Functional Analysis of α-DOX2, an Active α-Dioxygenase Critical for Normal Development in Tomato Plants

Abstract

Plant a-dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the a-methylene carbon atom of fatty acids. Previously, a-DOX1 has been shown to display a-dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second a-dioxygenase isoform, a-DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Sla-DOX2 and Ata-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Sla-DOX2 and Ata-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of a-DOX1, did not alter the expression of Sla-DOX2 or Ata-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Sla-DOX2 locus and was chosen for functional studies of a-DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of Ata-DOX2 and Sla-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between a-DOX2 from tomato and Arabidopsis. However, deletion of Ata-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of a-DOX2 in plant physiology is species specific.Plants have evolved elaborate signaling systems to regulate a variety of physiological responses to the environment and to facilitate intercellular cross talk in development and reproduction. Oxylipins comprise a large class of oxygenated fatty acid-derived lipid mediators that contribute to such signaling circuits (Weber, 2002;Farmer et al., 2003). A variety of functions have been ascribed to plant oxylipins, including critical roles in plant defense against microbial pathogens, as well as in reproduction and tissue development (Howe and Schilmiller, 2002;Browse, 2005;Kachroo and Kachroo 2009).The biosynthesis of oxylipins is initiated by hydroperoxide formation catalyzed by fatty acid oxygenases, among which the 9-and 13-lipoxygenases have been studied most intensively (Shibata and Axelrod, 1995;Feussner and Wasternack, 2002). a-Dioxygenase, first encountered about 10 years ago, also catalyzes primary fatty acid oxygenation. This enzyme was first identified in Nicotiana tabacum plants as a pathogeninduced protein showing homology to mammalian prostaglandin endoperoxide synthases (Sanz et al., 1998). Studies of the catalytic function of the recombinant tobacco protein and of a homologous protein from Arabidopsis (Arabidopsis thaliana) revealed that these plant enzymes, designated as a-dioxygenase-1 or a-DOX1, catalyze the incorporation of molecular oxygen at the a-methylene carbon atom of fatty ...

Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis

“…(1) Although a-DOX2 has a similar dioxygenase activity toward a-linolenic acid to a-DOX1 (Bannenberg et al, 2009), a-DOX2 did not contribute to 2-HOT production (Fig. 7).…”

“…The seeds were germinated and grown at 22°C under continuous light (100 mE s 21 m 22 ) for 3 weeks and then transferred to vermiculite for growth at 22°C using a 16-h-light (100 mE s 21 m 22 )/8-h-dark photoperiod. Four transfer DNA mutants (Col-0 background) were obtained from the Arabidopsis Biological Resource Center at Ohio State University: SALK_005633 (a-dox1-1), SM_3_27646 (a-dox1-4), SALK_029547 (a-dox2-1), and SALK_089649 (a-dox2-2); a-dox1-1, a-dox2-1, and a-dox2-2 have been described by Bannenberg et al (2009). The double mutants a-dox1-1 a-dox2-2 and a-dox1-4 a-dox2-1 were generated by crossing.…”

Identification of defense-related genes newly-associated with tomato flower abscission

“…Previous reports have already associated the expression of these groups of genes in the AZs of different plants with the activation of a preventive defense program against potential pathogen infections and oxidative stress. 1,5,[7][8][9][10] Our recent report 7 describes a set of AZ-specific genes, well known to be associated with different stress and defense processes in plants, which were found to fatty acids, 11,12 and was originally detected in tobacco as a pathogen-induced oxygenase (PIOX). 13 The oxilipins were suggested to play a role in protecting plant tissues from oxidative damage and cell death.…”

9-Lipoxygenase-derived oxylipins activate brassinosteroid signaling to promote cell wall-based defense and limit pathogen infection

“…The jasmonate pathway also regulates physiological processes such as fertility, senescence, and mechanotransduction (Santino et al, 2013). A number of other studies show that oxylipins produced by the a-DOX pathway participate in plant senescence and development (Obregón et al, 2001;Bannenberg et al, 2009) and that a-DOX enzymes localize in lipid bodies formed in senescent leaves and in leaves responding to pathogen infection (Shimada et al, 2014).…”