions

PLD-catalyzed hydrolysis of phospholipids has been observed during seed germi-nation, aging, and senescence and under a broad spectrum of stress conditions, in-cluding freezing, drought, wounding, pathogen infection, nutrient deficiency, andair pollution (5, 16, 95, 103). Most of the early studies linked the increase in PLDactivity to lipid catabolism and membrane degradation. Owing to recent advancesin the manipulation and analysis of PLD genes, proteins, and reaction products, aclearer picture has emerged of the cellular functions of PLD in plants. Specifically,PLD play pivotal roles in plant response to stresses, and one way in which they doso is through mediating the action and production of the stress-related hormones,abscisic acid (ABA) (15, 29, 65), jasmonic acid (93), and ethylene (39, 71). Inparticular, evidence is strong for the role of PLD in the ABA signaling pathway.Addition of PtdOH to protoplasts of barley aleurone andVicia fabaguard cellspartially mimics the effect of ABA (29, 65). In guard cells, ABA activates PLD,and PA triggers signaling events that lead to closure of the inward K+ channel andstomatal aperture. Activity and gene expression of PLD also increase in tissuestreated with ABA and in plants under a water deficit (16, 50, 102). The role of PLDin ABA action was indicated initially by the finding that genetic suppression ofPLDα in Arabidopsisdecreased the rate of ABA-promoted senescence in detachedleaves (15). Further studies using PLDα-depleted and PLDα-overexpressing plantsshowed that PLD play a crucial role in controlling plant water loss by regulatingstomatal closure induced by ABA and water deficit (Y Sang & X Wang, unpub-lished data). The expression and activities of the other PLDs are not altered in thePLDα-abrogated plants. These results identify the role of PLDα and also indicatethat the other PLDs cannot compensate for the loss of PLDα.

Results of gene expression studies suggest that PLD isoforms may have differ-ent roles in stress responses. The expression of PLDβ, γ1, andγ2 genes increasesin woundedArabidopsisleaves, whereas PLDα is activated by increased asso-ciation of the preexisting enzyme with membranes (93). The relative levels ofexpression of different PLDs also differ inArabidopsisexposed to low tempe-rature, heavy metals, salts, drought, and the stress-related hormones ABA and

P1: FXY


216 WANG

jasmonic acid (96). In addition, the various PLDs give distinct temporal responsesfor a given stressor. Of the twoα class PLDs inC. plantagineum, CpPLD1 isconstitutive, as is the PLDα characterized in other plant species, whereas CpPLD2is induced by dehydration (16). Such expression patterns suggest that differentPLDs may act in different steps in response to a specific stress; the constitu-tively expressed PLDs such as PLDα are likely to be activated first to initi-ate or prime signaling and metabolic events that may involve the stress-inducedisoforms.

Studies have begun to address the question of how PLDs carry out their cellularfunctions, particularly in animal and yeast systems. In yeast, PLD is required forthe late phases of meiosis and sporulation (68); its function in these processes mayresult from a role in membrane trafficking (100). In mammalian cells, PLD func-tion is important for various processes, including vesicular trafficking, secretion,mitogenesis, oxidative burst, and cytoskeletal rearrangement (reviewed in 42).Activation of PLD produces messengers activating various enzymes such as pro-tein kinases, lipid kinases, phosphatases, and phospholipases. PtdOH can stimu-late protein kinases, including Ca2+-dependent and -independent kinases, suchas protein kinase C, mitogen-activated protein kinases, and Raf-kinases (42). APtdOH-specific protein kinase mediating the activation of NADPH oxidase alsohas been identified in mammalian cells (91). In plants, a protein kinase activated byPtdOH in response to wounding has been found (Y Lee, personal communication),but the role of PLD in elicitor-induced reactive oxygen production is uncertain (86).PtdOH also stimulates PI-5 kinase, PLC, and PLA2 (Figure 2), which are involvedin signaling cascades. A recent study also suggests that PLD activation may pro-mote degradation of the translation factor eEF1A (63). In addition, PtdOH canbe phosphorylated to diacylglycerol (DAG) pyrophosphate, dephosphorylated toDAG, or deacylated to generate lysoPtdOH and free fatty acids (Figure 2). Suchmetabolism may attenuate the PtdOH effect or generate new lipid mediators in asignaling cascade (94). The head group released by PLD also may have regulatoryfunctions, and the formation ofN-acylethanolamine by PLD has been implicatedin plant responses to fungal elicitation (5, 89). Furthermore, some cellular rolesof PtdOH may result from its effect on membrane properties and configuration(8), rather than its direct effect on proteins, because of its non-bilayer-formingproperty. Thus, diverse targets may underlie the various effects of PLD in cellfunction.

PHOSPHOLIPASE C

Subfamilies

PLC hydrolyzes the glycerophosphate ester linkage of phospholipids to DAG andphosphorylated head groups (Figure 1). According to substrate specificity and cel-lular function, PLCs in plants can be divided into three groups: (a) the PI-PLC thathydrolyzes phosphoinositides, (b) the nonspecific-PLC (also referred to as PtdCho-PLC) that acts on the common phospholipid PtdCho and some other phospholipids,

P1: FXY



and (c) the glycosylphosphatidylinositol (GPI)-PLC that hydrolyzes GPI-anchorson proteins.

The PtdCho-hydrolyzing PLC has been found in particulate or soluble prepa-rations of various plant species and tissues (7, 32, 69, 83). However, detecting thePLC activity in plants requires caution because of the relatively high activity ofPLD and the possible interconversion of PLD and PLC products by phosphatasesand kinases (Figure 2). The identification and understanding of PtdCho-PLC func-tion in plants, as well as in animals, has been hindered by the lack of molecularinformation on this enzyme. On the other hand, multiple PLCs that use Ptd-Cho as a substrate have been cloned and characterized from gram-positive andgram-negative bacteria (reviewed in 88). AnArabidopsiscDNA with significantsequence similarity to the gram-negative bacterial PLC has been isolated, and theprotein is localized in plastids (D Ling & X Wang, unpublished data). In addition,theArabidopsisGenome Project has revealed sixArabidopsisgenes that show se-quence similarities to bacterial PLCs, but the catalytic identities of these putativeproteins await experimental verification.

The GPI-PLC cleaves the terminal lipid that links GPI-anchored proteins to theplasma membrane. GPI-anchored proteins are extracellular and function as en-zymes, such as phosphatases or nitrate reductase, to recruit nutrients, as receptorsto interact with extracellular ligands, or as matrix proteins, such as arabinogalactanproteins (reviewed in 87). GPI-PLC has been characterized in animals and mi-croorganisms (66). A GPI-PLC was purified partially from peanut seeds; it cleavedsolubilized GPI-anchor, but paradoxically did not act on membrane-bound GPI (4).A recent structural analysis of the lipid moiety of a GPI-anchored arabinogalactanhas raised the possibility of the presence of a GPI-PLD in plants (53). Extracel-lular GPI-PLD occurs in animal blood serum (66 and references therein), but noGPI-PLD activity has been characterized in plants.

