Reactive oxygen species production and signalling in the unicellular green algae Chlamydomonas reinhardtii – ROS-Signal

Life in an oxygen-rich atmosphere has to deal with the danger of oxidative stress. Plants are especially exposed to oxidative stress caused by photosynthetic processes. Reactive oxygen species (ROS) are produced during normal cell metabolism but their production is drastically enhanced when plants are exposed to stresses such as high light, low temperatures or drought. Despite the fact that ROS have been considered to be damaging molecules, it is recognized that they, especially H2O2, play a major role in cellular signaling pathways and regulation of gene expression in a wide range of organisms including plants. The aim of the project is to understand how the production and accumulation of ROS is controlled and how ROS can function as signaling molecules. Environmental stress is perceived primarily in the chloroplast by perturbations of the photosynthetic electron flow which lead to an increased ROS production. At the same time the redox state of the chloroplast is sensed by the reduction state of thioredoxin. According to our working hypothesis, thioredoxins and related thiolreactive molecules down-regulate the activity of ROS detoxifying enzymes and thereby allow a transient oxidative burst that triggers the expression of ROS responsive genes. We will study the molecular mechanism and kinetics of production and accumulation of ROS during photosynthesis, the role of thioredoxin in controlling photosynthetic flow and ROS production, the role of thioredoxins in the regulation of the activity of the ROS-detoxifying enzymes (ascorbate peroxidases and catalases) and the expression level of target genes in response to different ROS. A genetic dissection of ROS signaling pathways will also be performed taking advantage of recently developed molecular tools that allow monitoring in vivo ROS signaling in Chlamydomonas, based on the use of promoters responding specifically to 1O2 or H2O2 that are coupled to a luciferase reporter gene. In addition, a reverse genetic approach based on the use of RNA interference will be developed in Chlamydomonas to specifically knock down each component of the chloroplast TRX system. This will allow a functional analysis of chloroplastic thioredoxins in the control of the photosynthetic electron flow, the production of ROS, the regulation of ROS detoxifying enzymes and ROS signaling. This multidisciplinary project will combine techniques of spectroscopy, biochemistry, molecular biology and genetics to investigate the whole process of ROS production and signaling that allows adaptation of photosynthetic organisms to environmental conditions. The unicellular green alga Chlamydomonas reinhardtii will be used as model organism. Chlamydomonas is a haploid organism which facilitates genetic studies, mutants affected in photosynthesis are easy to grow and spectroscopic detection of ROS in vivo is easier in Chlamydomonas than in higher plants. Taken together these different approaches will give an integrated picture of the in vivo situation in a photosynthetic organism.