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Summary Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.

Natural rubber is usually synthesized in the rubber particles present in the latex of rubber-producing plants such as the Par&aacute, rubber tree (Hevea brasiliensis) and rubber dandelion (Taraxacum kok-saghyz). Since the detailed lipid compositions of fresh latex and rubber particles of the plants are poorly known, the present study reports detailed compound lipid composition, focusing on phospholipids and galactolipids in the latex and rubber particles of the plants. In the fresh latex and rubber particles of both plants, phospholipids were much more dominant (85&ndash, 99%) compared to galactolipids. Among the nine classes of phospholipids, phosphatidylcholines (PCs) were most abundant, at ~80%, in both plants. Among PCs, PC (36:4) and PC (34:2) were most abundant in the rubber tree and rubber dandelion, respectively. Two classes of galactolipids, monogalactosyl diacylglycerol and digalactosyl diacylglycerol, were detected as 12% and 1%, respectively, of total compound lipids in rubber tree, whereas their percentages in the rubber dandelion were negligible (&lt, 1%). Overall, the compound lipid composition differed only slightly between the fresh latex and the rubber particles of both rubber plants. These results provide fundamental data on the lipid composition of rubber particles in two rubber-producing plants, which can serve as a basis for artificial rubber particle production in the future.

Persimmon (Diospyros kaki Thunb.), which is a climacteric fruit, softens in 3–5 weeks after harvest. However, little is known regarding the transcriptional changes that underlie persimmon ripening. In this study, high-throughput de novo RNA sequencing was performed to examine differential expression between freshly harvested (FH) and softened (ST) persimmon fruit peels. Using the Illumina HiSeq platform, we obtained 259,483,704 high quality reads and 94,856 transcripts. After the removal of redundant sequences, a total of 31,258 unigenes were predicted, 1,790 of which were differentially expressed between FH and ST persimmon (1,284 up-regulated and 506 down-regulated in ST compared with FH). The differentially expressed genes (DEGs) were further subjected to KEGG pathway analysis. Several pathways were found to be up-regulated in ST persimmon, including “amino sugar and nucleotide sugar metabolism.” Pathways down-regulated in ST persimmon included “photosynthesis” and “carbon fixation in photosynthetic organisms.” Expression patterns of genes in these pathways were further confirmed using quantitative real-time RT-PCR. Ethylene gas production during persimmon softening was monitored with gas chromatography and found to be correlated with the fruit softening. Transcription involved in ethylene biosynthesis, perception and signaling was up-regulated. On the whole, this study investigated the key genes involved in metabolic pathways of persimmon fruit softening, especially implicated in increased sugar metabolism, decreased photosynthetic capability, and increased ethylene production and other ethylene-related functions. This transcriptome analysis provides baseline information on the identity and modulation of genes involved in softening of persimmon fruits and can underpin the future development of technologies to delay softening in persimmon.

Protein transport between organelles is an essential process in all eukaryotic cells and is mediated by the regulation of processes such as vesicle formation, transport, docking, and fusion. In animals, SCY1-LIKE2 (SCYL2) binds to clathrin and has been shown to play roles in trans-Golgi network-mediated clathrin-coated vesicle trafficking. Here, we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis (Arabidopsis thaliana) homologs of animal SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 isoforms using multiple single or double loss-of-function alleles show that SCYL2B is involved in root hair development and that SCYL2A and SCYL2B are essential for plant growth and development and act redundantly in those processes. Quantitative reverse transcription-polymerase chain reaction and a β-glucuronidase-aided promoter assay show that SCYL2A and SCYL2B are differentially expressed in various tissues. We also show that SCYL2 proteins localize to the Golgi, trans-Golgi network, and prevacuolar compartment and colocalize with Clathrin Heavy Chain1 (CHC1). Furthermore, bimolecular fluorescence complementation and coimmunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble NSF Attachment Protein Receptors (SNAREs): Vesicle Transport through t-SNARE Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip localization of Cellulose Synthase-Like D3 is dependent on SCYL2B. These findings suggest the role of SCYL2 genes in plant cell developmental processes via clathrin-mediated vesicle membrane trafficking.

The phospholipase A2 (PLA2) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA2s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA2s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA2 paralogs (PLA2-β, -γ, and -Δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA2 using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA2 inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA2s that are conserved in other organisms.