Compared with the other PLCs, the group of PI-PLCs is better understood. ThePI-PLCs in animal systems comprise at least ten different isoenzymes, which aredivided into three classes, PLCβ, γ , andδ, based on the sequence homology andmechanisms of activation (reviewed in 64, 98). Multiple PLCs have been clonedfrom plants, such asArabidopsis, soybean, tobacco, and potato (22, 25, 26, 35).Similar to yeast and mold PI-PLCs, the domain structure, size, and overall se-quence similarity of plant PI-PLCs characterized so far are related more closely tomammalian PLCδ than to the other PI-PLCs (reviewed in 49). Plant PI-PLCs allcontain domains X (∼170 amino acids) and Y (∼260 amino acids) that are neces-sary for the phosphoesterase activity, followed by a Ca2+-dependent phospholipidbinding a C2 domain toward the C terminus, but lack a pleckstrin homology do-main. The presence of N-terminal EF-hand motifs was reported for PI-PLCs fromsoybean andArabidopsis, but not from potato (35).

Catalysis and Regulation

Determination of the three-dimensional structure of a mammalian PI-PLCδ1 hasgiven insights into the catalysis of PI-PLCs (12). A two-step mechanism, tether

P1: FXY


218 WANG

and fix, has been proposed (12, 64). The PH domain of PI-PLCδ1 binds PtdInP2,and this binding tethers the enzyme to a membrane. In the presence of Ca2+, theC2 domain fixes the catalytic domain in the correct orientation. Another Ca2+ ionresides at the active site and, together with two His residues, mediates catalysis.The conservation of the C2 domain and the two His residues indicates that thecatalytic mechanism of plant PI-PLCs may be the same as that of mammalianPI-PLCs, but the lack of the PH domain in plant PI-PLCs suggests that some otherregions or mechanisms are involved in the initial interaction with membranes.This notion is consistent with a recent kinetic analysis of PtdInP2 hydrolysis,which indicates that plant PI-PLC binds lipid vesicles via a single binding site(24). It may first bind noncatalytically to PtdInP2 at the same site where catalysisoccurs (24). This is in contrast to mammalian PLCs that show multiple bindingsites for the interface and subsequent binding of the lipid substrate at the interface.

Ca2+ is required for plant PI-PLC activities (24), and, as described above, it mayregulate the PI-PLC via dual functions, catalysis and membrane binding (24). Thecloned PI-PLCs prefer PtdIn(4,5)P2 as a substrate at physiological concentrationsof Ca2+ (µM), but PtdIn is the preferred substrate at millimolar levels of Ca2+ (35).This decreased specificity toward PtdIn(4,5)P2 as Ca2+ levels increase resemblesthat of mammalian PI-PLCs. Both membrane-associated and soluble PI-PLCshave been found in plant extracts; the former hydrolyzes PtdIn(4,5)P2 and requiresmicromolar concentrations of Ca2+, whereas the latter acts primarily on PtdIn in thepresence of millimolar Ca2+ (49 and references therein). Expression of an epitope-tagged PI-PLC in transgenic tobacco indicates that both the membrane and solublePLC activities can be derived from the same PI-PLC (77). Activity of PI-PLCalso is modulated by other cations such as Mg2+ and Al3+. Al3+ inhibits PtdInP2hydrolysis (35), and the effect of Mg2+ on PI-PLC varies with the enzyme source.It stimulates PI-PLC activity associated with plant membranes (9), but not theE. coli-expressed potato PLC (35). These varied substrate specificities and cationeffects indicate that the cellular activity of PI-PLC is modulated by subcellularlocation, association with other cellular factors, and membrane environments, aproperty shared by other lipolytic enzymes (24, 55, 92, 95).

The G proteins are important regulators of mammalian PI-PLCs, and theireffects differ on the three classes of PI-PLCs (reviewed in 64, 98). PLCβs areactivated by theα subunit of heterotrimeric Gq proteins, whereas PLCγ s are ac-tivated by protein receptor-linked or nonreceptor tyrosine kinases. The activationof PLCδs is understood less than that of the other classes, and PLCδ1 may beactivated by the Ghα subunit and also by the small G protein RhoA. The regula-tion of plant PI-PLC by G proteins has been suggested mostly based on studiesusing mastoparan (6, 49). When used at concentrations above 10µM, however,mastoparan may activate PI-PLC via permeabilizing the plasma membrane that in-duces Ca2+ influx, rather than via G-protein activation. A recent study showed thatmastoparan stimulated PI-PLC activity without permeabilizingChlamydomonascells, and, interestingly, the level of mastoparan required for activating PI-PLCwas tenfold higher than that for activating PLD (90).

P1: FXY



The cellular levels of PI-PLC isoforms are regulated at the level of gene ex-pression (26, 35). Multiple PI-PLCs are expressed in most tissues, and the relativelevels of different isoforms can vary among tissues (35). In addition, different PI-PLCs are expressed differentially under various stress conditions. For example, ina drought treatment, the leaf transcript level of potato PLC1 decreased, whereasthat of PLC2 increased, and the level of PLC3 did not change. InArabidopsis,drought and low temperature increased the mRNA levels of AtPLC1S, but theAtPLC2 gene was expressed constitutively (26). These different patterns of ex-pression suggest that differentcisand/ortransfactors may regulate the expressionlevels of PI-PLC isoforms and raise the possibility that individual PI-PLCs mayserve particular functions.

Cellular Functions

PI-PLC-mediated signaling has been proposed to be important in the plant re-sponse to various stimuli, including osmotic stress, ABA, light, gravity, pathogenattack, and pollination (5, 40, 49, 60, 77, 81 and references therein). A recent re-port suggests that PI-PLC plays a role in the signaling cascade leading to thelight-dependent phosphorylation of C4 phosphoenolpyruvate carboxylase, a pro-cess that activates this enzyme that concentrates CO2 for C4 photosynthesis (9).The best-known function of PI-PLC in animal systems is its hydrolysis of PtdIn(4,5)P2 to produce the cellular messengers, inositol 1,4,5-trisphosphate (InP3) andDAG (64, 98) (Figure 2). InP3 binds to a receptor and mediates Ca2+ release to thecytoplasm, whereas DAG activates protein kinase C (PKC). Many components inthe animal PI-PLC cascade have been identified in plant cells; these include the oc-currence of PtdInP2, stimulus-induced production of InP3 and DAG, InP3-inducedCa2+ release, and PI-kinases involved in the production of PtdInP2 (reviewed in11, 49, 82). Compared with animal cells, the level of PtdInP2 in plants is quitelow, ranging from 0.05 to 0.5% of total phospholipids, depending on the typesof plants and cells. However, little is known about the plant InP3 receptor andDAG-activated protein kinase, and the immediate targets of InP3 and DAG remainelusive in plants.

