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 IKin 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 effecters 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 changes 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.