Cold acclimation diversity in Arabidopsis thaliana: CRISPR/Cas9 as a tool to fine analysis of Tandem Gene Arrays, application to CBF genes.

In this period of climate change, it is of major importance to increase knowledge about the mechanisms by whose plants adapt to their environment. Tandem gene arrays (TAG) are overrepresented in the pool of tandem duplicates involved in stress response and are consequently of special interest. Nevertheless, until recently, addressing questions about individual genes or fine regulations in such structures was very difficult due to the close arrangement of the genome, almost preventing the production of targeted simple or multiple mutants. The CRISPR/Cas9 new tool offers new opportunities as the setting of gene deletion strategies in various genetic backgrounds. Here, we used this technology on the cold acclimation CBF pathway in different accessions of Arabidopsis thaliana with the same set of guide RNAs. Deleted lines free of T-DNA have been produced for simple or multiple copies of CBF genes and evaluated for cold tolerance after acclimation. Expression levels of CBF genes and five COR genes have also been analyzed. Our data suggest first that two or three missing CBF genes are necessary to induce a strong reduction in cold tolerance and secondly that most deletions have a low impact on the expression of remaining CBF copies which contradicts the previous hypothesis in the literature. Our results thus show that the CRISPR/Cas9 deletion strategy is a useful performance tool to investigate how tandem gene arrays work.

Mapping of Quantitative Trait Loci (QTL) Associated with Plant Freezing Tolerance and Cold Acclimation.

Most agronomic traits are determined by quantitative trait loci (QTL) and exhibit continuous distribution in natural or especially built segregating populations. The genetic architecture and the hereditary characteristics of these traits are much more complicated than those of oligogenic traits and need adapted strategies for deciphering. The model plant Arabidopsis thaliana is widely studied for quantitative traits, especially via the utilization of genetic natural diversity. Here we describe a QTL-mapping protocol for analyzing freezing tolerance after cold acclimation in this species, based on its specific genetic tools. Nevertheless, this approach can be applied for the elucidation of complex traits in others species.

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens.

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

Mapping of quantitative trait loci (QTL) associated with plant freezing tolerance and cold acclimation.

Most agronomic traits are determined by quantitative trait loci (QTL) and exhibit continuous distribution in segregating populations. The genetic architecture and the hereditary characteristics of these traits are much more complicated than those of oligogenic traits and need adapted strategies for deciphering. The model plant Arabidopsis thaliana is widely studied for quantitative traits, especially via the utilization of natural genetic diversity. Here we describe a QTL-mapping protocol for analyzing freezing tolerance after cold acclimation in this species based on its specific genetic tools. Nevertheless, this approach can also be applied for the elucidation of complex traits in other plant species.

Natural variation in the freezing tolerance of Arabidopsis thaliana: effects of RNAi-induced CBF depletion and QTL localisation vary among accessions.

Plants from temperate regions are able to withstand freezing temperatures and to increase their freezing tolerance during exposure to low, but non-freezing, temperatures through a process known as cold acclimation. Key regulatory proteins in this process are the cold-induced CBF1, 2 and 3 transcription factors which control many cold regulated genes. Although much work has focused on this signal transduction pathway, the details of its regulation and of its quantitative contribution to cold acclimation are still unclear. Here, we have used the large natural variation present in the 48 accessions of the Versailles core collection of Arabidopsis thaliana to further elucidate the function of the CBF transcription factors. CBF gene expression studies showed that the freezing sensitive accessions had mostly low expression levels 2h after transfer of plants to 5°C, while the most tolerant accessions showed a wide range of CBF expression levels. To investigate the quantitative contribution of CBF expression to plant freezing tolerance and low temperature growth performance, RNAi lines targeting all three CBF genes were produced in eight different accessions. We observed striking differences between different accessions in the effects that reduced CBF expression had on freezing tolerance, while effects on growth were generally too small to draw firm conclusions. Analysis of CBF expression indicated a tight co-regulation between CBF1 and CBF3, while the relationship between the expression levels of CBF2 and CBF1 or CBF3 strongly depended on the genetic background of the RNAi lines. In agreement with the observed differences between the different accessions, QTL analyses with two different RIL populations indicated that QTL localisation varies strongly between populations. Collectively, these results show that both the regulation of the CBF genes and their relative contribution to freezing tolerance strongly depend on the accession studied. In addition, natural variation is suggested to be an interesting source of novel regulatory pathways and genes that may be useful in the future for improving plant freezing tolerance.