Strong evidence exists to support the involvement of the PI-PLC-mediatedsignaling in Ca2+ mobilization and oscillation in plants. One such process thathas received considerable attention is the osmotic regulation of guard cells (2, 81and references therein). InP3 was reported to increase in ABA-treated guard cellprotoplasts, to promote an increase in cytoplasmic Ca2+, and to attenuate the in-ward K+ channel of the plasma membrane. Inhibition of PI-PLC by U-73122also inhibited ABA-promoted Ca2+ oscillation and stomatal closure. The InP3-mediated Ca2+ oscillations have been proposed also to regulate pollen tube growth(17). In response to gravity, InP3 levels increased transiently on the lower sideof bent maize pulvini (60), and the levels of InP3 have been suggested alsoto oscillate in the early phase of gravistimulation (82). The InP3 fluctuations,which may result from a cascade effect of activation of PLC and the rate of

P1: FXY


220 WANG

InP3 metabolism, may regulate the oscillations and changes of cytoplasmic Ca2+

concentrations.In addition, the activity of PI-PLC decreases the levels of PtdIn(4,5)P2 in mem-

branes, and the decrease itself could be an important signal in cell regulation(reviewed in 64, 82). PtdIn(4,5)P2 is an activator of PLD and a substrate for PI-3kinase. It also serves as a membrane-attachment site for various proteins with pleck-strin homology domains and is required for membrane-trafficking events. Further-more, PtdIn(4,5)P2 modulates cytoskeletal dynamics by interacting with manyactin-binding proteins, including profilin, gelsolin, cofilin, and actinin. PI-PLCalso is affected by actin-depolymerizing factors. Two classes of maize profilin,an actin monomer binding protein, displayed different abilities to inhibit PI-PLC(37), which, in turn, may influence the levels of PtdInP2 that affect cytoskeletalorganization. Thus, the levels and locations of PtdInP2 in the cell are regulated dy-namically, and the activity of PI-PLC may play an important role in the regulation.

PHOSPHOLIPASE A2

Subfamilies

PLA2 hydrolyzes the sn-2 acylester bond of phospholipids to free fatty acids and1-acyl-2-lysophospholipids (Figure 1). Based on sequence data, PLA2s from ani-mals are classified into 10 groups, which can be simplified into three major typesbased on their biological properties: (a) the secretory, low-molecular-weight PLA2(sPLA2), (b) the cytosolic Ca2+-dependent PLA2 (cPLA2), and (c) the intracellularCa2+-independent PLA2 (iPLA2) (3). The presence of PLA2 in plants had beenpresumed for some time, and its activity has been associated with various physio-logical processes (reviewed in 49, 74). Only recently have sPLA2-like PLA2s beenpurified and cloned in plants (33, 79, 80), and intracellular, iPLA2-like PLA2s alsohave been reported in plants (31, 36, 45, 76). A database search in July 2000 re-vealed one iPLA2-like gene sequence inArabidopsis, but no cPLA2-like sequencehas been found yet in plants.

The sPLA2-like PLA2 was purified first from elm seed endosperm and has alow molecular weight of 14 kDa. It requires millimolar levels of Ca2+ for optimalactivity and exhibits specificity toward the sn-2 acyl group (79). Two cDNAscorresponding to the PLA2 were cloned from rice and one similar cDNA wasisolated from developing carnation flowers (33, 80). Sequence analysis revealedthat they possess several disulfide bonds and contain putative signal peptides forsecretion (33, 80). All these data indicate that these PLA2s resemble animal sPLA2.

The intracellular PLA2s reported to date show sequence similarities to patatin, agroup of closely related, vacuolar storage proteins in potato tubers that possess acylhydrolase activity (28 and references therein). These PLA2s include a membrane-associated enzyme from broad bean leaves (31), a latex allergen (Hev b 7) (36), aprotein associated with cucumber lipid bodies (45), and a potato cytosolic enzymeinduced by a late blight fungus (76). The sizes of these proteins range from 40

P1: FXY



to 48 kDa. The purified enzyme from broad bean was inhibited by inhibitors ofmammalian iPLA2 and cPLA2 (ETYA and an arachidonyl trifluoromethyl ketoneAACOCF3), but not by inhibitors of sPLA2. It prefers 2-linolenoyl-PtdCho to2-linoleoyl-PtdCho and has a lysoPLA activity, but no PLA1 activity. This enzymeis stimulated by calmodulin, but not by Ca2+, and, thus, is suggested to be aniPLA2-like PLA2 (31).

Identification and Catalytic Mechanisms

The identity of PLA2 in plants has been a common subject of confusion becauseof the overlapping specificities of PLA2 and nonspecific acyl hydrolases, whichare very active in some plant tissues (reviewed in 28). The acyl hydrolases removefatty acids at the sn-1 and -2 positions from several classes of lipids, includingglycolipids, sulfolipids, monoacylglycerols, and DAG, in addition to phospho-lipids, but they are inactive on triacylglycerols. Thus, these enzymes have thecombined capacities of PLA2, PLA1, PLB, lysoPLA, and galactolipases. One ex-ample of such proteins is patatin, which has been shown to have acyl-hydrolyzingactivities toward various glycerolipids (28 and references therein). As discussedearlier, patatin-like proteins from several plant sources recently have been re-ported to exhibit PLA2 activity. Depending on their sources, these enzymes alsodisplay other lipolytic activities, such as PLA1, monoacylglycerol esterase, and/orlysoPLA (31, 36, 45, 76). However, the substrate specificities of the establishedmammalian cPLA2 and iPLA2 also are not absolute (92 and references therein).For example, cPLA2 displays lysoPLA activities, and iPLA2 also exhibits PLA1,lysoPLA, transacylase, and platelet-activating factor acetylhydrolase activities.The relative level of these activities depends strongly on substrate presentation(43). Thus, establishing the identity of a PLA2 requires careful characterizationthat takes into account its substrate specificity and preferences, sequence andstructural similarities with known PLA2s, and, most important, the lipolytic ac-tivity in vivo. Enzymes with sequences related to the patatin family may act asPLAs to release free fatty acids and lysophospholipids involved in various plantresponses.

The broad substrate specificity may be related to the catalytic mechanism sharedby many acyl-hydrolyzing enzymes. The cPLA2 and iPLA2 have been proposedto function as serine hydrolases, with the active Ser residue located in the middleof the consensus sequence GxSxG (3, 92 and references therein). This sequencealso is found in PLA1, PLB, and lysoPLA, as well as many lipases. Both cPLA2and iPLA2 hydrolyze the ester bond via an acyl-enzyme intermediate. AlthoughCa2+ does not participate directly in the catalysis, cPLA2 needs Ca2+ to associatewith membranes (3). In contrast, Ca2+ has a direct role in the catalysis of sPLA2,whose catalytic mechanism is different from that of the other PLA2s. Ca2+ bindsto a conserved Ca2+ binding loop of sPLA2 and stabilizes the transition-stateintermediate. The sPLA2 catalysis does not involve the formation of the classicalacyl-enzyme intermediate of the serine hydrolases. Instead, sPLA2s use an His

P1: FXY


222 WANG

residue, aided by an Asp, to polarize an H2O, which then attacks the carbonylgroup. The low-molecular-weight PLA2s of plants conserve the His and Asp inthe catalytic site and other residues in Ca2+ binding (80) and, thus, may use thesame catalytic mechanism as sPLA2s.

