METABOLISM OF 3-PHOSPHORYLATED AND 4-PHOSPHORYLATED PHOSPHATIDYLINOSITOLS IN STOMATAL GUARD-CELLS OF COMMELINA-COMMUNIS L

Within the plant kingdom the stomatal guard cell is presented as a mod el system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3]-mediated sig nal transduction. Despite this it is only recently that the phosphoino sitide components of animal signal transduction pathways have been ide ntified in stomatal guard cells. Interestingly, stomatal guard cells c ontain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal o f the routes of synthesis and rates of turnover of these phosphatidyli nositols would appear timely as the in vivo biosynthesis of these comp onents is a much neglected facet of the phosphoinositide-mediated sign alling paradigm as purported to apply to plants. A non equilibrium [P- 32]P-i labelling strategy and enzymic and chemical dissection of label led phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L. The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [P-32]P-i. In separate experiments, isol ated guard cells were labelled over a 40-240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol pho sphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP(2) ) peaks were desalted and enzymically cleaved with alkaline phosphatas e and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90-97 % of the [P-32]P-i label while the 4- and 5-monoester phosphates of ph osphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] accounted for typ ically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P-2 by successive 4- and 5-phosphorylati on of phosphatidylinositol (PtdIns). This study therefore represents t he first report of the pathway of the synthesis of 4- and 5-phosphoryl ated phosphatidylinositols in a single defined hormone-responsive plan t cell type. The monoester phosphate in phosphatidylinositol 3-monopho sphate (PtdIns3P) accounted for 83-95% of the P-32 label. It was not p ossible, however, to determine the route of synthesis of phosphatidyli nositol 3,4-bisphosphate [PtdIns(3,4)P-2] owing to the rapid attainmen t of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3 ,4)P-2 each containing approximately 50% of the label at just 40 min o f labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. S imilarly, turnover of PtdIns(3,4)P-2 was quicker than that of PtdIns(4 ,5)P-2, and in mass terms PtdIns(3,4)P-2 appeared to predominate over PtdIns(4,5)P-2. By analogy with animal systems, in which signalling mo lecules such as PtdIns(4,5)P-2 show considerable basal turnover, the e vidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P-2 in addition to those previously indicated for PtdIns(4 ,5)P-2 in stomatal guard cells.