ESKIMO1 is a key gene involved in water economy as well as cold acclimation and salt tolerance.

BACKGROUND: Drought is a major social and economic problem resulting in huge yield reduction in the field. Today's challenge is to develop plants with reduced water requirements and stable yields in fluctuating environmental conditions. Arabidopsis thaliana is an excellent model for identifying potential targets for plant breeding. Drought tolerance in the field was successfully conferred to crops by transferring genes from this model species. While involved in a plant genomics programme, which aims to identify new genes responsible for plant response to abiotic stress, we identified ESKIMO1 as a key gene involved in plant water economy as well as cold acclimation and salt tolerance. RESULTS: All esk1 mutants were more tolerant to freezing, after acclimation, than their wild type counterpart. esk1 mutants also showed increased tolerance to mild water deficit for all traits measured. The mutant's improved tolerance to reduced water supply may be explained by its lower transpiration rate and better water use efficiency (WUE), which was assessed by carbon isotope discrimination and gas exchange measurements. esk1 alleles were also shown to be more tolerant to salt stress. Transcriptomic analysis of one mutant line and its wild-type background was carried out. Under control watering conditions a number of genes were differentially expressed between the mutant and the wild type whereas under mild drought stress this list of genes was reduced. Among the genes that were differentially expressed between the wild type and mutant, two functional categories related to the response to stress or biotic and abiotic stimulus were over-represented. Under salt stress conditions, all gene functional categories were represented equally in both the mutant and wild type. Based on this transcriptome analysis we hypothesise that in control conditions the esk1 mutant behaves as if it was exposed to drought stress. CONCLUSION: Overall our findings suggest that the ESKIMO1 gene plays a major role in plant response to water shortage and in whole plant water economy. Further experiments are being undertaken to elucidate the function of the ESKIMO1 protein and the way it modulates plant water uptake.

Natural variation in CBF gene sequence, gene expression and freezing tolerance in the Versailles core collection of Arabidopsis thaliana.

BACKGROUND: Plants from temperate regions are able to withstand freezing temperatures due to a process known as cold acclimation, which is a prior exposure to low, but non-freezing temperatures. During acclimation, a large number of genes are induced, bringing about biochemical changes in the plant, thought to be responsible for the subsequent increase in freezing tolerance. Key regulatory proteins in this process are the CBF1, 2 and 3 transcription factors which control the expression of a set of target genes referred to as the "CBF regulon". RESULTS: To assess the role of the CBF genes in cold acclimation and freezing tolerance of Arabidopsis thaliana, the CBF genes and their promoters were sequenced in the Versailles core collection, a set of 48 accessions that maximizes the naturally-occurring genetic diversity, as well as in the commonly used accessions Col-0 and WS. Extensive polymorphism was found in all three genes. Freezing tolerance was measured in all accessions to assess the variability in acclimated freezing tolerance. The effect of sequence polymorphism was investigated by evaluating the kinetics of CBF gene expression, as well as that of a subset of the target COR genes, in a set of eight accessions with contrasting freezing tolerance. Our data indicate that CBF genes as well as the selected COR genes are cold induced in all accessions, irrespective of their freezing tolerance. Although we observed different levels of expression in different accessions, CBF or COR gene expression was not closely correlated with freezing tolerance. CONCLUSION: Our results indicate that the Versailles core collection contains significant natural variation with respect to freezing tolerance, polymorphism in the CBF genes and CBF and COR gene expression. Although there tends to be more CBF and COR gene expression in tolerant accessions, there are exceptions, reinforcing the idea that a complex network of genes is involved in freezing tolerance and that the CBF genes alone cannot explain all differences in phenotype. Our study also highlights the difficulty in assessing the function of single transcription factors that are members of closely related gene families.