Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLDbeta, from Arabidopsis.

A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and nanomolar concentrations of calcium, has been identified in Arabidopsis This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLDβ for this PIP2-dependent PLD and PLDα for the previously characterized PIP2-independent PLD that requires millimolar Ca2+ for optimal activity. The PLDβ cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLDβ cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLDβ, but not PLDα, activity. The activity of the expressed PLDβ is dependent on PIP2 and submicromolar amounts of Ca2+, inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLDβ is evolutionarily divergent from PLDα and that its N terminus contains a regulatory Ca2+-dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.

Phospholipase D (PLD; EC 3.1.4.4) 1 -catalyzed hydrolysis of glycerophospholipids produces phosphatidic acid (PA) and a hydrophilic constituent. This activity was first identified in plants and since has been found in animals and microorganisms. PLD in plants was originally proposed to be important in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress injuries (1,2). Recent studies in plants, animals, and yeast indicate that PLD hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation (3)(4)(5)(6)(7)(8)(9). Activation of PLD has been proposed to mediate many cellular processes including cell proliferation, membrane trafficking, meiosis, and responses to external and internal stimuli. It has been suggested that multiple forms of PLD are involved in these diverse cellular processes since several studies have shown the presence of PLD variants that are expressed differently (9 -12). In castor bean (9) and rice (12), one PLD variant is constitutive whereas the appearance of other variants is associated with specific conditions such as rapid growth, wounding, and senescence. A distinct property shared by these variants is their in vitro requirement of millimolar Ca 2ϩ concentrations for optimal activity. Further analyses of the castor bean PLD variants have led to the suggestion that the catalytic activity of these variants results from the same gene product (9 -11).
A recent study has provided important evidence for the presence of two plant PLDs that are derived from different gene products and regulated distinctly (13). One PLD requires polyphosphoinositides and submicromolar concentrations of Ca 2ϩ for activity and the other is PIP 2 -independent and is most active in the presence of millimolar amounts of Ca 2ϩ . The latter is the prevalent form of PLD that has been purified and characterized from a number of plant species (14). Its cDNA has been recently cloned from castor bean (15), Arabidopsis (16), rice and maize (17). We have genetically suppressed the expression of this prevalent plant PLD by introducing a PLD antisense gene into Arabidopsis (13). While they showed less than 3% of the PIP 2 -independent, millimolar Ca 2ϩ -requiring PLD activity of wild-type Arabidopsis, the transgenic plants had PIP 2 -dependent PLD activity comparable to that of wild type at submicromolar calcium. In the present study, we provide molecular evidence for the presence of two distinct PLDs by isolating a new PLD cDNA encoding the PIP 2 -dependent PLD. Furthermore, analysis of the sequence and expressed protein from the PLD cDNA gives further insights to the activation and function of PLD␤ in plants. Because the regulatory and structural features of the newly identified PLD are distinct from those of the conventional PLD, we have given names of PLD␤ for this PIP 2 -dependent PLD and PLD␣ for the previously characterized PIP 2 -independent PLD that requires millimolar Ca 2ϩ for optimal activity.
PLD cDNA Cloning and Sequencing-Putative expression sequencetagged Arabidopsis PLD cDNAs were identified by searching the BLAST data base against the castor bean PLD cDNA sequence. These clones were kindly provided by the Ohio State University Arabidopsis Information Center. Strategies for isolating full-length PLD cDNAs are described in the text. The reactions for PCR amplification used DNA purified from an Arabidopsis PRL2 cDNA library (18) as DNA template, T7 sequence primer as the 5Ј primer, and 28 nucleotide bases corresponding to the near 5Ј end of the EST cDNA sequence as the 3Ј primer. The reaction mixture consisted of 50 pmol of each primer, 0.5 g of template DNA, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton, 2.5 mM MgCl 2 , 0.2 mM of each dNTP, 1 unit of Taq DNA polymerase in a 100-l volume. The thermal cycling was performed after an initial denaturing cycle of 5 min at 95°C. Then 25 to 30 cycles were completed using the following temperature profiles: denaturation at 95°C for 1 min, annealing for 30 s at 2-5°C lower than the calculated primer T m , and extension for 1 min at 72°C. PCR products were cloned into the pGEM-T vector (Promega) according to the manufacturer's instructions.
