Functional Analysis of AtVAMP714 gene in Arabidopsis

Abstract

An activation tagging screen was carried out to identify gain-of-function mutants showing potential auxin defects, with the aim of identifying the new genes regulating plant development. The conjoined (cnj) mutant isolated from the activation tagged screen exhibits duplication of the seedling axis. Sequencing the insertion locus revealed that the activation tag was positioned between ATP-binding protein and vesicle associated membrane protein genes. Cloned mutant locus and expression studies indicated that the vesicle associated membrane protein gene is upregulated in the activation tagging line. The aim of this work is to investigate the function of the vesicle associated membrane protein gene (AtVAMP714), which is a member of an R-SNARE protein family.
To determine the expression pattern of AtVAMP714, proAtVAMP714::GUS expression in seedlings was examined. GUS activity was observed in the hypocotyl and roots and the strongest expression was observed in the root vascular tissues. The AtVAMP714 gene is positively auxin regulated. VAMP714::GFP fusion protein localized to Golgi vesicles suggesting it may be involved in the Golgi secretory pathway. Auxin transport levels in roots and shoots found to be greatly reduced in AtVAMP714 overexpressors compared to the wild type plants. Quantitative real-time RT-PCR showed that transcript levels of IAA1 and IAA2 were significantly reduced in AtVAMP714 overexpressors. Immunolocalization of PIN1 and PIN2 showed strong defects in localization.
To characterize the development role of VAMP714, SALK null mutants of the AtVAMP714 gene were identified. The mutant phenotype showed an abnormal branching and short root phenotype and PIN1, PIN2 and PIN4 transcript levels were significantly reduced. Dominant negative transgenics of AtVAMP714 were also created as a method of knocking out the function of the protein and F2 generation plants were analysed for developmental defects. Consistent with the above results the dominant negative transgenics showed a short root phenotype with dwarfed, branchy shoots. PIN1 and PIN2 proteins were mislocalized in dominant negative transgenics. The results presented provide evidence for a role of AtVAMP714 in auxin transport through a requirement for correct PIN protein secretion and localization.