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

Summary.

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.