RESEARCH SUMMARY

A ubiquitous response of plants to pathogen attack is the generation of active lipid derivatives, collectively known as oxylipins, whose importance as regulators of plant defence is being established. Such compounds can be formed by enzymatic peroxidation of fatty acids, catalysed by the activities of 9- and 13-lipoxygenases and alfa-dioxygenases, or non-enzymatically in the presence of singlet oxygen and free radicals. The importance of the oxylipin pathway initiated by 13-lipoxygenases (13-LOX) and its main product, jasmonic acid (JA), in plant fertility and in controlling resistance to necrotrophic pathogens has been demonstrated. In addition, increasing experimental evidence indicates the participation of oxylipins produced by the 9-LOX and alpha-DOX pathways, as well as of non-enzymatically generated oxylipins in plant defence. Nevertheless, with the exception of JA, the signalling mechanisms by which distinct oxylipins exert their function remain poorly understood.

We aim to define the role of oxylipins in protecting plants against pathogen infection and to dissect the signalling pathways mediating their actions. During this period we undertook a genetic approach to investigate the signalling processes regulated by 9-HOT, a 9-LOX-derivative that induces production of callose deposits, initiation of oxidative stress and transcriptional changes of defence-related genes. A lox1 lox5 mutant, which is deficient in 9-LOX activity, and the mutant noxy22 (non-responding to oxylipins22), which is insensitive to 9-HOT, were used for this purpose. Map-based cloning positioned the noxy22 mutation at the ETO1 (ETHYLENE-OVERPRODUCER1) locus with constitutive ethylene (ET) production. As predicted from these results, we found that ET acts as a negative regulator of the 9-HOT signalling and, reciprocally, that 9-HOT interferes with the activation of the ethylene (ET) pathway.

We found that the 9-HOT and ET pathways play a critical role in controlling oxidative stress and the response to the lipid peroxidation-inducer singlet oxygen. Thus, the massive transcriptional changes seen in wild type plants in response to singlet oxygen were greatly affected in the mutants examined. Accordingly, lox1 lox5 and noxy22 displayed enhanced susceptibility to singlet oxygen. Further studies revealed the participation the 9-LOX and ET in the defence of plants against Pseudomonas infection. Results showing enhanced susceptibility of lox1 lox5 and noxy22 to Pseudomonas attack and altered ROS homeostasis supported the role of the 9-LOX oxylipin pathway in controlling oxidative stress during plant defence against biotrophic bacteria, and the negative role of ET in the defence response against this type of pathogens.

Given the participation of the 9-LOX oxylipin pathway in plant defence and in controlling the response to singlet oxygen (¹O₂), we are investigating the role of this reactive molecule and of the _¹O₂-formed oxylipins in the immune strategies of plants against microbial infection. Singlet oxygen is produced as part of an oxidative burst that takes place after pathogen attack, in which oxygen is converted in distinct reactive oxygen species (ROS), such as the superoxide anion radical (O^2-), the hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^•). Polyunsaturated fatty acids are a preferred target of ¹O₂ attack, and several of its oxidation products (non-enzymatically generated oxylipins) could act as secondary messengers to trigger defence responses. In support of this idea, we found that production of ¹O₂ (triggered by Rose Bengal) and application of ¹O₂-formed hydroxy acids (12-HOT, 10-HOT, 10-HOD) induce a strong accumulation of callose, a marker of the plant response to pathogen attack, in leaves and roots of Arabidopsis. Moreover, we found that application of 12-HOT provoked a strong transcriptional response in which ~50% of genes are defence-related. Further studies are underway to define the role of singlet oxygen and of ¹O₂-formed oxylipins in plant defence.