RACE for 5Ј-cDNA ends was performed according to the manufacturer's instructions (Life Technologies, Inc.). The first strand cDNA was synthesized from total RNA isolated from Arabidopsis flowers. After PCR amplification using nested gene-specific primers at the 3Ј end and a 5Ј-RACE anchor primer, the DNA products were cut with KpnI and PstI and were ligated into pBluescript (SK). The KpnI site was engineered into the 5Ј primer, and the PstI was an internal site of the PLD cDNA near the 3Ј end of the RACE product. To isolate full-length PLD cDNAs a ZapII cDNA library, constructed from 3 to 6 kb mRNA isolated from hypocotyls of 3-day-old Arabidopsis seedlings (19), was screened using the 5Ј cDNA fragment generated by the 5Ј-RACE procedure. The hybridization was conducted at 65°C, and the subsequent DNA manipulation of the positive clones was based on the previously described procedures (15).
To sequence PLD clones, cDNA inserts from positive clones were digested with various restriction enzymes and the fragments were subcloned into the pBluescript plasmids, SK and/or KS. The complete DNA sequence was determined by using the Sequenase 2 kit according to the manufacturer's instructions (U. S. Biochemical Corp.). Vector pBluescript-based primers, universal forward and reverse, T3, T7, SK, and KS primers were used in most sequencing reactions. PLD cDNA-based primers were also synthesized for clarifying ambiguities. The final sequence was determined from both strands. Phylogenic analyses, pI calculations, and comparison of PLD nucleic acid and amino acid sequences were done with the Genetics Computer Group software (University of Wisconsin).
Expression of PLD cDNA in Escherichia coli-Expression of the PLD␤ cDNA was performed using pBluescript SK(Ϫ) containing the cDNA insert in E. coli. The recombinant plasmid was transformed into E. coli JM 109. Fifty microliters of an overnight culture of the transformed E. coli were added to 25 ml of LB medium with 50 g/ml ampicillin. The cells were incubated at 37°C with shaking for 3 h, and then IPTG was added to a final concentration of 2 mM. After growing overnight at 30°C, the induced cells were pelleted by centrifugation and then resuspended in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.25 mM phenylmethylsulfonyl fluoride, 2 mM EDTA and then pelleted by centrifugation. The cells were lysed by sonication in the resuspension buffer and cell debris was removed by centrifugation at 10,000 ϫ g for 5 min. Proteins in the supernatant were assayed for PLD activity and also subjected to SDS-PAGE followed by immunoblot analysis using anti-PLD antibodies.