Cellular Functions

One extensively studied function of PLA2 in cell regulation is the release of arachi-donic acid, a rate-limiting step in the eicosanoid pathway in animal systems (3).An analogous process in plants is the octadecanoid pathway that uses linolenicacid to produce jasmonic acid and related compounds (52, 70 and referencestherein) (Figure 2). These oxylipins regulate many cellular processes, such aswound and defense responses. Evidence was provided recently for the presence ofa wound/systemin-inducible PLA2 activity. Wounding promoted systemic accu-mulation of lysoPtdCho and lysoPtEtn in several plant species (38). Systemin andoligosaccharide elicitors, which induce jasmonic acid synthesis, also increase aPLA2-like activity without wounding. The animal PLA2 inhibitors manoalide andAACOCF3 both decreased systemin-induced formation of lysoPtdCho (52). Theactivation of PLA2 may be mediated by a wound-inducible cell surface receptorto release polyunsaturated fatty acids for oxylipin synthesis (70).

The activities of PLA2 are important in other cellular processes, such as lipidmetabolism (5, 45), plant-pathogen interactions (49, 67, 76), and auxin-stimulatedgrowth (59, 74). A PLA-like activity preferentially removes uncommon fatty acids,such as ricinoleic and vernolic acids, from PtdCho, and this activity might channelthese fatty acids to triacylglycerols (5, 78). A patatin-related PLA2 in cucumber islocalized in lipid bodies and may be involved in lipid catabolism and mobilizationduring seed germination and seedling growth (45). In pathogen-infected or elicitor-treated cells, activation of PLA2 may be involved in reactive oxygen generation andalkaloid production (5, 49, 67, 76). In auxin-stimulated growth, PLA-released fattyacids may carry out second messenger functions, particularly in cell elongation,whereas lysoPtdCho and lysoPtEtn may have functions different from those of freefatty acids in auxin and stress responses (59). The iPLA2inhibitor HELSS inhibitedthe auxin-induced elongation of zucchini hypocotyls and maize coleoptils, but thePLA2 inhibitor AACOCF3, which inhibits systemin-inducible PLA, was not ef-fective (59). These results suggest that wounding and auxin signaling may involvedifferent types of PLA2, but little is known about the molecular nature of thesePLA activities.

PHOSPHOLIPASES A1 AND B; LYSOPHOSPHOLIPASES

PLA1 hydrolyzes the sn-1 acylester bond of phospholipids to free fatty acids and2-acyl-1-lysophospholipids. PLB sequentially removes two fatty acids from phos-pholipids and thus has both PLA and lysoPLA activities (Figure 1). MultiplePLA1s and PLBs have been identified and cloned from animals and yeast (46, 51),

P1: FXY



but their functions are not as well understood as those of PLA2. Simultaneousinactivation of the threePLBgenes in yeast resulted in total loss of PLB activity invitro, but no apparent growth defect occurred (46). Two forms of PtdSer-specificPLA1 have been identified in mammalian cells, and their activities are important incontrolling the levels of PtdSer and lysoPtdSer that are involved in the metastaticprocess in tumor cells (51). A PLA1 activity was found in the tonoplasts ofAcerpseudoplatanuscells (85). Although few reports deal directly with PLB in plants,several plant proteins possess PLB-like activities, such as the purified PLA2 frombroad bean (31) and the ricin B chain from castor bean (23).

PLBs, and some PLA1s, cPLA2s, and iPLA2s, also possess lysoPLA activity,which removes the last fatty acid from lysophospholipids (Figure 1). Togetherwith some transacylases and acyl transferases, these are grouped as large lyso-PLAs in animal cells (92). In addition, two types of small lysoPLAs, I and II, withmolecular weights of about 25 kDa, have been identified in mammalian cells (92).Lysophospholipids are found in low concentrations in biological membranes andhave many cellular functions, such as signal transduction and vesicular traffickingin mammalian cells (92 and references therein). LysoPtdCho modulates multiplegene expression, promotes secretion of growth factors, and induces cell adhesion.Receptors specific for lysoPtdOH, the simplest naturally occurring lysophospho-lipid, have been cloned and identified as G protein–coupled receptors (20). Theformation of lysoPtdOH and PtdOH occurs specifically at the neck of buddingsynaptic vesicles and is required for membrane budding (75). Lysophospholipidsin plants are produced in response to stress cues, as discussed earlier, and modulatea number of enzyme activities (5, 49, 59, 74 and references therein). LysoPtdChomay interact directly with plant plasma membrane H+-ATPase to stimulate pro-ton pumping (19). LysoPtdEtn retards senescence, possibly through inhibition ofPLD (71) (Figure 2). Thus, lysophospholipases are potentially important in regu-lating the levels of lysophospholipids and serve a major function in lipid signalingcascades and lipid metabolism (92).

Early studies reported the presence of acidic and basic lysoPLA activities ingerminating barley seeds, and these activities may be involved in lipid mobilization(18, 44). Database searches in July 2000 revealed multipleArabidopsisand ricegenes that encode proteins of 310–330 amino acids with sequence similarities tosmall animal lysoPLAs (X Wang, unpublished data). The catalytic mechanismof mammalian small lysoPLAs resembles the classical serine hydrolases (92).The deduced plant proteins contain the GXSXG and catalytic triad Ser-Asp-Hisconsensus sequences, although the lipolytic activity of these gene products remainsto be identified. Related to the catalytic mechanism of serine hydrolases are tworecently identified genes, EDS1 and PAD4; EDS1 is an essential component ofR gene–mediated disease resistance inArabidopsis(14) and PAD4 is importantfor salicylic acid signaling (30). But the identity of these defense-related genesas lipolytic enzymes has not been established. Nevertheless, the occurrence ofmultiple lysoPLA-like genes in the plant genomes and the functional revelation ofputative lipolytic enzymes warrant further study of these enzymes.

P1: FXY


224 WANG

NETWORK OF PHOSPHOLIPASESIN CELLULAR FUNCTION

It is increasingly clear that multiple lipid signaling enzymes often form complexnetworks that mediate a specific cellular response. The involvement of PLC, PLD,and PLA in stomatal movement presents an example for such interaction (Figure 2).Evidence has indicated that both PI-PLC and PLD are involved in mediating stom-atal closure (29, 81), whereas the activity of PLA may stimulate stomatal open-ing (5 and references therein). PI-PLC releases InP3 that promotes oscillationsand increases in cytoplasmic Ca2+ (81). The increase in Ca2+ may enhance PLDassociation with membranes, resulting in PLD activation (95, 104). On the otherhand, PLD-derived PA may activate PtdInP-5 kinase, producing the PI-PLC sub-strate PtdInP2, as shown in animal systems (42). PtdInP2 is a required activator forPLDs, and activations of PLD and PtdInP-5 kinase have been proposed to form apositive feedback loop that leads to rapid generation of PtdOH and PtdInP2, whichare involved in vesicular trafficking and cytoskeletal dynamics in animal systems(42). Active membrane trafficking and cytoskeletal rearrangements have been sug-gested to occur in stomatal movement (13, 27). Using U73122 to inhibit PI-PLCand 1-butanol to attenuate PtdOH formation by PLD, a recent study indicated thatPLC and PLD act on the same pathway in ABA-induced stomatal closure (29).On the other hand, lysophospholipids inhibit PLD activity (71) and, thus, the ac-tivation of PLA that promotes stomatal opening also may, in turn, down-regulatePLD (Figure 2). But the effect of PLA activation on PLD/PLC or vice versa hasnot been tested directly.

