Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide-mediated signalling paradigm as purported to apply to plants.
A non-equilibrium [32P]Pi labelling strategy and enzymic and chemical dissection of labelled 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 [32P]Pi. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP2) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90–97% of the [32P]Pi label while the 4- and 5-monoester phosphates of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P2 by successive 4- and 5-phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4- and 5-phosphorylated phosphatidylinositols in a single defined hormone-responsive plant cell type.
The monoester phosphate in phosphatidylinositol 3-monophosphate (PtdIns3P) accounted for 83–95% of the 32P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] owing to the rapid attainment of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3,4)P2, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P2 was quicker than that of PtdIns(4,5)P2, and in mass terms PtdIns(3,4)P2 appeared to predominate over PtdIns(4,5)P2. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P2 show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P2 in addition to those previously indicated for PtdIns(4,5)P2 in stomatal guard cells.