Comparative characterization of Arabidopsis Subfamily III beta-galactosidases
The Arabidopsis genome encodes 17 putative beta-galactosidases belonging to Glycosyl Hydrolase (GH) family 35, which have been classified into seven subfamilies based on sequence homology. The largest of these, Subfamily III, consists of six genes, Gal-1 (At3g13750), Gal-2 (At3g52840), Gal-3 (At4g36360), Gal-4 (At5g56870), Gal-5 (At1g45130), and Gal-12 (At4g26140) that share 60-81% sequence identity at the amino acid level. All six proteins have a signal peptide that may target them to the cell exterior.
We report purification and biochemical characterization of all six members of Subfamily III, each expressed as a recombinant protein in Pichia pastoris and one also in native form, purified from Arabidopsis leaves, with a special emphasis on substrate specificities. Organ specific expression of the six Gal genes was examined by analysis of the microarray databases and by semi-quantitative RT-PCR. The relative abundance and size of the Gal-1, Gal-2, Gal-5, and Gal-12 proteins was studied by immunoblotting using isoform-specific anti-peptide antibodies. The protein expression patterns of the Gal genes were generally consistent with microarray and RT-PCR data, though some discrepancies were observed suggesting distinct mechanisms of regulation for transcription and translation. Localization of total beta-galactosidase activity was visualized using the substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galatopyranoside (X-Gal), to stain whole plants. Subcellular localization of the four isoforms examined by immuno-dotblotting and western blotting showed that Gal-1, Gal-2, Gal-5 and Gal-12 are present in apoplastic and cell wall bound protein extracts. Immuno-EM analysis of Gal-1 and Gal-12 showed that these proteins are localized in the cell walls of vascular and epidermal tissues in mature root. Taken together, the biochemical properties, expression patterns, and subcellular localization of these isozymes indicate that the Subfamily III beta-galactosidases all have potential functions in restructuring the cell wall during plant growth and development.