Involvement of intracellular Ca2+ in blue light-dependent proton pumping in guard cell protoplasts from Vicia faba

Recent studies have suggested that Ca2+/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells, As the increase in cytosolic concentration of Ca2+ is required for the activation of CaM and CaM-like proteins, the origin of the Ca2+ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba. BL-dependent proton pumping was affected neither by Ca2+ channel blockers nor by changes of Ca2+ concentration in tint medium used for the GCPs. Addition of Ca2+ ionophores and an agonist to GCPs did not induce proton pumping, However, BL-dependent proton pumping was inhibited by 10 mill caffeine, which releases Ca2+ from the intracellular stores, and by 10 mu M 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) and 10 mu M cyclopiazonic acid (CPA), inhibitors of Ca2+ -ATPase in the sarcoplasmic and endoplasmic reticulum (ER), Bg contrast, the inhibitions were not observed by 10 pM thapsigargin, an inhibitor of animal ER-type Ca2+-ATPase. The inhibitions by caffeine and BNQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA, From these results, we conclude that the Ca2+ thought to be required for BL-dependent proton pumping may originate from intracellular Ca2+ stores, most likely from ER in guard cells, and that this origin of Ca2+ mag generate a stimulus-specific Ca2+ signal for stomatal opening.