Gα Protein Mediates High‐Concentration CO₂‐Induced Stomata Closure Through Interaction With Carbonic Anhydrase to Promote Guard Cell Hydrogen Peroxide Production in Arabidopsis

Guard cells detecting fluctuations in ambient CO₂ levels and modulating stomatal aperture constitute a crucial adaptive response to changes of atmospheric CO₂ in plants. CO₂ sensing in animals is closely related to G proteins. Nonetheless, the role of G proteins in high-concentration CO₂-regulated stomatal movement in plants remains unclear. Using NaHCO₃ as a CO₂ source, the results of this study showed that NaHCO₃-induced stomatal closure and guard cell hydrogen peroxide (H₂O₂) production in wild type plants were disrupted by Gα inhibitor pertussis toxin (PTX) and in mutants gpa1, cGα/DF, and wGα/DF but not in cGa and wGa lines. Furthermore, NaHCO₃ upregulated the expression of GPA1 during the early phases of NaHCO₃-induced stomatal closure. Additionally, Gα activator cholera toxin (CTX) failed to rescue NaHCO₃-induced stomatal closure in wild type plants treated with carbonic anhydrase (CA) inhibitors EZ and AZ, as well as in ca1ca4 mutant. NaHCO₃ also did not induce stomatal closure in cGα/ca1ca4 and wGα/ca1ca4 mutants. Notably, NaHCO₃-increased CA enzymatic activity was abolished in wild type plants treated with PTX and in gpa1 mutant but remained unaffected in cGa and wGa lines. Moreover, LCI, Pull-down and Co-IP assays displayed that GPA1 interacts with CA1 and CA4. And, NaHCO₃-induced guard cell H₂O₂ production impaired by ca1ca4, cGα/ca1ca4 and wGα/ca1ca4 mutants. These findings demonstrate that Gα subunit GPA1 mediates high-concentration CO₂-induced stomatal closure by interaction with CA1 and CA4 to promote H₂O₂ accumulation in Arabidopsis guard cells. This insight advances our understanding of CO₂-governing stomatal movement in plants.