Abstract
Cytoplasmic calcium elevations, transients, and oscillations
are thought to encode information that triggers a variety of physiological
responses in plant cells. Yet Ca2+ signals induced by a single stimulus
vary, depending on the physiological state of the cell and experimental
conditions. We compared Ca2+ homeostasis and stimulus-induced Ca2+
signals in guard cells of intact plants, epidermal strips, and isolated protoplasts.
Single-cell ratiometric imaging with the Ca2+-sensitive dye
Fura 2 was applied in combination with electrophysiological recordings.
Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing
a cytoplasmic free Ca2+ concentration of around 80 nM at
-47 mV. Upon hyperpolarization of the plasma membrane to -107 mV,
the Ca2+ concentration increased to levels exceeding 400 nM. Intact
guard cells were able to maintain much lower cytoplasmic free Ca2+
concentrations at hyperpolarized potentials, the average concentration at
-100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively.
Further hyperpolarization of the plasma membrane to
-160 mV induced a sustained rise of the guard cell cytoplasmic Ca2+
concentration, which slowly returned to the prestimulus level in intact
plants but not in epidermal strips. Our results show that cytoplasmic
Ca2+ concentrations are stringently controlled in guard cells of intact
plants but become increasingly more sensitive to changes in the plasma
membrane potential in epidermal strips and isolated protoplasts.
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