Vernalization Research Articles

Natural Variation in Brachypodium Links Vernalization and Flowering Time Loci as Major Flowering Determinants.

The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon yet identified considerable ambiguity surrounding the role of VRN2 To investigate the natural diversity governing flowering time pathways in a nondomesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a single-nucleotide polymorphism-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions, and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1 Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional quantitative trait loci suggests that greater complexity underlies flowering time in this nondomesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaceae as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.

Bioinformatic prediction of transcription factor binding sites at promoter regions of genes for photoperiod and vernalization responses in model and temperate cereal plants.

BackgroundMany genes involved in responses to photoperiod and vernalization have been characterized or predicted in Arabidopsis (Arabidopsis thaliana), Brachypodium (Brachypodium distachyon), wheat (Triticum aestivum) and barley (Hordeum vulgare). However, little is known about the transcription regulation of these genes, especially in the large, complex genomes of wheat and barley.ResultsWe identified 68, 60, 195 and 61 genes that are known or postulated to control pathways of photoperiod (PH), vernalization (VE) and pathway integration (PI) in Arabidopsis, Brachypodium, wheat and barley for predicting transcription factor binding sites (TFBSs) in the promoters of these genes using the FIMO motif search tool of the MEME Suite. The initial predicted TFBSs were filtered to confirm the final numbers of predicted TFBSs to be 1066, 1379, 1528, and 789 in Arabidopsis, Brachypodium, wheat and barley, respectively. These TFBSs were mapped onto the PH, VE and PI pathways to infer about the regulation of gene expression in Arabidopsis and cereal species. The GC contents in promoters, untranslated regions (UTRs), coding sequences and introns were higher in the three cereal species than those in Arabidopsis. The predicted TFBSs were most abundant for two transcription factor (TF) families: MADS-box and CSD (cold shock domain). The analysis of publicly available gene expression data showed that genes with similar numbers of MADS-box and CSD TFBSs exhibited similar expression patterns across several different tissues and developmental stages. The intra-specific Tajima D-statistics of TFBS motif diversity showed different binding specificity among different TF families. The inter-specific Tajima D-statistics suggested faster TFBS divergence in TFBSs than in coding sequences and introns. Mapping TFBSs onto the PH, VE and PI pathways showed the predominance of MADS-box and CSD TFBSs in most genes of the four species, and the difference in the pathway regulations between Arabidopsis and the three cereal species.ConclusionOur approach to associating the key flowering genes with their potential TFs through prediction of putative TFBSs provides a framework to explore regulatory mechanisms of photoperiod and vernalization responses in flowering plants. The predicted TFBSs in the promoters of the flowering genes provide a basis for molecular characterization of transcription regulation in the large, complex genomes of important crop species, wheat and barley.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2916-7) contains supplementary material, which is available to authorized users.

Evidence for an Early Origin of Vernalization Responsiveness in Temperate Pooideae Grasses.

The ability of plants to match their reproductive output with favorable environmental conditions has major consequences both for lifetime fitness and geographic patterns of diversity. In temperate ecosystems, some plant species have evolved the ability to use winter nonfreezing cold (vernalization) as a cue to ready them for spring flowering. However, it is unknown how important the evolution of vernalization responsiveness has been for the colonization and subsequent diversification of taxa within the northern and southern temperate zones. Grasses of subfamily Pooideae, including several important crops, such as wheat (Triticum aestivum), barley (Hordeum vulgare), and oats (Avena sativa), predominate in the northern temperate zone, and it is hypothesized that their radiation was facilitated by the early evolution of vernalization responsiveness. Predictions of this early origin hypothesis are that a response to vernalization is widespread within the subfamily and that the genetic basis of this trait is conserved. To test these predictions, we determined and reconstructed vernalization responsiveness across Pooideae and compared expression of wheat vernalization gene orthologs VERNALIZATION1 (VRN1) and VRN3 in phylogenetically representative taxa under cold and control conditions. Our results demonstrate that vernalization responsive Pooideae species are widespread, suggesting that this trait evolved early in the lineage and that at least part of the vernalization gene network is conserved throughout the subfamily. These results are consistent with the hypothesis that the evolution of vernalization responsiveness was important for the initial transition of Pooideae out of the tropics and into the temperate zone.

