A role for calcium-dependent protein kinases in differential CO₂- and ABA-controlled stomatal closing and low CO₂-induced stomatal opening in Arabidopsis

Low concentrations of CO₂ cause stomatal opening, whereas [CO₂] elevation leads to stomatal closure. Classical studies have suggested a role for Ca²⁺ and protein phosphorylation in CO₂-induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca²⁺ into specific phosphorylation events. However, Ca²⁺-binding proteins that function in the stomatal CO₂ 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 CO₂, 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 CO₂-induced stomatal closure and, unexpectedly, also in low CO₂-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, CO₂-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 CO₂ control of stomatal movements and support the results of classical studies showing a role for Ca²⁺ in this response.