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

Lisa A. Romano, Tobias Jacob, Simon Gilroy and Sarah M. Assmann

Biology Department, 208 Mueller Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802

Abstract.
The inward K+ channels (IKin) of guard cells are inhibited upon ABA application. It has been postulated that IKin inhibition requires an elevation in cytosolic free Ca2+ levels ([Ca2+]c) because: 1) experimental increases in [Ca2+] c can mimic the ABA effect, and; 2) 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 IKin 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 IKin inhibition. In the presence of extracellular Ca2+, the extent of [Ca2+]c elevation correlated with the extent of IKin 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 IKin currents were still strongly inhibited. These data illustrate that Ca2+-independent regulation is involved in ABA-inhibition of stomatal opening processes.