The continuing rise in the atmospheric carbon dioxide (CO 2 ) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO 2 sensor remains unknown. Here, we show that elevated CO 2 triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO 2 , HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO 2 . Dominant HT1 mutants abrogate the CO 2 /bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO 2 sensor function and CO 2 -triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO 2 /bicarbonate sensor upstream of the CBC1 kinase in plants.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO 2 /abscisic acid (ABA) was examined.We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophylldependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO 2 ]-shifts.According to our research, higher [CO 2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO 2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO 2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO 2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants.These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO 2 and ABA.