Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts

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.