Abstract
An alpha-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 alpha-xylosidase that we propose to call AtXYL1. It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM. AtXYL1 comprised 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 by AtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only alpha-xylosidases, but also alpha-glucosidases. The alpha-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 alpha-xylosidase activity and AtXYL1 expression 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 transform Saccharomyces cerevisiae, significantly higher alpha-xylosidase activity was detected in the yeast cells. alpha-Glucosidase activity was also increased in the transformed cells, although to a lesser extent. These results show that AtXYL1 encodes for an apoplastic alpha-xylosidase active against xyloglucan oligosaccharides that probably also has activity against p-nitrophenyl-alpha-D-glucoside.
Highlights
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 ␣-xylosidase that we propose to call AtXYL1
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 transform Saccharomyces cerevisiae, significantly higher ␣-xylosidase activity was detected in the yeast cells. ␣-Glucosidase activity was increased in the transformed cells, to a lesser extent. These results show that AtXYL1 encodes for an apoplastic ␣-xylosidase active against xyloglucan oligosaccharides that probably has activity against p-nitrophenyl-␣-d-glucoside
Both enzymes showed relatively high substrate specificity. They acted on xyloglucan oligosaccharides and released the unsubstituted side chain xylosyl residue attached to the backbone glucosyl residue situated farthest from the reducing end of the molecule
Summary
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 ␣-xylosidase that we propose to call AtXYL1. Xyloglucan is the main hemicellulosic polysaccharide present in the primary cell walls of dicotyledonous plants (Fry, 1989; Hayashi, 1989) It consists of a linear (134)--linked d-glucan backbone that carries ␣-d-xylosyl, -d-galactosyl-(132)-␣-d-xylosyl and ␣l-fucosyl-(132)--d-galactosyl-(132)-␣-d-xylosyl side chains attached to the OH-6 of -glucosyl residues. An ␣-d-xylosidase has been purified from auxin-treated pea epicotyls (O’Neill et al, 1989), and a second one from cotyledons of nasturtium seedlings (Fanutti et al, 1991) Both enzymes showed relatively high substrate specificity. They acted on xyloglucan oligosaccharides and released the unsubstituted side chain xylosyl residue attached to the backbone glucosyl residue situated farthest from the reducing end of the molecule. It should be noted that exo--glucosidase cannot act on the Glc at the nonreducing end of the oligosaccharide backbone unless
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