MPK12 in stomatal CO₂signaling: function beyond its kinase activity

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.