Discrete developmental roles for temperate cereal grass VERNALIZATION1/FRUITFULL-like genes in flowering competency and the transition to flowering

Members of the grass subfamily Pooideae are characterized by their adaptation to cool temperate climates. Vernalization is the process whereby flowering is accelerated in response to a prolonged period of cold. Winter cereals are tolerant of low temperatures and flower earlier with vernalization, whereas spring cultivars are intolerant of low temperatures and flower later with vernalization. In the pooid grasses wheat (Triticum monococcum, Triticum aestivum) and barley (Hordeum vulgare), vernalization responsiveness is determined by allelic variation at the VERNALIZATION1 (VRN1) and/or VRN2 loci. To determine whether VRN1, and its paralog FRUITFULL2 (FUL2), are involved in vernalization requirement across Pooideae, we determined expression profiles for multiple cultivars of oat (Avena sativa) and wheat with and without cold treatment. Our results demonstrate significant up-regulation of VRN1 expression in leaves of winter oat and wheat in response to vernalization; no treatment effect was found for spring or facultative growth habit oat and wheat. Similar cold-dependent patterns of leaf expression were found for FUL2 in winter oat, but not winter wheat, suggesting a redundant qualitative role for these genes in the quantitative induction of flowering competency of oat. These and other data support the hypothesis that VRN1 is a common regulator of vernalization responsiveness within the crown pooids. Finally, we found that up-regulation of VRN1 in vegetative meristems of oat was significantly later than in leaves. This suggests distinct and conserved roles for temperate cereal grass VRN1/FUL-like genes, first, in systemic signaling to induce flowering competency, and second, in meristems to activate genes involved in the floral transition.     

The molecular basis of vernalization-induced flowering in cereals

Genetic analyses have identified three genes that control the vernalization requirement in wheat and barley; VRN1, VRN2 and FT (VRN3). These genes have now been isolated and shown to regulate not only the vernalization response but also the promotion of flowering by long days. VRN1 is induced by vernalization and accelerates the transition to reproductive development at the shoot apex. FT is induced by long days and further accelerates reproductive apex development. VRN2, a floral repressor, integrates vernalization and day-length responses by repressing FT until plants are vernalized. A comparison of flowering time pathways in cereals and Arabidopsis shows that the vernalization response is controlled by different MADS box genes, but integration of vernalization and long-day responses occurs through similar mechanisms.     

The molecular mechanism of vernalization in Arabidopsis and cereals: role of Flowering Locus C and its homologs

Winter varieties of plants can flower only after exposure to prolonged cold. This phenomenon is known as vernalization and has been widely studied in the model plant Arabidopsis thaliana as well as in monocots. Through the repression of floral activator genes, vernalization prevents flowering in winter. In Arabidopsis, FLOWERING LOCUS C or FLC is the key repressor during vernalization, while in monocots vernalization is regulated through VRN1, VRN2 and VRN3 (or FLOWERING LOCUS T). Interestingly, VRN genes are not homologous to FLC but FLC homologs are found to have a significant role in vernalization response in cereals. The presence of FLC homologs in monocots opens new dimensions to understand, compare and retrace the evolution of vernalization pathways between monocots and dicots. In this review, we discuss the molecular mechanism of vernalization-induced flowering along with epigenetic regulations in Arabidopsis and temperate cereals. A better understanding of cold-induced flowering will be helpful in crop breeding strategies to modify the vernalization requirement of economically important temperate cereals.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

Effect of Ppd-1 on the expression of flowering-time genes in vegetative and reproductive growth stages of wheat

