Extensive allele mining discovers novel genetic diversity in the loci controlling frost tolerance in barley.

KEY MESSAGE: Exome sequencing-based allele mining for frost tolerance suggests HvCBF14 rather than CNV at Fr-H2 locus is the main responsible of frost tolerance in barley. Wild relatives, landraces and old cultivars of barley represent a reservoir of untapped and potentially important genes for crop improvement, and the recent sequencing technologies provide the opportunity to mine the existing genetic diversity and to identify new genes/alleles for the traits of interest. In the present study, we use frost tolerance and vernalization requirement as case studies to demonstrate the power of allele mining carried out on exome sequencing data generated from > 400 barley accessions. New deletions in the first intron of VRN-H1 were identified and linked to a reduced vernalization requirement, while the allelic diversity of HvCBF2a, HvCBF4b and HvCBF14 was investigated by combining the analysis of SNPs and read counts. This approach has proven very effective to identify gene paralogs and copy number variants of HvCBF2 and the HvCBF4b-HvCBF2a segment. A multiple linear regression model which considers allelic variation at these genes suggests a major involvement of HvCBF14, rather than copy number variation of HvCBF4b-HvCBF2a, in controlling frost tolerance in barley. Overall, the present study provides powerful resource and tools to discover novel alleles at relevant genes in barley.

Elucidation of molecular and hormonal background of early growth cessation and endodormancy induction in two contrasting Populus hybrid cultivars.

BACKGROUND: Over the life cycle of perennial trees, the dormant state enables the avoidance of abiotic stress conditions. The growth cycle can be partitioned into induction, maintenance and release and is controlled by complex interactions between many endogenous and environmental factors. While phytohormones have long been linked with dormancy, there is increasing evidence of regulation by DAM and CBF genes. To reveal whether the expression kinetics of CBFs and their target PtDAM1 is related to growth cessation and endodormancy induction in Populus, two hybrid poplar cultivars were studied which had known differential responses to dormancy inducing conditions. RESULTS: Growth cessation, dormancy status and expression of six PtCBFs and PtDAM1 were analyzed. The 'Okanese' hybrid cultivar ceased growth rapidly, was able to reach endodormancy, and exhibited a significant increase of several PtCBF transcripts in the buds on the 10th day. The 'Walker' cultivar had delayed growth cessation, was unable to enter endodormancy, and showed much lower CBF expression in buds. Expression of PtDAM1 peaked on the 10th day only in the buds of 'Okanese'. In addition, PtDAM1 was not expressed in the leaves of either cultivar while leaf CBFs expression pattern was several fold higher in 'Walker', peaking at day 1. Leaf phytohormones in both cultivars followed similar profiles during growth cessation but differentiated based on cytokinins which were largely reduced, while the Ox-IAA and iP7G increased in 'Okanese' compared to 'Walker'. Surprisingly, ABA concentration was reduced in leaves of both cultivars. However, the metabolic deactivation product of ABA, phaseic acid, exhibited an early peak on the first day in 'Okanese'. CONCLUSIONS: Our results indicate that PtCBFs and PtDAM1 have differential kinetics and spatial localization which may be related to early growth cessation and endodormancy induction under the regime of low night temperature and short photoperiod in poplar. Unlike buds, PtCBFs and PtDAM1 expression levels in leaves were not associated with early growth cessation and dormancy induction under these conditions. Our study provides new evidence that the degradation of auxin and cytokinins in leaves may be an important regulatory point in a CBF-DAM induced endodormancy. Further investigation of other PtDAMs in bud tissue and a study of both growth-inhibiting and the degradation of growth-promoting phytohormones is warranted.

Decreased R:FR Ratio in Incident White Light Affects the Composition of Barley Leaf Lipidome and Freezing Tolerance in a Temperature-Dependent Manner.

