Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.
Description: 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 PLDβ/γ (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 β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the characteristic catalytic and regulatory domains. The expression of Ghpldδ1b showed hydrolytic and transphosphatidylation activity toward radiolabelled phosphatidylcholine (PC) but it appears Ghpldδ1b does not utilize NAPE as a substrate to produce NAEs nor does it seem to be suppressed by NAEs.
Date: May 2005
Creator: McHugh, John
Item Type: Thesis or Dissertation