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PLANT PHYSIOLOGY , Vol 113, Issue 2 587-594, Copyright © 1997 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
Hypoosmotic Shock Induces Increases in Cytosolic Ca2+ in Tobacco Suspension-Culture Cells
K. Takahashi, M. Isobe, M. R. Knight, A. J. Trewavas and S. Muto
Graduate School of Agricultural Sciences (K.T., M.I., S.M.), School of Agricultural Sciences (M.I.), Nagoya University Bioscience Center (S.M.), Nagoya University, Chikusa, Nagoya, 464-01, Japan
Hypoosmotic shock treatment increased cytosolic Ca2+ ion concentration
([Ca2+]cyt) in tobacco (Nicotiana tabacum) suspension-culture cells.
[Ca2+]cyt measurements were made by genetically transforming these cells to
express apoaequorin and by reconstituting the Ca2+-dependent photoprotein,
aequorin, in the cytosol by incubation with chemically synthesized
coelenterazine. Measurement of Ca2+-dependent luminescence output thus
allowed the direct monitoring of [Ca2+]cyt changes. When cells were added
to a hypoosmotic medium, a biphasic increase in [Ca2+]cyt was observed; an
immediate small elevation (phase 1) was observed first, followed by a
rapid, large elevation (phase 2). Phase 1 [Ca2+]cyt was stimulated by the
V-type ATPase inhibitor bafilomycin A1. Phase 2 was inhibited by the
protein kinase inhibitor K-252a and required the continued presence of the
hypoosmotic stimulus to maintain it. Although Ca2+ in the medium was needed
to produce phase 2, it was not needed to render the cells competent to the
hypoosmotic stimulus. If cells were subject to hypoosmotic shock in Ca2+-
depleted medium, increases in luminescence could be induced up to 20 min
after the shock by adding Ca2+ to the medium. These data suggest that
hypoosmotic shock-induced [Ca2+]cyt elevation results from the activity of
a Ca2+ channel in the plasma membrane or associated hypoosmotic sensing
components that require Ca2+- independent phosphorylation and a continued
stimulus to maintain full activity.
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