Abstract
Low concentrations of CO(2) cause stomatal opening, whereas [CO(2) ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca(2+) and protein phosphorylation in CO(2) -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca(2+) into specific phosphorylation events. However, Ca(2+) -binding proteins that function in the stomatal CO(2) response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO(2) , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO(2) -induced stomatal closure and, unexpectedly, also in low CO(2) -induced stomatal opening. Furthermore, K(+) -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO(2) -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO(2) control of stomatal movements and support the results of classical studies showing a role for Ca(2+) in this response.