Molecular characterization of β-galactosidases from germinating rice (Oryza sativa)

Staining of germinating rice with X-Gal and immunohistochemical localization indicated that β-d-galactosidase (EC 3.2.1.23) is localized to the embryo, aleurone layer and surrounding tissues, and in the radicle and shoot after germination. The genes encoding two isoforms of β-galactosidase expressed in germinating rice and panicle, designated OsBGal1 and OsBGal2, were cloned and sequenced. The full-length cDNA sequence of OsBGal1 encodes 843 amino acids, including a signal peptide of 25 residues. The related OsBGal2 clone from panicle at flowering stage encodes 715 amino acid residues, including a signal peptide of 20 residues. The two genes were found to be relatively highly expressed in seedling roots and shoots and in leaf sheath in 15-30-day-old plants, which contained major immunoreactive proteins of approximately 90 kDa for OsBGal1 protein and 55 kDa for OsBGal2 in extracts of these tissues. Both were also expressed at low levels in flowers and immature seeds, but only the OsBGal2 transcript was found in mature seeds. The predicted mature proteins of the rice β-galactosidases were expressed as thioredoxin fusion proteins in E. coli strain OrigamiB (DE3) to produce β-galactosidase activities not found in control extracts. Purified OsBGal1 fusion protein hydrolyzed and transglycosylated p-nitrophenyl (pNP) β-d-galactopyranoside and hydrolyzed pNP β-d-fucopyranoside. Galactose was released by this enzyme from β-(1 → 3)-, β-(1 → 4)- and β-(1 → 6)-linked di- and trisaccharides of galactose and rice root and rice coleoptile alcohol-insoluble residue, but it did not hydrolyze larger [3H]oligo-(1 → 4)-galactans (penta-, octa- and undecasaccharides), lactose, a galactosylated xyloglucan oligosaccharide (XLLG), total rice hemicellulosic polysaccharides or pectin.