Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale,' UPRES EA 4358, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides 23, Plate-forme de Recherche en Imagerie Cellulaire de Haute Normandie (PRIMACEN), IBiSA, Université de Rouen, 76821 Mont-Saint Aignan Cedex, France.
Plant J. 2010 Dec;64(6):977-89. doi: 10.1111/j.1365-313X.2010.04388.x. Epub 2010 Nov 15.
Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.
木葡聚糖是双子叶植物初生细胞壁中主要的半纤维素多糖，在植物发育中起着关键作用。木葡聚糖是在高尔基堆中组装的，并通过高尔基衍生的小泡运输到细胞壁中，这一点已经得到很好的证实。人们还知道，木葡聚糖的生物合成需要糖苷转移酶的作用，包括负责向侧链添加木糖、半乳糖和岩藻糖残基的α-1,6-木糖基转移酶、β-1,2-半乳糖基转移酶和α-1,2-岩藻糖基转移酶活性。然而，对于这些酶如何在高尔基堆的亚区室中分布，人们知之甚少。我们进行了一项研究，旨在使用免疫金电子显微镜对高尔基堆中的这些糖苷转移酶进行定位。为此，我们生成了表达拟南芥的α-1,6-木糖基转移酶（AtXT1）、β-1,2-半乳糖基转移酶（AtMUR3）或α-1,2-岩藻糖基转移酶（AtFUT1）的烟草（Nicotiana tabacum）BY-2 悬浮培养细胞的转基因系，这些酶与绿色荧光蛋白（GFP）融合。使用共聚焦显微镜评估融合蛋白在内膜系统中的定位。此外，还对烟草细胞进行高压冷冻/冷冻置换，并使用抗 GFP 抗体进行定量免疫金标记，以确定酶在高尔基潴泡亚型中的定位模式。数据表明：（i）所有融合蛋白，AtXT1-GFP、AtMUR3-GFP 和 AtFUT1-GFP，都被特异性靶向到高尔基器；（ii）AtXT1-GFP 主要位于顺面和中间潴泡，AtMUR3-GFP 主要与中间潴泡相关，而 AtFUT1-GFP 主要检测到反式潴泡，这表明木葡聚糖侧链的起始发生在烟草细胞的早期高尔基区室中。