Phospholipase A2 (PLA2), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA2s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA2α, a PLA2 isoform, colocalized with the Golgi marker. Impairments of PLA2 function by PLA2α mutation, PLA2-RNA interference (RNAi), or PLA2 inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA2 hydrolytic product) restored the PM localization of PINs in the pla2α mutant and the ONO-RS-082–treated seedling. Suppression of PLA2 activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA2 inhibitor treatments, PLA2α mutation, or PLA2-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA2, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.

Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.

Summary Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes, including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE ( DGL ), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 ( DAD1 ), encodes a chloroplast-targeted lipase with strong galactolipase and weak phospholipase A 1 activity. DGL is expressed in the leaves and has a specific role in maintaining basal JA content under normal conditions, and this expression regulates vegetative growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant functions for JA production, but they show different induction kinetics, indicating temporally separated roles: DGL plays a role in the early phase of JA production, and DAD1 plays a role in the late phase of JA production. Whereas DGL and DAD1 are necessary and sufficient for JA production, phospholipase D appears to modulate wound response by stimulating DGL and DAD1 expression.

Plant secretory phospholipases A(2) (sPLA(2)s) probably play important roles in phospholipid signaling based on the data reported from other organisms, but their functions are poorly understood because of the lack of cloned sPLA(2) genes. In this study, we cloned and characterized an Arabidopsis secretory phospholipase A(2)-gamma (AtsPLA(2)-gamma) cDNA, and examined its enzymatic properties. The recombinant protein of AtsPLA(2)-gamma showed maximal enzyme activity at pH 8.0, and required Ca(2+) for activity. Moreover, AtsPLA(2)-gamma showed sn-2 position specificity but no prominent acyl preference, though it showed head group specificity to phosphatidylethanolamine rather than to phosphatidylcholine. AtsPLA(2)-gamma was found to predominate in the mature flower rather than in other tissues, and subcellular localization analysis confirmed that AtsPLA(2)-gamma is secreted into the intercellular space.

cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Despite potential significance ofcis-prenyltransferase in plant growth and development, no gene of the enzyme has been cloned from higher plants. Using sequence information of the conserved region ofcis-prenyltransferase cloned recently fromEscherichia coli, Micrococcus luteus, and yeast, we have isolated and characterized the first plantcis-prenyltransferase from Arabidopsis thaliana. Sequence analysis revealed that the protein is highly homologous in several conserved regions tocis-prenyltransferases from M. luteus, E. coli, and yeast. In vitro analyses using the recombinant protein overexpressed in E. coli revealed that the enzyme catalyzed the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130 with a predominance of C120. The enzyme exhibited a higher affinity for farnesyl diphosphate than for geranylgeranyl diphosphate, with theK m values being 0.13 and 3.62 μm, respectively, but a lower affinity for isopentenyl diphosphate, with aK m value of 23 μm. In vitro rubber biosynthesis analysis indicated that theArabidopsis cis-prenyltransferase itself could not catalyze the formation of higher molecular weight polyprenyl diphosphates similar to natural rubber. A reverse transcriptase-polymerase chain reaction analysis showed that the gene was expressed at low levels inArabidopsis plant, in which expression of thecis-prenyltransferase in leaf and root was higher than that in stem, flower, and silique. These results indicate the tissue-specific expression of cis-prenyltransferase and suggest a potential role and significance of the enzyme in the polyisoprenoid biosynthesis in plants.

Although the importance of phospholipase D (PLD) in signal transduction in mammalian cells is well documented, the negative regulation of PLD is poorly un- derstood. This is primarily due to a lack of known specific inhibitors of PLD. We herein report that the activity of par- tially purified rat brain PLD is inhibited by certain lysophos- pholipids, such as lysophosphatidylinositol, lysophosphati- dylglycerol, and lysophosphatidylserine in a highly specific manner. Inhibition of PLD by lysophospholipids was dose- dependent: the concentration of lysophosphatidylinositol required for half-maximal inhibition was about 3 m m . An analysis of the enzyme-kinetics suggested that lysophospho- lipids act as non-competitive inhibitors of PLD activity. As expected, PLD activity was stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The inhibition of PLD by lysophospholipids, however, was not affected by the presence or absence of Arf or by an in- crease in PIP 2 concentration. A protein-binding assay sug- gested that lysophospholipids bind directly to PLD. These results indicate that the observed inhibition of PLD by lysophospholipids is due to their direct interaction rather than to an interaction between lysophospholipids and either Arf or PIP 2 . The present study suggests that certain lyso- phospholipids are specific inhibitors of rat brain PLD in a cell-free system and may provide the new opportunities to investigate mechanisms by which PLD is regulated by lyso- phospholipids, presumably liberated by phospholipase A 2 activation, in mammalian cells. —Ryu, S. B. and J. P. Palta. Specific inhibition of rat brain phospholipase D by lyso- phospholipids. J. Lipid Res. 2000. 41: 940-944.

