Department of Plant Sciences, University of California, Davis, California 95616.
Light Microscopy Core, University of Kentucky, Lexington, Kentucky 40536.
Plant Cell. 2019 Mar;31(3):627-644. doi: 10.1105/tpc.18.00854. Epub 2019 Feb 13.
The plant endomembrane system facilitates the transport of polysaccharides, associated enzymes, and glycoproteins through its dynamic pathways. Although enzymes involved in cell wall biosynthesis have been identified, little is known about the endomembrane-based transport of glycan components. This is partially attributed to technical challenges in biochemically determining polysaccharide cargo in specific vesicles. Here, we introduce a hybrid approach addressing this limitation. By combining vesicle isolation with a large-scale carbohydrate antibody arraying technique, we charted an initial large-scale map describing the glycome profile of the SYNTAXIN OF PLANTS61 (SYP61) -Golgi network compartment in Arabidopsis (). A library of antibodies recognizing specific noncellulosic carbohydrate epitopes allowed us to identify a range of diverse glycans, including pectins, xyloglucans (XyGs), and arabinogalactan proteins in isolated vesicles. Changes in XyG- and pectin-specific epitopes in the cell wall of an Arabidopsis mutant corroborate our findings. Our data provide evidence that SYP61 vesicles are involved in the transport and deposition of structural polysaccharides and glycoproteins. Adaptation of our methodology can enable studies characterizing the glycome profiles of various vesicle populations in plant and animal systems and their respective roles in glycan transport defined by subcellular markers, developmental stages, or environmental stimuli.
植物内膜系统通过其动态途径促进多糖、相关酶和糖蛋白的运输。虽然已经鉴定出参与细胞壁生物合成的酶，但对于糖链成分在内膜基础上的运输知之甚少。这在一定程度上归因于在生化上确定特定囊泡中多糖货物的技术挑战。在这里，我们介绍了一种解决此限制的混合方法。通过将囊泡分离与大规模碳水化合物抗体阵列技术相结合，我们绘制了一个初始的大规模图谱，描述了拟南芥（Arabidopsis）中 SYP61-Golgi 网络隔室的聚糖图谱（）。一组识别特定非纤维素碳水化合物表位的抗体使我们能够鉴定出一系列不同的聚糖，包括果胶、木葡聚糖（XyGs）和阿拉伯半乳聚糖蛋白在分离的囊泡中。拟南芥突变体细胞壁中 XyG 和果胶特异性表位的变化证实了我们的发现。我们的数据提供了证据表明，SYP61 囊泡参与结构多糖和糖蛋白的运输和沉积。我们方法的适应性可以使研究能够描述植物和动物系统中各种囊泡群体的聚糖图谱及其在糖链运输中的各自作用，这些作用由亚细胞标记物、发育阶段或环境刺激来定义。
