| Totally twelve phospholipase D (PLD) genes have been identified in Arabidopsis thaliana and encode PLDs with distinct regulatory and catalytic properties. They are tentatively divided into five groups: four PLDαs, two PLDβs, three PLDγs, and one PLDδ, which all have the C2 domain, plus two mammalian-like PLDζs, which contain the phox homology (PX) and the pleckstrin homology (PH) domains. A detailed genomic analysis of the evolutionary relationships, sequence similarities, gene architectures, and domain structures is presented.; Three types of Arabidopsis PLDs were characterized in this study. Surface dilution kinetic analysis was carried out on the newly-cloned PLDδ. Two common regulatory factors, phosphatidylinositol 4,5-bisphosphate (PIP 2) and Ca2+, were found to stimulate PLDδ activity by promoting substrate binding to the enzyme. Phosphatidylethanolamine (PE) was indicated as a better substrate for PLDδ than phosphatidylcholine (PC).; The cDNA for the second member of the PLDγ group, PLDγ2 , was cloned and active protein was expressed. Its catalytic properties were examined and compared with the closely related PLDγ1 protein. An unexpected difference in Triton X-100 effect on enzyme activity was revealed.; The cDNAs for the PX/PH containing PLDζ1 and PLDζ2 were also cloned. PLDζ1 protein was expressed in E. coli, and it selectively hydrolyzed PC in a Ca2+-independent manner, similar to the mammalian PLDs but distinct from other plant PLDs. A series of transgenic plants for both PLDζ1 and PLDζ2 genes have been generated, including the single/double knock out mutants, plus the complementing and the overexpressing plants, in order to investigate the physiological roles these two PLDζ1 and PLDζ2s play in cellular processes. |
