Arabidopsis Circadian Clock Protein CCA1 Negatively Regulates the Calcium-Binding Protein CCaP1 to Modulate...

Stomatal conductance, which is crucial for plant carbon assimilation and water regulation, is modulated by Ca²⁺-binding proteins (CBPs). These proteins mediate Ca²⁺ 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 Ca²⁺-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 Ca²⁺ probe R-GECO1 has shown that CCaP1 affected cytosolic free Ca²⁺ ([Ca²⁺]_cyt) levels and potassium ion (K⁺) uptake in guard cells under dark conditions. Our findings indicate that CCA1-regulated CCaP1 is essential for maintaining Ca²⁺ dynamics and normal K⁺ flux in dark-induced stomatal closure.