Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity*.
Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.
El-Sharkawy
Islam
I
UMR 990 INRA/INPT-ENSAT Génomique et Biotechnologie des Fruits, Av. de l'Agrobiopole, BP 32607, F-31326, Castanet-Tolosan Cedex.
Manríquez
Daniel
D
Flores
Francisco B
FB
Regad
Farid
F
Bouzayen
Mondher
M
Latché
Alain
A
Pech
Jean-Claude
JC
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019662:Cucurbitaceae / Q000201:enzymology* / Q000235:genetics*; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D005638:Fruit / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D014835:Volatilization
D004952:Esters; D013912:Threonine; D000217:Acyltransferases