ABA and ABA signaling mediate Arabidopsis stomatal response to CO₂ via H₂O₂ and NO production

The augmentation of atmospheric CO₂ concentrations induces reduction in foliar stomatal aperture, thereby affecting photosynthetic capacity and transpiration- mediated cooling in plants. Elucidating the mechanism through which CO₂ modulates stomatal dynamics is crucial for understanding plant adaptability to future fluctuations in CO₂ levels. Although abscisic acid (ABA) is a pivotal phytohormone regulating stomatal movements, its role in CO₂-induced stomatal closure remains unclear. Here, we demonstrate that elevated CO₂ failed to induce stomatal closure in wild-type Arabidopsis treated with ABA biosynthesis inhibitor fluridon, as well as in the ABA-deficient mutant aba1, aba2 and aba3. Using β-glucuronidase (GUS) staining in pRD29B::GUS transgenic lines, we confirmed that the reporter gene controlled by ABA-responsive RD29B promoter in guard cells was strongly induced by elevated CO₂. Additionally, transcripts of the ABA-responsive gene RAB18 and ABA-synthetic gene ABA2 were upregulated during CO₂-induced stomatal closure in wild-type Arabidopsis. In contrast, fluridonand ABA receptor mutant pyr1pyl1pyl2pyl4 respectively inhibited CO₂-induced activation of RD29B promoter-driven GUS marker and RAB18 expression. The expression of green fluorescent protein (GFP) reporter gene driven by ABA2 and RAB18 promoters in tobacco leaves was enhanced under CO₂, and the fluorescence mainly distributed in non-guard cells. Furthermore, ABA, ABA receptor (PYR/RCARs) and OST1 kinase were identified as upstream cascade components of H₂O₂ and NO in stomatal response to CO₂. Notably, GHR1 was implicated as an intermediate between H₂O₂ and NO in this pathway. Additionally, NADPH oxidase subunits AtRBOHD and AtRBOHF, and nitrate reductase isoenzyme NIA1, were responsible for H₂O₂ and NO production in guard cells under high CO₂ conditions. Together, our findings propose that elevated CO₂ triggers an increase in ABA and activates ABA signaling to close stomata via H₂O₂ and NO production, providing comprehensive insights into CO₂ signaling in guard cells.