Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis

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.

Figure 1

Purification of α-xylosidase from young cabbage leaves. A, Selected region of gel permeation chromatography on Sephacryl S-100 HR column. Protein content (○) and α-xylosidase activity (●) of each fraction were measured. B, SDS-PAGE of fractions 48 to 50 from Sephacryl S-100 HR chromatography. The selected fractions (stripped area on A) were pooled, concentrated, and separated by SDS-PAGE. Left lane, Molecular markers; right lane, α-xylosidase-containing fractions.

Figure 2

Diagram showing the identification of two Arabidopsis ESTs (H8A7T7 and G10B11T7) and a bacterial artificial chromosome (BAC) clone (T12L6) containing sequences highly similar to those obtained from cabbage α-xylosidase. For Arabidopsis homologous sequences, dots mean conserved amino acids.

Figure 3

A, Tiling path of the region in chromosome I where F28A15 and F2A8 had been mapped. This diagram is based on Max Planck Institut für Moleculare Pflanzenphysiologie hybridization data. The initial F has been omitted from clone names. Five clones, including the two already mentioned, were selected for screening and are marked in black. Another clone, F24J5, has been recently sequenced by the SPP consortium (Stanford DNA sequencing and Technology Center, Plant Gene Expression Center at University of California-Berkeley, and the University of Pennsylvania). It includes the entire AtXYL1 gene (GenBank accession no. AC008075). B, Screening of the five IGF-BAC clones by PCR in search for AtXYL1.

Figure 4

Structure of AtXYL1 gene and its protein. A, Sequenced fragment from clone F22C21. Transcripted region is drawn as a set of boxes, stripped (5′- and 3′-untranslated regions), black (introns), or gray (coding regions). B, Protein diagram. ♦, Potential glycosylation sites. The asterisk marks the position of active cabbage α-xylosidase N terminus. SP, Signal peptide.

Figure 5

Schematic representation of AtXYL1 promoter. A, Sequence from −500 to the start codon. ▾, Transcription start site. A TATA-like sequence is underlined. Potential binding sites for SBF-1 and GT-1, transcription factors of the GT-family, are boxed. B, Concise view of binding sites arrangement in AtXYL1 and rbcS-3 promoters.

Figure 6

Partial alignment and phylogenetic relationship among family 31 α-glycosyl hydrolases. A, Alignment of the family 31 PROSITE signature sequences (labeled with the PROSITE accession no.) from known and putative plant α-xylosidases and two plant α-glucosidases. Dots mean conserved amino acids. B, Concise cladogram for family 31 glycosyl hydrolases. ●, The probable point where plant cell wall α-xylosidases started to diverge from α-glucosidases. ○, The independent origin of recently discovered prokaryotic α-xylosidases. Figures between brackets show sequence correspondence between A and B.

Figure 7

Developmental regulation of α-xylosidase. A, Photography of a typical 19-d-old Arabidopsis plant. The first eight true leaves were numbered according to their appearance order. B, Leaf relative expansion rate as function of developmental stage. The first eight leaves were sorted into four categories. Data are mean values with sd for 10 plants. C, α-Xylosidase-specific activity extracted from different leaf groups. D, Analysis of AtXYL1 expression using reverse transcriptase (RT)- PCR followed by Southern hybridization. rRNA stained with ethidium bromide was used as control.

Figure 8

Heterologous expression of AtXYL1. α-Xylosidase and α-glucosidase activities of extracts from S. cerevisiae cells transformed with AtXYL1 (white bars) or with a control plasmid that had no insert (black bars) grown in YP4 High Stability Expression medium. Both activities are referred to the fresh weight of harvested cells.