Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2

Extracellular calmodulin (ExtCaM) exerts multiple functions in animals and plants, but the mode of ExtCaM action is not well understood. In this paper, we provide evidence that ExtCaM stimulates a cascade of intracellular signaling events to regulate stomatal movement. Analysis of the changes of cytosolic free Ca2+ ([Ca2+](cyt)) and H2O2 in Vicia faba guard cells combined with epidermal strip bioassay suggests that ExtCaM induces an increase in both H2O2 levels and [Ca2+](cyt), leading to a reduction in stomatal aperture. Pharmacological studies implicate heterotrimeric G protein in transmitting the ExtCaM signal, acting upstream of [Ca2+](cyt) elevation, and generating H2O2 in guard cell responses. To further test the role of heterotrimeric G protein in ExtCaM signaling in stomatal closure, we checked guard cell responses in the Arabiclopsis (Arabidopsis thaliana) Ga-subunit-null gpa1 mutants and cGalpha overexpression lines. We found that gpa1 mutants were insensitive to ExtCaM stimulation of stomatal closure, whereas cGalpha overexpression enhanced the guard cell response to ExtCaM. Furthermore, gpa1 mutants are impaired in ExtCaM induction of H2O2 generation in guard cells. Taken together, our results strongly suggest that ExtCaM activates an intracellular signaling pathway involving activation of a heterotrimeric G protein, H2O2 generation, and changes in [Ca2+](cyt) in the regulation of stomatal movements.