Tomato Exo-(1->4)-[beta]-D-Galactanase (Isolation, Changes during Ripening in Normal and Mutant Tomato Fruit, and Characterization of a Related cDNA Clone)

Abstract

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.-G. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 105: 975–979; G.S. Ross, 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.G. 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 [beta]-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.