Generation of PLD Antibodies and Immunoblotting-A 13-amino acid peptide was synthesized that consisted of a cysteine and 12 amino acids corresponding to the C terminus of PLD␤. The cysteine was added to the N terminus of the peptide to provide a sulfhyryl group that was used for conjugation of the peptide to keyhole limpet hemocyanin by m-maleimidobenzoyl N-hydroxysuccinimide ester (20). The conjugated protein was used as an immunogen to raise antibodies in rabbits. For immunoblot analysis, proteins were separated by 8% SDS-PAGE gels, transferred onto a polyvinylidene difluoride membrane, and incubated with antiserum that contained JM109 lysate to remove nonspecific reactive bacterial proteins. The immunoblot analysis was performed as described (21). PIP 2 -dependent PLD Activity Assay-PLD activity was assayed by using either 1-palmitoyl-2-oleoyl-[oleoyl-1-14 C]glycero-3-P-choline or dipalmitoylglycerol-3-P-[methyl-3 H]choline as substrates. The acyl-labeled PC was used for assaying transphosphatidylation activity whereas the choline-labeled PC was used in all other studies. In both cases, 2.5 Ci of radiolabeled PC was mixed with 3.5 mol of PE, 0.3 mol of PIP 2 , and 0.2 mol of unlabeled PC in chloroform, and the solvent was evaporated under a stream of N 2 . In the phospholipidspecificity experiments, PIP 2 was replaced with 0.3 mol of PE, PA, PG, PS, PI, or PIP. The phospholipid substrate was dispersed in 1 ml of H 2 O by sonication at room temperature. Previously reported conditions were adapted to yield an enzyme assay mixture that contained 100 mM MES (pH 7.0), 5 M CaCl 2 , 2 mM MgCl 2 , 80 mM KCl, 0.4 mM lipid vesicles, and 5-15 g of expressed protein in a total volume of 100 l (22). In the Ca 2ϩ dependence experiments, the concentrations of free Ca 2ϩ and Mg 2ϩ in the reaction mixture were determined using Ca 2ϩ /Mg 2ϩ -EGTA buffers at pH 7.5 as described (23). The reaction was initiated by addition of substrate and incubated at 30°C for 30 min in a shaking water bath. When choline-labeled PC was used, the reaction was stopped by addition of 1 ml of 2:1 (v/v) chloroform:methanol and 100 l of 2 M KCl. After vortexing and centrifugation at 12,000 ϫ g for 5 min, a 200-l aliquot of the aqueous phase was mixed with 3 ml of scintillation fluid, and the release of [ 3 H]choline was measured by scintillation counting.
When acyl-labeled PC was used, the reaction mixture included ethanol to a final concentration of 0.5% (v/v) for assaying the transphosphatidylation activity of PLD. The reaction was stopped by adding 375 l of 1:2 (v/v) chloroform:methanol. Additionally, 100 l of chloroform and 100 l of 2 M KCl were added and the sample was vortexed. The chloroform and aqueous phases were separated by centrifugation at 12,000 ϫ g for 5 min. The aqueous phase was removed and the chloroform phase was dried. Thin layer chromatography was conducted as described previously using 65:35:5 chloroform:methanol:NH 4 OH as the developing solvent (21). Lipids separated on plates were visualized by exposure to iodine vapor. Spots corresponding to lipid standards, PA, PC, and phosphatidylethanol, were scraped and radioactivity was measured by scintillation counting.
High Ca 2ϩ -dependent PLD Activity Assay-This assay reaction mixture contained 100 mM MES (pH 6.5), 25 mM CaCl 2 , 0.5 mM SDS, 1% (v/v) ethanol, 5-15 g of protein, and 2 mM PC (egg yolk) containing dipalmitoylglycero-3-P-[methyl-3 H]choline. The substrate preparation, reaction conditions, and product separation were based on previously described procedures (21) with the following changes: the assay volume was reduced to 100 l and 100 l of 2 M KCl was added to the 2:1 (v/v) chloroform:methanol extraction. The release of [ 3 H]choline into the aqueous phase was quantitated by scintillation counting.
Arabidopsis Cytosolic and Membrane Fractionation; Heat and Protease Treatments-Leaves from PLD␣ antisense-suppressed Arabidopsis (4 weeks old) were homogenized in a buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM KCl, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol. The cytosolic protein fraction was the supernatant obtained after centrifugation of the homogenate at 100,000 ϫ g for 1 h. The pellet was extracted with 0.44 M KCl in the homogenization buffer to obtain the salt-solubilized membrane proteins (13). In typical PLD assays, the cytosol containing 3 g of protein was added to 10 g of the bacterially expressed PLD␤. In the heat denaturation treatment, the soluble fraction was boiled for 5 min followed by centrifugation for 5 min at 12,000 ϫ g to remove precipitates. The clarified fraction was used directly or treated with different proteases, thermolysin, trypsin, or proteinase K, to digest proteins. After incubating at 37°C for 30 min, trypsin and proteinase K were inactivated by adding phenylmethylsulfonyl fluoride, and thermolysin was inactivated by adding 2 mM EDTA.
