Next generation genetics to identify regulatory networks involved in responses to single stresses and their combination in Arabidopsis – stressnet

Understanding plants’ responses to environmental stresses is crucial to maintain agricultural production. In this proposal we focus on two of the most prominent threats for modern agriculture: water deficit and nitrogen deficit. Drought episodes are expected to increase in frequency and severity as a result of global warming, while there is a strong pressure to decrease the use of nitrogen-based fertilizers due to their toxicity for the environment and for humans. It is therefore likely that crops will suffer from water and nitrogen deficiencies in the near future. Furthermore, the coexistence of water and nitrogen deficit has disastrous outcomes: as the soil dries out the pore space fills with air, making nutrients even less available to the plant. Our goal is to dissect the molecular mechanisms underlying plant responses to water deficit, nitrogen deficit and to both stresses combined.
In the last decade technologies such as metabolomics, transcriptomics or proteomics have been used to study the effect of abiotic stresses in plants. A common theme in these works is the detection of a highly complex set of interconnected molecular networks that modulate stress responses. The emerging picture is that of a two-tier mechanism in which multiple transcriptional pathways integrate environmental signals and elicit changes in a higher set of proteins and metabolic networks that determine the plant’s physiological response. Activation of these networks is dependent on the genotype under study, the tissue, developmental stage and the strength and duration of the stress. Interestingly, the molecular networks activated in response to a combination of two stresses can be hardly predicted from the responses elicited by the individual stresses separately. The complexity of these systems is the reason why stress plasticity remains one of the worst understood signaling processes.
It has been pointed out that systems biology approaches may be our only chance to dissect complex biological networks. In this proposal we integrate a series of genetic, genomic and bioinformatic protocols to reconstruct the molecular networks underlying plant responses to single or combined stresses. The only plant species where these types of holistic approaches can be implemented at full power is Arabidopsis thaliana, where there are large amounts of publically available genomic, molecular and genetic tools. Key tools in this project are the variation in stress responses naturally occurring among Arabidopsis accessions, and the availability of segregating populations and genomic sequences for these accessions.
In short, we propose to use of a robotic facility that applies stresses with high precision and reproducibility to acquire transcriptomic and metabolomic datasets in multiple Arabidopsis accessions and their hybrids. Our innovative experimental design and analysis pipelines will allow us to detect eQTL using allele-specific expression in F1 hybrids and to reconstruct and compare regulatory networks activated under stress. We then propose to perform metabolic profiling of one full segregating population under various stress conditions. This will allow us to link variation in expression with variation in metabolite abundance, and to identify the molecules that have been selected in evolution to modify stress responses without compromising fitness. Unveiling the identity of these molecules, even when found in an undomesticated species, are of great interest for agriculture, where the goal is to obtain stress resistant lines without affecting production.
This project is expected to result in international publications with interest in fundamental biology, as well as a number of opportunities for translational research in crops. The project includes release of the most useful part of the results through a website that will be of interest to labs working with natural variation in Arabidopsis.