Gs. Ross et al., APPLE BETA-GALACTOSIDASE - ACTIVITY AGAINST CELL-WALL POLYSACCHARIDESAND CHARACTERIZATION OF A RELATED CDNA CLONE, Plant physiology, 106(2), 1994, pp. 521-528
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.