Institute of Biological Chemistry, Washington State University, Pullman, WA, 99164, USA.
Faculty of Bioresource Science, Akita Prefectural University, Shimoshinjo-Nakano, Akita-City, 010-0195, Japan.
Plant J. 2019 Mar;97(6):1073-1088. doi: 10.1111/tpj.14180. Epub 2019 Jan 14.
The CS8 transgenic rice (Oryza sativa L.) lines expressing an up-regulated glgC gene produced higher levels of ADPglucose (ADPglc), the substrate for starch synthases. However, the increase in grain weight was much less than the increase in ADPglc levels suggesting one or more downstream rate-limiting steps. Endosperm starch levels were not further enhanced in double transgenic plants expressing both glgC and the maize brittle-1 gene, the latter responsible for transport of ADPglc into the amyloplast. These studies demonstrate that critical processes within the amyloplast stroma restrict maximum carbon flow into starch. RNA-seq analysis showed extensive re-programming of gene expression in the CS8 with 2073 genes up-regulated and 140 down-regulated. One conspicuous gene, up-regulated ~15-fold, coded for a biochemically uncharacterized starch binding domain-containing protein (SBDCP1) possessing a plastid transit peptide. Confocal microscopy and transmission electron microscopy analysis confirmed that SBDCP1 was located in the amyloplasts. Reciprocal immunoprecipitation and pull-down assays indicated an interaction between SBDCP1 and starch synthase IIIa (SSIIIa), which was down-regulated at the protein level in the CS8 line. Furthermore, binding by SBDCP1 inhibited SSIIIa starch polymerization activity in a non-competitive manner. Surprisingly, artificial microRNA gene suppression of SBDCP1 restored protein expression levels of SSIIIa in the CS8 line resulting in starch with lower amylose content and increased amylopectin chains with a higher degree of polymerization. Collectively, our results support the involvement of additional non-enzymatic factors such as SBDCP in starch biosynthesis.
CS8 转基因水稻（Oryza sativa L.）系表达上调的 glgC 基因，产生更高水平的 ADPglucose（ADPglc），这是淀粉合酶的底物。然而，谷物重量的增加远低于 ADPglc 水平的增加，这表明存在一个或多个下游限速步骤。在同时表达 glgC 和玉米脆性-1 基因的双转基因植物中，淀粉水平并没有进一步提高，后者负责将 ADPglc 运入淀粉体。这些研究表明，淀粉体基质内的关键过程限制了碳进入淀粉的最大流量。RNA-seq 分析显示，CS8 中的基因表达发生了广泛的重新编程，有 2073 个基因上调，140 个基因下调。一个引人注目的基因上调了约 15 倍，编码一个生化上未鉴定的淀粉结合域蛋白（SBDCP1），具有质体转运肽。共聚焦显微镜和透射电子显微镜分析证实 SBDCP1 位于淀粉体中。免疫沉淀和下拉分析表明 SBDCP1 与淀粉合酶 IIIa（SSIIIa）相互作用，在 CS8 系中 SSIIIa 的蛋白水平下调。此外，SBDCP1 的结合以非竞争性方式抑制 SSIIIa 的淀粉聚合活性。令人惊讶的是，SBDCP1 的人工 microRNA 基因抑制在 CS8 系中恢复了 SSIIIa 的蛋白表达水平，导致淀粉的直链淀粉含量降低，支链淀粉链更长，聚合度更高。总的来说，我们的结果支持了其他非酶因素（如 SBDCP）在淀粉生物合成中的参与。
