Stomatal opening is induced by red light as well as blue light. Recently, we established an immunohistochemical technique using whole leaves to study plasma membrane (PM) H+-ATPase in guard cells, which is an important enzyme driving stomatal opening. Our technique revealed that red light illuminated to whole leaves induces photosynthesis-dependent phosphorylation of C-terminal penultimate residue of PM H+-ATPase, threonine, in guard cells, which has been considered to be important for activation of PM H+-ATPase, and we proposed that red light promotes stomatal opening via activation of PM H+-ATPase in guard cells in whole leaves. Here, using our new immunohistochemical technique, we investigated fluence rate dependence of red light-induced phosphorylation of PM H+-ATPase. We found that illumination of red light at 50 µmol m-2 s-1, which was suggested to initiate photosynthesis, saturates phosphorylation of PM H+-ATPase. Furthermore, we immunohistochemically confirmed decrease in the amount of PM H+-ATPase protein in a knock-out mutant of AHA1, an isogene encoding the major isoform of PM H+-ATPase in guard cells, implying the importance of AHA1 as the major PM H+-ATPase protein in guard cells for light-induced stomatal opening.
Keywords: guard cells; immunohistochemistry; phosphorylation; photosynthesis; plasma membrane H-ATPase; red light.