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.-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 (pTombetagal1) was isolated using the SR12 cDNA clone from carnation as a probe. This clone showed 73% identify at the amino acid level with beta-galactosidase-related sequences from apple and asparagus and 66% identity with SR12. pTombetagal1 is a member of a gene family. Northern analysis demonstrated that pTombetagal1 expression was ripening related in normal fruits, with lower levels apparent in the nonsoftening mutants.
Carey
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Horticulture Research International, Wellesborne, Warwick, United Kingdom.
Holt
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Picard
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Wilde
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Tucker
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Bird
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Schuch
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Seymour
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D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D006026:Glycoside Hydrolases; C066350:exo-1,4-beta-galactanase; D001616:beta-Galactosidase