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SummaryProtein phosphorylation by kinases is a major molecular switch mechanism involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements by changes in CO2concentration. However, whether MPK12 kinase activity is required for regulation of CO2-induced stomatal responses warrants in depth investigation.We apply genetic, biochemical, and structural modeling approaches to examining the non-catalytic role of MPK12 in guard cell CO2signaling that relies on allosteric inhibition of HT1.We show that CO2/HCO3−-enhanced MPK12 interaction with HT1 is independent of its phosphor-transfer activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line whereMPK12is deleted, we confirmed that CO2-dependent stomatal responses rely on MPK12’s ability to bind to HT1 but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2/HCO3−and present structural modeling that explains the MPK12:HT1 interaction interface.These data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which stomatal guard cells sense changes in atmospheric CO2concentration.

The continuing rise in the atmospheric carbon dioxide (CO

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.