In cereals, C-repeat binding factor genes have been defined as key components of the light quality-dependent regulation of frost tolerance by integrating phytochrome-mediated light and temperature signals. This study elucidates the differences in the lipid composition of barley leaves illuminated with white light or white light supplemented with far-red light at 5 or 15 °C. According to LC-MS analysis, far-red light supplementation increased the amount of monogalactosyldiacylglycerol species 36:6, 36:5, and 36:4 after 1 day at 5 °C, and 10 days at 15 °C resulted in a perturbed content of 38:6 species. Changes were observed in the levels of phosphatidylethanolamine, and phosphatidylserine under white light supplemented with far-red light illumination at 15 °C, whereas robust changes were observed in the amount of several phosphatidylserine species at 5 °C. At 15 °C, the amount of some phosphatidylglycerol species increased as a result of white light supplemented with far-red light illumination after 1 day. The ceramide (42:2)-3 content increased regardless of the temperature. The double-bond index of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine ceramide together with total double-bond index changed when the plant was grown at 15 °C as a function of white light supplemented with far-red light. white light supplemented with far-red light increased the monogalactosyldiacylglycerol/diacylglycerol ratio as well. The gene expression changes are well correlated with the alterations in the lipidome.

Identification of a redox-dependent regulatory network of miRNAs and their targets in wheat.

Reactive oxygen species and antioxidants have an important role in the regulation of plant growth and development under both optimal and stress conditions. In this study, we investigate a possible redox control of miRNAs in wheat (Triticum aestivum ssp. aestivum). Treatment of seedlings with 10 mM H2O2 via the roots for 24 h resulted in decreased glutathione content, increased half-cell reduction potential of the glutathione disulphide/glutathione redox pair, and greater ascorbate peroxidase activity compared to the control plants. These changes were accompanied by alterations in the miRNA transcript profile, with 70 miRNAs being identified with at least 1.5-fold difference in their expression between control and treated (0, 3, 6 h) seedlings. Degradome sequencing identified 86 target genes of these miRNAs, and 6722 possible additional target genes were identified using bioinformatics tools. The H2O2-responsiveness of 1647 target genes over 24 h of treatment was also confirmed by transcriptome analysis, and they were mainly found to be related to the control of redox processes, transcription, and protein phosphorylation and degradation. In a time-course experiment (0-24 h of treatment) a correlation was found between the levels of glutathione, other antioxidants, and the transcript levels of the H2O2-responsive miRNAs and their target mRNAs. This relationship together with bioinformatics modelling of the regulatory network indicated glutathione-related redox control of miRNAs and their targets, which allows the adjustment of the metabolism to changing environmental conditions.

Effects of ambient temperature in association with photoperiod on phenology and on the expressions of major plant developmental genes in wheat (Triticum aestivum L.).

In addition to its role in vernalization, temperature is an important environmental stimulus in determining plant growth and development. We used factorial combinations of two photoperiods (16H, 12H) and three temperature levels (11, 18 and 25 °C) to study the temperature responses of 19 wheat cultivars with established genetic relationships. Temperature produced more significant effects on plant development than photoperiod, with strong genotypic components. Wheat genotypes with PPD-D1 photoperiod sensitive allele were sensitive to temperature; their development was delayed by higher temperature, which intensified under non-inductive conditions. The effect of temperature on plant development was not proportional; it influenced the stem elongation to the largest extent, and warmer temperature lengthened the lag phase between the detection of first node and the beginning of intensive stem elongation. The gene expression patterns of VRN1, VRN2 and PPD1 were also significantly modified by temperature, while VRN3 was more chronologically regulated. The associations between VRN1 and VRN3 gene expression with early apex development were significant in all treatments but were only significant for later plant developmental phases under optimal conditions (16H and 18 °C). Under 16H, the magnitude of the transient peak expression of VRN2 observed at 18 and 25 °C associated with the later developmental phases.

Redox regulation of free amino acid levels in Arabidopsis thaliana.

