Cloning and Expression Pattern of a Gene Encoding an α-Xylosidase Active against Xyloglucan Oligosaccharides from Arabidopsis

Abstract

Abstract An α-xylosidase active against xyloglucan oligosaccharides was purified from cabbage (Brassica oleracea var. capitata) leaves. Two peptide sequences were obtained from this protein, the N-terminal and an internal one, and these were used to identify an Arabidopsis gene coding for an α-xylosidase that we propose to callAtXYL1. It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM. AtXYL1comprised three exons and encoded a peptide that was 915 amino acids long, with a potential signal peptide of 22 amino acids and eight possible N-glycosylation sites. The protein encoded byAtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only α-xylosidases, but also α-glucosidases. The α-xylosidase activity is present in apoplastic extractions from Arabidopsis seedlings, as suggested by the deduced signal peptide. The first eight leaves from Arabidopsis plants were harvested to analyze α-xylosidase activity and AtXYL1expression levels. Both increased from older to younger leaves, where xyloglucan turnover is expected to be higher. When this gene was introduced in a suitable expression vector and used to transformSaccharomyces cerevisiae, significantly higher α-xylosidase activity was detected in the yeast cells. α-Glucosidase activity was also increased in the transformed cells, although to a lesser extent. These results show thatAtXYL1 encodes for an apoplastic α-xylosidase active against xyloglucan oligosaccharides that probably also has activity againstp-nitrophenyl-α-d-glucoside.