Mechanism Underlying The Vesicle Trafficking Regulator AtSEC22 Controlling Plant Cell Morphogenesis

The cell morphogenesis is regulated by cytoskeleton dynamics.Plant cytoskeleton forms a stereoscopic network structure to guide cell wall deposition and cell expansion,and serves as a track for vesicle trafficking.Membrane fusion between transport vesicles and target membrane is mainly mediated by SNAREs and relating factors.R-SNARE Sec22 functions on anterograde and retrograde transport in ER-Golgi trafficking in yeast and mammals.Plant SEC22 regulates the early secretory pathway and plays an important role in Arabidopsis pollen maturation.However,the regulatory mechanism of SEC22 on plant cell morphogenesis and plant growth and development is unclear.In order to explore SEC22 function in plant growth and development,we used Arabidopsis AtSEC22-down regulated mutant atsec22-4,AtSEC22RNAi,and AtSEC22 overexpression transgenic lines as experimental materials to investigate the molecular mechanism of AtSEC22-regulating plant growth and development,using reverse genetics,cell biology,molecular biology and biochemistry etc.methods.The results are as below:1.The phenotypic analysis indicated that development of atsec22-4 mutant was delayed,the plants were dwarf,root length was shorter,and fertility was significantly decreased.Moreover,the morphology of pavement cells was abnormal,the number of trichomes was significantly reduced and the morphology was altered.The morphology of stomata was altered,number was increased and distribution was uneven in the leaves.These phenotypes indicated that cell morphogenesis was disturbed due to AtSEC22 defects.The phenotype of AtSEC22RNAi plant was similar to that of atsec22-4,but the phenotype of AtSEC22-overexpressing plants was not significantly different from that of wild type.2.Yeast two hybrid and pull down-LC-MS/MS analyses detected interaction between AtSEC22 and Golgi-localized syntaxin AtSYP32,suggesting that AtSEC22 coordinates with AtSYP32 to regulate ER-Golgi vesicle trafficking.Moreover,co-localization analysis indicated that GFP-AtSEC22 was partly co-localized with COPII marker,Sarla-GFP and Sec31B-GFP,suggesting that AtSEC22 may play a role in anterograde transport from the ER to the Golgi apparatus.3.In atsec22-4 cells,newly synthesized storage protein precursors were trapped in the ER lumen,forming a novel cell structure,and inducing severe ER stress.Distribution of the Golgi resident marker,ST-GFP altered obviously in atsec22-4 mutant,part of ST-GFP was re-distributed in the ER,indicating that the protein export from the ER was blocked.Moreover,plasma membrane-localized PIN1-GFP was partially aggregated inside the cells,indicating that the transport between the Golgi and plasma membrane was also affected.All these results suggested that AtSEC22 not only regulated the protein transport between the ER and the Golgi apparatus,probably also regulated the post-Golgi vesicle trafficking.4.The ultrastructural observation indicated that the morphology of the ER and the Golgi apparatus were abnormal.The ER was fragmented and expanded,width of the Golgi stacks became smaller and were composed of less cisternae.These findings indicated that AtSEC22 was essential for the integrity of endomembrane system in plant cells.5.Confocal images revealed that the cortical microtubules(MTs)in epidermal cells of atsec22-4 hypocotyl and leaves became thinner and denser,and the alignment direction also altered.The ratio of transverse,longitudinal and oblique alignment of actin filaments(AFs)altered significantly with increased frequency of thicker actin bundles.All these phenomena suggested that defect of AtSEC22 affected cytoskeletal organization.6.Under treatment of oryzalin,the microtubule depolymerizing agent,microtubule depolymerization was faster in atsec22-4 than that in wild type,and the recovery of microtubule alignment after washing was slower than that in wild type.And under treatment of LatB,the actin filament polymerization inhibitor,AFs were more stable in atsec22-4 mutant than those in wild type,and the recovery of AF alignment after washing was faster than that in wild type.Moreover,under normal conditions,the stomatal aperture in atsec22-4 was larger than that in wild type.Under oryzalin treatment,the stomatal closure in atsec22-4 was faster than that in wild type.These results suggested that the stability of microtubules and AFs in atsec22-4 mutant was altered and stomatal movement was affected.7.Some microtubule-and actin-associated proteins were identified in pull down-LC MS/MS analysis using myc-AtSEC22-overexpressing plants.And the expression levels of most of these protein coding genes altered significantly in atsec22-4.Among them,MAP65-1 significantly decreased not only in transcriptional but also in protein level.These results suggested that AtSEC22 might regulate the alignment and stability of cytoskeleton via affecting the expression of microtubule-and actin filament-associated proteins.In conclusion,AtSEC22 plays an important regulatory role in vesicle traffkcing in plant cells.Moreover,AtSEC22 regulates the organization and stability of cytoskeleton by affecting the expression level of cytoskeleton-associated proteins,thus controlling cell morphogenesis and regulating plant growth and development.