Abiotic stresses induce oxidative stress, which modifies the level of several metabolites including amino acids. The redox control of free amino acid profile was monitored in wild-type and ascorbate or glutathione deficient mutant Arabidopsis thaliana plants before and after hydroponic treatment with various redox agents. Both mutations and treatments modified the size and redox state of the ascorbate (AsA) and/or glutathione (GSH) pools. The total free amino acid content was increased by AsA, GSH and H2 O2 in all three genotypes and a very large (threefold) increase was observed in the GSH-deficient pad2-1 mutant after GSH treatment compared with the untreated wild-type plants. Addition of GSH reduced the ratio of amino acids belonging to the glutamate family on a large scale and increased the relative amount of non-proteinogenic amino acids. The latter change was because of the large increase in the content of alpha-aminoadipate, an inhibitor of glutamatic acid (Glu) transport. Most of the treatments increased the proline (Pro) content, which effect was due to the activation of genes involved in Pro synthesis. Although all studied redox compounds influenced the amount of free amino acids and a mostly positive, very close (r > 0.9) correlation exists between these parameters, a special regulatory role of GSH could be presumed due to its more powerful effect. This may originate from the thiol/disulphide conversion or (de)glutathionylation of enzymes participating in the amino acid metabolism.

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.

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-quality and temperature-dependent CBF14 gene expression modulates freezing tolerance in cereals.

UNLABELLED: C-repeat binding factor 14 (CBF14) is a plant transcription factor that regulates a set of cold-induced genes, contributing to enhanced frost tolerance during cold acclimation. Many CBF genes are induced by cool temperatures and regulated by day length and light quality, which affect the amount of accumulated freezing tolerance. Here we show that a low red to far-red ratio in white light enhances CBF14 expression and increases frost tolerance at 15°C in winter Triticum aesitivum and Hordeum vulgare genotypes, but not in T. monococcum (einkorn), which has a relatively low freezing tolerance. Low red to far-red ratio enhances the expression of PHYA in all three species, but induces PHYB expression only in einkorn. Based on our results, a model is proposed to illustrate the supposed positive effect of phytochrome A and the negative influence of phytochrome B on the enhancement of freezing tolerance in cereals in response to spectral changes of incident light. KEY WORDS: CBF-regulon, barley, cereals, cold acclimation, freezing tolerance, light regulation, low red/far-red ratio, phytochrome, wheat.

Pleiotropic effect of chromosome 5A and the mvp mutation on the metabolite profile during cold acclimation and the vegetative/generative transition in wheat.

BACKGROUND: Wheat is the leading source of vegetable protein in the human diet, and metabolites are crucial for both plant development and human nutrition. The recent advances in metabolomics provided an opportunity to perform an untargeted metabolite analysis in this important crop. RESULTS: Wheat was characterised at the metabolite level during cold acclimation and transition from the vegetative to the generative phase. The relationship between these changes and chromosome 5A and the maintained vegetative phase (mvp) mutation was also investigated. Samples were taken from the shoots and crowns during four developmental stages: plants grown at 20/17°C, after cold treatment but still during the vegetative phase, at the double ridge and during spikelet formation. The levels of 47 compounds were identified by gas chromatography-mass spectrometry, of which 38 were annotated. The cold treatment, in general, increased the concentrations of osmolites but not in all lines and not equally in the shoots and crowns. The accumulation of proline was not associated with the vernalisation process or with frost tolerance. The mvp mutation and chromosome 5A substitutions altered the amounts of several metabolites compared to those of the Tm and CS, respectively, during each developmental stage. The Ch5A substitution resulted in more substantial changes at the metabolite level than did the Tsp5A substitution. While Ch5A mainly influenced the sugar concentrations, Tsp5A altered the level of tricarboxylic acid cycle intermediates during the vegetative/generative transition. A much higher trehalose, proline, glutamine, asparagine, and unidentified m/z 186 content was detected in crowns than in shoots that may contribute to the frost tolerance of crowns. CONCLUSIONS: Substantial influences of chromosome 5A and the mvp mutation on metabolism during four different developmental stages were demonstrated. The distinct and overlapping accumulation patterns of metabolites suggest the complex genetic regulation of metabolism in the shoots and crowns.

