APPLE BETA-GALACTOSIDASE - ACTIVITY AGAINST CELL-WALL POLYSACCHARIDESAND CHARACTERIZATION OF A RELATED CDNA CLONE

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving a ffinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-p olyacrylamide gel electrophoresis indicated that two polypeptides of 4 4 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The en zyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monome ric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W. R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP 31 cDNA from asparagus (G. King, personal communication). Using the as paragus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-term inal signal sequence would leave a predicted polypeptide of 78.5 kD. G enomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosida se gene family. Northern analysis during fruit development and ripenin g showed accumulation of pABG1-homologous RNA during fruit ripening. E nzyme activity as measured in crude extracts increased during fruit de velopment to a level that was maintained during ripening.