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. S odium dodecyl sulfate-polyacrylamide gel electrophoresis of the most a ctive fraction revealed a predominant protein band at 75 kD and severa l minor bands. A 30-amino acid N-terminal sequence from this 75-kD pro tein showed a high degree of homology with other recently identified b eta-galactosidase/galactanase proteins from persimmon and apple fruits (I.-K. Kang, S.-C. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 1 05: 975-979; G.S. Boss, T. Wegrzyn, E.A. MacRae, R.J, Redgwell [1994] Plant Physiol 106: 521-528) and with the predicted polypeptide sequenc e encoded by the ethylene-regulated SR12 gene in carnation (K.C. Ragho thama, K.A. Lawton, P.B. Goldsbrough, W.R. Woodson [1991] Plant Mol Bi ol 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 specif ic for (1-->4)-beta-D-galactan substrates with a pH optimum of 4.5. Th e only reaction product detected was monomeric galactose, indicating t hat the enzyme was an exo-(1-->4)-beta-D-galactanase. beta-Galactanase activity increased at the onset of ripening in normal fruit, but no s imilar increase was detected in the nonripening mutants nor and rin. A tomato homolog (pTom beta gal 1) was isolated using the SR12 cDNA clo ne from carnation as a probe. This clone showed 73% identity at the am ino acid level with beta-galactosidase-related sequences from apple an d 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 ex pression was ripening related in normal fruits, with lower levels appa rent in the nonsoftening mutants.