Avaliação molecular da biossíntese de ácido ascórbico e possível participação da Oxidase alternativa em dois clones de aceroleira (Malpighia emarginata DC)

Abstract

The acerola, Malpiguia emarginata DC is a fruit small endowed with enviable nutritional qualities, being consumed both fresh and processed. The mail feature that stands out from other species is its enormous capacity to synthesize ascorbic acid. In this sense, Embrapa has developed four commercial clones with phenotypic and genetic characteristics well defined. The clones BRS 235 (Apodi), BRS 236 (Cereja), BRS 237 (Roxinha) e BRS 238 (Frutacor) have characteristics fully standardized. This study aimed to evaluate the clones BRS 236 (Cereja) and BRS 237 (Roxinha) were selected because of huge amount of ascorbic acid synthesized between then, approximately 50% in Cereja clone. For this study we analyzed the gene expression of enzymes of the way Wheeler /Smirnoff as well as the Alternative Oxidase (AOX). The last step this of this pathway ends in the inner mitochondrial membrane, due to the presence of transmembrane enzyme L-Galactono-1,4- Lactone dehydrogenase (L-GalLdh), located between the complex III and IV. Since the alternative oxidase is an enzyme uncoupling, no phosphorylating and insensitive to cyanide, among the complexes II and III of the inner mitochondrial membrane, responsible for alternative pathway of electrons. This study aimed to identify the enzymes that are crucial in this process and generate the difference between the clones, as well as the probable relationship between the biosynthesis of ascorbic acid and the role that the AOX could play. Acerola five years of age were harvested four tissues (flowers, unripe, semi mature and mature fruits). The first step of this work was the determination of ascorbic acid in unripe, semi mature and mature fruits of the two clones by titration of Tillman. Then, we performed the characterization of the AOX gene to define its isoforms. After isolation of DNA and carried out the reactions of PCR with degenerate primers the amplicons were purified and subjected to cloning, transformation and sequencing. The results revealed sequences that correspond to one AOX1 and one AOX2. The next step of work was the study of gene expression of enzymes to the Wheeler/Smirnoff path, AOX1 and AOX2 newly characterized. After the design of pairs of degenerate and specific primers for all the enzymes of the route beyond the AOX1 and AOX2 they were used in semiquantitative PCR reactions, using the elongation factor alpha as a constitutive elemento of reference. The results showed that both clones decrease their levels of ascorbic acid as the fruits develop, revealing that in all three stages of development, the Cereja clone has higher amounts of ascorbic acid that Roxinha clone. However, when comparing the levels of ascorbic acid between green and ripe fruits of both clones is evident that the Roxinha clone has a lower difference between these two stages. The gene expression of three enzymes stood out from others, these showed a synergism with the expression levels of vitamin C contained in each tissue. In addition, gene expression in two clones showed differences in tissues that favor of Cereja clone. They are: Mannose pyrophsphorylase, GDP-Mannose 3’5’ epimerase and GDP-Galactose phosphorylase. With greater emphasis on GDP-Mannose 3’5’ epimerase and GDP-Galactose phosphorylase. The enzymes that stood out showed a higher gene expression in tissues where exactly had more ascorbic acid accumulated, gradually reducing the expression with the degree of fruit development, revealing that the difference occurs not only between clones, but also in tissues clones. There were no differences in gene expression of other enzymes of the route Wheeler/Smirnoff to justfy the levels of ascorbic acid present in the tissues and also between clones. The analysis of gene expression of isoforms of AOX revealed that in both clones the AOX1 gene expression increases as the fruit ripens. However, the clone Roxinha has a higher gene expression than the clone Cereja, in all three stages of development. The analysis of transcripts of AOX2 revealed that in Cereja clone the gene expression decreased with fruit ripening, since in Roxinha clone the expression xx elevated in three developmental stages. The results cearly showed that three enzymes are essential for biosyntesis of ascorbic acid highlighting the GDP-Mannose 3’5’epimerase and GDP-Galactose phosphrylase. Another important conclusion was that the AOX may be owner a function not previously reported in the literature, that of assisting in the biosyntesis of vitamin C. This may be due uncoupling promoted by the activity of AOX causing a gradual rise of the relationship oxidized cytochrome C/rediced cytochrome C, where the cytochrome C oxidized acts of a GalLdh substrates in the syntesis of ascorbic acid. This fact may be explained by not only the highest levels of AOX1 transcripts present in Roxinha clone, but the increasing pattern of gene expression in AOX2 exatly in clone that produces less ascorbic acid and which has the smallest difference between the stages of fruit Green and ripe fruit. In the clone Cereja, one that synthesizes more ascorbic acid and has a huge difference in the levels between the stages of green fruit and ripe fruit of gene expression of AOX2 shown decresing during ripening. Thus, the concentration of products the Wheeler / Smirnoff path is still determining the amount of ascorbic acid produced, and in parallel, the increase in the AOX gene expression appears to contribute at some point to establish the total levels of vitamin C synthesized.