Arabidopsis Circadian Clock Protein CCA1 Negatively Regulates the Calcium‐Binding Protein CCaP1 to Modulate Dark‐Responsive Stomatal Movement

ABSTRACT Stomatal conductance, which is crucial for plant carbon assimilation and water regulation, is modulated by Ca 2+ ‐binding proteins (CBPs). These proteins mediate Ca 2+ signaling transduction, transport and homeostasis, influencing various physiological processes including stomatal movement, pollen tube growth and hypocotyl elongation. Despite their significance, the involvement of CBPs in dark‐induced stomatal closure remains elusive. Here, we investigated the role of Arabidopsis cytosolic Ca 2+ ‐binding protein 1 (CCaP1) in dark‐induced stomatal closure. β‐glucuronidase (GUS) staining and in situ hybridization confirmed its predominant expression in guard cells. Yeast one‐hybrid and transient expression assays demonstrated that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) directly binds to the CCaP1 promoter to negatively regulate its expression. Genetic studies demonstrated that CCaP1 regulated dark‐induced stomatal closure. Furthermore, the use of the Ca 2+ probe R‐GECO1 has shown that CCaP1 affected cytosolic free Ca 2+ (Ca 2+ cyt ) levels and potassium ion (K + ) uptake in guard cells under dark conditions. Our findings indicate that CCA1‐regulated CCaP1 is essential for maintaining Ca 2+ dynamics and normal K + flux in dark‐induced stomatal closure.