Tomato exo-(1 leads to 4)-beta-D-galactanase: isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related cDNA clone

• Published in: Plant physiology (Bethesda) 1995-07, Vol.108 (3), p.1099-1107
• Main Authors: Carey, A.T. (Horticulture Research International, Wellsbourne, Warwick, UK.), Holt, K, Picard, S, Wilde, R, Tucker, G.A, Bird, C.R, Schuch, W, Seymour, G.B
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN; Amino Acid Sequence; Amino acids; beta-Galactosidase - genetics; beta-Galactosidase - isolation & purification; beta-Galactosidase - metabolism; Biochemistry and Enzymology; Biological and medical sciences; Carbohydrate Sequence; Catalysis; Cell walls; Chromatography, Gel; Chromatography, Ion Exchange; CLONE; CLONES; Cloning, Molecular; Complementary DNA; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Electrophoresis, Polyacrylamide Gel; Enzymes; ETAPAS DE DESARROLLO; FRUITS; FRUTAS; Fundamental and applied biological sciences. Psychology; Galactans; GALACTOSIDASAS; GALACTOSIDASE; Gels; GENETICA; GENETIQUE; Glycoside Hydrolases; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; LYCOPERSICON ESCULENTUM; Lycopersicon esculentum - enzymology; MADURAMIENTO; MEDIO DE CULTIVO; Metabolism; MILIEU DE CULTURE; Molecular Sequence Data; MURISSAGE; MUTANT; MUTANTES; Plant physiology and development; Plants; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PROTEINAS; PROTEINE; Proteins; PURIFICACION; PURIFICATION; REACCIONES QUIMICAS; REACTION CHIMIQUE; Ripening; Sequence Homology, Amino Acid; STADE DE DEVELOPPEMENT; Substrate Specificity; VARIACION GENETICA; VARIATION GENETIQUE
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.108.3.1099

An exo-(1 to 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, I.-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 products 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