The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.
The Golgi apparatus of eukaryotic cells is known for its central role in the processing, sorting, and transport of proteins to intra- and extra-cellular compartments. In plants, it has the additional task of assembling and exporting the non-cellulosic polysaccharides of the cell wall matrix including pectin and hemicelluloses, which are important for plant development and protection. In this review, we focus on the biosynthesis of complex polysaccharides of the primary cell wall of eudicotyledonous plants. We present and discuss the compartmental organization of the Golgi stacks with regards to complex polysaccharide assembly and secretion using immuno-electron microscopy and specific antibodies recognizing various sugar epitopes. We also discuss the significance of the recently identified Golgi-localized glycosyltransferases responsible for the biosynthesis of xyloglucan (XyG) and pectin.
SUMMARYXyloglucan 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 a-1,6-xylosyltransferase, b-1,2-galactosyltransferase and a-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 a-1,6-xylosyltransferase, AtXT1, the b-1,2-galactosyltransferase, AtMUR3, or the a-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.
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