Caractérisation de gènes de la famille des alcool deshydrogénases et alcool acyl transférases chez le melon cantaloup charentais - INRA - Institut national de la recherche agronomique Accéder directement au contenu
Thèse Année : 2006

Characterisation of genes of the alcohol deshydrogenase and alcohol acyltransferase families in cantaloupe charentais melon

Caractérisation de gènes de la famille des alcool deshydrogénases et alcool acyl transférases chez le melon cantaloup charentais

Résumé

Aroma is one of the principal attributes of quality in fruits. Volatile compounds are synthesized during ripening. In climacteric fruit such as melon the synthesis of aroma volatiles is dependant upon ethylene. Esters represent a significant percentage of the volatile compounds participating in the flavor of many fruits. The esters biosynthesis pathway from fatty acids and amino acids is controlled by two key enzymes, alcohol dehydrogenase (ADH) and alcohol acyltransferase (AAT). ADHs perform the interconversion of aldehydes into alcohols thus providing substrates for the formation of esters. In this thesis, we have isolated two highly divergent ADH genes (15 % identity and the amino acid level) in cantaloupe Charentais melon (Cucumis melo var.cantalupensis). Cm-ADH1 belongs to the class of medium-chain ADHs and Cm-ADH2 to the class of short chain ADHs. The two proteins are enzymatically active when expressed in the yeast. Cm-ADH1 has strong preference for NAPDH as a cofactor, while Cm-ADH2 employs preferentially NADH. Both of them exhibit better efficiency as reductases with Kms 10 to 20 times lower for the conversion of aldehydes into alcohols than for the dehydrogenation of alcohols into aldehydes. They show strong preference for aliphatic aldehydes but Cm-ADH1 is able to reduce branched aldehydes such as 3-methylbutyraldehyde, while Cm-ADH2 cannot. The two ADH genes are expressed specifically in fruit and the expression increases during ripening. Gene expression and total ADH activity are strongly reduced in antisense-ACO (AS) melon and in wild type (WT) melons treated with the ethylene inhibitor 1-MCP, indicating a positive regulation by ethylene. AAT is involved in the acylation of alcohols to produce esters. We demonstrate in this thesis that cantaloupe Charentais melon express at least four members of the AAT gene family : Cm-AAT1, Cm-AAT2, Cm-AAT3 and Cm-AAT4. All the encoded proteins, except Cm-AAT2, were active in the production of esters when expressed in the yeast. Each of them shows specific preferences for the formation of esters. Cm-AAT1 is able to produce short and long-chain esters with different acyls-CoA, with a marked preference for the production of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a great number of substrates but has a very strong preference for the production of benzyl acetate. Cm-AAT4 produces almost exclusively acetates, with a strong preference for cinnamoyl acetate. A site directed mutagenesis showed that the failure of Cm-AAT2 to produce esters is related to the presence of a 268-alanine residue instead of a threonine. Threonine is present in all other AATs so far characterized for being active in the formation of ester volatiles. The activity of each of the three proteins increases during ripening of untransformed wild type (WT) fruit. However, antisense-ACO (AS) melon in which ethylene production is inhibited show very low AAT activty. The expression of all three genes is strongly reduced in AS melon and in WT melon treated with 1-MCP, indicating a positive regulation by ethylene. A biochemical study of the recombinant proteins has been performed. It shows that proteins have a molecular mass of around 200 kDa corresponding to tetramers. Native proteins extracted from the melon have the same molecular mass. The Km of all three recombinant proteins (Cm-AAT1, Cm-AAT3 and Cm-AAT4) are of 1.23, 1.9 et 0.15 mM towards acetyl CoA and 0.56, 0.67 and 0.32 mM towards the alcool. Depending on the level, CoA-SH resulting from the reaction can be either activator or inhibitor. Kinetic studies performed in the presence of an enzyme capable of removing the interference of the CoA-SH (phosphotransacetylase) show in general a very strong decrease of the Km towards acetyl CoA , except for Cm-AAT3. The Ki related to the inhibition by CoA-SH is close to -0.90 mM for the three enzymes. The data of this thesis suggest that the AAT and ADH protein plays a specific role in the biosynthesis of odorant ester volatiles in melon.
Cette thèse est consacrée à la caractérisation de gènes impliqués dans la synthèse d'esters volatils, composés importants de l'arôme de melon cantaloup Charentais (Cucumis melo var. cantalupensis). La biosynthèse des esters est contrôlée par deux enzymes clés, l'alcool déshydrogénase (ADH) qui participe à l'interconversion des aldéhydes en alcools et l'alcool acyl-transférase (AAT) qui transfère un résidu acyl sur des alcools pour former des esters. Nous avons isolé et caractérisé deux gènes fortement divergents codant pour une ADH à moyenne et courte chaîne. Nous démontrons également que le melon exprime au moins quatre gènes correspondent à l'AAT: Cm-AAT1, Cm-AAT2, Cm-AAT3 et Cm-AAT4. Toutes les protéines codées par ces gènes, excepté Cm-AAT2, sont enzymatiquement actives lorsqu'elles sont exprimées dans la levure et sont sous forme de tetramères (200 kDa). Des melons antisens-ACC Oxydase (AS) ne produisant pas d'éthylène ont une très faible activité ADH et AAT et ont une expression très réduite des gènes Cm-ADH et Cm-AAT. On conclut que chaque gène joue un rôle spécifique dans la production d'arômes et que l'éthylène est le régulateur principal de leur expression.
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Dates et versions

tel-04580997 , version 1 (21-05-2024)

Identifiants

  • HAL Id : tel-04580997 , version 1
  • OATAO : 7517

Citer

Daniel Manriquez. Caractérisation de gènes de la famille des alcool deshydrogénases et alcool acyl transférases chez le melon cantaloup charentais. Alimentation et Nutrition. Institut National Polytechnique (Toulouse), 2006. Français. ⟨NNT : 2006INPT010A⟩. ⟨tel-04580997⟩
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