Functional Studies Of SNARE Proteins VAMP721 And VAMP722 In Intracellular Transport Of Auxin Transporters

SNARE proteins mediate the complex vesicle trafficking by which the dynamic protein delivery is accomplished in endomembrane system of living cells.In plant cells,the research on SNARE protein mainly focuses on Q-SNARE protein,and there are relatively less reports on the roles of R-SNARE protein in vesicle transport and its physiological function.Previous studies have shown that the dynamic transport of auxin transporters in plant cells regulates the flow direction and gradient distribution of auxin in tissues.The polar localization of auxin transporters on the plasma membrane is regulated by vesicle-mediated endocytosis and recycling trafficking.At present,researches on the regulation of intracellular transport of auxin transporters have mostly focused on upstream regulatory proteins,such as RabGTPase,ARF,ARF-GEF and so on.However,it is unclear how the SNARE protein is involved in the intracellular transport of auxin transporters.In this paper,we used Arabidopsis thaliana as an experimental system to study the role of R-SNARE proteins VAMP721 and VAMP722-mediated vesicle trafficking in the polar localization and intracellular transport of auxin transporters.The main findings of our study are listed as follows:1.First,we observed and analyzed the phenotypes related to auxin polar transport defects in vamp721vamp722 homozygous double mutants.The results showed that the cotyledon development of homozygous double mutant seedlings was abnormal,resulting in a single,three,fused cotyledon.The leaf vein appeared many abnormalities such as fusion,disorder,irregularity,and discontinuity.The arrangement of starch grains in root cap was disordered.In addition,the lateral root formation induced by NAA was seriously inhibited in vamp721vamp722 mutant.According to the responses of DR5rev: 3XVENUS-N7 and DR5rev: GFP reporters to auxin treatment,it was found that the distribution of auxin in the homozygous double mutant was affected compared with that in wild type,indicating that VAMP721 and VAMP722 play an important role in auxin polar transport.Moreover,the expression patterns of the transcription factors SCR,SHR and WOX5 in the root tissue of homozygous double mutants were changed.Combined with the phenotype of auxin trafficking defects previously reported,our results suggest that VAMP721 and VAMP722-mediated vesicle trafficking is involved in polar auxin trafficking.2.We investigated the role of VAMP721 and VAMP722 in the subcellular localization and intracellular transport of auxin transporters.The abnormal subcellular localization of the auxin efflux transporters PIN1,PIN2 and the influx transporter AUX1 in the vamp721vamp722 mutant indicates that the change in the auxin distribution in the homozygous double mutant is due to the disruption of the polar localization of the auxin transporters.Secondly,the results of fluorescence recover after photobleaching(FRAP)and treatment with vesicle trafficking inhibitor BFA showed that the vamp721vamp722 mutant affected the transport of PIN2-GFP to the plasma membrane.In addition,Wm and dark treatment showed that homozygous double mutations did not affect the transport of PIN2-GFP to vacuoles.Our results indicate that VAMP721 and VAMP722 mediate the transport of auxin transporters,such as PIN2 protein,from cytoplasm to the plasma membrane.3.VAMP721 and VAMP722 participate in maintaining the structure of TGN.The intracellular localization of organelles labeled with fluorescent proteins showed that YFP-RabA2 a and SYP61-CFP-labeled TGNs partially formed aggregation in different degrees in the mutant root cells without affecting the morphology of Golgi and prevacuolar marker proteins,indicating that VAMP721 and VAMP722 are involved in maintaining the structure of TGN.Previous studies have shown that VAMP721 and VAMP722 are localized in TGN and are involved in the secretory transport of PM proteins.Therefore,our results suggest that the vamp721vamp722 mutant may partially affect the sorting transport of auxin transports from TGN to the plasma membrane by changing the structure of TGN.In summary,we make the following conclusions:(1)VAMP721 and VAMP722 proteins participate in auxin polar transport;(2)VAMP721 and VAMP722-labeled vesicles are loaded with auxin transporter cargo proteins;(3)VAMP721 and VAMP722-mediated vesicle transport is involved in the transport of PIN2 to the plasma membrane;(4)VAMP721 and VAMP722 play roles in maintaining the structure of partial TGN,and then likely regulate the sorting transport of auxin transporter from TGN to plasma membrane.