BACKGROUND Seed size has been extensively studied in crop plants, as it determines crop yield. However, the mechanism of seed development remains elusive. In this study, we explored the mechanism of seed development in rice (Oryza sativa L.), and identified proteins affecting seed size. RESULTS Proteomic analysis showed that glyceraldehyde 3-phosphate dehydrogenase, chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) accumulated to high levels in the seeds of the natural japonica rice mutant Oochikara, which carries a loss-of-function mutation in the grain width 2 (GW2) gene; GW2 encodes a RING-type E3 ubiquitin ligase. In vitro pull-down and ubiquitination assays showed that CHT14 and PGK directly interacted with GW2 but were not ubiquitinated by GW2. Immunoblot analysis revealed that protein disulfide isomerase-like 1-1 accumulated to high levels in young developing seeds of the gw2 mutant compared with the wild type. Histochemical β-glucuronidase staining showed strong expression of GW2 in leaf and root tissues but weak expression in leaf sheaths and internodes. In addition, transformation of the green fluorescent protein (GFP) gene under the control of the GW2 promoter in rice revealed GFP expression in the aleurone layer of seeds. CONCLUSIONS Collectively, these results suggest that GW2 regulates seed size through direct interactions with proteins involved in carbohydrate metabolism by modulating their activity or stability and controlling disulfide bond formation in various proteins during seed development. Additionally, GW2 participates in vegetative as well as reproductive growth, and protects the seed from pathogen attack.

中文翻译：

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籽粒宽度2（GW2）及其相互作用蛋白调节水稻（Oryza sativa L.）的种子发育。

背景技术已经在农作物中广泛研究了种子大小，因为种子大小决定了作物的产量。但是，种子发育的机制仍然难以捉摸。在这项研究中，我们探讨了水稻（Oryza sativa L.）种子发育的机制，并确定了影响种子大小的蛋白质。结果蛋白质组学分析表明，天然粳稻突变体Oochikara的种子中甘油三磷酸3-磷酸脱氢酶，几丁质酶14（CHT14）和磷酸甘油酸激酶（PGK）积累到高水平，该突变在谷物中带有功能丧失突变宽度2（GW2）基因; GW2编码RING型E3泛素连接酶。体外下拉和泛素化试验表明，CHT14和PGK与GW2直接相互作用，但未被GW2泛素化。免疫印迹分析显示，与野生型相比，gw2突变体的年轻发育种子中蛋白质二硫键异构酶1-1的积累水平很高。组织化学β-葡萄糖醛酸苷酶染色显示，GW2在叶片和根组织中表达强，而在叶鞘和节间表达弱。此外，在水稻中GW2启动子的控制下绿色荧光蛋白（GFP）基因的转化揭示了GFP在种子糊粉层中的表达。结论总体而言，这些结果表明，GW2通过与糖代谢中涉及的蛋白质直接相互作用来调节种子大小，方法是调节其活性或稳定性并控制种子发育过程中各种蛋白质中二硫键的形成。此外，GW2参与营养和生殖生长，