Another interplay among the lipid-signaling enzymes occurs in wound-inducedlipid hydrolysis and oxylipin production. Activation of PLA has been suggested torelease linolenic acid for the synthesis of jasmonic acid and related oxylipins (52).However, in studies where the temporal patterns were analyzed, increases in PtdOHpreceded those of other lipid metabolites such as DAG, free fatty acids, peroxidizedfatty acids, or lysophospholipids (38, 73). The activation of PLD may generatePtdOH, stimulating other lipolytic activities such as PLA, as shown in mammaliansystems (42) (Figure 2). A recent study showed that antisense suppression of PLDα

in Arabidopsisresulted in decreases in wound-induced accumulation of linolenicacid and jasmonic acid (93), providing evidence that activation of PLD modulateswound induction of jasmonic acid.

CONCLUDING REMARKS

Over the past several years, significant advances have been made toward under-standing the biochemistry and molecular biology of PLD, PLC, and PLA2. Theseenzymes have been connected with various facets of cellular processes, particu-larly responses to hormones and abiotic and biotic stresses. Based on the availableinformation, the functions of phospholipases as a whole may be considered in

P1: FXY



three categories: (a) cell regulation, such as signal transduction, vesicular traf-ficking, and cytoskeletal dynamics; (b) lipid catabolism, such as membrane lipiddegradation during cell differentiation, senescence, aging, and stress injuries andremoval of undesirable lipids from membranes; and (c) membrane remodeling,such as changing acyl and head group compositions of membrane lipids duringgrowth and development and in response to stresses. One underexplored, importantarea is the detailed molecular and cellular mechanisms by which phospholipasesmediate these cellular functions in plants. Little is known about the cellular ac-tivators and targets of PLD, PLC, and PLAs. Understanding the mechanisms inwhich phospholipases are involved will require identification and knowledge ofthe molecules that interact with phospholipases and the reactions or processes thatlie directly downstream of these enzymes and their reaction products.

PLD, PI-PLC, and PLA2 have been demonstrated to have multiple isoforms,and the same is anticipated for other classes of plant phospholipases. This raisesimportant questions about what roles particular phospholipase isoforms play inplant growth and development and how the properties of a specific isoform areexploited in the cell. The locations and timing of expression and activation arelikely to be keys to determining the function of each isoform, and decipheringthese questions requires approaches directed toward specific isoenzymes. Ge-netic depletion of a PLDα represents the first alteration of a specific phospho-lipase isoform in plants; this manipulation has improved understanding of thePLD family (58, 95). Genetic manipulations, including suppression, point andinsertional mutagenesis, and overexpression in plant cells, should be forthcom-ing for other phospholipases and specific isoforms. Future studies should capi-talize on the vast information on phospholipase-like genes revealed by thegenomic sequencing and the approaches of functional genomics, proteinomics,and metabolomics. Results should shed light on the diverse cellular roles of phos-pholipases in plants.

ACKNOWLEDGMENTS

I thank Dr R Welti for critically reading the manuscript, Drs W Boss and B Drobakfor discussion on PI-PLC, and Drs T Munnik and Y Lee for sharing unpublisheddata. Work in the author’s laboratory is supported by grants from the US NationalScience Foundation and US Department of Agriculture and this is contribution01-59-J of the Kansas Agricultural Experiment Station.

Visit the Annual Reviews home page at www.AnnualReviews.org

LITERATURE CITED

1. Abousalham A, Nari J, Teissere M, FerteN, Noat G, Verger R. 1997. Study of fattyacid specificity of sunflower phospholipase

D using detergent/phospholipid micelles.Eur. J. Biochem.248:374–79

2. Assmann SM, Shimazaki K. 1999. The

P1: FXY


226 WANG

multisensory guard cell. Stomatal responseto blue light and abscisic acid.Plant Phys-iol. 119:809–15

3. Balsinde J, Balboa MA, Insel PA, Den-nis EA. 1999. Regulation and inhibition ofphospholipase A2.Annu. Rev. Pharmacol.Toxicol.39:175–89

4. Butikofer P, Brodbeck U. 1993. Partial pu-rification and characterization of a (gly-cosyl) inositol phospholipid-specific phos-pholipase C from peanut.J. Biol. Chem.268:17794–802

5. Chapman KD. 1998. Phospholipase acti-vity during plant growth and developmentand in response to environmental stress.Trends Plant Sci.11:419–26

6. Cho MH, Tan Z, Erneux C, Shears SB, BossWF. 1995. The effects of mastoparan onthe carrot cell plasma membrane polyphos-phoinositide phospholipase C.Plant Phys-iol. 107:845–56

7. Chrastil J, Parrish FW. 1987. Phospholi-pases C and D in rice grains.J. Agric. FoodChem.35:624–27

8. Cornell RB, Arnold RS. 1996. Modulationof the activities of enzymes of membranelipid metabolism by non-bilayer-forminglipids. Chem. Phys. Lipids81:215–27

9. Coursol S, Giglioli N, Vidal J, Pierre J-N. 2000. An increase in phosphoinositidespecific phospholipase C activity precedesinduction of C4 phosphoenolpyruvate car-boxylase phosphorylation in illuminatedand NH4Cl-treated protoplasts fromDig-itaria sanguinalis. Plant J.23:497–506

10. Cui X, Brown RM Jr. 1999. Molecularcloning of a phospholipase D gene fromcotton fibers (accession no. AF159139).Plant Physiol.120:1207

11. Drobak BK, Dewey RE, Boss WF. 1999.Phosphoinositide kinase and the synthe-sis of polyphosphoinositide in higher plantcells.Int. Rev. Cytol.189:95–130

12. Essen LO, Perisic O, Cheung R, KatanM, Williams RL. 1996. Crystal structureof a mammalian phosphoinositide-specificphospholipase Cδ. Nature380:595–602

13. Eun SO, Lee Y. 1997. Actin filaments ofguard cells are reorganized in responseto light and abscisic acid.Plant Physiol.115:1491–98

14. Falk A, Feys BJ, Frost LN, Jones JD,Daniels MJ, Parker JE. 1999. EDS1, anessential component of R gene-mediateddisease resistance inArabidopsishas ho-mology with eukaryotic lipases.Proc. Natl.Acad Sci. USA96:3292–97

15. Fan L, Zheng S, Wang X. 1997. Anti-sense suppression of phospholipase Dα re-tards abscisic acid- and ethylene-promotedsenescence of postharvestArabidopsisleaves.Plant Cell9:2916–19

