Microtubule Plus-End Binding Protein AUGMIN8 Regulates PIN2 Recycling In Arabidopsis

Auxin is an important phytohormone in higher plants and regulates plant growth and development,such as embryonic formation,the plant organogenesis,and morphogenesis.The polar auxin transportation promotes the formation of an auxin gradient which is critical for the regulation of plant organogenesis and morphogenesis.Auxin polar transport between cells replies on auxin transport carriers.PINs proteins are auxin efflux carriers that play important roles in polar auxin transport.Many PIN proteins have polar distribution to the plasma membrane in cells,which is controlled by vesicle trafficking.Microfilaments were reported to be involved in PINs-associated vesicle trafficking.Recently,investigations showed that microtubules may also participate in PINs-associated vesicle trafficking.However,the regulatory mechanism between PINs-associated vesicle tranfficking and microtubules remains unresolved.A previous study from our lab shows that a plant-specific microtubule-binding protein AUG8 is a microtubule plus-end binding protein that regulates hypocotyls growth by promoting microtubule reorientation.GUS activity analysis shows that AUG8 gene is expressed in all tissues and organs in Arabidopsis,including primary roots and lateral roots.In this study,phenotype analysis indicated that the primary roots in aug8 mutant were longer and the density of lateral roots was higher compared with wild-type Arabidopsis.In addition,aug8 mutant exhibited less sensitivity to gravitational stimulus compared to wild type.Auxin redistribution labelled by DR5-GFP was also influenced in aug8 mutant after gravitational stimulus.These results suggested that AUG8 might be involved in the regulation of auxin polar transportation.PIN2 is an auxin efflux carriear involved in auxin polar transportation.The polar localization of PIN2 is regulated by vesicle trafficking.PIN2 localizes at the upper side of epidermal cells(towards stem apex)and the basal side of cortex cells(towards root apex)in roots.BFA treatment and BFA wash-out experiments showed that both endocytosis and exocytosis of PIN2 proteins were affected in aug8 mutant.This indicated that AUG8 participated in the process of PIN2-associated vesicle trafficking.Pull dwon and Co-IP experiments revealed that AUG8 protein binded to SNARE protein SYP61 directly in vitro and in vivo.Fluorescence observation showed that SYP61 and AUG8 colocalized occasionally and moved together in cells.When microtubules were disrupted by oryzalin treatment,SYP61 protein localization and PIN2-associated vesicle trafficking were influenced,indicating that PIN2-associated vesicle trafficking was dependent on microtubules.According to these results,we hypothesize that microtubule plus-end binding protein AUG8 regulates PIN2-associated vesicle trafficking by recognizing SYP61 protein,and then participates in the process of auxin polar transportion.In this study,we discovered that microtubule plus-end binding protein AUG8 mediated the microtubular regulation of PIN2-associated vesicle trafficking to regulate auxin polar transportation,root growth,and root response to gravity.Our finding revealed a new mechanism for the regulation of PIN2-associated vesicle trafficking to control auxin polar transportation,and suggested that microtubules-mediated vesicle trafficking may present as a common mechanism participating in plant development and responding to environment signals.