Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L.) seedlings

N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDbeta/y (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products---three putative PLD beta isoforms and three putative PLD delta isoforms. The PLD beta isoforms, designated Ghpldbeta1a, GHpldbeta1b and a truncated Ghpldbeta1b isoform. Both the full-length PLD beta proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. (Abstract shortened by UMI.)...