Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells
Résumé
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.