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Summary Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

Activation of phospholipase D (PLD) has been linked to accumulation of nonhydratable phosphatides and lipid degradation leading to soybean seed deterioration during preharvest and postharvest events. This study examined the changes in PLD activity, protein, and mRNA in soybeans during seed development and germination. RNA blotting analysis indicated that expression of the gene that encodes PLD was highest during the early and middle stages of seed development. However, the amount of PLD activity accumulated per cotyledon reached the highest level in mature seeds. During germination and early seedling growth, PLD mRNA was not detected one day after imbibition, while a significant increase in PLD expression occurred in the cotyledons of three- and seven-day seedlings. Similarly, PLD activity and protein concentration showed little change during the first day of imbibition and increased afterward in three- and seven-day seedlings. These results suggested that expression of PLD is developmentally regulated and that the changes in its amount of activity and protein are controlled primarily at the mRNA level. Immunoblotting analysis further revealed the presence of PLD variants that were associated with specific stages of seed development and seedling growth. The PLD variants present in the cotyledons of mature seeds appeared to be distinct from those observed in the early stage of seed development and in young seedlings.

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.

Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase. Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified. A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis. Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H. brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP. A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized. Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity). In vitro functional analysis of the recombinant protein overexpressed in E. coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does. These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees.

To elucidate the cellular functions of phospholipase A(2) in plants, an Arabidopsis cDNA encoding a secretory low molecular weight phospholipase A(2) (AtsPLA(2)beta) was isolated. Phenotype analyses of transgenic plants showed that overexpression of AtsPLA(2)beta promotes cell elongation, resulting in prolonged leaf petioles and inflorescence stems, whereas RNA interference-mediated silencing of AtsPLA(2)beta expression retards cell elongation, resulting in shortened leaf petioles and stems. AtsPLA(2)beta is expressed in the cortical, vascular, and endodermal cells of the actively growing tissues of inflorescence stems and hypocotyls. AtsPLA(2)beta then is secreted into the extracellular spaces, where signaling for cell wall acidification is thought to occur. AtsPLA(2)beta-overexpressing or -silenced transgenic plants showed altered gravitropism in inflorescence stems and hypocotyls. AtsPLA(2)beta expression is induced rapidly by auxin treatment and in the curving regions of inflorescence stems undergoing the gravitropic response. These results suggest that AtsPLA(2)beta regulates the process of cell elongation and plays important roles in shoot gravitropism by mediating auxin-induced cell elongation.

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.

Ethephon [2-(chloroethyl) phoshonic acid] is used widely to maximize the yield of ripe tomato fruits. However, ethephon causes rapid and extensive defoliation, overripening, and promotes sunscald damage to the fruit. Recent studies from our laboratory have provided evidence that lysophosphatidylethanolamine (LPE) can reduce leaf senescence. We investigated the potential use of LPE to reduce damaging effect of ethephon on tomato foliage and influence on the activity of phospholipase D (PLD). Disruption of membrane integrity has been suggested as a primary cause of senescence in plants. PLD is known to be a key enzyme, which initiates the selective degradation of membrane phospholipids in senescing tissues. Two-month-old tomato plants (`Mountain Spring') grown in greenhouse condition were sprayed with water, 200 ppm LPE, and 1000 ppm ethephon. In addition, LPE spray prior to ethephon or mixture with ethephon were also tested. Leaves were sampled after 0, 2, 5, 24, 72, and 168 h of spray application, for PLD activity measurements. Spray of LPE prior to ethephon spray or inclusion of LPE in the ethephon spray reduced foliar injury by ethephon. Activity of soluble PLD was increased dramatically in leaves sprayed with ethephon initially and than dropped by 7 days. We also found that LPE-treated leaves had lower PLD activity than the ethephon-treated leaves. Plants treated with LPE-ethephon mixture also showed significantly lower PLD activity. These results suggest that LPE treatments mitigate ethephon injury to tomato plants. Furthermore, it appears that this mitigation involves modulation of the activity of PLD.

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electrophoresis, isoelectric focusing, and size-exclusion chromatography. PLD was detected with both a PLD activity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1, 2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism is discussed.