Light is essential for plants through photosynthetic carbon assimilation. However, when solar energy is absorbed in excess to what can be utilized by the photosynthetic processes (e.g. under abiotic stress conditions), reactive oxygen species (ROS), including singlet oxygen (1O2), are produced in the chloroplasts, causing oxidative damage to macromolecules. Singlet oxygen is generated in chloroplasts by interaction of molecular oxygen with triplet state chlorophyll molecules in the reaction center and the chlorophyll antennae of the photosystems. This project is centered on 1O2 signalling in plants (Arabidopsis thaliana) during high light stress and involves two partners with complementary expertises. One of the two partners has developed new biochemical and biophysical methods to analyze and visualize lipid peroxidation and also to quantify the relative contribution of 1O2 and the other ROS produced during light stress. Combining these new analytical tools with the use of appropriate Arabidopsis mutants and well-chosen growth conditions, we will identify a physiological model that selectively produces high amounts of 1O2. The mutants that we will screen are affected in their capacity to detoxify 1O2 (deficiency in 1O2 quenchers and/or nonphotochemical energy quenching (NPQ)). By modulating the stress response, this physiologically relevant plant model should allow us to investigate different levels of 1O2 production leading to (i) cell death or (ii) photoacclimation. A transcriptomic analysis of our 1O2-generating model plants will be performed after different stress conditions [(i) and (ii) described above] and a number of genes identified as strongly upregulated will be studied in detail. In the context of our studies on signalling, a special attention will be given to enzymes involved in the oxidation of lipids and carotenoids. Because of its high reactivity and short lifetime, it is unlikely that the message transmitted to the nucleus to regulate gene expression is conveyed by 1O2 itself. More probably, the initial molecular messengers are natural target molecules of 1O2 in the chloroplast membranes: polyunsaturated fatty acids, lipid-soluble antioxidants or chlorophyll. This is the working hypothesis of this project. Lipid oxidation products (oxylipins) and carotenoid oxidation products are regarded as bioactive molecules with signalling functions, particularly in animals and during plant-pathogen interactions. The proposed research concerns the possible involvement of this type of molecules in 1O2 signalling in Arabidopsis plants exposed to high light stress. Different approaches, ranging from (bio)chemistry and biophysics to molecular biology and physiology will be used to tackle this problem. We will characterize the products generated in vitro by the oxidation of lipids, carotenoids and possibly tocopherols by 1O2. Subsequently, we will search for those molecules in vivo in Arabidopsis plant leaves exposed to light stress. We will also analyze conjugation of the lipid/carotenoid oxidation products to glutathione. Glutathione conjugates are known to be transported across membranes by transporters and therefore they are potential signal messengers from the chloroplast to the nucleus. Since lipid and carotenoids can be also oxidized by enzymatic systems (e.g. lipoxygenases (LOX), carotenoid cleavage dioxygenases (CCD)), we will study the regulation of those enzymes during high light stress and we will try to detect their products in high light-stressed plants. The possible signalling function of oxylipins and carotenoid/tocopherol oxidation products, generated either enzymatically or directly by 1O2 attack, will be investigated in Arabidopsis cell cultures. These in vitro analyses will be completed by studies on whole plants exposed to volatile oxidized carotenoids or lipids. Singlet oxygen signalling will also be analyzed in a number of Arabidopsis mutants (or in crossings between those mutants and our 1O2-overproducing plant model), such as those affected in the 13-LOX pathway (e.g. OPDA and jasmonate mutants: opr3/dd1, jar1, coi1), in carotenoid cleavage enzymes (CCDs) and in carotenoid, vitamin E or lipid composition (e.g. lut2, npq2, fad mutants). The proposed study is expected to provide new data on light stress in plants, on the way plants respond and acclimate to this stress by gene expression regulation and on the pathway that transmits signals from 1O2 to the nucleus to regulate gene expression.