Critical Review of Plant Cell Wall Matrix Polysaccharide Glycosyltransferase Activities Verified by Heterologous Protein Expression

Abstract

The life cycle and development of plants requires the biosynthesis, deposition, and degradation of cell wall matrix polysaccharides. The structures of the diverse cell wall matrix polysaccharides influence commercially important properties of plant cells, including growth, biomass recalcitrance, organ abscission, and the shelf life of fruits. This review is a comprehensive summary of the matrix polysaccharide glycosyltransferase (GT) activities that have been verified using in vitro assays following heterologous GT protein expression. Plant cell wall (PCW) biosynthetic GTs are primarily integral transmembrane proteins localized to the endoplasmic reticulum and Golgi of the plant secretory system. The low abundance of these enzymes in plant tissues makes them particularly difficult to purify from native plant membranes in quantities sufficient for enzymatic characterization, which is essential to study the functions of the different GTs. Numerous activities in the synthesis of the major cell wall matrix glycans, including pectins, xylans, xyloglucan, mannans, mixed-linkage glucans (MLGs), and arabinogalactan components of AGP proteoglycans have been mapped to specific genes and multi-gene families. Cell wall GTs include those that synthesize the polymer backbones, those that elongate side branches with extended glycosyl chains, and those that add single monosaccharide linkages onto polysaccharide backbones and/or side branches. Three main strategies have been used to identify genes encoding GTs that synthesize cell wall linkages: analysis of membrane fractions enriched for cell wall biosynthetic activities, mutational genetics approaches investigating cell wall compositional phenotypes, and omics-directed identification of putative GTs from sequenced plant genomes. Here we compare the heterologous expression systems used to produce, purify, and study the enzyme activities of PCW GTs, with an emphasis on the eukaryotic systems Nicotiana benthamiana, Pichia pastoris, and human embryonic kidney (HEK293) cells. We discuss the enzymatic properties of GTs including kinetic rates, the chain lengths of polysaccharide products, acceptor oligosaccharide preferences, elongation mechanisms for the synthesis of long-chain polymers, and the formation of GT complexes. Future directions in the study of matrix polysaccharide biosynthesis are proposed.

Keywords: enzyme mechanism; glycosyltransferase; heterologous expression; matrix glycan; plant cell wall; polysaccharide biosynthesis; protein complex.

Figure 1

Methods for identifying CWGTs for in vitro activity verification. The activities of plant cell wall glycosyltransferases have been identified using three main strategies as outlined in this review: activity enrichment, mutational genetics, and omics-directed GT selection. Heterologous expression and in vitro assays are critical steps in the process of verifying the synthesis of specific cell wall linkages associated with different families of CWGTs. Purification of enzymes following heterologous expressional enables advanced studies of GT mechanism, including enzyme kinetic, and polysaccharide chain length analyses.

Figure 2

Polysaccharide chain elongation patterns for processive, distributive, and two-phase distributive glycosyltransfer. Glycosyltransferases synthesize polysaccharides by different mechanisms that result in characteristic product profiles over the progress of an elongation reaction. The hypothetical product profiles shown correspond to the elongation of a short-chain acceptor (left) into longer-chain products (right). Three panels are shown for each proposed mechanism corresponding to the early, middle, and late stages of reaction progress. Product profiles are representative of several methods that are used to obtain chain length information, including HPLC, MALDI-MS, and polyacrylamide gel electrophoresis. In processive elongation, formation of an enzyme-substrate catalytic complex that is maintained through many rounds of monosaccharide transfer leads to a bimodal distribution of low and high MW products (Levengood et al., ; Raga-Carbajal et al., 2016). In distributive elongation, dissociation of the acceptor substrate following each round of addition and the lack of acceptor length bias leads to a Poisson distribution of products over time (Keys et al., ; Urbanowicz et al., 2014). A two-phase distributive mechanism accumulates short-chain products during the early phase of the reaction followed by rapid elongation of high MW polysaccharides resulting from large increases in catalytic efficiency for acceptors with chain lengths longer than a “Critical DP” (Vionnet and Vann, ; Amos et al., 2018). The product distribution of a two-phase distributive mechanism can resemble the bimodal distribution observed by processive glycosyltransferases. Chain length analysis may be suggestive of a particular elongation mechanism, but processivity cannot necessarily be inferred without direct evidence, such as the multi-TM translocation pore found in crystal structures of the BcsA:BscB cellulose synthase complex and proposed for other GT2-family structures (Morgan et al., ; Bi et al., 2015).