Central role of the flowering repressor ZCCT2 in the redox control of freezing tolerance and the initial development of flower primordia in wheat.

BACKGROUND: As both abiotic stress response and development are under redox control, it was hypothesised that the pharmacological modification of the redox environment would affect the initial development of flower primordia and freezing tolerance in wheat (Triticum aestivum L.). RESULTS: Pharmacologically induced redox changes were monitored in winter (T. ae. ssp. aestivum cv. Cheyenne, Ch) and spring (T. ae. ssp. spelta; Tsp) wheat genotypes grown after germination at 20/17°C for 9 d (chemical treatment: last 3 d), then at 5°C for 21 d (chemical treatment: first 4 d) and subsequently at 20/17°C for 21 d (recovery period). Thiols and their disulphide forms were measured and based on these data reduction potentials were calculated. In the freezing-tolerant Ch the chemical treatments generally increased both the amount of thiol disulphides and the reduction potential after 3 days at 20/17°C. In the freezing-sensitive Tsp a similar effect of the chemicals on these parameters was only observed after the continuation of the treatments for 4 days at 5°C. The applied chemicals slightly decreased root fresh weight and increased freezing tolerance in Ch, whereas they increased shoot fresh weight in Tsp after 4 days at 5°C. As shown after the 3-week recovery at 20/17°C, the initial development of flower primordia was accelerated in Tsp, whereas it was not affected by the treatments in Ch. The chemicals differently affected the expression of ZCCT2 and that of several other genes related to freezing tolerance and initial development of flower primordia in Ch and Tsp after 4 d at 5°C. CONCLUSIONS: Various redox-altering compounds and osmotica had differential effects on glutathione disulphide content and reduction potential, and consequently on the expression of the flowering repressor ZCCT2 in the winter wheat Ch and the spring wheat Tsp. We propose that the higher expression of ZCCT2 in Ch may be associated with activation of genes of cold acclimation and its lower expression in Tsp with the induction of genes accelerating initial development of flower primordia. In addition, ZCCT2 may be involved in the coordinated control of the two processes.

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.

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.

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.

Modulated Light Dependence of Growth, Flowering, and the Accumulation of Secondary Metabolites in Chilli.

Chili is widely used as a food additive and a flavouring and colouring agent and also has great importance in health preservation and therapy due to the abundant presence of many bioactive compounds, such as polyphenols, flavonoids, carotenoids, and capsaicinoids. Most of these secondary metabolites are strong antioxidants. In the present study, the effect of light intensity and spectral composition was studied on the growth, flowering, and yield of chilli together with the accumulation of secondary metabolites in the fruit. Two light intensities (300 and 500 µmol m-2 s-1) were applied in different spectral compositions. A broad white LED spectrum with and without FR application and with blue LED supplement was compared to blue and red LED lightings in different (80/20 and 95/5%) blue/red ratios. High light intensity increased the harvest index (fruit yield vs. biomass production) and reduced the flowering time of the plants. The amount of secondary metabolites in the fruit varied both by light intensity and spectral compositions; phenolic content and the radical scavenging activity were stimulated, whereas capsaicin accumulation was suppressed by blue light. The red colour of the fruit (provided by carotenoids) was inversely correlated with the absolute amount of blue, green, and far-red light. Based on the results, a schematic model was created, representing light-dependent metabolic changes in chilli. The results indicated that the accumulation of secondary metabolites could be modified by the adjustment of light intensity and spectral composition; however, different types of metabolites required different light environments.

The Effect of White Light Spectrum Modifications by Excess of Blue Light on the Frost Tolerance, Lipid- and Hormone Composition of Barley in the Early Pre-Hardening Phase.

