The microtubule-associated protein WDL3 mediates ABA-induced stomatal closure in Arabidopsis

Stomatal movements control CO2 uptake for plant photosynthesis and water loss by transpiration, then determine plant productivity and water utilization efficiency. The microtubule dynamics is widely recognized to be essential for guard cell function. However, the molecular mechanisms underlying this process remain largely unknown. WDL3 belongs to the microtubule-associated protein WAVE-DAMPENED 2(WVD2)/WVD2-LIKE (WDL) family, which binds to and stabilizes microtubules against low-temperature and dilution-induced depolymerization. In the presence of WDL3, tubulins assemble into large microtubule bundles in vitro, otherwise only single filament patterns form. It has been intensively investigated that WDL3 participates in COP1(CONSTITUTIVE PHOTOMORPHOGENIC 1)-mediated hypocotyl cell elongation in darkness, but whether and how it modulates the microtubule behaviors during guard cell signaling transduction is still an open question. In this study, we dissected the interplay among WDL3, microtubule dynamics and Ca2+ in ABA-induced stomatal closure. We found that transpirational water loss from detached leaves occurred slowly in the WDL3 RNA interference transgenic line. Stomatal bioassay revealed that guard cell sensitivity to ABA was promoted in the WDL3 RNAi seedlings, and this phenotype could be partially blocked by paclitaxel, a microtubule stabilizing agent. On the other hand, the microtubule-disrupting drug oryzalin, enhanced ABA-triggered stomatal closure even further, and the WDL3 RNAi guard cells were more sensitive to oryzalin treatment. Based on the pharmacological results above, we next tested the effect of WDL3 on cortical microtubules in guard cells directly by Confocal microscopy. The differences in the configurations of microtubule filaments between WDL3 RNAi and wild type (WT) were analyzed after ABA treatment. Microscopic images showed that ABA-induced microtubule disassembly took faster in WDL3 RNAi guard cells than in WT, and the extent of filament bundling decreased significantly in WDL3 RNAi seedlings, as evaluated by the Image J software. This was consistent with the rapid ABA-induced stomatal closing in WDL3 RNAi material. Moreover, we examined the potential role of Ca2+ in this signal pathway. The cytosolic Ca2+ chelator-BAPTA generally alleviated ABA effects in both WT and WDL3 RNAi materials. ABA-induced stomatal closure and microtubule remodeling were much more delayed in WDL3 RNAi seedlings, indicating that Ca2+ acted upstream in the WDL3 mediated-ABA signaling pathway for stomatal movement. In addition, when exogenous ABA was applied, the Ca2+ influx monitored by the non-invasive micro-test technique (NMT) in WDL3 RNAi guard cells was more pronounced than in WT. Taken together, our results suggest that WDL3, probably coordinated with Ca2+, is involved in the precise regulation of microtubule architecture and dynamics, to accurately execute ABA-stimulated signaling transduction during stomatal movement. Thus it contributes to the proper control of leaf transpiration.