The mvp2 mutation affects the generative transition through the modification of transcriptome pattern, salicylic acid and cytokinin metabolism in Triticum monococcum

Wild type and mvp2 (maintained vegetative phase) deletion mutant T. monococcum plants incapable of flowering were compared in order to determine the effect of the deleted region of chromosome 5A on transcript profile and hormone metabolism. This region contains the vernalization1 (VRN1) gene, a major regulator of the vegetative/generative transition. Transcript profiling in the crowns of T. monococcum during the transition and the subsequent formation of flower primordia showed that 306 genes were affected by the mutation, 198 by the developmental phase and 14 by the interaction of these parameters. In addition, 546 genes were affected by two or three factors. The genes controlled by the deleted region encode transcription factors, antioxidants and enzymes of hormone, carbohydrate and amino acid metabolism. The observed changes in the expression of the gene encoding phenylalanine ammonia lyase (PAL) might indicate the effect of mvp2 mutation on the metabolism of salicylic acid, which was corroborated by the differences in 2-hydroxycinnamic acid and cinnamic acid contents in both of the leaves and crowns, and in the concentrations of salicylic acid and benzoic acid in crowns during the vegetative/generative transition. The amount and ratio of active cytokinins and their derivatives (ribosides, glucosides and phosphates) were affected by developmental changes as well as by mvp2 mutation, too.

Heading Date QTL in Winter Wheat (Triticum aestivum L.) Coincide with Major Developmental Genes VERNALIZATION1 and PHOTOPERIOD1.

In wheat (Triticum aestivum L.), time from planting to spike emergence is influenced by genes controlling vernalization requirement and photoperiod response. Characterizing the available genetic diversity of known and novel alleles of VERNALIZATION1 (VRN1) and PHOTOPERIOD1 (PPD1) in winter wheat can inform approaches for breeding climate resilient cultivars. This study identified QTL for heading date (HD) associated with multiple VRN1 and PPD1 loci in a population developed from a cross between two early flowering winter wheat cultivars. When the population was grown in the greenhouse after partial vernalization treatment, major heading date QTLs co-located with the VRN-A1 and VRN-B1 loci. Copy number variation at the VRN-A1 locus influenced HD such that RIL having three copies required longer cold exposure to transition to flowering than RIL having two VRN-A1 copies. Sequencing vrn-B1 winter alleles of the parents revealed multiple polymorphisms in the first intron that were the basis of mapping a major HD QTL coinciding with VRN-B1. A 36 bp deletion in the first intron of VRN-B1 was associated with earlier HD after partial vernalization in lines having either two or three haploid copies of VRN-A1. The VRN1 loci interacted significantly and influenced time to heading in field experiments in Louisiana, Georgia and North Carolina. The PPD1 loci were significant determinants of heading date in the fully vernalized treatment in the greenhouse and in all field environments. Heading date QTL were associated with alleles having large deletions in the upstream regions of PPD-A1 and PPD-D1 and with copy number variants at the PPD-B1 locus. The PPD-D1 locus was determined to have the largest genetic effect, followed by PPD-A1 and PPD-B1. Our results demonstrate that VRN1 and PPD1 alleles of varying strength allow fine tuning of flowering time in diverse winter wheat growing environments.

Allelic state and effects of VRN genes on soft wheat in in vivo and in vitro systems

The article investigates the effects of the main manifestations of the genetic system controlling the type and rate of wheat development – VRN (vernalization) at the level of an integrated system of plant organism in vivo and callus certain population of cells in vitro. The paper studies the system of allelic VRN genes, which determine the need or insensitivity Triticum aestivum L. to vernalization and predetermination process of callusogenesis by the present system in vitro. In the experiments the modern model system of nearly isogenic lines (NILs) of spring type was used. The NILs were created in genotypes of the winter varieties Myronivska 808 and Olvia. Molecular genetic analysis of allelic loci of VRN genes was carried out by PCR analysis on grains and secondary callus culture using five pairs of specific primers (Grain Gene Mass Wheat). In the course of the experiments, it was found that the genetic system controlling the wheat rate determined the frequency of callusogenesis. However, the studied genetic system did not affect the morphological characteristics of primary and secondary callus tissue. In both hexaploid wheat cultivars the maximum frequency of callusogenesis appeared to be characterized in the Vrn 2 isogenic line and the original variety, slowly developing and intensely accumulating vegetative mass in vivo. The minimal frequency of callusogenesis was determined in the VRN 1 and VRN 3 isogenic lines, characterized by the rapid development of vegetation. The callus was derived from immature wheat embryos by morphological features analysis. Calluses with low water content, mainly, amorphous, compact, transparent, white or with yellowish tint were identified. Using PCR analysis, in grains in vivo and in the callus culture in vitro the almost identical allelic status of VRN genes was revealed. In grains and callus of Vrn 1 isogenic lines of both wheat varieties the presence of a dominant gene VRN A1 and recessive VRN B1 and VRN D1 was detected, whereas in VRN 2 – a dominant gene VRN B1 and recessive VRN A1 and VRN D1. However, the dominant allele VRN D1 in the studied NILs was detected. Therefore, in varieties of grains and callus cultures all genes are represented only by recessive alleles VRN A1, VRN B1 and VRN D1. Differences were found in the callus culture of VRN 3 isogenic line of Myronivska 808 on the allelic state of theVRN B1 gene. The obtained results could be associated with genomic reconstructions during callusogenesis induction. Our studies indicate the unidirectional system of VRN genes functioning, which appears to be the main control system of type and rate of soft wheat development in the system in vivo and in vitro. This allows us to assume the role of the VRN genetic system in the determination of callusogenesis, and also the adequacy of the functioning of the system in vitro processes, determining the system of integration of plants. Thus, our study confirms that the callus tissues cells of higher plants are able to preserve the cells properties of the whole organism along with the acquisition of new specific properties. Moreover, the culture in vitro is an adequate system for the study of the plant organism properties as a system.

