Caracterizacion bioquimica y funcional del enzima a-dioxigenasa2

Resumen

Plant ¿-dioxygenases constitute a family of fatty acid-metabolizing enzymes that initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the ¿- methylene carbon atom of fatty acids. Previously, ¿-DOX1 has been shown to display ¿- dioxygenase activity and to be implicated in plant defense. In the present Ph.D. study we investigated the function of a second ¿-dioxygenase isoform, ¿-DOX2, in Arabidopsis thaliana (At¿-DOX2) and Solanum lycopersicum (Sl¿-DOX2). The activity of these enzymes was studied both under in vitro conditions, through recombinant Sl¿-DOX2 and At¿-DOX2 proteins, and in vivo conditions, establishing the presence of the lipid derivatives products of At¿-DOX2 in the cases in which we observed higher expression levels of the corresponding gene. The analysis showed that recombinant Sl¿-DOX2 and At¿-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives in vitro, and an increase in 2-hydroxy derivatives in vivo. Expression of Sl¿-DOX2 and At¿-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. The expression of At¿-DOX2 during detachment is compromised in ein2-1 mutants, pointing to a role of ethylene in the control of At¿-DOX2 expression. In contrast, microbial infection, earlier known to increase the expression of ¿-DOX1, did not alter the expression of Sl¿-DOX2 or At¿-DOX2. In tomato, mutation, of Sl¿-DOX2 also known as FEEBLY result in severe defects in plant development and anthocyanin accumulation pointing to a critical role of the tomato ¿-dioxygenase-2 in plant development. (van der Biezen et al., 1996). A second allele named DIVARICATA carries a mutation at the same locus, which results in a similar but milder compromised growth pointing to a critical role of the tomato ¿-dioxygenase-2 in plant development. This mutant was chosen for functional studies of ¿-DOX2. Transgenic expression of At¿-DOX2 and Sl¿-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between ¿-DOX2 from tomato and Arabidopsis. However, deletion of At¿-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of ¿-DOX2 in plant physiology is species-specific.