Department of Plant Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea.
Department of Plant Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea; Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu, Jeongeup, 56212, Republic of Korea.
J Plant Physiol. 2023 May;284:153981. doi: 10.1016/j.jplph.2023.153981. Epub 2023 Apr 7.
The importance of oats has increased because of their high nutritional value and health benefits in the human diet. High-temperature stress during the reproductive growth period has a detrimental effect on grain morphology by changing the structure and concentration of several seed-storage proteins. DA1, a conserved ubiquitin-proteasome pathway component, plays an important role in regulating grain size by controlling cell proliferation in maternal integuments during the grain-filling stage. However, there have been no reports or studies on oat DA1 genes. In this study, we identified three DA1-like genes (AsDA1-2D, AsDA1-5A, and AsDA1-1D) using genome-wide analysis. Among these, AsDA1-2D was found to be responsible for high-temperature stress tolerance via a yeast thermotolerance assay. The physical interaction of AsDA1-2D with oat-storage-globulin (AsGL-4D) and a protease inhibitor (AsPI-4D) was observed using yeast two-hybrid screening. A subcellular localization assay revealed that AsDA1-2D and its interacting proteins are localized in the cytosol and plasma membrane. An in vitro pull-down assay showed that AsDA1-2D forms a complex with both AsPI-4D and AsGL-4D. An in vitro cell-free degradation assay showed that AsGL-4D was degraded by AsDA1-2D under high-temperature conditions and that AsPI-4D inhibited the function of AsDA1-2D. These results suggest that AsDA1-2D acts as a cysteine protease and negatively regulates oat-grain-storage-globulin under heat stress.
由于燕麦具有高营养价值和对人类饮食健康的益处，其重要性日益增加。在生殖生长期间，高温胁迫通过改变几种种子贮藏蛋白的结构和浓度对谷物形态产生不利影响。DA1 是一种保守的泛素-蛋白酶体途径组成部分，通过在灌浆阶段控制母体内果皮中的细胞增殖，在调节粒大小方面发挥重要作用。然而，目前还没有关于燕麦 DA1 基因的报道或研究。在本研究中，我们使用全基因组分析鉴定了三个 DA1 样基因（AsDA1-2D、AsDA1-5A 和 AsDA1-1D）。其中，AsDA1-2D 通过酵母耐热性测定被发现负责高温胁迫耐受。通过酵母双杂交筛选观察到 AsDA1-2D 与燕麦贮藏球蛋白（AsGL-4D）和蛋白酶抑制剂（AsPI-4D）的物理相互作用。亚细胞定位测定表明 AsDA1-2D 及其相互作用蛋白定位于细胞质和质膜中。体外下拉测定表明，AsDA1-2D 与 AsPI-4D 和 AsGL-4D 形成复合物。体外无细胞降解测定表明，AsGL-4D 在高温条件下被 AsDA1-2D 降解，而 AsPI-4D 抑制了 AsDA1-2D 的功能。这些结果表明，AsDA1-2D 作为半胱氨酸蛋白酶起作用，并在热应激下负调控燕麦谷贮藏球蛋白。