The photoperiod sensitivity gene Ppd-1 influences the timing of flowering in temperate cereals such as wheat and barley. The effect of Ppd-1 on the expression of flowering-time genes was assessed by examining the expression levels of the vernalization genes VRN1 and VRN3/WFT and of two CONSTANS-like genes, WCO1 and TaHd1, during vegetative and reproductive growth stages. Two near-isogenic lines (NILs) were used: the first carried a photoperiod-insensitive allele of Ppd-1 (Ppd-1a-NIL), the other, a photoperiod-sensitive allele (Ppd-1b-NIL). We found that the expression pattern of VRN1 was similar in Ppd-1a-NIL and Ppd-1b-NIL plants, suggesting that VRN1 is not regulated by Ppd-1. Under long day conditions, VRN3/WFT showed similar expression patterns in Ppd-1a-NIL and Ppd-1b-NIL plants. However, expression differed greatly under short day conditions: VRN3/WFT expression was detected in Ppd-1a-NIL plants at the 5-leaf stage when they transited from vegetative to reproductive growth; very low expression was present in Ppd-1b-NIL throughout all growth stages. Thus, the Ppd-1b allele acts to down-regulate VRN3/WFT under short day conditions. WCO1 showed high levels of expression at the vegetative stage, which decreased during the phase transition and reproductive growth stages in both Ppd-1a-NIL and Ppd-1b-NIL plants under short day conditions. By contrast to WCO1, TaHd1 was up-regulated during the reproductive stage. The level of TaHd1 expression was much higher in Ppd-1a-NIL than the Ppd-1b-NIL plants, suggesting that the Ppd-1b allele down-regulates TaHd1 under short day conditions. The present study indicates that down-regulation of VRN3/WFT together with TaHd1 is the cause of late flowering in the Ppd-1b-NIL plants under short day conditions.     

Cloning and characterization of SOC1 homologs in barley (Hordeum vulgare) and their expression during seed development and in response to vernalization

A number of genes are involved in the vernalization pathway, such as VRN1, VRN2 and VRN3/FT1, whose function has been studied in barley and wheat. However, the function of the flowering and vernalization integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) has not been well studied in Triticeae, and particularly in barley. Herein, we cloned and characterized two barley SOC1-like homologs, HvSOC1-like1 and HvSOC1-like2. Primary sequence analysis of the predicted HvSOC1-like1 and HvSOC1-like2 proteins showed that they are members of the type II MADS-box protein family. Phylogenetic analysis placed the predicted proteins with other SOC1 and SOC1-like proteins from different species neighboring those from other cereal plant species. Primary and secondary structures of the predicted proteins are conserved to each other and more distant to the recently identified barley ODDSOC1 proteins. Genomic organization of HvSOC1-like1 is very similar to the Arabidopsis and Brachypodium SOC1 genes and localized in highly syntenic chromosomal regions. Regulatory cis-acting elements detected in the HvSOC1-like1 promoter include the CArG-box, implicated in the regulation of SOC1 expression in Arabidopsis. Both HvSOC1-like1 and HvSOCI-like2 are expressed in vegetative and reproductive tissues and at different stages of seed development. Both are upregulated in a particular seed developmental stage suggesting their possible implication in seed development. Furthermore, HvSOC1-like1 was induced in two winter barley cultivars after vernalization treatment pointing to its probable involvement in the vernalization process. The study of the SOC1 genes reported here opens the way for a better understanding of both the vernalization process and seed development and germination in this important cereal crop.     

Natural variation of barley vernalization requirements: implication of quantitative variation of winter growth habit as an adaptive trait in East Asia

In many temperate plant species, prolonged cold treatment, known as vernalization, is one of the most critical steps in the transition from the vegetative to the reproductive stage. In contrast to recent advances in understanding the molecular basis of vernalization in Arabidopsis non-vernalization mutants or the spring growth habits of cereal crops such as wheat and barley, natural variations in winter growth habits and their geographic distribution are poorly understood. We analyzed varietal variation and the geographic distribution of the degree of vernalization requirements in germplasms of domesticated barley and wild barley collections. We found a biased geographic distribution of vernalization requirements in domesticated barley: Western regions were strongly associated with a higher degree of spring growth habits, and the extreme winter growth habits were localized to Far Eastern regions including China, Korea and Japan. Both wild accessions and domesticated landraces, the regions of distribution of which overlapped each other, mainly belonged to the moderate class of winter growth habit. As a result of quantitative evaluations performed in this study, we provide evidence that the variation in the degree of winter growth habit in recombinant inbred lines was controlled by quantitative trait loci including three vernalization genes (VRN1, VRN2 and VRN3) that account for 37.9% of the variation in vernalization requirements, with unknown gene(s) explaining the remaining two-thirds of the variation. This evidence implied that the Far Eastern accessions might be a genetically differentiated group derived for an evolutionary reason, resulting in their greater tendency towards a winter growth habit.     