Frost-tolerance genes Fr-A2 and Fr-B2 in Australian wheat and their effects on days to heading and grain yield in lower rainfall environments in southern Australia

FROST RESISTANCE 2 (FR2) genes of wheat are C-Repeat Binding Factor (CBF) genes with two major alleles known for both Fr-A2 (‘T’ and ‘S’) and Fr-B2 (‘WT’ and ‘DEL’). VERNALIZATION 1 (VRN1) genes have a regulatory role on CBF genes, with known epistatic interactions between Vrn-A1 and Fr-A2 for tolerance to freezing temperatures during vegetative growth. VRN1 genes were also known to affect days to heading and grain yield. Therefore, FR2 genes might also affect these traits. A wide range of cultivars was characterised for VRN1, Fr-A2 and Fr-B2 genes. A third allele of Fr-A2 was found in cvv Excalibur and Axe. The winter cultivar Norstar, which was known to have a high level of frost tolerance during vegetative growth, had the combination Vrn-A1w + Fr-A2T + Fr-B2WT, as did a spring landrace from Afghanistan that was known to have superior tolerance to frost during reproductive development. No Australian spring cultivar was found with this combination, but it could be selected from crosses between adapted cultivars. This would enable the role of VRN1 and FR2 alleles in reproductive frost tolerance to be evaluated in an adapted background. Using large, existing, plant-breeding datasets, the T allele of Fr-A2 delayed heading relative to the S allele, and the WT allele of Fr-B2 delayed heading relative to the DEL allele, but only in combination with particular alleles of the VRN1 genes. Fr-B2 affected grain yield, with the highest grain yields for spring lines produced by Fr-B2DEL in combination with the spring allele of Vrn-B1.

Gene expression profiling in shoot apical meristem of Gossypium hirsutum

Early maturity is a particularly important agronomic trait for cotton breeding in China and is determined by many morphological and phenological traits. The time of floral initiation is one of most important factors related to early maturation of cotton. The aim of this study was to identify differentially expressed (DE) genes related to floral initiation using the Arabidopsis thaliana GeneChip® system on the shoot apical meristems (SAMs) of an early-maturing cotton cultivar. Compared with SAMs at 10 days, 365 genes were differentially expressed in the SAMs at 20 days. Of these, 210 and 155 transcripts were up- and down- regulated, respectively. The results of Gene Ontology (GO) annotation indicated that most genes fell into the four largest functional groups: metabolism, transposable elements, protein binding or cofactor requirements, and protein fate. These groups constituted 18.9, 11.8, 12.8 and 6.8% of the total DE genes, respectively. Many DE genes were identified, including those encoding the transcription factors SOC1-like floral activator MADS4, B3-domain containing transcription factor, MYB2, MYB85 and GHMADS-1. Our research found that the B3-domain containing transcription factor was similar to Arabidopsis genes encoding auxin response factor 36 and VERNALIZATION 1 (VRN1) and was one of 155 down-regulated ‘Apex-unique’ transcripts. The B3-domain containing transcription factor was 1128 bp long and was named GhV1 (GenBank accession No. GU929695). The induction of the transcripts we identified in the cotton SAM after 10 days of SD revealed that the transition to reproductive development occurred at this particular time point. These results allowed for a detailed description of temporal gene expression changes in the cotton SAM as it undergoes the floral initiation process.

