Lee Kyu Ho, Park Sang Woo, Kim Yeon Jeong, Koo Yeon Jong, Song Jong Tae, Seo Hak Soo
Department of Plant Science, College of Agriculture and Life Sciences, Research Institute of Agriculture and Life Sciences, Seoul National University, Gwanakro 200, Gwanak-gu, Seoul, 151-921, South Korea.
Department of Biological Chemistry, Chonnam National University, Gwangju, 61186, South Korea.
Bot Stud. 2018 Oct 26;59(1):23. doi: 10.1186/s40529-018-0240-z.
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.
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.
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.
种子大小在农作物中已得到广泛研究，因为它决定作物产量。然而，种子发育的机制仍然不清楚。在本研究中，我们探索了水稻（Oryza sativa L.）种子发育的机制，并鉴定了影响种子大小的蛋白质。
蛋白质组学分析表明，在天然粳稻突变体大粒（Oochikara）的种子中，甘油醛-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，该突变体在粒宽2（GW2）基因中携带功能缺失突变；GW2编码一种环状E3泛素连接酶。体外下拉和泛素化分析表明，CHT14和PGK直接与GW2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，蛋白质二硫键异构酶样1-1在gw2突变体发育中的幼嫩种子中积累到高水平。组织化学β-葡萄糖醛酸酶染色显示，GW2在叶和根组织中强烈表达，但在叶鞘和节间中弱表达。此外，在水稻中由GW2启动子控制的绿色荧光蛋白（GFP）基因转化显示，GFP在种子糊粉层中表达。
总体而言，这些结果表明，GW2通过在种子发育过程中调节参与碳水化合物代谢的蛋白质的活性或稳定性以及控制各种蛋白质中二硫键的形成，与这些蛋白质直接相互作用来调节种子大小。此外，GW2参与营养生长和生殖生长，并保护种子免受病原体侵袭。
