Caracterizacion bioquimica y funcional del enzima a-dioxigenasa2

Supervised by:
  1. Carmen Castresana Fernández Director

Defence university: Universidad Autónoma de Madrid

Fecha de defensa: 13 January 2010

  1. José Juan Sánchez Serrano Chair
  2. Marta Martín Basanta Secretary
  3. Carmen Aragón Rueda Committee member
  4. Óscar Lorenzo Sánchez Committee member
  5. Isabel Allona Alberich Committee member
  6. Manuela Pérez Gilabert Committee member
  7. Luis Carlos Sanz Martínez Committee member

Type: Thesis


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.