The group is exploiting genomic methods as well as classical and quantitative genetics to understand the genetic networks that underly complex characters such as abiotic stress responses. In order to undertake this work, the department has invested in high performance tools needed to understand genetic variation: the department has created and manages series of genetic resources for Arabidopsis thaliana, it has put in place a high-throughput genotyping platform, and it is developping a high-throughput phenotyping tool.
The study of the response to abiotic stress is of both fundamental and agronomic interest. Abiotic stress is a major cause of lower yield, for all types of crops, and in diverse environments. Plants, as organisms dependent on their surroundings, have more or less developed capacities to tolerate episodes of climatic extremes. Certain plant species or varieties within a species are capable of continued growth and reproduction in physical conditions that vary from their norms ; for this they take advantage of the plasticity of their genomes. Others restrict their life cycle in a series of narrower parameters ; they are adapted to a particular surrounding. This diversity in behavior is exploited to understand the mechanisms of tolerance to abiotic stress, in particular, drought, osmotic and cold stress. To breed crops adapted to their evolving culture conditions -a major challenge for the XXIst century- it is necessary to get a better understanding of stress signal pathways, their regulation and the mechanisms of the physiological responses.

Water deficit and osmotic stress

We are looking for variable factors (genes) that intervenes in the tolerance to these stresses. To achieve this, several approaches have been taken :

The identification of major and minor QTLs (Quantitative Trait Loci) will allow us to update the list of genes involved in the responses to these stresses as well as the interesting alleles and their interactions within a network of genes. In parallel, association studies, relying on statistical methods, between the genotype of candidate genes and the behavior of the plant under stress, will be performed. This approach will confirm or not the importance of the candidate genes in the expression of the trait. When the QTLs define new candidate genes, positional cloning of the gene(s) will be conducted. This key step will benefit from our access to the high-throughput genotyping and phenotyping platforms.

Concerning water deficit, we have determined conditions for a moderate stress to obtain an interaction between the response of the genotypes and the treatment. The plants are grown on a commercially available substrate, in sub-optimal conditions of hydration. We have determined the vegetative and reproductive traits that vary with the genotype and with the hydration conditions.
Osmotic stress is studied in vitro by the application of a moderate osmotic stress (mannitol, PEG). The effect on the growth of the leaves and the seedling is used as a model for cellular growth and the integration of signals from the environment. The QTLs detected at this stage will also be studied individually in conditions of water deficit.
Root system architecture is another important factor that needs to be studied in details to understand a genotype's response to drought stress. We use in vitro displays to do so, allowing us also to measure the response to osmotic stress.

In parallel to these quantitative genetics studies, our team is involved in a large genomics program aiming at identifying mutants in genes with unknown function, named “orphan genes”, altered in their response to drought stress. In a list of mutants that were selected in silico, a systematic screen allowed us to retain about forty mutants for deeper genetic, physiological and molecular analysis. Once genes are validated on their role in response to drought, a complete functional analysis is engaged to unravel their function.

More information on www.inra.fr/vast

Cold tolerance

Our team is working on mechanisms involved in cold tolerance, mainly on the model plant Arabidopsis thaliana.
Some mechanisms allowing plants to survive at freezing temperature have been partially elucidated recently. A lot of studies dealing with the acclimation process have conducted to identify some response pathway, especially the CBF one. On the other hand, transcriptome analyses suggest that other pathways exist and have to be unravelled.
Several strategies are currently being done in our team to identify such new genes.
The first one consists in exploring natural variability in order to characterize accessions exhibiting variable response to cold stress. Based on this first screen and using the genetic resources of the Centre of Resources in Versailles (mainly RIL), a QTL approach could allow identification of new genes. 8 accessions of the core collection, showing variable degree of tolerance have been characterised in this purpose.
In parallel, we have participated to a genomic project dealing with orphan genes. Mutants affected in such genes and impaired in the cold response have been searched.
At last we are studying natural variability for CBF gene family. Natural diversity is analysed by sequencing, specific gene expression and phenotypic test for cold tolerance. Combining all these different data should elucidate the importance of this pathway in the cold response in relation with genetic background and produce data on the role of each member of this gene family. Moreover RNAi approach is used to inactivate CBF genes in different accessions. Phenotypic test on these lines will complete data on previous analysis.
All these approaches need a robust phenotypic test. We have elaborated a freezing test including or not an acclimation phase. Plants are grown in soil and the test is realised in climate rooms able to freeze. This is destructive for the most sensitive plants. Damages are evaluated on a scale from o (no damage) to 6 (dead plants). (Protocol is available by asking).

observations on 2 types of seed sowing (in groups of seeds or individually) after the same cold stress.

Results

The phenotypic test is reliable, reproducible, and allows revealing high variability. It has been used to evaluate the cold response of the 48 accessions of the core collection. Among these, 4 are very sensitive, apparently unable to acclimate, and 4 are very tolerant until –8°C after acclimation.
We didn’t identify any accessions able to survive to freezing period without acclimation as the Eskimo mutant does. Crosses between sensitive and resistant accessions indicate that resistance is mostly dominant. Crosses between sensitive lines are mostly sensitive indicating that common genes are involved. A plasmid has been constructed to induce depletion of CBF by RNAi and has been introduced in 9 different accessions. Phenotypic analysis of the transformed lines is presently realised.
Expression analysis of CBF1, CBF2 and CBF3 has been performed on accessions exhibiting contrasted responses to cold stress. A synthetic analysis of phenotypic data and expression results is under study.
In the program on orphan genes: among 225 mutant lines that have been selected in silico, a mass screening has revealed 80 interesting ones. These will be submitted to deeper genetic analyses. The best candidates will then enter a functional analysis.

Epigenetic regulations

The importance of epigenetic processes in Eucaryotic gene regulation was established in the past years, and experiments undertaken on plants played a key role in the discovery of the mechanisms underlying these processes. It is now clear that gene expression is not regulated only throughout interactions between promoters and transcriptional activators, and that it can also be modulated epigenetically via chromatin remodelling, DNA methylation, histone modifications, mRNA degradation or translational repression. Epigenetic modifications do not alter the DNA sequence, but are heritable mitotically and/or meiotically and are potentially reversible. Most epigenetic regulations depend upon DNA/RNA or RNA/RNA interactions involving small RNAs (20-25 nucleotides long).
Phenotypic changes have been observed in plants exposed to extreme environments as well as in theirprogenies. This led to the hypothesis that environmental stimuli could induce heritable epigenetic modifications. The occurence of epialleles in response to environmental stresses, followed by selection, would allow a rapid adaptation to new conditions, without changes in the DNA sequence. The potentially reversible nature of epialleles allows some plasticity of gene expression. This feature would be advantageous if the inducing environmental conditions were relatively short-termed.
Our aim is to determine if epigenetic regulations have an impact on the adaptability and evolution of plant species. To do so, we will use the model plant species Arabidopsis thaliana, and will focus our research on its tolerance to cold and moderate drought.