State Key Laboratory of Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China.
National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
Plant Physiol. 2021 Dec 4;187(4):2192-2208. doi: 10.1093/plphys/kiab099.
Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H+-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.
致密小泡（DVs）是高尔基体衍生的植物特异性载体，介导被子植物中种子贮藏蛋白的高尔基体后运输。这个过程是如何被调控的仍然难以捉摸。在这里，我们报告了一个水稻（Oryza sativa）突变体，名为谷蛋白前体积累 8 号（gpa8），它在成熟胚乳中异常积累 57kDa 的 proglutelins。gpa8 突变体的细胞学分析表明，含有 proglutelin 的 DV 被错误靶向质外体，在发育中的胚乳细胞中形成电子致密聚集体和周质体。与以前报道的具有高尔基体后转运缺陷的 gpa 突变体不同，gpa8 突变体表现出弯曲的高尔基体体、有缺陷的转高尔基网络（TGN）和扩大的 DV，表明 GPA8 在 DV 生物发生中具有特定的作用。我们证明 GPA8 编码液泡 H+-ATPase（OsVHA-E1）的亚基 E 同工型 1，主要定位于 TGN 和液泡膜。进一步的分析表明，gpa8 突变体中转高尔基网络和液泡的内腔 pH 值显著增加。此外，在 gpa8 原生质体中，GPA1 和 GPA3 与 TGN 标记蛋白的共定位明显减少。我们的数据表明，OsVHA-E1 参与内膜腔 pH 稳态，以及高尔基体形态和 TGN 的维持，这是水稻胚乳细胞中 DV 生物发生和随后蛋白质运输所必需的。
