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

doi:10.1111/j.1365-313X.2007.03292.x

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Wiley InterScience

The Plant Journal

Volume 52 Issue 6, Pages 1014 - 1026

Published Online: 1 Oct 2007

Published in association with the Society for Experimental Biology

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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

Wessel van Leeuwen ^1,†,‡ , Joop E.M. Vermeer ^1,2,3,† , Theodorus W.J. Gadella Jr ^2,3 and Teun Munnik ^1,*

¹ Section Plant Physiology, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands ,
² Section Molecular Cytology, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands , and
³ Centre for Advanced Microscopy, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands

Correspondence to ^*(fax +31 205257934; e-mail munnik@science.uva.nl).

^† These authors contributed equally to this work.

^‡ Present address: Laboratory of Genetics, Wageningen University, Arboretumlaan 4, NL-6703 BD, Wageningen, The Netherlands.

KEYWORDS

polyphosphoinositide • lipid binding proteins • pleckstrin homology • salt stress • stress signalling

ABSTRACT

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] 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)P₂in 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)P₂. 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)P₂ 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)P₂/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)P₂. 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)P₂, 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)P₂ 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)P₂ in plant cell signalling are discussed.