An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis

Abstract

Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of alpha-xylosyltransferase and beta-glucan synthase activities. Here we report that etiolated pea microsomes contain an alpha-xylosyltransferase that catalyzes the transfer of xylose from UDP-[(14)C]xylose onto beta(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode alpha-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the (14)C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an alpha(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an alpha-xylosyltransferase activity involved in xyloglucan biosynthesis.

Figure 1

Transfer of ¹⁴C-labeled xylose from UDP-[¹⁴C]Xyl to various acceptors by pea microsomal membranes treated with 0.4% digitonin. The reactions were stopped by addition of 4 volumes of water before addition of a strong basic ion exchange resin and centrifugation, as described in Materials and Methods. The supernatants containing the radiolabel incorporated into cellotriose (■), cellotetraose (□), and G5 (○) were counted. The values are the average of three assays, and the bars show SE.

Figure 2

Characterization of radiolabeled products formed by digitonin-soluble pea enzyme in the presence of G5 and UDP-[¹⁴C]Xyl. (A) The products produced in the absence (□) or presence (■) of G5 were fractionated on a Bio-gel P2 column. The numbers along the top correspond to the size (DP) as estimated with malto-oligosaccharides (DP 2-7) and glucose as standards. (B) The products that eluted as hexasaccharides from A were treated with β-glucosidase (●), followed by XylS (○), then refractionation on a P2 column. (C) In another experiment, products the size of the hexasaccharides from A were digested with Driselase (▴) then with XylS (○) before separation on a P2 column. Reaction conditions for each treatment are described in Materials and Methods. The structure of the product shown in boxes presents the conclusions deduced from the product characterization (see text). Boxed X, isoprimeverose (17). Vo, void volume; Vt, total volume of the column.

Figure 3

Structural characteristics and phylogenetic tree of AtXT1 and AtGT proteins. The molecular phylogenetic tree was based on the deduced amino acid sequences of AtXT1 and AtGTs and their alignment with the CLUSTAL W program. Lengths of lines indicate the relative distance between nodes. Accession numbers of each gene are indicated on the right. Because AtXT1 was confirmed to be a xylosyltransferase, we are using “AtXT” for its designation. Because computer predictions support the hypothesis that the other genes might be putative glycosyltransferases, we are using “AtGT” to designate them.

Figure 4

Cello-oligosaccharide-dependent α-xylosyltransferase activity from UDP-[¹⁴C]Xyl in detergent-soluble extracts from P. pastoris-expressing candidate genes. (A) Screening for activity in five colonies but only three colonies (X, Y, and Z) are shown for each clone: AtXT1 (■), AtGT2 (░⃞), AtGT3 (▧), AtGT4 (▤), AtGT5 (▩), and AtGT6 (□). The cells were grown under induction conditions (3- 4 days) before solubilization in EB containing 0.4% Triton X-100, and then the soluble fraction was used as the source of enzyme. (B) A single colony showing high activity with AtXT1 was tested for xylosyltransferase activity with cellobiose (▵), cellotriose (■), cellotetraose (□), and G5 (○). C shows the time-dependent incorporation of Xyl into G5 (6 mM). The values are the average of three assays, and the error bars show the SE.

Figure 5

Characterization of the radiolabeled products formed from G5 and UDP-[¹⁴C]Xyl by 0.4% Triton X-100 extract from Pichia cells expressing full-length AtXT1 (see Fig. 4C). (A) Products produced in the absence (□) or presence (■) of G5 were fractionated on a Bio-gel P2 column. (B) The radiolabeled product that eluted as hexasaccharide from A was treated with β-glucosidase (●) and then with Xyls (○). (C) Products the size of pentasaccharides were treated with Driselase (▴) and then with Xyls (○), as described in Materials and Methods and as in Fig. 2. The structure of the product shown in boxes presents the conclusions deduced from the product characterization (see text). Boxed X, isoprimeverose (17). Vo, void volume; Vt, total volume of the column.