Southern and Northern Blotting-Total RNA and genomic DNA were isolated from Arabidopsis tissues (15). Full-length cDNAs of PLD␣ and PLD␤ were used as probes to hybridize the genomic DNA digested with various restriction enzymes at 65°C under the previously described conditions (15). Total RNA was separated by denaturing formaldehydeagarose gel electrophoresis, transferred onto a nylon membrane, and hybridized with a full-length PLD␤ cDNA at 65°C (15).

RESULTS
Cloning and Sequences of PLD cDNAs-Arabidopsis expression sequence-tagged cDNA clones were identified as putative PLD cDNAs by searching the BLAST data base using the castor bean PLD cDNA sequence. These clones were about 1 kb in length and incomplete PLD cDNAs. The first complete Arabidopsis PLD cDNA was cloned by PCR using nested primers and encodes a protein of 809 amino acids (16). This cDNA has been shown to be PLD␣, because introduction of this cDNA as an antisense gene almost completely abolishes the PLD␣ activity of transgenic plants. The antisense plants lost the millimolar Ca 2ϩ -responsive PLD activity, but showed PIP 2 -dependent PLD activity comparable to that of wild-type plants at nanomolar Ca 2ϩ concentrations (13). In addition, the amino acid sequence of the Arabidopsis PLD cDNA shares a high level of identity (about 80%) with the previously cloned castor bean PLD cDNA whose product displays PIP 2 -independent activity.
The cloning of PLD␤ cDNA proved to be much more difficult than that of PLD␣. The cloning strategies involved nested PCR, 5Ј-RACE, and screening of cDNA libraries. A 1.5-kb 5Ј-fragment of the cDNA was first cloned by PCR amplification using an Arabidopsis PRL2 cDNA library (18) as a DNA template, an internal fragment of the EST cDNA as the 3Ј primer, and T7 sequence primer as the 5Ј primer. Further attempts at using nested PCR were not fruitful, so 5Ј-RACE was performed to generate the missing 5Ј end. The full-length cDNA was finally isolated by using the 5Ј-fragments to screen a ZapII Arabidopsis cDNA library constructed using 3-6 kb mRNA isolated from hypocotyls of 3-day-old Arabidopsis seedlings (19). The newly cloned PLD cDNA consists of 3309 nucleotides with an open reading frame from 178 to 3081. The deduced amino acid sequence of this protein is about 40% identical and 60% similar to that of Arabidopsis PLD␣ (Fig. 1) as well as to PLDs cloned from castor bean, maize, and rice (15)(16)(17). The sequence identity of this new cDNA is in contrast to previously cloned PLD cDNAs from Arabidopsis, castor bean, maize, and rice, which share about 75-90% amino acid sequence identity. All of these previously cloned PLDs display PIP 2 independent activity at millimolar Ca 2ϩ concentrations and thus belong to the class of PLD␣. Additionally, the newly cloned PLD differs from PLD␣ in size and pI. PLD␣s from different plant species are very similar in size, ranging from 92 to 96 kDa, whereas the predicted polypeptide from this cDNA has a calculated molecular mass of 109 kDa. This PLD protein consists of 968 amino acids, which is 148 amino acids longer at the N terminus than that of Arabidopsis PLD␣ (Fig. 1). The newly cloned PLD has a calculated pI of 7.9 whereas all previously cloned plant PLDs display acidic pI values (15)(16)(17). Prior to this report, only PLDs from non-plant sources had been reported to have basic pI values (4,24). This newly cloned cDNA contains a duplicated HXKXXXXD motif (amino acids 484 -491 and 818 -826, Fig. 1) that has been conserved in all PLDs and is proposed to be involved in catalysis (24,25). These results indicate that the newly cloned PLD is a distinct isoform.
