Identification, Structural and Functional Characterization of Dormancy Regulator Genes in Apricot (Prunus armeniaca L.).

In the present study, we identified and characterized the apricot (Prunus armeniaca L.) homologs of three dormancy-related genes, namely the ParCBF1 (C-repeat binding factor), ParDAM5 (dormancy-associated MADS-BOX) and ParDAM6 genes. All highly conserved structural motifs and the 3D model of the DNA-binding domain indicate an unimpaired DNA-binding ability of ParCBF1. A phylogenetic analysis showed that ParCBF1 was most likely homologous to Prunus mume and Prunus dulcis CBF1. ParDAM5 also contained all characteristic domains of the type II (MIKCC) subfamily of MADS-box transcription factors. The homology modeling of protein domains and a phylogenetic analysis of ParDAM5 suggest its functional integrity. The amino acid positions or small motifs that are diagnostic characteristics of DAM5 and DAM6 were determined. For ParDAM6, only a small part of the cDNA was sequenced, which was sufficient for the quantification of gene expression. The expression of ParCBF1 showed close association with decreasing ambient temperatures in autumn and winter. The expression levels of ParDAM5 and ParDAM6 changed according to CBF1 expression rates and the fulfillment of cultivar chilling requirements (CR). The concomitant decrease of gene expression with endodormancy release is consistent with a role of ParDAM5 and ParDAM6 genes in dormancy induction and maintenance. Cultivars with higher CR and delayed flowering time showed higher expression levels of ParDAM5 and ParDAM6 toward the end of endodormancy. Differences in the timing of anther developmental stages between early- and late-flowering cultivars and two dormant seasons confirmed the genetically and environmentally controlled mechanisms of dormancy release in apricot generative buds. These results support that the newly identified apricot gene homologs have a crucial role in dormancy-associated physiological mechanisms.

Genome-wide association study and genetic diversity analysis on nitrogen use efficiency in a Central European winter wheat (Triticum aestivum L.) collection.

To satisfy future demands, the increase of wheat (Triticum aestivum L.) yield is inevitable. Simultaneously, maintaining high crop productivity and efficient use of nutrients, especially nitrogen use efficiency (NUE), are essential for sustainable agriculture. NUE and its components are inherently complex and highly influenced by environmental factors, nitrogen management practices and genotypic variation. Therefore, a better understanding of their genetic basis and regulation is fundamental. To investigate NUE-related traits and their genetic and environmental regulation, field trials were evaluated in a Central European wheat collection of 93 cultivars at two nitrogen input levels across three seasons. This elite germplasm collection was genotyped on DArTseq® genotypic platform to identify loci affecting N-related complex agronomic traits. To conduct robust genome-wide association mapping, the genetic diversity, population structure and linkage disequilibrium were examined. Population structure was investigated by various methods and two subpopulations were identified. Their separation is based on the breeding history of the cultivars, while analysis of linkage disequilibrium suggested that selective pressures had acted on genomic regions bearing loci with remarkable agronomic importance. Besides NUE, genetic basis for variation in agronomic traits indirectly affecting NUE and its components, moreover genetic loci underlying response to nitrogen fertilisation were also determined. Altogether, 183 marker-trait associations (MTA) were identified spreading over almost the entire genome. We found that most of the MTAs were environmental-dependent. The present study identified several associated markers in those genomic regions where previous reports had found genes or quantitative trait loci influencing the same traits, while most of the MTAs revealed new genomic regions. Our data provides an overview of the allele composition of bread wheat varieties anchored to DArTseq® markers, which will facilitate the understanding of the genetic basis of NUE and agronomically important traits.

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley.

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light-including its spectra-also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeumvulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance.

Light and Temperature Signalling at the Level of CBF14 Gene Expression in Wheat and Barley.

