Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-L-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-L-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-LRha/UDP-D-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-L-Rha and UDP-D-Gal for matrix polysaccharide biosynthesis. membrane transport | proteoliposomes | glycan biosynthesis | galactan

Hemicelluose polysaccharides influence assembly and properties of the plant primary cell wall (PCW), perhaps by interacting with cellulose to affect the deposition and bundling of cellulose fibrils. However, the functional differences between plant cell wall hemicelluloses such as glucomannan, xylan and xyloglucan (XyG) remain unclear. As the most abundant hemicellulose, XyG is considered important in eudicot PCWs, but plants devoid of XyG show relatively mild phenotypes. We report here that a patterned β-galactoglucomannan (β-GGM) is widespread in eudicot PCWs and shows remarkable similarities to XyG. The sugar linkages forming the backbone and side chains of β-GGM are analogous to those that make up XyG, and moreover, these linkages are formed by glycosyltransferases from the same CAZy families. Solid-state nuclear magnetic resonance indicated that β-GGM shows low mobility in the cell wall, consistent with interaction with cellulose. Although Arabidopsis β-GGM synthesis mutants show no obvious growth defects, genetic crosses between β-GGM and XyG mutants produce exacerbated phenotypes compared to XyG mutants. These findings demonstrate a related role of these two similar but distinct classes of hemicelluloses in PCWs. This work opens avenues to study the roles of β-GGM and XyG in PCWs.

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All members of the DUF579 family characterized so far have been described to affect the integrity of the hemicellulosic cell wall component xylan: GXMs are glucuronoxylan methyltransferases catalyzing 4‐O–methylation of glucuronic acid on xylan; IRX15 and IRX15L, although their enzymatic activity is unknown, are required for xylan biosynthesis and/or xylan deposition. Here we show that the DUF579 family members, AGM1 and AGM2, are required for 4‐O–methylation of glucuronic acid of a different plant cell wall component, the highly glycosylated arabinogalactan proteins (AGPs).

The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered.