Search

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH). Upon enzymatic treatment, XGA oligosaccharides were primarily produced from pectin extracts obtained from the young and mature leaves and to a lesser extent from those originating from the stem of A. thaliana. The oligosaccharide GalA(3)Xyl was predominantly formed from these pectin extracts. No XGA oligosaccharides were detected in digests of pectin extracts from the seeds and roots. A low number of XGA oligosaccharides was obtained from pectins of A. thaliana. This indicates a uniform distribution of xylose in XGA from A. thaliana. The predominant production of GalA(3)Xyl, as well as the release of linear GalA oligosaccharides pointed to a lower degree of xylose substitution in XGA from A. thaliana than in XGA from apple and potato. The estimated amount of XGA accounted for approximately 2.5%, 7% and 6% (w/w) of the total carbohydrate in the pectin fraction of the stem, young leaves and mature leaves, respectively.

XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of alpha-D-galacturonic acid, highly substituted with beta-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were derived from an XGH digestion of gum tragacanth, then fractionated, and analysed for their sugar composition and structure by matrix-assisted laser-desorption ionization-time-of-flight MS and nanospray MS. Several oligosaccharides from XGA were identified with different galacturonic acid/xylose ratios including five oligosaccharide isomers. Although XGH can act as an endo-enzyme, product-progression profiling showed that the disaccharide GalAXyl was predominantly produced from XGA by XGH, which indicated also an exolytic action. The latter was further supported by degradation studies of purified oligosaccharide GalA4Xyl3. It was shown that XGH acted from the non-reducing end towards the reducing end of this oligosaccharide, and showed the processive character of XGH. The results from this study further show that although XGH prefers to act between two xylosidated GalA units, it tolerates unsubstituted GalA units in its -1 and +1 subsites.