16. Frank W, Munnik T, Kerkmann K,Salamini F, Bartels D. 2000. Water deficittriggers phospholipase D activity in theresurrection plantCraterostigma plan-tagineum. Plant Cell12:111–24

17. Franklin-Tong VE, Drobak BK, Allan AC,Watkins PAC, Trewavas AJ. 1996. Growthof pollen tubes ofPapaver rhoeasis reg-ulated by a slow-moving calcium wavepropagated by inositol 1,4,5-trisphosphate.Plant Cell8:1305–21

18. Fujikura Y, Baisted D. 1985. Purificationand characterization of a basic lysophos-pholipase in germinating barley.Arch.Biochem. Biophys.243:570–78

19. Gomes E, Venema K, Simon-Plas F, Mi-lat ML, Palmgren MG, Blein JP. 1996.Activation of the plant plasma membraneH+-ATPase. Is there a direct interactionbetween lysophosphatidylcholine and theC-terminal part of the enzyme?FEBS Lett.398:48–52

20. Guo Z, Liliom K, Fischer DJ, Bathurst IC,Tomei LD, et al. 1996. Molecular cloningof a high-affinity receptor for the growthfactor-like lipid mediator lysophosphatidicacid from Xenopusoocytes.Proc. Natl.Acad. Sci. USA93:14367–72

21. Hammond SM, Alshuller YM, Sung T,Rudge SA, Rose K, et al. 1995. HumanADP-ribosylation factor-activated phos-phatidylcholine-specific phospholipase D

P1: FXY



defines a new and highly conserved genefamily J. Biol. Chem.270:29640–43

22. Hartweck LM, Llewellyn DJ, Dennis ES.1997. TheArabidopsis thalianagenomehas multiple divergent forms of phos-phoinositol-specific phospholipase C1.Gene202:151–56

23. Helmy M, Lombard S, Pieroni G. 1999.Ricin RCA60: evidence of its phospholi-pase activity.Biochem. Biophys. Res. Com-mun.258:252–55

24. Hernandez-Sotomayor SMT, Santos-Briones CDL, Munoz-Sanchez JA, Loyola-VargasVM. 1999. Kinetic analysis of phos-pholipase C fromCatharanthus roseustransformed roots using different assays.Plant Physiol.120:1075–82

25. Hirayama T, Mitsukawa N, Shibata D, Shi-nozaki K. 1997. AtPLC2, a gene encodingphosphoinositide-specific phospholipaseC, is constitutively expressed in vegetativeand floral tissues inArabidopsis thaliana.Plant Mol. Biol.34:175–80

26. Hirayama T, Ohto C, Mizoguchi T,Shinozaki K. 1995. A gene encoding aphosphatidylinositol-specific phospholi-pase C is induced by dehydration and saltstress inArabidopsis thaliana. Proc. Natl.Acad. Sci. USA92:3903–7

27. Homann U, Thiel G. 1999. Unitary exo-cytic and endocytotic events in guard-cellprotoplasts during osmotically driven vol-ume changes.FEBS Lett.460:495–99

28. Huang AHC. 1993. Lipases. InLipidMetabolism in Plants, ed. TS Moore, pp.473–503. Boca Raton, FL: CRC Press

29. Jacob T, Ritchie S, Assmann SM, GilroyS. 1999. Abscisic acid signal transductionin guard cells is mediated by phospholi-pase D activity.Proc. Natl. Acad. Sci. USA96:12192–97

30. Jirage D, Tootle TL, Reuber TL, Frost LN,Feys BJ, et al. 1999.Arabidopsis thalianaPAD4 encodes a lipase-like gene that is im-portant for salicylic acid signaling.Proc.Natl. Acad. Sci. USA96:13583–88

31. Jung KM, Kim DK. 2000. Purification and

characterization of a membrane-associated48-kilodalton phospholipase A2 in leavesof broad bean.Plant Physiol.123:1057–68

32. Kates M. 1955. Hydrolysis of lecithin byplant plastid enzymes.Can. J. Biochem.Physiol.33:575

33. Kim JY, Chung YS, Ok SH, Lee SG,Chung WI, et al. 1999. Characterizationof the full-length sequences of phospho-lipase A2 induced during flower develop-ment.Biochim. Biophys. Acta1489:389–92

34. Kim DU, Roh TY, Lee J, Noh JY, JangYJ, et al. 1999. Molecular cloning andfunctional expression of a phospholipase Dfrom cabbage (Brassica oleraceavar.cap-itata). Biochim. Biophys. Acta.1437:409–14

35. Kopka J, Pical C, Gray JE, Muller-Rober B.1998. Molecular and enzymatic character-ization of three phosphoinositide-specificphospholipase C isoforms from potato.Plant Physiol.116:239–50

36. Kostyal DA, Hickey VL, Noti JD, SussmanGL, Beezhold DH. 1998. Cloning and char-acterization of a latex allergen (Hev b 7):homology to patatin, a plant PLA2.Clin.Exp. Immunol.112:355–62

37. Kovar DR, Drobak BK, Staiger CJ. 2000.Maize profilin isoforms are functionallydistinct.Plant Cell12:583–98

38. Lee S, Suh S, Kim S, Crain RC, Kwak JM,et al. 1997. Systemic elevation of phospha-tidic acid and lysophospholipid levels inwounded plants.Plant J.12:547–56

39. Lee SH, Chae TK, Kim SH, Shin SH, ChoBH, et al. 1998. Ethylene-mediated phos-pholipid catabolism pathway in glucose-starved carrot suspension cells.Plant Phys-iol. 116:223–29

40. Legendre L, Yueh YG, Crain R, HaddockN, Heinstein PF, Low PS. 1993. Phospho-lipase C activation during elicitation of theoxidative burst in cultured plant cells.J.Biol. Chem.268:24559–63

41. Leiros I, Secundo F, Zambonelli C, Servi S,Hough E. 2000. The first crystal structure

P1: FXY


228 WANG

of a phospholipase D.Struct. Fold Des.8:855–67

42. Liscovitch M, Czarny M, Fiucci G, Tang X.2000. Phospholipase D: molecular and cellbiology of a novel gene family.Biochem.J. 345:401–15

43. Loo RW, Conde-Frieboes K, Reynolds LJ,Dennis E. 1997. Activation, inhibition,and regiospecificity of the lysophospholi-pase activity of the 85-kDa group IV cy-tosolic phospholipase A2.J. Biol. Chem.272:19214–19

44. Lundgard R, Baisted D. 1986. Secre-tion of a lipolytic protein aggregate bybarley aleurone and its dissociation bystarch endosperm.Arch. Biochem. Bio-phys.249:447–54

45. May C, Preisig-Muller R, Hohne M, GnauP, Kindl H. 1998. A phospholipase A2 istransiently synthesized during seed ger-mination and localized to lipid bodies.Biochim. Biophys. Acta1393:267–76

46. Merkel O, Fido M, Mayr JA, Pruger H,Raab F, et al. 1999. Characterization andfunction in vivo of two novel phospho-lipases B/lysophospholipases fromSac-charomyces cerevisiae. J. Biol. Chem.274:28121–27

