SUMMARY OF THE DOCTORAL THESIS "CHANGES IN THE GENE EXPRESSION ASSOCIATED TO THE INTERNAL RIPENING OF CITRUS FRUITS: IDENTIFICATION OF METABOLIC PATHWAYS INVOLVED IN THE ACCUMULATION AND ELIMINATION OF ACIDS ".

Presented by: D. Guillermo Soler Fayos

In Clementines, and specially in the Clementina de Nules, degradation of acids supposes a disadvantage when, because of the market, the harvest is delayed. The main problem is that these fruits suffer a great descent of the acidity that diminishes their internal quality, up to becoming unacceptable for the consumption.
The most of the studies realized on the internal ripening of citrus fruits (Sinclair and Ramsey, 1944; Bain, 1958; Erickson, 1968; Davies and Albrigo, 1994), have focused towards the determination of the principal metabolites that compose the juice of the fruits and the changes that take place during the last phases of ripening, just when it begins to increase the concentration of sugars and to diminish the acids.
In spite of the already mentioned importance of the metabolism of the acids in the citrus fruits, scarcely studies exist about the genes that regulate the accumulation of the citric acid.
The general target of this work is to reveal for the first time the changes in the gene expression that take place during the development and ripening of the pulp of the citrus fruits, and in particular those associates to the mechanisms of regulation of the acidity, identifying the set of induced and suppressed genes that regulate the synthesis and the degradation of the citric acid during the internal ripening of the citrus fruits.
From a metabolic point of view, we have studied the evolution of the principal acids of the fruits in four varieties that present differences during their ripening period, with the intention of determining the corresponding patterns of accumulation and degradation. On the other hand, we have focussed on the principal genes involved in the metabolism of the acids, studying the synthesis regulation mechanism during the phases I and II of the development of the fruit, and the degradation during phases II and III of fruit ripening.
At the same time, we have identified three genes that codify three enzymes of aconitase, one probably located in the mitochondria and others two in the citosol. The aconitase located in the mitochondria presents a constant expression during the development and the ripening of the fruit, whereas at least one of the aconitases located in the citosol is induced during the ripening.
Regarding with the information of the microarrays and the parallel analysis of metabolites, it was possible to suggest that the citric acid is metabolized via 2-oxoglutarato to glutamine and to succinate, in the GABA shunt, so this pathway seems to be a priority route of catabolism of the citric acid.
According to the studies done, the accumulation of citric acid in the fruits is directly related to a decrease of the level of the ATP citrate liase transcript, because low levels of this enzyme diminishes the degradation of citrate in the citosol, so there is more citrate available to be stored in the vacuole.
The reduction of acidity observed in the fruits after the treatment with arsanilic acid, a well-known acidity reducer of the citrus fruits, is associated with the repression of the glycolysis and the Calvin's cycle. These results suggest that the participation of these pathways are necessary to canalize the carbon towards the synthesis of acids.
At the same time, the Fortune variety that presents a high content of citric acid, showed a major expression of the Calvin's cycle, the sucrose hydrolysis and the glycolysis, and a minor expression of the pentoses phosphate cycle and the oxidative phosphorylation than the Clementina de Nules, a variety with a normal acid content.