Abstract: INTRODUCTION: Continuously increasing atmospheric CO₂ concentrations 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 the 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 the CO₂ response.
RESULTS: (1) Pertussis toxin (PTX, Gα inhibitor) and gpα1 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 gpα1 on NaHCO₃-induced stomatal closure. c-PTIO (NO scavenger), Na₂WO₄ (nitrate reductase inhibitor), and the nia1-2 mutant all disrupted NaHCO₃-induced stomatal closure and guard cell NO generation, effects that 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 the 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 that 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