Microtubule Growth-Driven Vesicle Trafficking Regulates Arabidopsis Stomatal Movement

The respiration and photosynthesis of plant are regulated by stomatal opening and closure.Guard cells control the stomatal movement in response to various stimulations.Study of the mechanisms of stomatal movement has scienctic importance and potential applicaitons for the improvement of plant resistance to stress.The ion channels and transports that control the ion efflux/influx of guard cells also play important roles in stomatal movement.In addition to regulation of the activity of ion channels and transports,the redistribution of channels and transports by endocytosis and exocytosis plays important role in stomatal movement.Microtubules are involved in stomatal movement.The regulation of the microtubule organization and dynamics is involved in stomatal movement in response to light.Also,the role of microtubule in vesicle trafficking have been reported.Therefore,investigation of the relationship between microtubules and vesicle trafficking will deepen our understanding of the mechanisms controling stomatal movement.Arabidopsis AUGMIN subunit 8(AUG8)is a microtubule-binding protein that localizes at the plus end of microtubules,functioning in regulating cell growth in Arabidopsis hypocotyls in response to light.GUS activity assay and bioinformation data showed that AUG8 is expressed in guard cells,suggesting that AUG8 may also be involved in stomatal movement.Therefore,the reaseach was carried out in this study.In this study,observations showed that stomatal opening was delayed in the aug8 mutant during light treatment,suggesting that AUG8 is required for normal stomatal opening.Infrared thermal imaging analysis showed that leaf temperature of aug8 mutant was higher than wild-type leaves,indicating that transpiration rate was reduced in aug8 mutant.These data indicate that AUG8 functions in light-induced stomatal opening.Further observations showed that AUG8 also localized at the plus end of growing microtubules in guard cells.To further reveal the relationship of microtubules and vesicle trafficking during stomatal movement,the plasma membrane(PM)distribution of KAT1 in guard cell was observed.The PM redistribution of KAT1 was delayed in aug8 guard cells during stomatal opening induced by light,some KAT1 protein was detained in the cytosol of guard cells.Previous research indicated that SYP121 facilitates stomatal reopening and KAT1 distribution in guard cells,suggesting that the functions of AUG8 and SYP121 may be related during stomatal opening.Further biochemistry and cell biology assays were performed to test the relationship of AUG8 and SYP121 during stomatal opening.The results of in vitro pull-down,yeast two-hybrid assay,and Co-Ip assay indicated that AUG8 directly interacts with SYP121.Observations of the sub-celluar localization of SYP121 showed that the PM distribution of SYP121 in guard cells relied on AUG8 and microtubules during light-induced stomatal opening.Knockdown of AUG8 or disruption of microtubules resulted in a reduction of SYP121 at the PM,and more SYP121-associated vesicles were detected in the cytosol of guard cells.Moreover,the exocytosis of SYP121 was delay in aug8 mutant,suggesting that AUG8 is involved in the exocytosis of SYP121 to the PM.Further analysis showed that SYP121-associated vesicles could co-localize with AUG8 at the growing ends of microtubules,trafficking along with the growth of microtubules,and transported to the PM for the exocytosis.This study revealed that microtubule-plus-end binding protein AUG8 is involved in light-induced stomatal opening by regulating the exocytosis of SYP121-associated vesicles that affects stomatal opening.In conclusion,this study proposed a novel a mechanism of microtubule-based vesicle trafficking powered by microtubule growth,which has significant importance for revealing the regulation of stomatal movement.