Microtubules and heterotrimeric G-protein regulate ABA-induced stomatal movement synergistically

Abstract

Stomata, formed by a pair of specialized guard cells, are present on the epidermis of most plant leaves. The regulation of stomatal apertures controls the exchange of moisture and gas between plants and the environment, and balances the carbon dioxide (CO2) entry into the plant for photosynthesis with water loss via transpiration. G-protein, a ubiquitous signaling molecule in eukaryotic cells, is an indispensable participator in the mechanism of stomatal movement. It was widely recognized that G-protein α subunit (GPA1) can regulate a majority of signals during stomatal movement, such as Ca2+, reactive oxygen species (ROS), H2O2, ethylene, and so on. On the other hand, guard cell microtubule (MT) dynamics play a comparably critical role in regulating stomatal movement, and MTs rearrangement has been observed in stomatal responses to a variety of stimuli, like light, dark, nitric oxide (NO). Both heterotrimeric G-protein and MTs are essential for abscisic acid (ABA) signaling in guard cells, but whether and how these two signaling components work together is poorly understood.Here, the interplay between MTs and G-protein during ABA-induced stomatal movement was investigated in Arabidopsis thaliana .It was found that the heterotrimeric G-protein α subunit mutant, gpa1 , had a significantly higher water loss ratio than the wild type under the same conditions.Stomatal bioassay analyses revealed that guard cell sensitivity to ABA was attenuated in gpa1 , and the mutant′s ABA-insensitive phenotype could be partially restored by Oryzalin, an MT-specific inhibitor. However, exogenously applied Ca2+ chelator BAPTA-AM together with Oryzalin impaired the inhibition of stomatal opening by ABA. These results suggest that both MTs dynamics and Ca2+ flux may participate in the process of G-protein-mediated regulation of ABA-induced stomatal movement. Confocal microscopic images showed that after ABA treatment, MTs arrays in wild type underwent rapid disruption from radial arrays to disassembled fragments, whereas those in gpa1 largely remained bundled.A similar behavior was observed in BAPTA-AM plus ABA treatment, which is consistent with the stomatal bioassay results. In other words, G protein can control MTs rearrangement by adjusting the Ca2+ level in ABA-induced stomatal movement.In addition, non-invasive micro-test (NMT) demonstrated that the ABA-induced transmembrane Ca2+ flux was abolished in gpa1 guard cells, but Oryzalin enhanced the ABA response; Ca2+ efflux was suppressed dramatically when Oryzalin was added in gpa1. Therefore, our data clearly indicate that it exists a possitive crosstalking among G-protein, MTs and Ca2+ during ABA-induced stomatal movement. In this signal pathway, both MTs and Ca2+ act downstream of G-protein. Intracellular Ca2+ of guard cells relays signal information from G-protein to MTs; in turn, the changes of MTs dynamics in guard cells also have a feedback to Ca2+ flux. Finally the ABA signalling transduction results in stomatal movement, as well as the regulation of transpirational water loss and drought resistance.