Investigation Of The Mechanisms Underlying Arabidopsis SNARE AtSYP71 Regulating Polar Auxin Transport

Auxin is an important plant hormone.The polar transport of auxin is mainly achieved through auxin transporters.Auxin transporters,PIN proteins are key regulators of auxin polar transport and asymmetric distribution.Vesicle transport is critical for polar transport of PIN proteins.Vesicle transport mainly includes:budding of transport vesicles,directional movement,tethering,anchoring and membrane fusion.Tethering,anchoring and membrane fusion mediated by SNAREs,SM proteins,Rabs and tethering factors,etc.are critical step for vesicle transport.Among them,SNARE proteins are key factors for anchoring and membrane fusion.SNARE proteins include Q-SNAREs on the target membrane and R-SNAREs on the transport vesicles.Q-SNAREs include Qa-SNAREs,Qb-SNAREs and Qc-SNAREs.In Arabidopsis,ER-localized Qa-SNAREs and Qb-SNAREs are known,while Qc-SNAREs are uncertain.The plant-specific S YP7 family proteins are candidates of Qc-SNAREs.In this study,atsyp71-1,atsyp71-2,atsyp71-3,atsyp71-4 mutants and SYP71-overexpressing plants were used as experimental materials.The molecular mechanisms underlying AtSYP71 regulating auxin transport was preliminarily investigated.The results are below:1.Four mutants are isolated in this study.AtSYP71 protein level in atsyp71-1 mutant is slightly down-regulated,those in atsyp71-2 and atsyp71-4 mutants are significantly down-regulated,and AtSYP71 protein is completely depleted in atsyp71-3 mutant.2.The root length of 7-day-old seedlings of atsyp71-1 is slightly shorter than that of wild type;the root length of atsyp71-2 and atsyp71-4 are significantly shorter than those of wild type,indicating that the more AtSYP71 protein reduced,the shorter the root length becomes.atsyp71-3 mutant in which SYP71 protein is completely depleted is homozygous lethal.These phenotypes indicate that AtSYP71 is essential for plant growth and development.3.The expression levels of DR5::GUS and DR5::RFP in the root tips of atsyp71-2 mutant are higher than those of wild type,while the expression levels of DII-Venus decreased more than that of wild type,indicating that the auxin accumulation in the atsyp71-2 mutant are higher than that in wild type.4.The expression levels of PIN3-GFP in the root tips of atsyp71-2 and atsyp71-4 mutants are higher than those of Col-0,indicating that AtSYP71 regulates auxin distribution via regulating distribution of auxin transporter,PIN3,as well.5.Under 70 nM NAA treatment,the root length of Col-0,atsyp71-2 and SYP71-overexpressing plants are shortened.While,the root length of atsyp71-2 is shorter than that of wild type,indicating that atsyp71 mutant is insensitive to exogenous auxin compared with wild type.Under 3 ?M NPA conditions,the root length of Col-0,atsyp71-2 and SYP71-overexpressing plants are shortened.While,the root length of atsyp71-2 is shorter than that of wild type,indicating atsyp71 mutant is more sensitive to auxin transport inhibitors.This indicates that the atsyp71 mutant has defects in exogenous auxin response.6.Under normal conditions,the expression levels of DR5::GUS and DR5::GFP in the root tips of atsyp71-2 mutants are significantly higher than those of wild type.Under 70 nM NAA treatment,expression levels of DR5::GUS and DR5::GFP in Col-0 and atsyp71-2 root tips increased,but increased range in atsyp71-2 is smaller than that in wild type,and the final expression level in atsyp71-2 is lower than that in wild type.Under 3 ?M NPA treatment,the expression levels of DR5::GUS and DR5::GFP in Col-0 and atsyp7l-2 root tips are increased,and the expression level in atsyp71-2 is significantly higher than that in wild type.These results indicate that the defective auxin transport in atsyp71-2 affect response to exogenous auxin and inhibitor.