No abstract

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

Wheat chromosome 5A plays a key role in cold acclimation and frost tolerance. The major frost tolerance gene Fr-A1 (formerly Fr1) and two loci that regulate the transcription of cold-regulated genes (Cor) have previously been mapped on the long arm of this chromosome. In this study we report the discovery of a new locus for frost tolerance designated Fr-A2. This new locus was mapped on the long arm of chromosome 5A of diploid wheat (T. monococcum), 40 cM from the centromere and 30 cM proximal to the major frost tolerance locus Fr-A1. We found also that frost-tolerant and frost-susceptible T. monococcum parental lines differed in the transcription level of the cold induced gene Cor14b when plants were grown at 15°C. Transcription levels of this gene were measured in each of the recombinant inbred lines and mapped as a QTL that perfectly overlapped the QTL for frost survival at the Fr-A2 locus. This result suggested that frost tolerance in this cross was mediated by differential regulation of the expression of the Cor genes. In a previous study in hexaploid wheat (T. aestivum) we had shown that Cor14b was regulated by two loci located on chromosome 5A, one in the same chromosome region as the T. monococcum Fr-A2 locus and the other one closely linked to Fr-A1. Since CBF transcriptional activators in Arabidopsis regulate Cor genes and are involved in frost tolerance, we decided to localize the cold-regulated CBF-like barley gene Cbf3 on the T. monococcum map. This gene was mapped on the peak of the Fr-A2 QTL for frost tolerance. This result suggests that the observed differential regulation of Cor14b at the Fr-A2 locus is due to allelic variation at the XCbf3 locus, and that this transcriptional activator gene might be a candidate gene for the Fr-A2 frost tolerance locus on wheat chromosome 5A.

The C-repeat binding factor (Cbf) gene family has been shown to have a critical role in the regulation of low-temperature stress response in Arabidopsis. In Triticum monococcum, a locus carrying a family of Cbflike genes, orthologs of Arabidopsis Cbf genes, is tightly linked to the frost tolerance locus Fr-A m 2, representing candidates for the differences in frost tolerance mapped at this locus. In this work we show that several Cbf genes have dramatically different levels of induction after cold exposure in hexaploid wheat. The Cbf-transcription levels differ between substitution and single chromosome recombinant lines carrying different 5A chromosomes or chromosome segments of the chromosome 5A from frost-tolerant and frost-sensitive wheat varieties. When the expression of eight Cbf genes, previously mapped at the Fr-A2 locus was investigated with gene specific primers using real-time RT-PCR, three Cbf sequences (Cbf1A, Cbf1C, Cbf7) showed a significantly higher relative transcription level (more than fourfold change) in lines differing for the Fr-A2 region. Differences in Cbf expression were also associated with a variation in frost tolerance. These results suggest that the amount of some Cbf mRNAs might be a critical factor for determining the level of frost tolerance in wheat.

Although cold acclimation in cereals involves the expression of many cold-regulated genes, genetic studies have shown that only very few chromosomal regions carry loci that play an important role in frost tolerance. To investigate the genetic relationship between frost tolerance and the expression of cold-regulated genes, the expression and regulation of the wheat homolog of the barley cold-regulated gene cor14b was studied at various temperatures in frost-sensitive and frost-tolerant wheat genotypes. At 18/15°C (day/night temperatures) frost-tolerant plants accumulated cor14b mRNAs and expressed COR14b proteins, whereas the sensitive plants did not. This result indicates that the threshold temperature for induction of the wheat cor14b homolog is higher in frost-resistant plants, and allowed us to use this polymorphism in a mapping approach. Studies made with chromosome substitution lines showed that the polymorphism for the threshold induction temperature of the wheat cor14b homolog is controlled by a locus(i) located on chromosome 5A of wheat, while the cor14b gene was mapped in Triticum monococcum on the long arm of chromosome 2A m . The analysis of single chromosome recombinant lines derived from a cross between Chinese Spring/Triticum spelta 5A and Chinese Spring/Cheyenne 5A identi®ed two loci with additive eects that are involved in the genetic control of cor14b mRNA accumulation. The ®rst locus was tightly linked to the marker psr911, while the second one was located between the marker Xpsr2021 and Frost resistance 1 (Fr1).

The enhancement of winter hardiness is one of the most important tasks facing breeders of winter cereals. For this reason, the examination of those regulatory genes involved in the cold acclimation processes is of central importance. The aim of the present work was the functional analysis of two wheat CBF transcription factors, namely TaCBF14 and TaCBF15, shown by previous experiments to play a role in the development of frost tolerance. These genes were isolated from winter wheat and then transformed into spring barley, after which the effect of the transgenes on low temperature stress tolerance was examined. Two different types of frost tests were applied; plants were hardened at low temperature before freezing, or plants were subjected to frost without a hardening period. The analysis showed that TaCBF14 and TaCBF15 transgenes improve the frost tolerance to such an extent that the transgenic lines were able to survive freezing temperatures several degrees lower than that which proved lethal for the wild-type spring barley. After freezing, lower ion leakage was measured in transgenic leaves, showing that these plants were less damaged by the frost. Additionally, a higher Fv/Fm parameter was determined, indicating that photosystem II worked more efficiently in the transgenics. Gene expression studies showed that HvCOR14b, HvDHN5, and HvDHN8 genes were up-regulated by TaCBF14 and TaCBF15. Beyond that, transgenic lines exhibited moderate retarded development, slower growth, and minor late flowering compared with the wild type, with enhanced transcript level of the gibberellin catabolic HvGA2ox5 gene.

A cluster of eleven CBF genes was recently mapped to the Frost resistance-2 (Fr-Am2) locus on chromosome 5 of diploid wheat (Triticum monococcum) using a cross between frost tolerant accession G3116 and frost sensitive DV92. The Fr-Am2 locus was mapped at the peak of two overlapping quantitative trait loci (QTL), one for frost survival and the other for differential expression of the cold regulated gene COR14b. Seven lines with recombination events within the CBF cluster were used to identify CBF candidate genes for these QTL. The lines carrying the critical recombination events were tested for whole plant frost survival and for differential transcript levels of cold induced COR14b and DHN5 genes. The strongest effect for these traits was associated to the linked TmCBF12, TmCBF14 and TmCBF15 genes, with the G3116 allele conferring improved frost tolerance and higher levels of COR14b and DHN5 transcript at mild cold temperatures (12-15 degrees C) than the DV92 allele. Comparison of CBF protein sequences revealed that the DV92 TmCBF12 protein contains a deletion of five amino acids in the AP2 DNA binding domain. Electrophoretic Mobility Shift Assays (EMSA) confirmed that the protein encoded by this allele cannot bind to the CRT/DRE (C-repeat/ dehydration-responsive element) motif present in the promoters of several cold induced genes. A smaller effect on frost tolerance was mapped to the distal group of CBF genes including TmCBF16. Transcript levels of TmCBF16, as well as those of TmCBF12 and TmCBF15 were up-regulated at mild cold temperatures in G3116 but not in DV92. Higher threshold induction temperatures can result in earlier initiation of the cold acclimation process and better resistance to subsequent freezing temperatures. The non-functional TmCBF12 allele in DV92 can also contribute to its lower frost tolerance.

The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chromosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [(35)S]sulfate. In Ch and CS (Ch 5A) the total cysteine, gamma-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat.