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Caracterização de melões transgênicos acc oxidase antisense e estudo bioquímico de álcool aciltransferases envolvidas na biossíntese de aromas

Lucchetta, Luciano. Caracterização de melões transgênicos acc oxidase antisense e estudo bioquímico de álcool aciltransferases envolvidas na biossíntese de aromas. PhD, Institut National Polytechnique de Toulouse, 2007

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Official URL: http://ethesis.inp-toulouse.fr/archive/00000483/

Abstract

The increase of ethylene production in ‘Cantaloupe Vedrantais’ melon is one of the main causes of the pre and post harvest alterations. In order to extend the shelf life of this fruit, melon plants were transformed with an ACC oxidase DNA sequence of "Royal Gala" apple in antisense orientation (pAP4). Three transformants were obtained. One of them showing the greatest reduction in ethylene production (AS3) was analysed further and used as a model to discriminate between ethylene- dependent and ethylene-independent processes during fruit ripening. AS3 melons exhibited a delay of about 10 days for ripening and accumulated more soluble solids than untransformed melons. Besides, fruit firmness, chlorophyll content and titratable acidity were higher in AS3 melons than in untransformed melons. However, the carotenoid content was unaffected and the aroma intensity was reduced in AS3 fruits. In AS3 plants the senescence of the leaves was reduced and the formation of lateral branching was increased. In order to confirm that chlorophyll degradation, cell wall degradation and synthesis of volatile compounds were ethylene-dependent events, ethylene was applied on AS3 fruits. However, the reversion of the process was limited. While the reduction of pulp firmness occurred, the production of volatiles compounds and the yellowing of the skin were not achieved. Considering that the phenotype of AS3 plants resembled a response to cytokinins action, the concentration of this phytohormone was monitored in both AS3 and untransformed melons. This hypothesis was confirmed as the cytokinins concentration in the pulp and skin of AS3 melons was fourth to eight fold higher. This result suggests that reduction in ethylene production induced changes in the vegetative cycle of the plant, and favoured the synthesis of cytokinins, hormones that are known as retardants of senescence and ethylene action. Using plants from a different lineage, "Cantaloupe" "Charentais", transformed with an ACC oxidase DNA sequence from melon, in antisense orientation (pMEL1), the responses to ethylene action were all observed. For this reason, AAT clones (Cm-AAT1, Cm-AAT3 and Cm-AAT4) were isolated for biochemical characterization. All recombinant proteins were active to synthesize sulfur-containing esters but Cm-AAT1 was the most active. Only the tetrameric form of these proteins, with molecular mass of 200 kDa, were active. The kinetic analysis indicated that CoA-SH, a product of the reaction, is an activator at low concentration and inhibitor at higher concentration. The removal of CoA-SH from the reaction medium, by adding phosphotransacetylase, resulted in Km values two to three fold lower for the co-substrate acyl-CoA. The site directed mutation of a few amino acids in the Cm-AAT sequences affected the selectivity of the original protein and the number of esters produced

Item Type:PhD Thesis
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Institution: Université de Toulouse > Institut National Polytechnique de Toulouse - INPT
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Research Director:
Rombaldi, César and Latché, Alain
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Deposited On:21 Nov 2012 13:49

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