Arabidopsis R-SNARE Proteins VAMP721 and VAMP722 Are Required for Cell Plate Formation

BackgroundCell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited. Methodology/Principal FindingsWe report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins. Conclusion/SignificanceThese observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.背景：植物胞质分裂过程中的细胞板形成，依赖于SNARE复合体介导的囊泡在细胞分裂平面处的融合。然而，目前学界对于细胞板形成所需膜融合机制中的R-SNARE组分的认知仍十分有限。 研究方法与主要结果：本研究报道了拟南芥中两个序列高度同源的R-SNARE蛋白VAMP721与VAMP722在细胞板形成过程中的体内功能。双纯合突变体vamp721vamp722的幼苗表现出致死性矮化表型，其根系、子叶与下胚轴发育极不完善。此外，在该双突变体幼苗中常可观察到细胞壁残体与不完全胞质分裂现象。共聚焦显微镜成像结果显示，绿色荧光蛋白（GFP）标记的VAMP721与VAMP722会优先定位在分裂细胞正在扩展的细胞板上。药物处理与共定位分析表明，被VAMP721和VAMP722标记的点状细胞器为早期内体，其与VHA-a1标记的反式高尔基体网络（TGN）存在重合，且该类细胞器与高尔基体堆及前液泡区室存在明显区别。进一步通过标记蛋白的亚细胞定位实验发现，vamp721vamp722双突变体中，蛋白质向质膜的运输过程受到严重破坏，但向液泡的运输未受显著影响。 结论与意义：上述实验结果表明，VAMP721与VAMP722通过反式高尔基体网络/早期内体区室参与蛋白质向质膜的分泌运输途径，这一过程对于植物胞质分裂中的细胞板形成具有重要贡献。