CHromatin and ROS: Role of Topoisomerase VI-mediated chromatin remodelling in the transcriptional response of plants to singlet oxygen, ROS and abiotic stresses. – CHROS

The aim of the proposed experiments is to understand the role of the Topoisomerase VI in the plant adaptive response to adverse environmental conditions and, more specifically, the molecular mechanisms by which the Topo VI complex and associated chromatin remodeling factors mediate stress-related gene expression changes in response to oxidative and abiotic stresses. We work with the model species Arabidopsis thaliana to benefit from all the studies, data, microarray facilities, mutant collections, methods related to chromatin analysis, etc... that are available to the scientific community and makes it possible to use state-of-the-art technologies and assays to understand aspects of gene regulation at the chromatin level and at the genome scale. The expected results should be easily transposable to crops and other plants, however. Arabidopsis lines that allow the selective and controlled generation of reactive oxygen species (ROS) are analyzed for their expression profiles using whole genome expression microarray technology. To identify the ROS-responsive genes that are direct targets of Topo VI - physically bound by Topo VI -, and hence distinguish between direct effects and those mediated by signal transduction pathways, we examine in vivo the association of Topo VI and other chromatin-related factors with ROS-responsive gene promoters and other genomic locations by chromatin immunoprecipitation (ChIP) assays at the genome scale.

No results to be disseminated broadly at this stage of the project

This project proposes an original approach to improve abiotic stress tolerance via the study of the topoisomerase VI and its role in regulating transcription of genes involved in the adaptive response of the plant to stress. The results obtained in Arabidopsis can be transferred in species of agronomic interest (search for favorable alleles of these genes by TILLING or EcoTILLING). A project with this outlook application has been submitted for a PACA Region Doctoral Scholarship with a private partner (Gautier Semences, Eyragues, Bouches-du-Rhone, France). This request was rejected in 2012 but will be resubmitted.

Publications :
• Simkova, K, Moreau, F, Pawlak, P, Vriet, V, Baruah, A, Alexandre, C, Hennig, L, Apel, K and Laloi, C. The integration of stress-related and ROS-mediated signals by Topoisomerase VI in Arabidopsis thaliana. En revision
Communications (invited speaker) :
• Conference ‘Molecular Basis of Plant Stress’, Sunny Day Black Sea resort, Varna, Bulgaria, 21-23.9.2011. « The role of the Arabidopsis Topoisomerase VI in the expression of singlet oxygen-regulated genes ».
• Journées de la société française de Photosynthèse, Ecole Normale Supérieure, Paris, 24-25.05.2012. « Singlet oxygen signaling in Arabidopsis ».

Plants are constantly exposed to environmental changes that adversely affect their growth and development and may ultimately result in the death of the plant. Thus, abiotic stresses constitute a major limitation to agricultural production and, according to the International Service for the Acquisition of Agri-biotech Applications (ISAAA), are the primary cause of crop loss worldwide, reducing average yields by over 50% (www.isaaa.org). Most of these stress conditions disrupt the metabolic balance of cells, resulting in the enhanced production of reactive oxygen species (ROS), primarily in chloroplasts. The generated ROS may be toxic and cause oxidative damage, or they may act as signalling molecules and activate the plant’s defenses against environmental stress.
Singlet oxygen, a non-radical ROS, produced inside the chloroplast, has been shown to be involved in plastid-to-nucleus retrograde signalling and to activate nuclear genes that are distinct from those activated by other ROS. We used a genetic approach to identify extraplastidic components involved in retrograde plastid-to-nucleus signalling that may directly regulate nuclear gene expression. A transgenic line expressing a singlet oxygen reporter gene was mutagenized and then mutants were isolated that are deregulated in the expression of the reporter gene. One of these mutants that have lost the capacity to activate the reporter gene following the release of singlet oxygen, carries a mutation in the chromatin remodelling factor Topoisomerase VI. We could show that Topo VI works both as a positive regulator of singlet oxygen-responsive genes and a negative regulator of hydrogen peroxide-responsive genes. These results suggested a central role of Topo VI as an integrator of multiple signals generated by different ROS that are released in response of plants to adverse environmental conditions.
The CHROS project is aimed at elucidating the molecular mechanisms by which the topoisomerase VI and associated chromatin remodelling activities regulate singlet oxygen and ROS-responsive gene expression changes in response to abiotic stresses. Knowing that the production of ROS is a common feature of most abiotic stresses, results from the study of this model system may disclose widely-spread molecular mechanisms of the plant response to adverse environmental conditions.
Firstly, we will identify the singlet oxygen-responsive genes that are genuine direct targets of Topo VI by comparing genome-wide expression and ChIP-on-chip profiles of WT and a Topo VI knock-down mutant line under conditions that release singlet oxygen. Secondly, once several genuine singlet oxygen-responsive Topo VI target genes have been identified, we will investigate the molecular mechanisms by which Topo VI and associated chromatin remodelling factors change a quiescent target gene into an actively transcribed gene in response to singlet oxygen; we will examine the modifications of Topo VI, the chromatin modifications and study other candidate Topo VI-associated chromatin remodelling factors. Thirdly, since Arabidopsis Topo VI seems to act as an integrator of multiple signals generated by different ROS and that acclimation processes can be triggered by ROS, we will examine the role of Topo VI in acclimation processes and ultimately we will study transgenerational inheritance of ROS-induced chromatin modifications.