Identification and distribution of VERNALIZATION1 alleles in Chinese barley (Hordeum vulgare) germplasm

VERNALIZATION1 (VRN1) is a central regulator of the vernalization requirement, which affects the yield and geographical distribution of Triticeae crops through regulation of the flowering time. The first intron (Intron I) of VRN1 contains putative cis-elements that mediate repression of VRN1 expression. Allelic variations in Intron I are closely associated with increased VRN1 expression in vernalization-responsive accessions in the absence of vernalization. In this study, diagnostic molecular markers were developed to distinguish the main 12 known alleles, including a new allele, of barley (Hordeum vulgare) VRN1 (HvVRN1), and to investigate HvVRN1 alleles in 373 barley varieties from major agroecological regions in China. The new allele, HvVRN1-11, with a 3172-bp deletion in Intron I of HvVRN1, was identified in a Chinese barley landrace. Analysis of eight polymorphic sites flanking major variations in Intron I of HvVRN1 classified the 12 HvVRN1 alleles into five distinct groups. Compared with modern commercial barley, landraces possessed greater genetic diversity and contained the majority of HvVRN1 alleles, especially in the winter barley regions. HvVRN1 diversity in terms of allele number reduced significantly (P < 0.01) along with higher growing latitudes, but increased in accessions carrying the winter HvVRN2 allele. Wild-type HvVRN1 and the HvVRN1-8 allele showed the most widespread distribution in most regions whereas HvVRN1-1 & HvVRN1-8, and HvVRN1-5 are predominant in the Inner Mongolian Plateau spring barley region and the Northeast Plain spring barley region, respectively, with relatively higher latitudes. Compared with landraces, the frequency of HvVRN1-8 decreased while wild-type HvVRN1 increased significantly (P < 0.01) in commercial varieties. Our study classified HvVRN1 genetic diversity in a large collection of Chinese germplasm and provided a tool for identifying HvVRN1 alleles in barley breeding populations.

The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs.

Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein basic pentacysteine6 (BPC6) interacts with like heterochromatin protein1 (LHP1), a PRC1 component, and associates with vernalization2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution.

Effect of the hope FT-B1 allele on wheat heading time and yield components.

Precise regulation of flowering time is critical for plant reproductive success and, in cereals, to maximize grain yields. Seasonal cues including temperature and day length are integrated to regulate the timing of flowering. In temperate cereals, extended periods of cold (vernalization) release the repression of FLOWERING LOCUS T1 (FT1), which is upregulated in the leaves in response to inductive long-day photoperiods. FT1 is a homolog of rice HD3a, which encodes a protein transported from leaves to the shoot apical meristem to induce flowering. A rare FT-B1 allele from the wheat variety "Hope" has been previously shown to be associated with an early flowering phenotype under long-day photoperiods. Here, we demonstrate that the Hope FT-B1 allele accelerates flowering even under short days, and that it is epistatic to the VERNALIZATION 1 (VRN1) gene. On average, the introgression of Hope FT-B1 into 6 genetic backgrounds resulted in 2.6 days acceleration of flowering (P<0.0001) and 4.1% increase in spike weight (P=0.0093), although in one variety, it was associated with a decrease in spike weight. These results suggest that the Hope FT-B1 allele could be useful in wheat breeding programs to subtly accelerate floral development and increase adaptation to changing environments.

Copy number and haplotype variation at the VRN-A1 and central FR-A2 loci are associated with frost tolerance in hexaploid wheat.

