Stomatal CO 2 /bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity requiring MPK12/MPK4

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.