Summary: Genetic analyses have identified two loci in wheat and barley that mediate the capacity to overwinter in temperate climates. One locus co‐segregates with VRN‐1, which affects the vernalization requirement. This locus is known as Frost resistance‐1 (Fr‐1). The second locus, Fr‐2, is coincident with a cluster of more than 12 Cbf genes. Cbf homologs in Arabidopsis thaliana play a key regulatory role in cold acclimatization and the acquisition of freezing tolerance. Here we report that the Hordeum vulgare (barley) locus VRN‐H1/Fr‐H1 affects expression of multiple barley Cbf genes at Fr‐H2. RNA blot analyses, conducted on a ‘Nure’בTremois’ barley mapping population segregating for VRN‐H1/Fr‐H1 and Fr‐H2, revealed that transcript levels of all cold‐induced Cbf genes at Fr‐H2 were significantly higher in recombinants harboring the vrn‐H1 winter allele than in recombinants harboring the Vrn‐H1 spring allele. Steady‐state Cbf2 and Cbf4 levels were also significantly higher in recombinants harboring the Nure allele at Fr‐H2. Additional experiments indicated that, in vrn‐H1 genotypes requiring vernalization, Cbf expression levels were dampened after plants were vernalized, and dampened Cbf expression was accompanied by robust expression of Vrn‐1. Cbf levels were also significantly higher in plants grown under short days than under long days. Experiments in wheat and rye indicated that similar regulatory mechanisms occurred in these plants. These results suggest that VRN‐H1/Fr‐H1 acts in part to repress or attenuate expression of the Cbf at Fr‐H2; and that the greater level of low temperature tolerance attributable to the Nure Fr‐H2 allele may be due to the greater accumulation of Cbf2 and Cbf4 transcripts during normal growth and development.

In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are: What determines the size and scope of the CBF regulon, and is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to −4°C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived −14°C. Both species possessed low-temperature-induced CBFs but differed in the expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a promoter structure comprising modules proximal and distal to the coding sequence. CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However, fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low-temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, their proximity to one another is likely key to regulatory output in Medicago, and they may be located kilobases distal to the transcriptional start site. We hypothesize that these differences in CRT/DRE copy numbers in CAS30/CAS31 upstream regions combined with differences in gene copy numbers may be a factor in determining differences in low-temperature tolerance between M. truncatula and M. falcata.

Background: Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. Results: Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. Conclusions: We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.