It is well established that cold acclimation processes are highly influenced, apart from cold ambient temperatures, by light-dependent environmental factors. In this study we investigated whether an extra blue (B) light supplementation would be able to further improve the well-documented freezing tolerance enhancing effect of far-red (FR) enriched white (W) light. The impact of B and FR light supplementation to white light (WFRB) on hormone levels and lipid contents were determined in winter barley at moderate (15 °C) and low (5 °C) temperatures. Low R:FR ratio effectively induced frost tolerance in barley plantlets, but additional B light further enhanced frost hardiness at both temperatures. Supplementation of WFR (white light enriched with FR light) with B had a strong positive effect on abscisic acid accumulation while the suppression of salicylic acid and jasmonic acid levels were observed at low temperature which resembles the shade avoidance syndrome. We also observed clear lipidomic differences between the individual light and temperature treatments. WFRB light changed the total lipid content negatively, but monogalactosyldiacylglycerol (MGDG) content was increased, nonetheless. Our results prove that WFRB light can greatly influence phytohormone dynamics and lipid contents, which eventually leads to more efficient pre-hardening to avoid frost damage.

Light Intensity- and Spectrum-Dependent Redox Regulation of Plant Metabolism.

Both light intensity and spectrum (280-800 nm) affect photosynthesis and, consequently, the formation of reactive oxygen species (ROS) during photosynthetic electron transport. ROS, together with antioxidants, determine the redox environment in tissues and cells, which in turn has a major role in the adjustment of metabolism to changes in environmental conditions. This process is very important since there are great spatial (latitude, altitude) and temporal (daily, seasonal) changes in light conditions which are accompanied by fluctuations in temperature, water supply, and biotic stresses. The blue and red spectral regimens are decisive in the regulation of metabolism because of the absorption maximums of chlorophylls and the sensitivity of photoreceptors. Based on recent publications, photoreceptor-controlled transcription factors such as ELONGATED HYPOCOTYL5 (HY5) and changes in the cellular redox environment may have a major role in the coordinated fine-tuning of metabolic processes during changes in light conditions. This review gives an overview of the current knowledge of the light-associated redox control of basic metabolic pathways (carbon, nitrogen, amino acid, sulphur, lipid, and nucleic acid metabolism), secondary metabolism (terpenoids, flavonoids, and alkaloids), and related molecular mechanisms. Light condition-related reprogramming of metabolism is the basis for proper growth and development of plants; therefore, its better understanding can contribute to more efficient crop production in the future.

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.

The Impact of Far-Red Light Supplementation on Hormonal Responses to Cold Acclimation in Barley.

Cold acclimation, the necessary prerequisite for promotion of freezing tolerance, is affected by both low temperature and enhanced far-red/red light (FR/R) ratio. The impact of FR supplementation to white light, created by artificial LED light sources, on the hormone levels, metabolism, and expression of the key hormone metabolism-related genes was determined in winter barley at moderate (15 °C) and low (5 °C) temperature. FR-enhanced freezing tolerance at 15 °C was associated with promotion of abscisic acid (ABA) levels, and accompanied by a moderate increase in indole-3-acetic acid (IAA) and cis-zeatin levels. The most prominent impact on the plants' freezing tolerance was found after FR pre-treatment at 15 °C (for 10 days) followed by cold treatment at FR supplementation (7 days). The response of ABA was diminished in comparison with white light treatment, probably due to the elevation of stress tolerance during FR pre-treatment. Jasmonic acid (JA) and salicylic acid (SA) were transiently reduced. When the plants were exposed directly to a combination of cold (5 °C) and FR supplementation, ABA increase was higher than in white light, and was associated with enhanced elevation of JA and, in the longer term (after 7 days), with IAA and cis-zeatin increase, which indicates a stronger stress response and better acclimation. Cold hardening was more efficient when FR light was applied in the early developmental stage of the barley plants (three-leaf stage, 18 days), rather than in later stages (28-days). The dynamics of the phytohormone changes are well supported by the expression profiles of the key hormone metabolism-related genes. This series of treatments serves as evidence for the close relationship between plant hormones, light quality, and low temperature at the beginning of cold acclimation. Besides the timing of the FR treatments, plant age also represents a key factor during light spectrum-dependent cold acclimation.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.