Incorporation of UDP-[¹⁴C]Glucose into Xyloglucan by Pea Membranes

Abstract

The water-insoluble 1,4-,-linked products formed from UDP-[14CJglucose by pea membranes were dissolved in hot dimethylsulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [14C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP-xylose at concentrations equal to or less than UDP-[14CJglucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-fl-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc4Xy13, which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-a-D-glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[14C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-,8-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass

“…Such coupling of backbone synthesis and substitution is also observed, for example, in xyloglucan synthesis (31). It is therefore surprising that in the gux1 gux2 mutants, despite the loss of GuxT activity, there was no change in quantity of xylan in the walls.…”

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…There are at least nine clades of nucleotide sugar pyrophosphorylases ( Figure 1A), which have different activities and physiological functions (Kotake et al, 2010). UDP-Glc pyrophosphorylases generate UDP-Glc, the starting substrate of UDP-sugar metabolism, which is used for the synthesis of cellulose and b-1,3-glucan and for the b-1,4-glucan backbone of xyloglucan (Hayashi et al, 1987;Gordon and Maclachlan, 1989;Cocuron et al, 2007;Park et al, 2010). A plant-specific enzyme, UDP-sugar pyrophosphorylase (USP), converts various monosaccharide 1-Ps to their respective UDP-sugars in the salvage pathway and is required for pollen development (Kotake et al, 2004(Kotake et al, , 2007Schnurr et al, 2006).…”

The Maize Mixed-Linkage (1→3),(1→4)-β-d-Glucan Polysaccharide Is Synthesized at the Golgi Membrane

“…Using several plant systems, including grass species, autoradiography and membrane fractionation showed that monosaccharides from 14 C-labeled substrates accumulated in cell wall polysaccharides in Golgi vesicles during a pulse were subsequently transferred to the cell wall when chased with unlabeled substrates (Northcote and Pickett-Heaps, 1966;Pickett-Heaps, 1967;Jilka et al, 1972). Early studies showed that labeled sugars from nucleotide-sugar substrates could be incorporated into alcohol-insoluble polysaccharides using microsomal membranes, and later refined by isolation of Golgi membranes and the synthesis of defined polysaccharides with combinations of nucleotide sugars (Bailey and Hassid, 1966;Ray et al, 1969Ray et al, , 1976Smith and Stone, 1973;Ray, 1980;Hayashi and Matsuda, 1981a;Gordon and Maclachlan, 1989;Gibeaut and Carpita, 1993).…”

“…For example, xyloglucan oligomers with the characteristic a-D-Xyl-(1/6)-Dglucosyl unit, isoprimeverose, were synthesized with isolated microsomal membranes and low concentrations of UDP-Glc and UDP-Xyl (Ray et al, 1976;Hayashi and Matsuda, 1981b). When concentrations of each nucleotide sugar were increased to millimolar concentrations, then polysaccharides of about 250 kD were synthesized containing the characteristic XXXG heptasaccharide unit structure (Gordon and Maclachlan, 1989). Immunocytochemical evidence with antibodies directed against the terminal nonreducing xylosyl and fucosyl residues confirm that synthesis of the xyloglucan backbone begins in the cis-Golgi membrane and culminates with fucosylation in the trans-Golgi membrane and trans-Golgi network (Moore et al, 1991;Lynch and Staehelin, 1992;Zhang and Staehelin, 1992).…”