Increases in cytosolic Ca²⁺ are not required for abscisic acid-inhibition of inward K⁺ currents in guard cells of Vicia faba L.

The inward K⁺ channels (I(Kin)) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I(Kin) inhibition requires an elevation in cytosolic free Ca²⁺ levels ([Ca²⁺](c)) because: (i) experimental increases in [Ca²⁺](c) can mimic the ABA effect, and: (ii) ABA can trigger an elevation of [Ca²⁺](c) in guard cells. However, not all guard cells respond to ABA with a [Ca²⁺](c) increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca²⁺ in the regulation of downstream effectors of ABA response, such as the I(Kin) channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca²⁺ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced chances in [Ca²⁺](c) and I(Kin) inhibition. In the presence of extracellular Ca²⁺ the extent of [Ca²⁺](c) elevation correlated with the extent of I(Kin) inhibition. However, upon chelation of either extracellular Ca²⁺, [Ca²⁺ ](c) or both, extracellular Ca²⁺ and [Ca²⁺](c). [Ca²⁺](c) elevation did not occur in response to ABA yet I(Kin) currents were still strongly inhibited. These data illustrate that Ca²⁺-independent regulation is involved in ABA-inhibition of stomatal opening processes.