PIN1 Functional Motifs-Mediated Polar Auxin Transport Regulate The Growth And Development Of Arabidopsis Thaliana

The synthesis and distribution of auxin play an important role in plant growth,morphogenesis and responses to environmental stimuli.Its nonuniform distribution between cells and tissues underlies the development events and responses to environmental stimuli.The auxin efflux carriers PIN(PIN-FORMED)-mediated polar auxin transport(PAT)regulates Arabidopsis growth and development.PIN controls auxin transport,which largely depends on PIN activity and polarity.Recent studies have shown that motifs(phosphorylation motifs,endocytic sorting motifs and ubiquitination motifs)in the intracellular loop in PIN proteins can mediate regulation of PIN protein activity and localization in Arabidopsis.Previous studies have shown that the LL53::54?Y145?Y245 site on PIN2 protein of mutant Arabidopsis could not largely effectively rescued the phenotype of pin2 mutant,indicating that the LL53::54?Y145?Y245 site is critical for PIN2 protein to exercise function.Bioinformatic analysis showed that PIN1 protein contain conserved homologous site,including LL53::54?Y145?Y239.To explore the biological functions of the LL53::54?Y145 and Y239 motifs on PIN1 protein in mediating auxin polar transport,we performed reverse genetics,cell biology,and physiological biochemistry assays,combined with site directed mutagenesis,Arabidopsis transgenesis,live cell fluorescence imaging,and yeast two hybrid assays to dissect biological functions of motifs.The specific findings are as follows:(1)The PIN1-GFP expression vector was constructed by Gateway and site directed mutagenesis.The site mutation PIN1-GFP expression vector was obtained by site-directed mutagenesis of LL53::54AA?Y145A and Y239 A.(2)The site mutation PIN1-GFP expression vectors were transformed into pin1 mutant of Arabidopsis thaliana by Agrobacterium tumefaciens mediated flower staining method.Genetic analysis showed that the PIN1-GFP expression vectors with site directed mutations LL53::54AA and Y145 A could not effectively complement the phenotype of pin1 mutant,indicating that LL53::54 and Y145 sites were the key sites for PIN1 protein to perform its function.(3)Cell biology experiments showed that site directed mutation of LL53::54AA and Y145 A reduced the polar localization of PIN1-GFP at the base of plasma membrane in root stele cells,and site directed mutation of LL53::54AA reduced the endocytosis rate of PIN1-GFP,while site directed mutation of Y145 A did not affect the endocytosis process of PIN1-GFP.(4)Yeast two hybrid experiments showed that the 239 fragment(169-259aa)of PIN1 protein interacted with AP3-? subunit of adaptor protein,but the interaction was not related to Y239 site.In summary,the following preliminary conclusions can be drawn: The conserved PIN1 Motifs(LL53::54 and Y145)mediate PIN1 polarity and polar auxin transport and thus regulate the plant growth and development.In addition,the LL53::54 is involved in the regulation of PIN1 endocytosis.These results together provide a new insight in the molecular mechanism underlying PIN activity and polarity,and have a great importance for a deeper understanding of PIN regulation in plants.