Abstract: INTRODUCTION: The continuously increasing atmospheric CO2concentration affects plant stomatal movement and water use efficiency. Unveiling the molecular mechanisms underlying CO2-regulated stomatal closure is of great significance for understanding plant adaptation to high CO2 environments and ensuring agricultural productivity. Previous studies have indicated that Gα protein, carbonic anhydrase (CA), and nitric oxide (NO) are all involved in high CO2-induced stomatal closure, but the signaling relationships among these three components remain unclear. RATIONALE: Gα mediates stomatal movement in response to various stimuli by regulating NO generation in guard cells; CA is a key enzyme for CO2 perception in guard cells and participates in early CO2 signal transduction; NO acts as a gaseous signaling molecule crucial for stomatal movement, with its synthesis primarily dependent on nitrate reductase NIA1. Previous research suggests that Gα may interact with CA, and both likely function upstream of NO to jointly regulate CO2 response. RESULTS: (1) Pertussis toxin (PTX, Gα inhibitor) and gpa1 mutants impaired NaHCO3-induced stomatal closure and NO production in guard cells of wild-type plants, but not in cGα and wGα lines. Sodium nitroprusside (SNP, NO donor) eliminated the blocking effects of PTX and gpa1 on NaHCO3-induced stomatal closure. c-PTIO (NO scavenger), Na2WO4(nitrate reductase inhibitor), and nia1-2 mutant all disrupted NaHCO3-induced stomatal closure and guard cell NO generation, effects which were not reversed by cholera toxin (CTX, Gα activator). Furthermore, NaHCO3 failed to induce stomatal closure and NO production in guard cells of cGα/nia1-2 and wGα/nia1-2. These results indicate that Gα is located upstream of NO. (2) The CA inhibitors acetazolamide (AZ) and ethoxzolamide (EZ), as well as ca1ca4 mutant, disrupted NaHCO3-induced stomatal closure and guard cell NO generation, while SNP restored NaHCO3-induced stomatal closure in these samples. These results demonstrate that CA is also situated upstream of NO. (3) The failure of NaHCO3 to induce stomatal closure and guard cell NO generation in wGα/ca1ca4 and cGα/ca1ca4 was consistent with the phenotype of ca1ca4 but opposite to that of wGα and cGα. This indicates that CA is genetically located downstream of Gα. CONCLUSION: This study elucidates a sequential signaling pathway of Gα-CA-NO, which collectively regulates high CO2-induced stomatal closure in Arabidopsis. These findings deepen our understanding of CO2 signal transduction in guard cells and provide a theoretical basis for modulating crop adaptation to future high-CO2 environments.

Key words: CO2, carbonic anhydrase, G protein, Nitric oxide, stomatal movement