The molecular biology of seasonal flowering-responses in Arabidopsis and the cereals

Background

In arabidopsis (Arabidopsis thaliana), FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC) play key roles in regulating seasonal flowering-responses to synchronize flowering with optimal conditions. FT is a promoter of flowering activated by long days and by warm conditions. FLC represses FT to delay flowering until plants experience winter.

Scope

The identification of genes controlling flowering in cereals allows comparison of the molecular pathways controlling seasonal flowering-responses in cereals with those of arabidopsis. The role of FT has been conserved between arabidopsis and cereals; FT-like genes trigger flowering in response to short days in rice or long days in temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare). Many varieties of wheat and barley require vernalization to flower but FLC-like genes have not been identified in cereals. Instead, VERNALIZATION2 (VRN2) inhibits long-day induction of FT-like1 (FT1) prior to winter. VERNALIZATION1 (VRN1) is activated by low-temperatures during winter to repress VRN2 and to allow the long-day response to occur in spring. In rice (Oryza sativa) a VRN2-like gene Ghd7, which influences grain number, plant height and heading date, represses the FT-like gene Heading date 3a (Hd3a) in long days, suggesting a broader role for VRN2-like genes in regulating day-length responses in cereals. Other genes, including Early heading date (Ehd1), Oryza sativa MADS51 (OsMADS51) and INDETERMINATE1 (OsID1) up-regulate Hd3a in short days. These genes might account for the different day-length response of rice compared with the temperate cereals. No genes homologous to VRN2, Ehd1, Ehd2 or OsMADS51 occur in arabidopsis.

Conclusions

It seems that different genes regulate FT orthologues to elicit seasonal flowering-responses in arabidopsis and the cereals. This highlights the need for more detailed study into the molecular basis of seasonal flowering-responses in cereal crops or in closely related model plants such as Brachypodium distachyon.Key words: Flowering, vernalization, photoperiod, day length, VRN1, VRN2, FLC, FT, cereals, arabidopsis, MADS     

Epigenetic regulation of flowering

The acceleration of flowering by prolonged low temperature treatment (vernalization) has unique properties including the floral transition occurring at a time separate from the vernalization treatment. This implies the vernalization condition is inherited through mitotic divisions, but this vernalized state is not inherited from one generation to the next. FLC, the key gene mediating this response in the Arabidopsis is repressed by histone modifications involving the VRN2 protein complex. Other protein complexes participate in activating the gene. While many plant species depend on vernalization for optimising flowering time, the genes involved differ between dicot and monocot plants in both Arabidopsis and cereals, vernalization regulates photoperiod control of flowering by preventing the induction of the floral promoter FT by long days in autumn but allowing induction of FT in spring and hence flowering occurs at an optimal time in the annual life cycle.     

Two MADS-box genes from perennial ryegrass are regulated by vernalization and involved in the floral transition

Many plants in temperate regions have a requirement for vernalization in order to initiate the reproductive growth phase. In cereals, this requirement has been linked to the VRN1 locus, which encodes an APETALA1 -like ( AP1 -like) MADS-box gene. In perennial ryegrass ( Lolium perenne L.), we have isolated two MADS-box genes that are regulated by vernalization, LpMADS1 , which co-localize to the VRN1 locus in ryegrass, and LpMADS10 , which is an SVP -like MADS-box gene. In the shoot apex, LpMADS1 is increasingly induced by cold exposure, whereas LpMADS10 is increasingly repressed. Comparison of LpMADS1 promoter regions from several ryegrass varieties, with and without vernalization requirement, suggests that a putative MADS-box protein-binding site (CArG-box) might be important for the vernalization-regulated expression of LpMADS1 . Although the LpMADS10 expression pattern suggests it to be involved in floral repression, ectopic expression of LpMADS10 did neither affect flowering time significantly in Arabidopsis thaliana nor in L. perenne . Interestingly, we found that LpMADS1 interacts with LpMADS10 in a yeast two-hybrid assay. This finding is discussed in regard to the regulation of vernalization response in perennial ryegrass.     

