Expression and characterization of Arabidopsis phospholipase Dγ2

The phospholipase D (PLD) family of Arabidopsis thaliana has 12 identified members, including three highly homologous PLD gamma s. The enzymatic and molecular properties of PLD gamma 2 were characterized and compared with those of PLD gamma 1. Two variants of PLD gamma 2 cDNAs, designated PLD gamma 2a and PLD gamma 2b, were isolated, and they differ in the presence of a 96-nucleotide fragment at the beginning of the open reading frame. Catalytically active PLD gamma 2a was expressed in E. coli. PLD gamma 2a and gamma 1 both require phosphatidylinositol 4,5-bisphosphate (PIP2) and calcium for activity, but they differ in the effect Of PIP2 and Triton X-100 on their activities. While Triton X-100 could greatly activate PLD gamma 1 activity and served only as a neutral diluent in the substrate vesicles, it totally abolished PLD-gamma 2a activity and prohibited any stimulation effect from PIP2. PLD gamma 2a misses one of the basic, PIP2-interacting residues, which may weaken the binding of PIP2 to PLD gamma 2a. In addition, PLD gamma 2 and PLD gamma 1 displayed different patterns of expression in different tissues, and the transcript of PLD gamma 2a differs from that of PLD gamma 1 by having a longer 5'-UTR. These differences in biochemical and molecular properties suggest that the highly homologous PLD gamma s are subjected to unique regulations and might have distinguishable functions. (c) 2006 Elsevier B.V. All rights reserved.