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Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLD ␣ , PLD ␤ , and PLD ␥ differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD , PLD ␣ , decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein.Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLD ␣ attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLD ␣ -suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA. INTRODUCTIONJasmonic acid (JA) and related compounds are a new class of plant hormones that play an important role in regulating many cellular processes, such as wound and defense responses (Farmer and Ryan, 1992;Bell et al., 1995;Creelman and Mullet, 1997;McConn et al., 1997). The production of JA is a tightly regulated process, and the concentrations of JA in unperturbed plant tissues are often very low. However, JA accumulates in wounded plants or in plants and cultured cells treated with pathogen elicitors; it acts as a signal activating the expression of various genes, such as proteinase inhibitors, thionin, and enzymes in phytoalexin metabolism . The pathway for de novo JA biosynthesis, beginning with free ␣ -linolenic acid, has been well elucidated (Vick, 1993;Creelman and Mullet, 1997; also see Figure 1). But when and how linolenic acid is made available for JA synthesis is not well understood. Linolenic acid, the most abundant fatty acid in leaves, is mostly present in esterified glycerolipid form (Browse and Somerville, 1991). Free fatty acids are not generally found in large amounts in healthy, intact plant cells. The release of linolenic acid from membranes has been thought to be an important step in controlling JA synthesis. An increase in free linolenic acid was observed in cultured cells of several plant species after treatment with fungal wall elicitors (Gundlach et al., 1992) and in wounded plants (Conconi et al., 1996;Ryu and Wang, 1998). A phospholipase A (PLA)-like activity has been proposed to mediate the release of linolenic acid from membranes (Farmer and Ryan, 1992), and the presence of such a wound-inducible PLA activity has been noted in tomato and other plant species (Lee et al., 1997;Narváez-Vásquez et al., 1999).Recent studies have suggested that activation of phospholipase D (PLD) also may play an important role in mediating wound-induced lipid hydrolysis Wang, 1996, 1998;Lee et al., 1997). PLD hydrolyzes phospholipids at the terminal phosphoesteric bond, generating phosphatidic acid (PA) and free head groups, such as choline . T...

Summary Pathogen infection of higher plants often induces a rapid production of phosphatic acid (PA) and changes in lipid profiles, but the enzymatic basis and the function of the lipid change in pathogen-plant interactions are not well understood. Infection of PLDβ1-deficient plants by Pseudomonas syringae pv. DC3000 resulted in less bacterial growth than in wild-type plants, and the effect was more profound in virulent Pst DC3000 than avirulent Pst DC3000 (avrRpt2) infection. The expression levels of salicylic acid (SA)-inducible genes were higher, but those inducible by jasmonic acid (JA) were lower in PLDβ1 mutants than in wild-type plants. However, PLDβ1-deficient plants were more susceptible than wild-type plants to the fungus Botrytis cinerea. The PLDβ1-deficient plants had lower levels of PA, JA and JA-related defense gene expression after B. cinerea inoculation. PLDβ1 plays a positive role in pathogen-induced JA production and plant resistance to necrotrophic fungal pathogen B. cinerea, but a negative role in the SA-dependent signaling pathway and plant tolerance to the infection of biotrophic Pst DC3000. PLDβ1 is responsible for the major part of PA increased in response to necrotrophic B. cinerea and virulent Pst DC3000 infection, but contributes less to the avirulent Pst DC3000 (avrRpt2)-induced PA production.

Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown. Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity. Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis. This PLD, designated PLD, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques. A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLD was expressed from its cDNA in Escherichia coli. PLD was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mm. Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective. Phosphatidylinositol 4,5-bisphosphate stimulated PLD to a lesser extent than oleate. Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLD, indicating that separate sites mediate the oleate regulation of PLD. Oleate stimulated PLD's binding to phosphati-dylcholine. Mutation at Arg-399 resulted in a decrease in oleate binding by PLD and a loss of PLD activity. However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding. These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.

