MPK12 in stomatal CO2 signaling: function beyond its kinase activity

Abstract

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 caused by changes in CO₂ concentration. 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 noncatalytic role of MPK12 in guard cell CO₂ signaling that relies on allosteric inhibition of HT1. We show that CO₂ /HCO₃ ^- -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 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 guard cells sense changes in atmospheric CO₂ concentration.

Keywords: CO2 signaling; HT1; MAP kinase 12; allosteric inhibition; guard cell; noncatalytic activity; stomata.