TOMATO EXO-(1-]4)-BETA-D-GALACTANASE - ISOLATION, CHANGES DURING RIPENING IN NORMAL AND MUTANT TOMATO FRUIT, AND CHARACTERIZATION OF A RELATED CDNA CLONE

An exo-(1-->4)-beta-D-galactanase was isolated from ripe tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig and cv Better Boy) using anion-exchange, gel filtration, and cation-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction revealed a predominant protein band at 75 kD and several minor bands. A 30-amino acid N-terminal sequence from this 75-kD protein showed a high degree of homology with other recently identified beta-galactosidase/galactanase proteins from persimmon and apple fruits (I.-K. Kang, S.-C. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 105: 975-979; G.S. Boss, T. Wegrzyn, E.A. MacRae, R.J, Redgwell [1994] Plant Physiol 106: 521-528) and with the predicted polypeptide sequence encoded by the ethylene-regulated SR12 gene in carnation (K.C. Raghothama, K.A. Lawton, P.B. Goldsbrough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71). The enzyme focused to a single band of P-galactosidase activity on an isoelectrofocusing gel at pH 9.8. The enzyme was specific for (1-->4)-beta-D-galactan substrates with a pH optimum of 4.5. The only reaction product detected was monomeric galactose, indicating that the enzyme was an exo-(1-->4)-beta-D-galactanase. beta-Galactanase activity increased at the onset of ripening in normal fruit, but no similar increase was detected in the nonripening mutants nor and rin. A tomato homolog (pTom beta gal 1) was isolated using the SR12 cDNA clone from carnation as a probe. This clone showed 73% identity at the amino acid level with beta-galactosidase-related sequences from apple and asparagus and 66% identity with SR12. pTom beta gal 1 is a member of a gene family. Northern analysis demonstrated that pTom beta gal 1 expression was ripening related in normal fruits, with lower levels apparent in the nonsoftening mutants.