Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLC delta 1 that specifically binds PtdIns(4,5)P-2. In this way, YFP-PHPLC delta 1 has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P-2 is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PHPLC delta 1 to try monitoring PtdIns(4,5)P-2/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P-2. Confocal imaging revealed that most of the YFP-PHPLC delta 1 fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PHPLC delta 1 was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P-2, as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PHPLC delta 1. Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PHPLC delta 1 is a valuable plant PtdIns(4,5)P-2 reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P-2 in plant cell signalling are discussed.