Development of the aleurone layer of maize grains requires the activity of the Defective kernel 1 (Dek1) gene, encoding a predicted 240-kDa membrane-anchored protein with a C terminus similar to animal calpain domain II&III. Three-dimensional modeling shows that DEK1 domain II contains a conserved calpain catalytic triad and that domain II&III has a predicted structure similar to m-calpain. Recombinant DEK1 domain II&III exhibits activity in the caseinolytic assay in the absence of calcium, although the activity is enhanced by calcium. This is in sharp contrast to animal calpains, which require Ca 2؉ to be active. Bacterially expressed DEK1 domain II does not display caseinolytic activity, suggesting an important role for DEK1 domain III. Mutation of the catalytic Cys residue to Ser leads to a loss of caseinolytic activity of DEK1 domain II&III. Two features of DEK1 calpain may contribute to maintaining the active site triad in an "active" configuration in the absence of Ca 2؉ , both of which are predicted to keep m-calpain domains IIa and IIb apart. First, DEK1 lacks key charged residues in the basic loop of domain II, and secondly, the absence of an acidic loop in domain III, both of which are predicted to be neutralized upon Ca 2؉ binding. The Dek1 transcript is present in all cell types in developing maize endosperm, suggesting that the activity of the DEK1 calpain is regulated at the posttranscription level. The role of DEK1 in aleurone signaling is discussed.In cereal grains, the aleurone layer consists of densely cytoplasmic cells covering the surface of the endosperm, the grain storage tissue that is used for feed, food and industrial raw material (1). Aleurone cells contain a large numbers of protein and oil bodies and are cytologically and biochemically distinct from the storage cells of the underlying starchy endosperm. Upon imbibition of the grain, aleurone cells secrete enzymes that mobilize stored starch and protein reserves for seedling growth (2). The endosperm develops from the central cell after double fertilization through a cellularization process that results in a peripheral layer of aleurone cell initials (3). From genetic evidence, the tumor necrosis factor receptor-like kinase CRINKLY4 (CR4) is implicated in aleurone cell fate specification (4 -7). Based on the identity of the Cr4 gene product, as well as the peripheral position of the aleurone layer, we proposed a model in which the CR4 receptor is activated by a ligand in the periphery of the endosperm (8). Recently, we isolated the defective kernel 1 (dek1) 1 gene, which is also essential for aleurone cell development (9). Homozygous recessive dek1 endosperm initiates aleurone cell fate specification, but fails to maintain aleurone cell fate, resulting in grains that lack aleurone cells (9). Revertant sector analysis has demonstrated that Dek1 function is essential for the maintenance of aleurone cell fate throughout grain development (10, 11). Sequence analysis predicts that the Dek1 gene encodes a 240 kDa protein with 21 membrane spa...

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Aluminum (Al) toxicity is the major stress in acidic soil that comprises about 50% of the world's arable land. The complex molecular mechanisms of Al toxicity have yet to be fully determined. As a barrier to Al entrance, plant cell membranes play essential roles in plant interaction with Al, and lipid composition and membrane integrity change significantly under Al stress. Here, we show that phospholipase Dγs (PLDγs) are induced by Al stress and contribute to Al-induced membrane lipid alterations. RNAi suppression of PLDγ resulted in a decrease in both PLDγ1 and PLDγ2 expression and an increase in Al resistance. Genetic disruption of PLDγ1 also led to an increased tolerance to Al while knockout of PLDγ2 did not. Both RNAi-suppressed and pldγ1-1 mutants displayed better root growth than wild-type under Al stress conditions, and PLDγ1-deficient plants had less accumulation of callose, less oxidative damage, and less lipid peroxidation compared to wild-type plants. Most phospholipids and glycolipids were altered in response to Al treatment of wild-type plants, whereas fewer changes in lipids occurred in response to Al stress in PLDγ mutant lines. Our results suggest that PLDγs play a role in membrane lipid modulation under Al stress and that high activities of PLDγs negatively modulate plant tolerance to Al.

Flowering is regulated by a network integrated from four major pathways, including the photoperiod, vernalization, gibberellin, and autonomous pathways. RNA processing within the autonomous pathway is well known to regulate Arabidopsis thaliana flowering time. Here we identify a novel Arabidopsis gene, designated AT PRP39-1, that affects flowering time. Based on observations that homozygous at prp39-1 plants are late flowering under both long and short days and responsive to GA and vernalization treatment, we tentatively conclude that AT PRP39-1 may represent a new component of the autonomous pathway. Consistent with previous studies on genes of the autonomous pathway, knockout of AT PRP39-1 in Arabidopsis displays an upregulation of the steady state level of FLC, and simultaneous downregulation of FT and SOC1 transcript levels in adult tissues. AT PRP39-1 encodes a tetratricopeptide repeat protein with a similarity to a yeast mRNA processing protein Prp39p, suggesting that the involvement of these tetratricopeptide repeat proteins in RNA processing is conserved among yeast, human, and plants. Structure modeling suggests that AT PRP39-1 has two TPR superhelical domains suitable for target protein binding. We discuss how AT PRP39-1 may function in the control of flowering in the context of the autonomous pathway.