Variation in its C-terminal amino acids determines whether endo-beta-mannanase is active or inactive in ripening tomato fruits of different cultivars.
Endo-beta-mannanase cDNAs were cloned and characterized from ripening tomato (Lycopersicon esculentum Mill. cv Trust) fruit, which produces an active enzyme, and from the tomato cv Walter, which produces an inactive enzyme. There is a two-nucleotide deletion in the gene from tomato cv Walter, which results in a frame shift and the deletion of four amino acids at the C terminus of the full-length protein. Other cultivars that produce either active or inactive enzyme show the same absence or presence of the two-nucleotide deletion. The endo-beta-mannanase enzyme protein was purified and characterized from ripe fruit to ensure that cDNA codes for the enzyme from fruit. Immunoblot analysis demonstrated that non-ripening mutants, which also fail to exhibit endo-beta-mannanase activity, do so because they fail to express the protein. In a two-way genetic cross between tomato cvs Walter and Trust, all F(1) progeny from both crosses produced fruit with active enzyme, suggesting that this form is dominant and homozygous in tomato cv Trust. Self-pollination of a plant from the heterozygous F(1) generation yielded F(2) plants that bear fruit with and without active enzyme at a ratio appropriate to Mendelian genetic segregation of alleles. Heterologous expression of the two endo-beta-mannanase genes in Escherichia coli resulted in active enzyme being produced from cultures containing the tomato cv Trust gene and inactive enzyme being produced from those containing the tomato cv Walter gene. Site-directed mutagenesis was used to establish key elements in the C terminus of the endo-beta-mannanase protein that are essential for full enzyme activity.
Bourgault
Richard
R
Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Bewley
J Derek
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000254:growth & development; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D015151:Immunoblotting; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008361:Mannosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017386:Sequence Homology, Amino Acid
D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase