Wannitikul Pitchaporn, Dachphun Issariya, Sakulkoo Jenjira, Suttangkakul Anongpat, Wonnapinij Passorn, Simister Rachael, Gomez Leonardo D, Vuttipongchaikij Supachai
Department of Genetics, Faculty of Science, Kasetsart University, 50 Ngarm Wong Wan Road, Chattuchak, Bangkok 10900, Thailand.
Center of Advanced Studies for Tropical Natural Resources, Kasetsart University, Ngam Wong Wan Road, Chattuchak, Bangkok 10900, Thailand.
ACS Omega. 2024 Jul 11;9(29):31438-31454. doi: 10.1021/acsomega.4c00534. eCollection 2024 Jul 23.
Identification of proteins involved in cell wall matrix polysaccharide biosynthesis is crucial to understand plant cell wall biology. We utilized cross-linking and immunoprecipitation with cell wall antibodies that recognized xyloglucan, xylan, mannan, and homogalacturonan to capture proteins associated with matrix polysaccharides in protoplasts. The use of cross-linkers allowed us to capture proteins actively associated with cell wall polymers, including those directly interacting with glycans via glycan-protein (GP) cross-linkers and those associated with proteins linked to glycans via a protein-protein (PP) cross-linker. Immunoprecipitations led to the identification of 65 protein IDs localized in the Golgi, ER, plasma membrane, and others without subcellular localization data. Among these, we found several glycosyltransferases directly involved in polysaccharide synthesis, along with proteins related to cell wall modification and vesicle trafficking. Protein interaction networks from DeepAraPPI and AtMAD databases showed interactions between various IDs, including those related to cell-wall-associated proteins and membrane/vesicle trafficking proteins. Gene expression and coexpression analyses supported the presence and relevance of the proteins to the cell wall processes. Reverse genetic studies using T-DNA insertion mutants of selected proteins revealed changes in cell wall composition and saccharification, further supporting their potential roles in cell wall biosynthesis. Overall, our approach represents a novel approach for studying cell wall polysaccharide biosynthesis and associated proteins, providing advantages over traditional immunoprecipitation techniques. This study provides a list of putative proteins associated with different matrix polysaccharides for further investigation and highlights the complexity of cell wall biosynthesis and trafficking within plant cells.
鉴定参与细胞壁基质多糖生物合成的蛋白质对于理解植物细胞壁生物学至关重要。我们利用与识别木葡聚糖、木聚糖、甘露聚糖和同型半乳糖醛酸的细胞壁抗体进行交联和免疫沉淀，以捕获原生质体中与基质多糖相关的蛋白质。交联剂的使用使我们能够捕获与细胞壁聚合物积极相关的蛋白质，包括那些通过聚糖-蛋白质（GP）交联剂直接与聚糖相互作用的蛋白质以及那些通过蛋白质-蛋白质（PP）交联剂与连接到聚糖的蛋白质相关的蛋白质。免疫沉淀导致鉴定出65个蛋白质ID，它们定位于高尔基体、内质网、质膜以及其他没有亚细胞定位数据的部位。在这些蛋白质中，我们发现了几种直接参与多糖合成的糖基转移酶，以及与细胞壁修饰和囊泡运输相关的蛋白质。来自DeepAraPPI和AtMAD数据库的蛋白质相互作用网络显示了各种ID之间的相互作用，包括那些与细胞壁相关蛋白和膜/囊泡运输蛋白相关的ID。基因表达和共表达分析支持了这些蛋白质在细胞壁过程中的存在和相关性。使用所选蛋白质T-DNA插入突变体的反向遗传学研究揭示了细胞壁组成和糖化的变化，进一步支持了它们在细胞壁生物合成中的潜在作用。总体而言，我们的方法代表了一种研究细胞壁多糖生物合成及相关蛋白质的新方法，比传统免疫沉淀技术具有优势。本研究提供了一份与不同基质多糖相关的推定蛋白质列表以供进一步研究，并突出了植物细胞内细胞壁生物合成和运输的复杂性。