The wheat and barley CBF14 genes have been newly defined as key components of the light quality-dependent regulation of the freezing tolerance by the integration of phytochrome-mediated light and temperature signals. To further investigate the wavelength dependence of light-induced CBF14 expression in cereals, we carried out a detailed study using monochromatic light treatments at an inductive and a non-inductive temperature. Transcript levels of CBF14 gene in winter wheat Cheyenne, winter einkorn G3116 and winter barley Nure genotypes were monitored. We demonstrated that (1) CBF14 is most effectively induced by blue light and (2) provide evidence that this induction does not arise from light-controlled CRY gene expression. (3) We demonstrate that temperature shifts induce CBF14 transcription independent of the light conditions and that (4) the effect of temperature and light treatments are additive. Based on these data, it can be assumed that temperature and light signals are relayed to the level of CBF14 expression via separate signalling routes.

The expression of CBF genes at Fr-2 locus is associated with the level of frost tolerance in Bulgarian winter wheat cultivars.

The regulation of the majority of cold-regulated genes in plants is mediated by CBF (C-repeat binding factors) transcription factor family. Natural differences in frost tolerance (FT) of wheat have been mapped to the Fr-2 (Frost Resistance-2) locus on chromosome group 5 and are associated with variation in threshold induction temperatures and/or transcript levels of CBF genes. This study used real-time reverse-transcription polymerase chain reaction (qRT-PCR) to compare the relative expression levels of four T. aestivum CBF genes (TaCBF15.2, TaCBFA19, TaCBFA2 and TaCBFD21) in crown tissue of two Bulgarian hexaploid winter wheat cultivars (Milena and Russalka) with distinct levels of low-temperature (LT) tolerance but same vernalization requirement, and the spring cultivar Chinese Spring. The transcription profiles of the selected TaCBF genes showed that they are induced by cold treatment at 2 °C. Analysis of transcript abundance revealed that the four TaCBF genes were expressed at higher levels in the frost tolerant Milena than in the susceptible Russalka. Largest differences (fivefold and fourfold) in expression levels between both winter cultivars were observed in two of the analysed genes, TaCBF15.2 and TaCBFA19, respectively. The higher steady-state expression levels of TaCBF genes before the onset of the LT treatment in Milena, combined with stronger induction by cold treatment, suggest that these molecular responses to LT are associated with superior FT development capacity. The results expand our understanding of the molecular mechanisms underlying LT acclimation in Bulgarian wheat and can be used for development of functional markers for improvement of FT wheat-breeding programmes.

Large deletions in the CBF gene cluster at the Fr-B2 locus are associated with reduced frost tolerance in wheat.

Wheat plants which are exposed to periods of low temperatures (cold acclimation) exhibit increased survival rates when they are subsequently exposed to freezing temperatures. This process is associated with large-scale changes in the transcriptome which are modulated by a set of tandemly duplicated C-repeat Binding Factor (CBF) transcription factors located at the Frost Resistance-2 (Fr-2) locus. While Arabidopsis has three tandemly duplicated CBF genes, the CBF family in wheat has undergone an expansion and at least 15 CBF genes have been identified, 11 of which are present at the Fr-2 loci on homeologous group 5 chromosomes. We report here the discovery of three large deletions which eliminate 6, 9, and all 11 CBF genes from the Fr-B2 locus in tetraploid and hexaploid wheat. In wild emmer wheat, the Fr-B2 deletions were found only among the accessions from the southern sub-populations. Among cultivated wheats, the Fr-B2 deletions were more common among varieties with a spring growth habit than among those with a winter growth habit. Replicated freezing tolerance experiments showed that both the deletion of nine CBF genes in tetraploid wheat and the complete Fr-B2 deletion in hexaploid wheat were associated with significant reductions in survival after exposure to freezing temperatures. Our results suggest that selection for the wild-type Fr-B2 allele may be beneficial for breeders selecting for varieties with improved frost tolerance.

Transgenic barley lines prove the involvement of TaCBF14 and TaCBF15 in the cold acclimation process and in frost tolerance.

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.

The rice Osmyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants.

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at -11°C and -12°C. Frost tolerance was assessed measured the F(v)/F(m) parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

Regulation of freezing tolerance and flowering in temperate cereals: the VRN-1 connection.

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.

Identification of candidate CBF genes for the frost tolerance locus Fr-Am2 in Triticum monococcum.

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 expression of several Cbf genes at the Fr-A2 locus is linked to frost resistance in wheat.

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 Cbf-like 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.