Establishing That the cDNA Encodes a New PIP 2 -dependent PLD␤-To establish unequivocally that the newly cloned cDNA encodes a PLD, protein from this cDNA was expressed in E. coli using pBluescript SK(Ϫ) as expression vector. After IPTG induction, the production of a protein encoded by the cDNA was detected by immunoblotting using antibodies raised against a synthetic peptide corresponding to the 12 C-terminal amino acids of this protein (Fig. 2). No immunoreactive proteins were detected in the protein extracts from E. coli containing vector alone, and a trace amount of PLD was expressed without IPTG induction in the SK construct.
The expressed protein was assayed for both PIP 2 -dependent and PIP 2 -independent PLD activity. There were only trace levels of PLD activities in protein extracts from E. coli JM109 harboring the SK alone or the vector containing the cDNA insert without IPTG induction. A significant increase in PIP 2dependent PLD activity was observed after IPTG induction (Fig. 2). The levels of PLD activity were in agreement with the presence or absence of PLD protein detected by immunoblotting. On the other hand, the expressed PLD showed no PIP 2independent, conventional PLD activity.
The transphosphatidylation activity of PLD␤ was examined in order to verify that the activity monitored in the bacterial extracts was due to the action of PLD. IPTG-induced samples assayed in the presence of 0.5% ethanol showed a 13-fold increase in phosphatidylethanol production compared to uninduced samples (data not shown). Similarly, phosphatidic acid production increased 11-fold in IPTG-induced samples relative to the uninduced samples, showing that the expressed protein is indeed a member of the PLD family.
Stimulation of PLD by a Cytosolic Factor-Recent studies have shown that some mammalian PLDs are stimulated by cytosolic factors (22,24). To examine whether PLD␤ could be activated by plant soluble factors, the expressed PLD␤ was assayed in the presence or absence of a soluble fraction from Arabidopsis. The soluble extract was obtained from transgenic Arabidopsis plants in which the expression of PLD␣ was antisense suppressed. These plants were used because the 100,000 ϫ g supernatant contained virtually no detectable PIP 2 -dependent nor PIP 2 -independent PLD activity (13). The soluble extract alone showed little PLD␤ activity (Fig. 3A), but its inclusion increased the PIP 2 dependent activity of the expressed protein and this enhancement was dependent upon the concentration of cytosol added (Fig. 3B). These results suggest that the PIP 2 -dependent PLD expressed in E. coli is stimulated by a soluble factor. In comparison, the cytosolic fraction had no stimulatory effect on PLD␣ expressed from its cDNA in E. coli (data not shown). Similarly, the PIP 2 -dependent PLD extracted from the PLD␣ antisense membranes was insensitive to the addition of cytosol (Fig. 3B). It is possible that the cytosolic stimulator remains bound with the membrane-associated PLD from the antisense plants.
The cytosolic factor was examined by various means to determine its nature. The cloned human PLD is stimulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family (24). We tested the ability of recombinant human ARF1 to substitute for the cytosolic factor from Arabidopsis. The amino acid sequences of human ARF1 and Arabidopsis ARF are 88% identical and 95% similar (26). While it stimulated the expressed human PLD greatly, the recombinant human ARF1 had no effect on the expressed plant PLD␤ (data not shown). The inclusion of GTP␥S or GDP␤S, which are nonhydrolyzable analogues of GTP and GDP, respectively, in the activity assays neither stimulated nor inhibited the level of PIP 2 -dependent PLD activity (Fig. 3A). To examine further the nature of the cytosolic stimulatory factor, proteins in the soluble fraction were heat-denatured. The stimulation of the expressed PLD by the cytosol remained unchanged after the soluble fraction was boiled for 5 min (Fig. 3A). Furthermore, treatment of the heat-denatured cytosol with the proteases thermolysin, trypsin, and proteinase K also did not inactivate the stimulatory effect of the cytosol (data not shown). However, when the cytosol was passed over a gel filtration column with a molecular mass cut-off of 6,000 Da, it lost its ability to stimulate PLD␤ activity (Fig. 3A). Taken together, these results suggest that the stimulating factor is a heat-stable small molecule. The exact nature of the cytosolic factor remains to be determined.
