Functional Analysis Of Arabidopsis Gene VAMP714 And Phylogenetic Analysis Of SNARE Proteins

The plant hormone auxin and its directional intercellular transport play a major role in diverse aspects of plant growth and development,including the formation of primary and lateral apices,the differentiation of vascular tissue,tropisms,and embryo development.Auxin efflux carrier-PIN-FORMED(PIN)proteins determine the directionality of intercellular auxin flow.An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes,providing a mechanism for dynamic localization,this is turn requires the regulated movement and fusion of membrane systems with their associated cargo.N-ethylmaleimide-sensitive factor adaptor protein receptors(SNAP receptors,SNAREs)mediate fusion between vesicles and target membranes and are classed as Q-or R-SNAREs based on their sequence.To date,very little is know about the function of R-SNARE VAMP714(Vesicle Associated Membrane Protein 714).Here we demonstrate that an R-SNARE-dependent exocytic pathway is essential for the insertion of PINs into the plasmamembrane from the Golgi,through the analysis of vamp714 T-DNA insertional mutants,dominant negative mutants and vamp714 over-expressors.And using recent public expression and transcriptomic data from nine representative green plants,we investigated the evolution of the SNARE classes and linked protein changes to expression patterns.The concrete results are as following:1.Seedlings of all vamp714 related mutants were smaller than wildtype and showed a reduced root system,less erect,dwarfed and excessive branching phenotype,and altered root gravitropism and morphogenesis.And vamp714 related mutant roots reveals a more disorganized tissue patterning compared with Col-0.And compared to wildtype,both the roots of vamp714 mutant and misexpressing seedlings exhibit the disorganized columella and lower auxin expression.The spatial expression pattern of the VAMP714 gene was examined in seedlings expressing a promoter reporter fusion(pro VAMP714::GUS)using histochemical localization of GUS activity.In the absence of exogenous auxin,GUS activity was detected in roots vascular tissues and cortex,and at relatively low levels in cotyledon veins and the QC of roots but not in leaf and root tips.While exogenous auxin induced GUS activity in root tips,and in cotyledon vascular tissues and young leaf veins.2.vamp714 T-DNA insertional mutants,dominant negative mutants and vamp714 over-expressors roots showed an abnormal patterning of columella cells,lacking both the columella tier and specification of the QC,and the columella stem cells showed evidence of differentiation(starch accumulation),suggesting a failure of QC activity.And we investigated that the transcript levels of the meristem genes SHORTROOT(SHR)and SCARECROW(SCR)were reduced in all three vamp714 mutants.VAMP714 and also of related gene family members genes expression is upregulated by auxin.And the transcript levels of auxin-regulated genes PIN1,PIN2 and PIN4 genes were reduced in all vamp714 related mutants.3.All VAMP714 fusions locate to vesicles and plasma membrane localization seen in both tobaco leaf and stable transformants.All three mutants exhibit aberrant PIN1 and PIN2 localization.In transgenic plants expressing low levels of VAMP714:m Cherry,both PIN1:GFP and VAMP714:m Cherry,and PIN2:GFP and VAMP714:m Cherry,co-localize at the plasmamembrane.By using vesicle-trafficking inhibitor-Brefeldin A(BFA),we found that VAMP714 and PIN accumulation in BFA bodies does not occur in vamp714 T-DNA insertional mutants,dominant negative mutants and vamp714 over-expressors.This suggests that VAMP714 is required for PIN endosome recycling.And by using latrunculin B(Lat B),we found that VAMP714 associated vesicle occur in cytoplasm.These results showed that VAMP714,PIN1 and PIN2 exhibit the same BFA body formation and recycling from BFA compartments to plasma membrane,VAMP714 forms part of both the exocytic vesicle trafficking pathway and the actin dependent endocytic recycling pathway,which together regulate PIN protein concentrations at the plasma membrane.4.The analysis of phylogenetic relationships between different classes of SNARE proteins across plant species.We identified two Qc-SNARE homologues,AT1G16225 and AT1G16230;and an R-SNARE homologue,AT3G25013 in Arabidopsis.Each SNARE class could be broken down into subclades,many of which had genes representating all studied species,attesting to their ancient evolutionary origins.To understand the evolutionary relationships between the functional domains of different Arabidopsis SNAREs.The results of this analysis are presented where the 46 protein motifs identified across all Arabidopsis SNAREs are presented alongside maximum likelihood trees of each of the gene classes to highlight evolutionary patterns of loss and gain of motifs.The expression of 64 SNARE protein-coding genes in 79 organs and developmental stages of Arabidopsis shows that SNARE-encoding genes show a wide range of expression levels and distinct regulation during Arabidopsis development.5.We compared genetic distances to expression differences within and between each gene class.Except Qa,no significant positive relationships were found at either the within-or between gene class levels suggesting that the hypothesis of rapid sub-functionalization of gene duplicates cannot be ruled out within-or between gene class.We also found that all SNARE classes have expanded in number to a greater or lesser degree alongside the evolution of multicellularity.Thus,we proposed that the vesicle associated membrane protein gene AtVAMP714 plays an essential role in the auxin transport and signalling,and VAMP714 is essential for the transport and localization of PIN proteins to the plasma membrane.The innovation points of this thesis are: Our systematically research showed that VAMP714 is required for both exocytosis of PIN vesicles to the plasma membrane and for PIN cycling between the plasma membrane and endosomes,and that this forms part of a positive regulatory loop in which auxin activates a VAMP714-dependent PIN/auxin transport system for the first time.And this is the first thesis to systematically study phylogenetic relationships of the whole SNARE family proteins.Our phylogenetic analysis provides insights into patterns and drivers of SNARE family gene diversification across green plants.Understanding these phylogenetic relationships will help address our understanding of the evolution of SNARE function.The work provides a platform for future research into the precise functions of these proteins in plant development and responses to the environment.