Identification of candidate genes important for frost tolerance in Festuca pratensis Huds. by transcriptional profiling
Introduction
The Pooideae grasses (temperate grasses) is a large and economically important plant sub-family, including cereals (Triticeae tribe) and forage grasses (Poaeae tribe). Meadow fescue (Festuca pratensis Huds.) is the second most important forage grass species in Northern Europe. Species within the genera Festuca have a higher level of general stress tolerance compared with perennial ryegrass and is therefore excellent for studying plant adaptations to cold environment because of their adaptation to a life in northern most Europe.
Exposure to low, non-freezing temperatures induces a process in plants known as cold acclimation (CA). This process improves frost tolerance (FT) and survival of over-wintering plants through the expression of cold-responsive genes, which ultimately leads to altered physiological state of the plant [1]. The CA process is multigenic and drives major metabolic changes including changes in carbohydrates, proteins, nucleic acids, amino acids, growth regulators, phospholipids, and fatty acids [2]. Expression analyses using the Arabidopsis whole-genome array have shown that about 4–14% of the Arabidopsis genome is cold-responsive [3], [4]. Similar results have been reported in Pooideae grasses [5], [6], [7].
Low temperature specific gene expression is mediated by different parallel signal transduction pathways. In Arabidopsis two main signaling pathways are known; one is depending on the involvement of the phytohormone abscisic acid (ABA) and one is not [8]. Both pathways trigger the expression of a range of transcription factors (TFs) that binds to CRT/DRE, ABRE, and MYCR/MYBR binding sites, amongst others, and regulates downstream transcription of CA and FT associated genes [9].
Many molecular processes which happen during CA are conserved between the dicot and monocots lineages [10]. However, most studies on CA transcriptional responses have been carried out on species that are not adapted to a perennial life in extreme winter climates such as the Pooideae forage grasses. This is important because adaptation to a perennial life history in harsh winter climates must have required changes at the genetic level which cannot be studied using an annual model species. If we constrict our research on cold and frost stress transcriptional responses to Arabidopsis or annual Triticeae species, we can only have limited insights into the genetic mechanisms underlying frost tolerance in important agricultural forage grass species. Some investigations into CA transcriptional responses in perennial forage grasses have been limited to L. perenne [11], [12], a species which does not represent a typical frost tolerant forage grass.
Furthermore, identical CA conditions can result in large differences in interspecific FT levels. Such differences have been linked to several QTLs in Pooideae species [13], [14], unpublished results including one QTL which co-locate with CBF transcription factors upstream in the regulation of CA-associated genes [15], [16]. This might suggests that natural variation in regulation of downstream targets during CA may be a common cause for differences in FT levels. The relative importance of regulatory mutations versus structural mutations in adaptive evolution is a debated topic [17], however there are many examples of natural variation in gene regulation contributing to phenotypic evolution [18], [19]. An attractive hypothesis is therefore that selection acts on variation in expression levels of important CA genes that are involved in minimizing damaging effects of cold stress.
The aim of this work was to identify genes differentially regulated in response to cold acclimation (CA) in the frost tolerant forage grass F. pratensis and to establish links between differences in CA induced gene expression and levels of frost tolerance (FT). This was achieved using a combination of experiments involving artificial selection for frost tolerance, phenotyping of FT using freezing tests, SSH-EST library construction, and microarray analysis.
Section snippets
Plant materials used to generate SSH cDNA libraries
Plant material used to construct the two SSH cDNA libraries were developed from a pair-cross between a genotype from a Yugoslavian cultivar (B14/16) and a genotype from a Norwegian cultivar (HF2/7). A total of 138 plants from the F1 population were freeze tested by Alm et al. [20] and the two most extreme genotypes with the lowest and highest FT was selected. Five clonal ramets of the two genotypes were pre-grown at 17/12 °C day/night, 16/8 h light/darkness for 12 weeks. The light intensity was
Divergent selection for frost tolerance
The results of the divergent selections for frost tolerance demonstrate good responses to selection especially for selection in direction high frost tolerance (Table 1). Additive genetic variation for freezing tolerance is thus large within the ‘B14/16 × HF2/7′ meadow fescue mapping population, illustrated by realized heritability estimates of h2 = 0.89 and 0.39 for selection in direction high and low frost tolerance, respectively. The difference in realized heritability is explained by differences
Functional groups involved in CA
In this study we have successfully combined the technologies of SSH and cDNA microarrays to identify genes differentially expressed between cold acclimated and non-acclimated plant tissues. In general, the results are comparable to other studies on CA as regards the distribution of functional classes in our EST libraries [30], [31], [32]. There was relatively high abundance of differentially expressed genes involved in cell signaling i.e. receptor-like protein kinases, calcium-modulated protein
Acknowledgements
We thank Drs. Lidia Skuza and Magdalena Achrem for excellent help with the qRT-PCR and BAC sequencing work, and Øyvind Jørgensen and Britta Fromm for excellent technical assistance with the plant propagation and freezing tests. We are also indebted to Dr. Oene Dolstra and Carole Boucoiran, Plant Research International, Wageningen, The Netherlands, for performing the microarray hybridizations. We also thank Dr. Iain Donnison and Dr. Kerrie Farrar, IBERS, Aberystwyth University, UK, for the
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