Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 beta-glucosidase

Abstract

Background: Glycosyl hydrolase family 1 (GH1) beta-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 beta-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.

Results: Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed beta-(1,4)-linked oligosaccharides of 3-6 glucose residues and laminaribiose.

Conclusion: Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 beta-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.

Figure 1

Sequence Logos for the residues surrounding the catalytic acid/base (A) and catalytic nucleophile (B) in rice GH1 genes. The logos show the size of the different amino acids at each position in proportion to their relative abundance within the 40 rice Glycosyl Hydrolase 1 gene protein sequences. The logos were drawn with the weblogo facility [73].

Figure 2

Phylogenetic tree of predicted protein sequences of rice and Arabidopsis Glycosyl Hydrolase Family 1 genes. The tree was derived by the Neighbor-joining method from the protein sequence alignment in the Supplementary Data Additional File 2 made with Clustalx with default settings, followed by manual adjustment. Large gap regions were removed for the tree calculation. The tree is drawn as an unrooted tree, but is rooted by the outgroup, Os11bglu36, for the other sequences. The bootstrap values are shown at the nodes. The clusters supported by a maximum parsimony analysis are shown as bold lines, and the loss and gain of introns are shown as open and closed diamonds, respectively. The 7 clusters that contain both Arabidopsis and rice sequences that are clearly more closely related to each other than to other Arabidopsis or rice sequences outside the cluster are numbered 1–7, while the outgroup cluster for which the Arabidopsis orthologue is not shown in numbered (8). Two Arabidopsis clusters that are more distantly diverged from the clusters containing both rice and Arabidopsis are numbered At I and At II, while rice genes and groups of genes that appear to have diverged before subclusters containing both rice and Arabidopsis are marked with stars.

Figure 3

Relationship between rice and other plant GH1 protein sequences described by a phylogenetic tree rooted by Os11bglu36. The sequences were aligned with ClustalX, then manually adjusted, followed by removal of N-terminal, C-terminal and large gap regions to build the data model. The tree was produced by the neighbor joining method and analyzed with 1000 bootstrap replicates. The internal branches supported by a maximum parsimony tree made from the same sequences are shown as bold lines. The sequences other than rice include: ME AAB71381, Manihot esculenta linamarase; RSMyr BAB17226, Raphanus sativus myrosinase; BJMyr AAG54074, Brassica juncea myrosinase; BN CAA57913, Brassica napus zeatin-O-glucoside-degrading β-glucosidase; HB AAO49267, Hevea brasiliensis rubber tree β-glucosidase; CS BAA11831, Costus speciosus furostanol glycoside 26-O-β-glucosidase (F26G); PS AAL39079Prunus serotina prunasin hydrolase isoform PH B precursor; PA AAA91166, Prunus avium ripening fruit β-glucosidase; TR CAA40057, Trifolium repens white clover linamarase; CA CAC08209, Cicer arietinum epicotyl β-glucosidase with expression modified by osmotic stress; DC AAF04007, Dalbergia cochinchinensis dalcochinin 8'-O-β-glucoside β-glucosidase; PT BAA78708, Polygonum tinctorium β-glucosidase; DL CAB38854, Digitalis lanata cardenolide 16-O-glucohydrolase; OE AAL93619, Olea europaea subsp. europaea β-glucosidase; CR AAF28800, Catharanthus roseus strictosidine β-glucosidase; RS AAF03675, Rauvolfia serpentina raucaffricine-O-β-D-glucosidase; CP AAG25897, Cucurbita pepo silverleaf whitefly-induced protein 3; AS CAA55196, Avena sativa β-glucosidase; SC AAG00614, Secale cereale β-glucosidase; ZM AAB03266, Zea mays cytokinin β-glucosidase; ZM AAD09850, Zea mays β-glucosidase; SB AAC49177, Sorghum bicolor dhurrinase; LE AAL37714, Lycopersicon esculentum β-mannosidase; HV AAA87339, barley BGQ60 β-glucosidase; HB AAP51059, Hevea brasiliensis latex cyanogenic β-glucosidase; PC AAC69619Pinus contorta coniferin β-glucosidase; GM AAL92115, Glycine max hydroxyisourate hydrolase; CS BAC78656, Camellia sinensis β-primeverosidase.

Figure 4

Predicted gene structure patterns for putative rice GH1 β-glucosidase genes. Exons are shown as boxes with corresponding exons having the same pattern. Introns, represented as simple lines, are drawn in proportion to their length. Note that 5 gene organization patterns can be seen in rice genes, those with 13, 12, 11, or 9 exons and intronless patterns, with the splice sites conserved in each group and between groups for common exons and introns.

Figure 5

SDS-PAGE profiles of Os4bglu12 recombinant protein expressed in OrigamiB (DE3)E. coli after incubation in the presence of 0.3 mM IPTG, at 20°C for 8 h. Lanes: 1, standard marker (Bio-RAD); 2, total protein in E. coli cells containing pET32a(+) without an insert; 3, total protein of E. coli cells containing pET32a(+)/DEST-Os4bglu12; 4, soluble fraction of E. coli cells containing pET32a(+)/DEST-Os4bglu12; 5, purified Os4bglu12 recombinant protein. The arrow points to the position of thioredoxin fusion protein monomer.

Figure 6

Hydrolysis of oligosaccharide substrates by Os4bglu12 detected by TLC. The Os4bglu12 was incubated with 5 mM substrates for 30 min and the products were detected after TLC by the carbohydrate staining. Samples were incubated with (+) or without (-) enzyme. Lanes: 1, glucose (G) and cello-oligosaccharides of DP 2–4 (C2-C4) marker; 2 and 3, cellobiose; 4 and 5, cellotriose; 6 and 7, cellotetraose, 8 and 9, cellopentaose, 10 laminari-oligosaccharides of DP 2–4 (L2-L4) marker; 11 and 12, laminaribiose; 13–14 laminaritriose.