The interaction between VRN - A1 and FR - A2 largely affect the frost tolerance of hexaploid wheat. Frost tolerance is critical for wheat survival during cold winters. Natural variation for this trait is mainly associated with allelic differences at the VERNALIZATION 1 (VRN1) and FROST RESISTANCE 2 (FR2) loci. VRN1 regulates the transition between vegetative and reproductive stages and FR2, a locus including several tandemly duplicated C-REPEAT BINDING FACTOR (CBF) transcription factors, regulates the expression of Cold-regulated genes. We identified sequence and copy number variation at these two loci among winter and spring wheat varieties and characterized their association with frost tolerance. We identified two FR-A2 haplotypes-'FR-A2-S' and 'FR-A2-T'-distinguished by two insertion/deletions and ten single nucleotide polymorphisms within the CBF-A12 and CBF-A15 genes. Increased copy number of CBF-A14 was frequently associated with the FR-A2-T haplotype and with higher CBF14 transcript levels in response to cold. Factorial ANOVAs revealed significant interactions between VRN1 and FR-A2 for frost tolerance in both winter and spring panels suggesting a crosstalk between vernalization and cold acclimation pathways. The model including these two loci and their interaction explained 32.0 and 20.7 % of the variation in frost tolerance in the winter and spring panels, respectively. The interaction was validated in a winter wheat F 4:5 population segregating for both genes. Increased VRN-A1 copy number was associated with improved frost tolerance among varieties carrying the FR-A2-T allele but not among those carrying the FR-A2-S allele. These results suggest that selection of varieties carrying the FR-A2-T allele and three copies of the recessive vrn-A1 allele would be a good strategy to improve frost tolerance in wheat.

Level of &lt;i&gt;VERNALIZATION 1&lt;/i&gt; expression is correlated with earliness in &lt;i&gt;extra early-flowering&lt;/i&gt; mutant wheat lines

Four extra early-flowering mutants, named extra early-flowering1 (exe1), exe2, exe3, and exe4, were identified in Triticum monococcum strain KU104-1 following heavy-ion beam mutagenesis. The four exe mutants fell into two groups, namely Type I (moderately extra early-flowering type; exe1 and exe3) and Type II (extremely extra early-flowering type; exe2 and exe4). Analysis of plant development in a growth chamber showed that the speed of leaf emergence was accelerated in exe mutants at the reproductive stage compared to wild-type (WT) plants. The speed of leaf emergence was faster in Type II than Type I plants. Analysis of VERNALIZATION 1 (VRN1), a flowering promoter gene, showed that it was more highly expressed in seedlings at early developmental stages in Type II mutants than Type I mutants. These findings indicate that the difference in earliness between Type I and Type II mutants is associated with the level of VRN1 expression. The original KU104-1 is an einkorn wheat strain that carries a null allele of the VRN2 gene, a repressor of flowering. Thus, our results indicate that the level of VRN1 expression controls earliness in exe mutants independently of VRN2.

Vernalization treatment induces site-specific DNA hypermethylation at the VERNALIZATION-A1 (VRN-A1) locus in hexaploid winter wheat

BackgroundCertain temperate species require prolonged exposure to low temperature to initiate transition from vegetative growth to flowering, a process known as vernalization. In wheat, winter cultivars require vernalization to initiate flowering, making vernalization requirement a trait of key importance in wheat agronomy. The genetic bases of vernalization response have been largely studied in wheat, leading to the characterization of a regulation pathway that involves the key gene VERNALIZATION1 (VRN1). While previous studies in wheat and barley have revealed the functional role of histone modification in setting VRN1 expression, other mechanisms might also be involved. Here, we were interested in determining whether the cold-induced expression of the wheat VRN-A1 gene is associated with a change in DNA methylation.ResultsWe provide the first DNA methylation analysis of the VRN-A1 gene, and describe the existence of methylation at CG but also at non CG sites. While CG sites show a bell-shape profile typical of gene-body methylation, non CG methylation is restricted to the large (8.5 kb) intron 1, in a region harboring fragments of transposable elements (TEs). Interestingly, cold induces a site-specific hypermethylation at these non CG sites. This increase in DNA methylation is transmitted through mitosis, and is reset to its original level after sexual reproduction.ConclusionsThese results demonstrate that VRN-A1 has a particular DNA methylation pattern, exhibiting rapid shift within the life cycle of a winter wheat plant following exposure to particular environmental conditions. The finding that this shift occurs at non CG sites in a TE-rich region opens interesting questions onto the possible consequences of this type of methylation in gene expression.

Exogenous Gibberellins Induce Wheat Spike Development under Short Days Only in the Presence of VERNALIZATION1

