Vesicular Trafficking Mediates Stomata Movement In Response To Environmental Stimuli

The opening and closure of a pair of guard cells, i.e. stomatal movement, plays crucial roles in maintaining sustainable environment by mediating water use efficiency, photobiosynthesis, responses to biotic and abiotic stresses of terrestrial plants. Environmental cues, including light conditions, CO2 concentrations, humidity, and most importantly, the phytohormone abscisic acid(ABA), all affect stomatal movement. Among the environmental factors, ABA and light have been studied most intensively for their roles in stomatal movement. Specifically, ABA promotes stomatal closure whiles light induces stomatal opening.Stomatal movement is not only important for plant fitness, but also serves as an excellent system to answer fundamental questions, such as cellular responses to environmental stimuli, ion homeostasis, endomembrane dynamics, and so on. Membrane transporters, such as calcium channels, potassium channels, and proton pumps, play crucial roles in stomatal movement by regulating the homeostasis of K+, H+, Cl-, NO3- and so on. As transmembrane proteins, transporters are subject to the regulation of vesicular trafficking. Eukaryotic cells contain complex endomembrane compartments whose resident proteins are delivered through vesicles. Vesicle trafficking plays critical roles in determining the subcellular localization of proteins within the endomembrane system. The reward of Nobel prize(2013) to three researchers who discovered key components regulating vesicle trafficking exemplified the importance of vesicle trafficking in eukaryotic cells. Small GTPases, including Arf GTPases and Rab GTPases, are key players regulating vesicle trafficking. Small GTPases act as molecular switches that convert between a GDP-bound inactive form and a GTP-bound active form. Genetic approaches, i.e. expressing dominant negative forms of small GTPases, together with pharmacological approaches, revealed that Arf1 mediates post-Golgi trafficking while Rab5 mediates a Golgi to vacuolar trafficking route. However, the role of vesicle trafficking on environment-stimulated stomatal movement is obscure.This study applied both genetic and pharmacological approaches to dissect the role of vesicle trafficking on ABA-induced stomatal closure and light-induced stomatal opening. Specifically, this study focused on two pathways, i.e. Arf1-mediated post-Golgi secretion and Rab5-mediated vacuolar trafficking. In addition, a key plasma membrane H+-ATPase(AHA1) was examined for its dynamic membrane localization upon environmental stimuli.Results and Conclusions:(1) The expression of Arf1-DN specifically in guard cells severely compromised plant growth. The guard cells of the transgenic plants were defective in light-induced stomatal opening. This result suggested that Arf1-mediated post-Golgi secretion plays a key role in stomatal movement.(2) The expression of Rab5-DN specifically in guard cells did not impair plant growth. Although fully opened stomata of Rab5-DN transgenic plants have reduced aperture, its movement nevertheless responded to ABA and light comparably with wild type. This result suggested that Rab5-mediated vacuolar trafficking does not play a major role in ABA- and light-induced stomatal movement.(3) Upon light stimulation, AHA1 showed strong labeling at the plasma membrane(PM) of wild-type guard cells. By contrast, the expression of Arf1-DN significantly caused a reduced PM association of AHA1 upon light. On the other hand, the expression of Rab5-DN did not substantially affect the light-induced PM accumulation of AHA1. These results suggested that light-induced PM accumulation of AHA1 relies on Arf1-mediated post-Golgi secretion but not Rab5-mediated vacuolar trafficking.(4) In addition to genetic manipulation, pharmacological approaches were applied to investigate the effect of vesicle trafficking routes to the dynamic localization of AHA1. Brefeldin A(BFA) causes the accumulation of trans-Golgi network/early endosomes(TGN/EE) into BFA compartments whereas wortmannin specifically inhibits the fusion of prevacuolar compartments(PVC) with the tonoplast. BFA caused the accumulation of AHA1 into BFA compartments, indicating that AHA1 is present at the TGN/EE. BFA washout resulted in vacuolar delivery of AHA1-positive signals, indicating that internalized AHA1 underwent vacuolar degradation. Light-induced PM association of AHA1 is inhibited by BFA, suggesting that BFA-sensitive post-Golgi trafficking contributes to AHA1 accumulation at the PM upon light induction.To summarize, results reported here demonstrated a key role of post-Golgi secretion on stomatal movement in response to ABA and light. Specifically, stomatal movement and AHA1 dynamic targeting rely on Arf1-mediated, BFA-sensitive vesicle trafficking but not Rab5-mediated vacuolar trafficking.