MULTIPLE FORMS OF PHOSPHOLIPASE-D FOLLOWING GERMINATION AND DURING LEAF DEVELOPMENT OF CASTOR BEAN

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were ide ntified and partially characterized in endosperm of germinated seeds a nd leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electroph oresis, isoelectric focusing, and size-exclusion chromatography. PLD w as detected with both a PLD activity assay and immunoblots with PLD-sp ecific antibodies. There were three major forms of PLD, designated typ es 1, 2, and 3, based on their mobility during nondenaturing polyacryl amide gel electrophoresis. Molecular masses of the PLD variants were e stimated at 330, 230, and 270 kD for the types 1, 2, and 3, respective ly. Isoelectric points of the native type 1, 2, and 3 PLDs were approx imately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, t he three forms of PLD exhibited the same substrate specificity, hydrol yzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and pho sphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidy linositol (PI). The three forms of PLD differed in their substrate pre ferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The K-m values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were ex pressed differentially following seed germination and during leaf deve lopment. Type 1 was found in the early stages of seedling growth and i n young leaves, type 2 was present in all the tissues and growth stage s examined, and type 3 was expressed in senescent tissues. The PLDs sh ifted from largely cytosolic to predominantly membrane-associated form s during leaf development. The present studies demonstrate the structu ral heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of mu ltiple molecular forms of PLD in cellular metabolism is discussed.