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

Authors

  • Wessel Van Leeuwen,

    1. Section Plant Physiology, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands
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    • These authors contributed equally to this work.

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

  • Joop E.M. Vermeer,

    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
    3. Centre for Advanced Microscopy, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands
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    • These authors contributed equally to this work.

  • Theodorus W.J. Gadella Jr,

    1. Section Molecular Cytology, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands
    2. Centre for Advanced Microscopy, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands
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  • Teun Munnik

    Corresponding author
    1. Section Plant Physiology, University of Amsterdam, Swammerdam Institute for Life Sciences, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands
      *(fax +31 205257934; e-mail munnik@science.uva.nl).
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*(fax +31 205257934; e-mail munnik@science.uva.nl).

Summary

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-PHPLCδ1 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-PHPLCδ1 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-PHPLCδ1 fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PHPLCδ1 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-PHPLCδ1. Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PHPLCδ1 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.

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