PLD␤ Dependence on Polyphosphoinositides, Ca 2ϩ , and pH-The requirement of specific phospholipids for PLD␤ activity was examined by substituting PIP 2 in the lipid substrate vesicles with PIP, PI, PG, PS, PE, or PA. PIP also was capable of stimulating PLD␤ activity but its stimulation was only a third of the level observed in the presence of PIP 2 (Fig. 4A). The maximal stimulation of PLD␤ activity by PIP 2 was achieved when PIP 2 was included in the substrate vesicles at an amount of 7.6 mol % (Fig. 4B). PIP 2 levels much higher or lower than this resulted in a significant decrease in activity. Further evidence for the requirement of PIP 2 for this PLD was obtained by studying the influence of neomycin on its activity. Neomycin is a high affinity cationic ligand for polyphosphoinositides and has been used to demonstrate the activity dependence of various enzymes by it ability to sequester PIP 2 (27,28). PLD␤ was inhibited by neomycin in a concentration-dependent manner (Fig. 4C). PLD␤ activity was inhibited by greater than 50% at 500 M neomycin and was nearly abolished at 2 mM neomycin.
To determine the influence of Ca 2ϩ on PIP 2 -dependent PLD activity, free Ca 2ϩ and Mg 2ϩ in the reaction mixture were controlled using Ca 2ϩ /Mg 2ϩ -EGTA buffers at pH 7.5 (23). PIP 2 dependent activity was undetectable in the absence of Ca 2ϩ , with little activity observed at or below a concentration of 50 nM (Fig. 4D). At 500 nM calcium, PLD␤ activity increased to a maximum and gradually tapered off as millimolar levels of calcium were approached. Under the optimal PIP 2 and Ca 2ϩ conditions, the expressed PIP 2 -dependent PLD showed the highest activity between pH 7.0 and 7.5.
Presence of a Ca 2ϩ /Phospholipid-binding (C2) Domain in PLD-PLD␣ from castor bean was reported to contain a C2 domain at its N terminus (29). The C2 domain is a Ca 2ϩ / phospholipid-binding domain present in a number of different proteins involved in signal transduction and membrane trafficking (29,30). The three-dimensional structure of a C2 domain from the neuronal protein synaptotagmin has been resolved recently by x-ray crystallography and NMR (30, 31). The crystal structure of a phosphoinositide-specific phospholipase C, which also contains a C2 domain, has been reported recently (32). The C2 domains of synaptotagmin and PLC are comprised of an eight-strand sandwich containing 4 -5 acidic residues involved in Ca 2ϩ binding. While the eight strands are conserved, the PLD␣s from castor bean and Arabidopsis possess substitutions within the C2 Ca 2ϩ -binding site, indicating a potential loss of Ca 2ϩ affinity (29) (Fig. 5).
An approach similar to that described previously (29) was used to align the sequence of PLD␤ with those of synaptotagmin and PLD␣. The PIP 2 -dependent PLD␤ contains a C2 domain near its N terminus stretching from amino acid 158 to 279 (Fig. 5). The two most highly conserved segments in different C2-containing proteins are PYV and NPVFNEXF (30). These two regions have been proposed to maintain the structural integrity of the C2 fold because their residues are largely hydrophobic. In PLD␤ the first segment is completely conserved and the second segment, NPVWMQHF, is largely the same with several conservative amino acid substitutions. Furthermore, PLD␤ contains the conserved acidic residues (underlined in Fig. 5) that serve to coordinate Ca 2ϩ -binding in the C2 domain. This is in contrast to PLD␣ in which two of the acidic residues are substituted with positively charged or neutral amino acids.
