Plant Molecular Biology

, Volume 59, Issue 2, pp 345–362 | Cite as

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*

  • Islam El-Sharkawy
  • Daniel Manríquez
  • Francisco B. Flores
  • Farid Regad
  • Mondher Bouzayen
  • Alain Latché
  • Jean-Claude Pech
Article

Abstract

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.

Keywords

alcohol acyl-transferase aromas melon site-directed mutagenesis volatile esters 

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Copyright information

© Springer 2005

Authors and Affiliations

  • Islam El-Sharkawy
    • 1
  • Daniel Manríquez
    • 1
  • Francisco B. Flores
    • 1
  • Farid Regad
    • 1
  • Mondher Bouzayen
    • 1
  • Alain Latché
    • 1
  • Jean-Claude Pech
    • 1
  1. 1.UMR 990 INRA/INPT-ENSAT “Génomique et Biotechnologie des Fruits”Castanet-Tolosan CedexFrance

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