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Visualization of phosphatidylinositol 4,5‐bisphosphate in the plasma membrane of suspension‐cultured tobacco BY‐2 cells and whole Arabidopsis seedlings
Author(s) -
Van Leeuwen Wessel,
Vermeer Joop E.M.,
Gadella Theodorus W.J.,
Munnik Teun
Publication year - 2007
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2007.03292.x
Subject(s) - pleckstrin homology domain , arabidopsis , yellow fluorescent protein , phosphatidylinositol 4,5 bisphosphate , microbiology and biotechnology , phosphatidylinositol , cytoplasm , phospholipase c , second messenger system , fluorescence recovery after photobleaching , chemistry , biochemistry , biology , intracellular , signal transduction , membrane , gene , mutant
Summary 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 PLCδ1 that specifically binds PtdIns(4,5) P 2 . In this way, YFP‐PH PLCδ1 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‐PH PLCδ1 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‐PH PLCδ1 fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP‐PH PLCδ1 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‐PH PLCδ1 . Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP‐PH PLCδ1 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.