47. Morioka S, Ueki J, Komari T. 1997. Char-acterization of two distinctive genomicclones (accession nos. AB001919 andAB001920) for phospholipase D from rice(PGR 97-076).Plant Physiol.114:396

48. Munnik T, Arisz SA, de Vrije T, MusgraveA. 1995. G-protein activation stimulatesphospholipase D signaling in plants.PlantCell 7:2187–210

49. Munnik T, Irvine RF, Musgrave AP. 1998.Phospholipid signalling in plants.Biochim.Biophys. Acta1389:222–72

50. Munnik T, Meijer HJ, Ter Riet B, HirtH, Frank W, et al. 2000. Hyperosmoticstress stimulates phospholipase D activityand elevates the levels of phosphatidic acidand diacylglycerol pyrophosphate.Plant J.22:147–54

51. Nagai Y, Aoki J, Sato T, Amano K,

Matsuda Y, et al. 1999. An alterna-tive splicing form of phosphatidylserine-specific phospholipase A1 that exhibitslysophosphatidylserine-specific lysophos-pholipase activity in humans.J. Biol.Chem.274:11053–59

52. Narvaez-Vasquez J, Florin-Christensen J,Ryan CA. 1999. Positional specificityof a phospholipase A activity inducedby wounding, systemin, and oligosaccha-ride elicitors in tomato leaves.Plant Cell11:2249–60

53. Oxley D, Bacic A. 1999. Structure ofthe glycosylphosphatidylinositol anchor ofan arabinogalactan protein fromPyruscommunissuspension-cultured cells.Proc.Natl. Acad. Sci. USA96:14246–51

54. Pannenberg P, Mansfeld J, Ulbrich-Hofmann R. 1998. Identification of twoisoenzymes (accession nos. AF09044 and09045) of phospholipase D from cabbage(Brassica oleraceavar. capitata). PlantPhysiol.118:1102

55. Pappan K, Austin-Brown S, Chapman KD,Wang X. 1998. Substrate selectivities andlipid modulation of phospholipase Dα, β,andγ from plants.Arch. Biochem. Biophys.353:131–40

56. Pappan K, Wang X. 1999. Plant phospho-lipase Dα is an acidic phospholipase activeat near-physiological Ca2+ concentrations.Arch. Biochem. Biophys.368:347–53

57. Pappan K, Qin W, Dyer JH, Zheng L, WangX. 1997. Molecular cloning and func-tional analysis of polyphosphoinositide-dependent phospholipase D, PLDβ, fromArabidopsis. J. Biol. Chem.272:7055–61

58. Pappan K, Zheng S, Wang X. 1997. Iden-tification and characterization of a novelphospholipase D that requires polyphos-phoinositide and submicromolar calciumfor activity in Arabidopsis. J. Biol. Chem.272:7048–54

59. Paul RU, Holk A, Scherer GF. 1998. Fattyacids and lysophospholipids as potentialsecond messengers in auxin action. Rapidactivation of a phospholipase A2 activity

P1: FXY



by auxin in suspension-cultured parsleyand soybean cells.Plant J.16:601–11

60. Perera IY, Heilmann I, Boss WF. 1999.Transient and sustained increases in inos-itol 1,4,5-trisphosphate precede the differ-ential growth response in ravistimulatedmaize pulvini.Proc. Natl. Acad. Sci. USA96:5838–43

61. Ponting CP, Kerr ID. 1996. A novel fam-ily of phospholipase D homologues thatincludes phospholipid synthases and puta-tive endonucleases: identification of du-plicated repeats and potential active siteresidues.Protein Sci.5:914–22

62. Qin W, Pappan K, Wang X. 1997.Molecular heterogeneity of phospholipaseD(PLD): cloning of PLDγ and regulationof plant PLDα, β, and γ by polyphos-phoinositides and calcium.J. Biol. Chem.272:28267–73

63. Ransom-Hodgkins WD, Brglez I, Wang X,Boss WF. 2000. Calcium-regulated prote-olysis of eEF1A.Plant Physiol.122:957–65

64. Rhee SG, Bae YS. 1997. Regulation ofphosphoinositide-specific phospholipaseC isozymes.J. Biol. Chem.272:15045–48

65. Ritchie SM, Gilroy S. 1998. Abscisic acidsignal transduction in the barley aleuroneis mediated by phospholipase D activity.Proc. Natl. Acad. Sci. USA95:2697–702

66. Roberts MF. 1996. Phospholipases: struc-tural and functional motifs for working atan interface.FASEB J.10:1159–72

67. Roos W, Dordschbal B, Steighardt J, HiekeM, Weiss D, Saalbach G. 1999. A redox-dependent, G-protein-coupled phospholi-pase A of the plasma membrane is involvedin the elicitation of alkaloid biosynthesisin Eschscholtzia californica. Biochim. Bio-phys. Acta1448:390–402

68. Rose K, Rudge SA, Frohman MA, Mor-ris AJ, Engebrecht J. 1995. PhospholipaseD signaling is essential for meiosis.Proc.Natl. Acad. Sci. USA92:12151–55

69. Rouet-Mayer MA, Valentova O, Simond-Cote E, Daussan J, Thevenot C. 1995.

Critical analysis of phospholipid hy-drolyzing activities in ripening tomatofruits. Study by spectrofluorimetry andhigh-performance liquid chromatography.Lipids30:739–46

70. Ryan CA. 2000. The systemin signalingpathway: differential activation of plantdefensive genes.Biochim. Biophys. Acta1477:112–21

71. Ryu SB, Karlsson BH, Ozgen M, PaltaJP. 1997. Inhibition of phospholipase Dby lysophosphatidylethanolamine, a lipid-derived senescence retardant.Proc. Natl.Acad. Sci. USA94:12717–21

72. Ryu SB, Wang X. 1996. Activation of phos-pholipase D and the possible mechanism ofactivation in wound-induced lipid hydroly-sis in castor bean leaves.Biochim. Biophys.Acta1303:243–50

73. Ryu BS, Wang X. 1998. Increase in freelinolenic and linoleic acids associated withphospholipase D-mediated hydrolysis ofphospholipids in wounded castor beanleaves.Biochim. Biophys. Acta1393:193–202

74. Scherer GF. 1995. The functional relation-ship of plant lipid-derived second messen-gers and plant lipid-activated protein ki-nase.Biochem. Soc. Trans.23:871–75

75. Schmidt A, Wolde M, Thiele C, Fest W,Kratzin H, et al. 1999. Endophilin I medi-ates synaptic vesicle formation by transferof arachidonate to lysophosphatidic acid.Nature401:133–41

76. Senda K, Yoshioka H, Doke N, KawakitaK. 1996. A cytosolic phospholipase A2from potato tissues appears to be patatin.Plant Cell Physiol.37:347–53

77. Shi J, Gonzales RA, BhattacharyyaMK. 1995. Characterization of a plasmamembrane-associated phopshinositide-specific phospholipase C from soybean.Plant J.8:381–90