Genomic Organization of PLD␤ and PLD␣-The molecular organization of PLD␤ and PLD␣ in the Arabidopsis genome was examined by Southern blotting analysis (Fig. 6, A and B). Total genomic DNA was digested with restriction enzymes and hybridized with probes made from full-length PLD␤ and PLD␣ cDNAs. Hybridization of the same DNA with PLD␣ or PLD␤ probes gave unique banding patterns, indicating that the PLD␤ and PLD␣ sequences do not cross-hybridize with each other at high stringency conditions. The PLD␣ cDNA has one XhoI site, but no BamHI, KpnI, and XbaI restriction sites, and the digested genomic DNA gave one strong hybridization band (Fig.  6A). The simple banding pattern of hybridization by the PLD␣ cDNA indicates that the Arabidopsis genome may contain one gene copy of PLD␣. When the same DNA was probed with PLD␤, which contains one KpnI and two BamHI recognition sites, but no XbaI nor XhoI site, the number of hybridization bands were more than that predicted from the BamHI, KpnI, and XhoI digestion of PLD␤ cDNA (Fig. 6B). These results could be caused by the presence of these restriction sites in intron sequences of the PLD␤ gene and/or by the presence of of PLD activity. PLD was assayed in the presence of 7.6 mol % PIP 2 and 0 -2 mM neomycin. D, dependence of PLD␤ activity on Ca 2ϩ . Free Ca 2ϩ and Mg 2ϩ in reaction mixtures were controlled using Ca 2ϩ /Mg 2ϩ -EGTA buffers at pH 7.5. PLD␤ was expressed from its cDNA in E. coli, and each assay used 10 g of soluble protein containing PLD␤ and 3 g of cytosol proteins from PLD␣ antisense Arabidopsis. Values are means Ϯ S.E. of three experiments. another or closely related PLD␤ gene. Northern blot analysis using the full-length PLD␤ cDNA as a probe detected one RNA band of approximately 3.5 kb (Fig. 6C). The estimated size was in agreement with that of the cloned PLD␤ cDNA. However, it was unclear if this band was composed of one PLD␤ mRNA species or different PLD transcripts with similar sizes. It is worth noting that another study has shown that two protein bands from Arabidopsis extracts were recognized by the antibodies raised against a PLD␤ peptide, suggesting the presence of other PLD(s) closely related to the cloned PLD␤ in Arabidopsis (13). DISCUSSION The present results provide molecular evidence for the presence of two plant PLD isoforms that are distinctly regulated and expressed. PLD␣, which was previously cloned and characterized from several plant species, requires millimolar Ca 2ϩ , but no PIP 2 , for activity. Our study involving antisense suppression of the PLD␣ gene in Arabidopsis has unmasked the presence of a PIP 2 -regulated PLD in plants. In this study, we have cloned and functionally expressed the PIP 2 -dependent PLD which is designated PLD␤. The biochemical properties of the PIP 2 -dependent PLD expressed from the PLD␤ cDNA are almost the same as those identified in Arabidopsis protein extracts (13). Specifically, the PIP 2 requirement by the PLD from the plant extracts and the cDNA expressed in E. coli can be partially substituted by PIP, but not by PI, PS, PG, PE, or PA. The optimal pH for the PIP 2 -dependent PLD from both sources is around 7 to 7.5. The PLD obtained from both sources requires Ca 2ϩ and is fully active at submicromolar ranges of Ca 2ϩ . These similarities suggest that the cloned PLD is the isoform responsible for the PIP 2 dependent activity measured in the extracts of Arabidopsis.
The only difference between the two enzymes appears in the Ca 2ϩ effect at higher concentrations; the activity from PLD expressed in E. coli decreased at millimolar Ca 2ϩ whereas the PLD examined in the PLD␣-antisense plants showed a sigmoidal response to the increase of Ca 2ϩ concentration. However, the plant extract contains more than one type of PLD, and PLD␣ is known to be most active at millimolar Ca 2ϩ . Therefore, the stable PLD activity observed in millimolar Ca 2ϩ could result from residual PLD␣ or from any other PLD that is stimulated by millimolar Ca 2ϩ and not suppressed by PLD␣ antisense.
