Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis.
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.
Sampedro
J
J
Departamento de Biología Vegetal, Laboratorio de Fisiología Vegetal, Facultad de Biología, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain.
Sieiro
C
C
Revilla
G
G
González-Villa
T
T
Zarra
I
I
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology; D003001:Cloning, Molecular; D018744:DNA, Plant; D020224:Expressed Sequence Tags; D005786:Gene Expression Regulation; D005936:Glucans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D011134:Polysaccharides / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D014990:Xylans; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
D018744:DNA, Plant; D005936:Glucans; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D014995:Xylosidases