Table 8_OsAPSE modulates non-covalent interactions between arabinogalactan protein O-glycans and pectin in rice cell walls.xlsx

Flexibility of cell walls is crucial to accommodate cell elongation and growth, typically associated with the reorganization of cell wall polysaccharides. Seed germination is a fast-paced developmental process in which cell wall adaptability is highly required. The plant cell utilizes multiple strategies to obtain a flexible cell wall and in part relies on cell wall-active enzymes to loosen both covalent and non-covalent interactions between cell wall polysaccharides. OsAPSE is an example of a cell wall-active enzyme originating from Japanese rice (Oryza sativa subsp. Japonica) belonging to the glycoside hydrolase family 27 (GH27), potentially active on the pectin–arabinogalactan protein O-glycan junction. We provide insights into the biochemical and enzymatic properties of this protein, characterized by the presence of a GH27 domain linked to a ricin-B-like domain. Using small-scale production experiments in a cell-free protein synthesis system, we demonstrated the catalytic activity of the recombinant OsAPSE towards synthetic and natural substrates. Furthermore, subcellular localization analysis and in silico data suggest that OsAPSE may undergo unconventional secretion to the cell surface. We hypothesize that OsAPSE plays a role during rice seed germination by removing terminal α-D-Galp and β-L-Arap moieties along the pectin–arabinogalactan protein O-glycan network. This activity may abolish non-covalent interactions between pectic rhamnogalacturonan I and O-glycans of arabinogalactan proteins, contributing to cell wall relaxation for growth during germination.细胞壁柔性对于适配细胞伸长与生长至关重要，这一过程通常与细胞壁多糖的重组密切相关。种子萌发是一类快速进行的发育过程，其间对细胞壁适应性有着极高的要求。植物细胞可通过多种策略获取柔性细胞壁，其中部分依赖于细胞壁活性酶来松解细胞壁多糖间的共价与非共价相互作用。OsAPSE是一种源自日本粳稻（Oryza sativa subsp. Japonica）的细胞壁活性酶，隶属于糖苷水解酶家族27（glycoside hydrolase family 27，GH27），其潜在底物为果胶-阿拉伯半乳聚糖蛋白的O-糖基连接位点。本研究阐明了该蛋白的生化与酶学特性：其结构包含一个与蓖麻毒素B样结构域相连的GH27结构域。通过无细胞蛋白质合成系统开展的小规模蛋白制备实验，我们证实重组OsAPSE对合成底物与天然底物均具有催化活性。此外，亚细胞定位分析与计算机模拟数据显示，OsAPSE或通过非常规分泌途径转运至细胞表面。我们推测，OsAPSE在水稻种子萌发过程中发挥关键作用，通过移除果胶-阿拉伯半乳聚糖蛋白O-糖基网络末端的α-D-半乳糖吡喃糖基（α-D-Galp）与β-L-阿拉伯糖吡喃糖基（β-L-Arap）残基。该活性可破坏果胶鼠李半乳糖醛酸I与阿拉伯半乳聚糖蛋白O-糖基之间的非共价相互作用，进而促进萌发过程中细胞壁松弛以支持细胞生长。