The ability of PLD␤ to shift from an inactive state to a highly active state over a narrow range of calcium concentration strongly suggests that changing intracellular concentrations could be a major form of regulation for the enzyme in vivo. This observation is particularly relevant considering that 100 nM and 1 M are the respective resting and stimulated intracellular calcium concentrations of plants and animals (33,34). It remains to be elucidated how Ca 2ϩ is involved in the PLDmediated hydrolysis of phospholipids. The finding of a C2 domain near the N-terminal region of several PLDs suggests that one of the roles of Ca 2ϩ may be to regulate the enzymes' binding to phospholipids. The predominant feature of the C2 domain is its ability to mediate Ca 2ϩ -dependent phospholipid binding. The Ca 2ϩ /phospholipid-binding domain was first identified in Ca 2ϩ -dependent protein kinase C isoforms and has since been found in a number of different proteins including intracellular PLA 2 and PIP 2 -PLC isoforms. It is believed that the binding of membrane phospholipids by a C2 domain represents a Ca 2ϩ -dependent translocation mechanism whereby cytosolic proteins become associated with the membrane in a highly regulated manner. Thus for some C2-containing enzymes, phospholipid binding could represent one mechanism of cellular activation. The presence of a C2 domain in PLDs raises the question of whether Ca 2ϩ -dependent phospholipid binding is involved in the enzyme's activation, catalysis, or both. Comparison of the C2 domains of PLD␣ and PLD␤ reveals an important difference. The PLD␤ C2 domain conserves the acidic residues needed to coordinate Ca 2ϩ binding whereas the PLD␣ C2 domain possesses substitutions, potentially indicating a loss of Ca 2ϩ affinity. The difference in the amount of Ca 2ϩ needed for activity is one of the most distinct in vitro properties that distinguishes between PLD␣ and PLD␤. PLD␣ requires millimolar amounts of Ca 2ϩ whereas PLD␤ is fully active at low micromolar levels of Ca 2ϩ (13). An ongoing study in this laboratory is to determine whether or not the differences in the C2 domain underlie the different Ca 2ϩ requirements observed for PLD␣ and PLD␤. Such studies should help understand the regulatory and catalytic mechanisms for these PLD isoforms.
Sequence analysis indicates that PLD␤ and PLD␣ are evolutionarily divergent and that PLD␤ is more closely related to the PLDs cloned from yeast (4) and human (24) than is PLD␣. Alignments of these PLD sequences reveals two distinct groups: PLDs from plants and those from human and yeast. Within the plant group PLD␤ forms a subgroup distinct from that of PLD␣s from Arabidopsis, castor bean, maize, and rice. The grouping of PLDs from Arabidopsis, castor bean, maize, and rice suggests that these are more closely related evolutionarily. Phylogram analysis in the unrooted phylogeny places the Arabidopsis PLD␤ with the yeast and human PLDs. Furthermore, the phylogenic groupings are supported by comparing the calculated pI values and catalytic properties of these PLDs. PLD␣ of different plant species all have acidic pI values around 5-6 whereas PLD␤ and the yeast and human PLDs have basic pI values of 7.9, 7.6, and 9.3, respectively. PLD␣ activity is PIP 2 -independent and requires millimolar concentrations of Ca 2ϩ whereas PLD␤ and the cloned PLDs from human and yeast are activated by PIP 2 . Both PLD␤ and the human PIP 2dependent PLD are regulated by physiological concentrations of Ca 2ϩ and cytosolic factors.
These analyses have clearly shown that there are PLD isoforms in plants that are encoded by different genes and are regulated in a distinct manner. The distinct regulatory mechanisms suggest that these PLDs have different cellular functions. PLD␣ is the most prevalent plant PLD (13) and it seems to be unique to plants based on the comparison of its catalytic properties with those of mammalian and yeast PLDs. PLD␤, on the other hand, shares some properties with the recently cloned human and yeast PLDs. It has been shown that the yeast PLD is required for meiosis (4 -6). The cloned human PLD is thought to be involved in membrane trafficking and secretion but its role in these processes is unclear (3,24). We have produced PLD␣-suppressed transgenic plants that should be instrumental in defining the function of PLD␣. Efforts are underway to produce plants deficient in PLD␤ activity and to use these systems to sort out the roles of the different PLDs in growth and development.