Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1) -: comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B-napus

The cloning and identification of full-length cDNA fragments coding for the Brassica napus phosphatidylinositol-specific phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase (BnPtdIns S1) is described. In addition, two complementary fragments (120 nucleotides long) corresponding to Arabidopsis PtdIns 4-kinase (PtdIns 4-K) and PtdIns-4-phosphate 5-kinase (PtdIns4P 5-K) sequences were chemically synthesized. These, as well as the cDNA clones, were used as probes to study the corresponding steady state mRNA levels in different tissues and developmental stages of B. napus, as well as in response to different environmental conditions. Transcripts corresponding to BnPLC2, BnPtdIns S1, BnVPS34 and PtdIns 4-K were found constitutively expressed at different levels in most tissues, with young leaves, siliques, and developing seeds showing the lowest levels. No detectable PtdIns4P 5-K transcripts were found in buds or flowers. Up-regulation of BnPLC2 was seen in response to low temperature stress, which was notably accompanied by a parallel down-regulation of BnPtdIns S1, while BnVPS34 and PtdIns 4-K remained at control levels. A moderate increase in PtdIns4P 5-K levels was noted. In high salinity conditions BnPtdIns S1, BnVPS34 and BnPLC2 transcripts had similar responses but at different levels, with no major changes detected for PtdIns 4-K or PtdIns4P 5-K. Significantly, all five transcripts increased under drought stress conditions and all stressed plants clearly showed relatively higher levels of total inositol trisphosphate.