78. Stahl U, Banas A, Stymne S. 1995. Plantmicrosomal acyl hydrolases have selec-tivities for uncommon fatty acids.PlantPhysiol.107:953–62

P1: FXY


230 WANG

79. Stahl U, Ek B, Stymne S. 1998. Purificationand characterization of a low-molecular-weight phospholipase A2 from developingseeds of elm.Plant Physiol.117:197–205

80. Stahl U, Lee M, Sjodahl S, Archer D,Cellini F, et al. 1999. Plant low-molecular-weight phospholipase A2S (PLA2s) arestructurally related to the animal secretoryPLA2s and are present as a family of iso-forms in rice (Oryza sativa). Plant Mol.Biol. 41:481–90

81. Staxen I, Pical C, Montgomery LT, GrayJE, Hetherington AM, McAinsh MR.1999. Abscisic acid induces oscillations inguard-cell cytosolic free calcium that in-volve phosphoinositide-specific phospho-lipase C. Proc. Natl. Acad. Sci. USA96:1779–84

82. Stevenson JM, Perera IY, Heilmann I, Pers-son S, Boss WF. 2000. Inositol signalingand plant growth.Trends Plant Sci.5:252–58

83. Strauss H, Leibovitz-Ben Gershon Z,Heller M. 1976. Enzymatic hydrolysisof 1-monoacyl-SN-glycerol-3-phosphory-l-choline (1-lysolecithin) by phospholi-pases from peanut seeds.Lipids11:442–48

84. Stuckey JA, Dixon JE. 1999. Crystal struc-ture of a phospholipase D family member.Nat. Struct. Biol.6:278–84

85. Tavernier E, Pugin A. 1995. Phospholi-pase activities associated with the tonoplastfrom Acer pseudoplatanuscells: identi-fication of a phospholipase A1 activity.Biochim. Biophys. Acta1233:118–22

86. Taylor AT, Low SP. 1997. PhospholipaseD involvement in the plant oxidative burst.Biochem. Biophys. Res. Commun.237:10–15

87. Thompson GA, Okuyama H. 2000. Lipid-linked proteins of plants.Prog. Lipid Res.39:19–39

88. Titball RW. 1998. Bacterial phospholi-pases.J. Appl. Microbiol. Symp. Suppl.84:127S–37S

89. Tripathy S, Venables BJ, Chapman KD.1999. N-Acylethanolamines in signal

transduction of elicitor perception. At-tenuation of alkalinization response andactivation of defense gene expression.Plant Physiol.121:1299–308

90. Van Himbergen JAJ, ter Riet B, Mei-jer HJG, van den Ende H, Musgrave A,Munnik T. 1999. Mastoparan analoguesstimulate phospholipase C and phospho-lipase D-activity in Chlamydomonas: acomparative study.J. Exp. Bot.50:1735–42

91. Waite KA, Wallin R, Qualliotine-MannD, McPhail LC. 1997. Phosphatidic acid-mediated phosphorylation of the NADPHoxidase component p47-phox. Evidencethat phosphatidic acid may activate a novelprotein kinase.J. Biol. Chem.272:15569–78

92. Wang A, Dennis EA. 1999. Mamma-lian lysophospholipases.Biochim. Bio-phys. Acta1439:1–16

93. Wang C, Zien C, Afitlhile M, WeltiR, Hildebrand DF, Wang X. 2000. In-volvement of phospholipase D in wound-induced accumulation of jasmonic acid inArabidopsis. Plant Cell12:

94. Wang X. 1999. The role of phospholi-pase D in signaling cascade.Plant Physiol.120:645–51

95. Wang X. 2000. Multiple forms of phospho-lipase D in plants: the gene family, cat-alytic and regulatory properties, and cellu-lar functions.Prog. Lipid Res.39:109–49

96. Wang X, Wang C, Sang Y, Zheng L, QinC. 2000. Determining functions of multi-ple phospholipase Ds in stress response ofArabidopsis.Biochem. Soc. Trans.28:000–00

97. Wang X, Xu L, Zheng L. 1994. Cloningand expression of phosphatidylcholine-hydrolyzing phospholipase D fromRicinuscommunisL. J. Biol. Chem.269:20312–17

98. Williams RL. 1999. Mammalian phospho-inositide-specific phospholipase C.Bio-chim. Biophys. Acta1441:255–67

99. Wissing JB, Grabo P, Kornak B. 1996.

P1: FXY



Purification and characterization of multi-ple forms of phosphatidylinositol-specificphospholipases D from suspension cul-tured Catharanthus roseuscells. PlantSci.117:17–31

100. Xie Z, Fang M, Rivas MP, Faulkner AJ,Sternweis PC, et al. 1998. PhospholipaseD activity is required for suppression ofyeast phosphatidylinositol transfer pro-tein defects.Proc. Natl. Acad. Sci. USA95:12346–51

101. Xie Z, Ho WT, Exton JH. 2000. Associ-ation of the N- and C-terminal domainsof phospholipase D: contribution of theconserved HKD motifs to the interactionand the requirement of the association for

Ser/Thr phosphorylation of the enzyme.J. Biol. Chem.275:24962–69

102. Xu L, Zheng S, Zheng L, Wang X. 1997.Promoter analysis and expression of aphospholipase D gene from castor bean.Plant Physiol.115:387–95

103. Young S, Wang X, Leach JE. 1996.Changes in the plasma membrane dis-tribution of rice phospholipase D duringresistant interactions withXanthomonasoryzaepv oryzae. Plant Cell8:1079–90

104. Zheng L, Krishnamoorthi R, ZolkiewskiM, Wang X. 2000. Distinct Ca2+ bindingproperties of novel C2 domains of plantphospholipase Dα andβ. J. Biol. Chem.275:19700–6

P1: FQP

April 18, 2001 12:33 Annual Reviews AR129-08-COLOR

Figure 2 A model depicting networking of phospholipases and lipid mediators in plantcells. PLC produces DAG and InP3 that increases the level of cytoplasmic Ca2+ (81).Ca2+ and PtdInP2 both stimulate the activity of PLD (95, 104). The activation of PLDgenerates PtdOH, which enhances the activities of PLC, PLA, and PI5-kinase, based onmammalian studies (42). The role of PLD on PLA activity and oxylipin synthesis is indi-cated also by the effect of PLD-suppression on wound-induced accumulation of jasmonicacid (93). LysoPtdEtn, a potential PLA product, inhibits PLD activity (71). Kinases andphosphatases involved in the interconversion of DAG, PtdOH, and DAG-PPi have beencharacterized in plants (49).⊕ denotes stimulation, and — denotes inhibition. DAG-PPi,DAG-pyrophosphate; InP3, inositol 1,4,5-trisphosphate; lysoPL, lysophospholipids; PI5-kinase, phosphatidylinositol 4-phosophate 5-kinase; PL, phospholipid; PPtase, phospho-lipid phosphatase; PUFAs, polyunsaturated fatty acids; X-OH, free head group.

plant phospholipasesarquivo.ufv.br/.../phospholipasesreview.pdf · abstract phospholipases are a...

Documents