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.