Confocal Fluorescence Ratio Imaging of pH in Plant Cells
Fluorescent probes offer unparalleled opportunities to visualise dynamic events within living cells with a minimum of perturbation. Combined with ultra-sensitive imaging systems and powerful computer processing, quantitative measurements of ephemeral gradients, transients and oscillations are possible. Studies in plants have concentrated on measurements of cytosolic calcium ([Ca 2+]i) and protons ([H+]i) to assess their role in signal transduction and ion homeostasis (see reviews by Hepler and Callaham, 1991; Flicker et al., 1992; Read et al., 1992). Results from stomatal guard cells indicate highly heterogeneous, spatially localised changes in [Ca2+]i of varying amplitude in response to a number of stimuli (e.g. Gilroy et al., 1991; Fricker et al., 1991). In many plant systems, including guard cells, [Ca2+]i and [H+]i directly regulate membrane ion transporters at the tonoplast and plasma membrane as an integral part of a wider signalling network (Hepler and Wayne, 1985; Johannes et al., 1991; Blatt, 1992). Our knowledge of these transport mechanisms draws heavily on electrophysiological studies using patch-clamp and whole-cell voltage clamp techniques (Hedrich and Schroeder, 1989; Tester, 1990; Blatt, 1992). A major thrust of our work is to combine optical and electrophysiological approaches to allow unambiguous interpretation of signalling sequences during transduction of external stimuli. We have adopted confocal microscopy as the optimum solution for fluorescence quantitation in living tissue, and voltage-clamping in intact cells for electrophysiological analysis of membrane transporters. Voltage clamping was chosen in preference to patch-clamping, as patching involves considerable trauma during protoplast isolation. With patching the cytoplasm is also massively diluted by the contents of the pipette, potentially disrupting signalling networks (Blatt, 1992).
KeywordsGuard Cell Acridine Orange Ratio Image Stomatal Guard Cell Stomatal Complex
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