MPK12 in stomatal CO 2 signaling: function beyond its kinase activity.

Protein phosphorylation is a major molecular switch 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 CO 2 concentration. However, whether MPK12 kinase activity is required for regulation of CO 2 -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 CO 2 signaling that relies on allosteric inhibition of HT1. We show that CO 2 /HCO 3 - -enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO 2 -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 CO 2 /HCO 3 - 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 guard cells sense changes in atmospheric CO 2 concentration.