The activation of the meristem identity gene VERNALIZATION1 (VRN1) is a critical regulatory point in wheat (Triticum spp.) flowering. In photoperiod-sensitive wheat varieties, VRN1 is expressed only under long days (LDs), but mutants carrying deletions in a regulatory element in its promoter show VRN1 transcription and early spike development under short days (SDs). However, complete spike development is delayed until plants are transferred to LDs, indicating the existence of an additional regulatory mechanism dependent on LDs. We show here that exogenous gibberellin (GA) application accelerates spike development under SDs, but only in wheat lines expressing VRN1. The simultaneous presence of GA and VRN1 results in the up-regulation of the floral meristem identity genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-1 and LEAFY, whereas inhibition of GA biosynthesis with paclobutrazol precludes the LD induction of these two genes. The inductive role of GA on wheat flowering is further supported by the up-regulation of GA biosynthetic genes in the apices of plants transferred from SDs to LDs and in photoperiod-insensitive and transgenic wheat plants with increased FLOWERING LOCUS T transcription under SDs. The up-regulation of GA biosynthetic genes was not observed in the leaves of the same genetic stocks. Based on these observations, we propose a model in which FLOWERING LOCUS T is up-regulated in the leaves under LDs and is then transported to the shoot apical meristem, where it simultaneously induces the expression of VRN1 and GA biosynthetic genes, which are both required for the up-regulation of the early floral meristem genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-1 and LEAFY and the timely development of the wheat spike.

Development of vegetative axillary branches after vernalization in Arabis alpina , a perennial plant

Development of vegetative axillary branches after vernalization in Arabis alpina , a perennial plant

The Arabidopsis B3 Domain Protein VERNALIZATION1 (VRN1) Is Involved in Processes Essential for Development, with Structural and Mutational Studies Revealing Its DNA-binding Surface

The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence-nonspecific binding, we crystallized VRN1(208-341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface.

Wheat TILLING Mutants Show That the Vernalization Gene VRN1 Down-Regulates the Flowering Repressor VRN2 in Leaves but Is Not Essential for Flowering

Most of the natural variation in wheat vernalization response is determined by allelic differences in the MADS-box transcription factor VERNALIZATION1 (VRN1). Extended exposures to low temperatures during the winter (vernalization) induce VRN1 expression and promote the transition of the apical meristem to the reproductive phase. In contrast to its Arabidopsis homolog (APETALA1), which is mainly expressed in the apical meristem, VRN1 is also expressed at high levels in the leaves, but its function in this tissue is not well understood. Using tetraploid wheat lines with truncation mutations in the two homoeologous copies of VRN1 (henceforth vrn1-null mutants), we demonstrate that a central role of VRN1 in the leaves is to maintain low transcript levels of the VRN2 flowering repressor after vernalization. Transcript levels of VRN2 were gradually down-regulated during vernalization in both mutant and wild-type genotypes, but were up-regulated after vernalization only in the vrn1-null mutants. The up-regulation of VRN2 delayed flowering by repressing the transcription of FT, a flowering-integrator gene that encodes a mobile protein that is transported from the leaves to the apical meristem to induce flowering. The role of VRN2 in the delayed flowering of the vrn1-null mutant was confirmed using double vrn1-vrn2-null mutants, which flowered two months earlier than the vrn1-null mutants. Both mutants produced normal flowers and seeds demonstrating that VRN1 is not essential for wheat flowering, which contradicts current flowering models. This result does not diminish the importance of VRN1 in the seasonal regulation of wheat flowering. The up-regulation of VRN1 during winter is required to maintain low transcript levels of VRN2, accelerate the induction of FT in the leaves, and regulate a timely flowering in the spring. Our results also demonstrate the existence of redundant wheat flowering genes that may provide new targets for engineering wheat varieties better adapted to changing environments.

Vernalization

Vernalization

The Promoter of the Cereal VERNALIZATION1 Gene Is Sufficient for Transcriptional Induction by Prolonged Cold

The VERNALIZATION1 (VRN1) gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization) to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP) reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare). A 2 kb segment of the promoter of VRN1 was sufficient for GFP expression in the leaves and shoot apex of transgenic barley plants. Fluorescence increased at the shoot apex prior to inflorescence initiation and was subsequently maintained in the developing inflorescence. The promoter was also sufficient for low-temperature induction of GFP expression. A naturally occurring insertion in the proximal promoter, which is associated with elevated VRN1 expression and early flowering in some spring wheats, did not abolish induction of VRN1 transcription by prolonged cold, however. A translational fusion of the promoter and transcribed regions of VRN1 to GFP, VRN1::GFP, was localised to nuclei of cells at the shoot apex of transgenic barley plants. The distribution of VRN1::GFP at the shoot apex was similar to the expression pattern of the VRN1 promoter-GFP reporter gene. Fluorescence from the VRN1::GFP fusion protein increased in the developing leaves after prolonged cold treatment. These observations suggest that the promoter of VRN1 is targeted by mechanisms that trigger vernalization-induced flowering in economically important temperate cereal crops.