Quantitative effects of vernalization on FLC and SOC1 expression

Prolonged exposure to cold results in early flowering in Arabidopsis winter annual ecotypes, with longer exposures resulting in a greater promotion of flowering than shorter exposures. The promotion of flowering is mediated through an epigenetic down-regulation of the floral repressor FLOWERING LOCUS C (FLC). We present results that provide an insight into the quantitative regulation of FLC by vernalization. Analysis of the effect of seed or plant cold treatment on FLC expression indicates that the time-dependent nature of vernalization on FLC expression is mediated through the extent of the initial repression of FLC and not by affecting the ability to maintain the repressed state. In the over-expression mutant flc-11, the time-dependent repression of FLC correlates with the proportional deacetylation of histone H3. Our results indicate that sequences within intron 1 and the activities of both VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2) are required for efficient establishment of FLC repression; however, VRN1 and VRN2 are not required for maintenance of the repressed state during growth after the cold exposure. SUPPRESSOR OF OVER-EXPRESSION OF CO 1 (SOC1), a downstream target of FLC, is quantitatively induced by vernalization in a reciprocal manner to FLC. In addition, we show that SOC1 undergoes an acute induction by both short and long cold exposures.     

The PHD finger protein VRN5 functions in the epigenetic silencing of Arabidopsis FLC

Vernalization, the acceleration of flowering by the prolonged cold of winter, ensures that plants flower in favorable spring conditions. During vernalization in Arabidopsis, cold temperatures repress FLOWERING LOCUS C (FLC) expression in a mechanism involving VERNALIZATION INSENSITIVE 3 (VIN3), and this repression is epigenetically maintained by a Polycomb-like chromatin regulation involving VERNALIZATION 2 (VRN2), a Su(z)12 homolog, VERNALIZATION 1 (VRN1), and LIKE-HETEROCHROMATIN PROTEIN 1. In order to further elaborate how cold repression triggers epigenetic silencing, we have targeted mutations that result in FLC misexpression both at the end of the prolonged cold and after subsequent development. This identified VERNALIZATION 5 (VRN5), a PHD finger protein and homolog of VIN3. Our results suggest that during the prolonged cold, VRN5 and VIN3 form a heterodimer necessary for establishing the vernalization-induced chromatin modifications, histone deacetylation, and H3 lysine 27 trimethylation required for the epigenetic silencing of FLC. Double mutant and FLC misexpression analyses reveal additional VRN5 functions, both FLC-dependent and -independent, and indicate a spatial complexity to FLC epigenetic silencing with VRN5 acting as a common component in multiple pathways.     

Vernalization and epigenetics: how plants remember winter

One of the remarkable aspects of the promotion of flowering by vernalization is that plants have evolved the ability to measure a complete winter season of cold and to 'remember' this prior cold exposure in the spring. Recent work in Arabidopsis demonstrates the molecular basis of this memory of winter: vernalization causes changes in the chromatin structure of a flowering repressor gene, FLOWERING LOCUS C (FLC), that switch this gene into a repressed state that is mitotically stable. A key component of the vernalization pathway, VERNALIZATION INSENSITIVE3 (VIN3), which is a PHD-domain-containing protein, is induced only after a prolonged period of cold. VIN3 is involved in initiating the modification of FLC chromatin structure. The stable silencing of FLC also requires the DNA-binding protein VERNALIZATION1 (VRN1) and the polycomb-group protein VRN2.     

The quantitative response of wheat vernalization to environmental variables indicates that vernalization is not a response to cold temperature

The initiation of flowering is a crucial trait that allows temperate plants to flower in the favourable conditions of spring. The timing of flowering initiation is governed by two main mechanisms: vernalization that defines a plant's requirement for a prolonged exposure to cold temperatures; and photoperiod sensitivity defining the need for long days to initiate floral transition. Genetic variability in both vernalization and photoperiod sensitivity largely explains the adaptability of cultivated crop plants such as bread wheat (Triticum aestivum L.) to a wide range of climatic conditions. The major genes controlling wheat vernalization (VRN1, VRN2, and VRN3) and photoperiod sensitivity (PPD1) have been identified, and knowledge of their interactions at the molecular level is growing. However, the quantitative effects of temperature and photoperiod on these genes remain poorly understood. Here it is shown that the distinction between the temperature effects on organ appearance rate and on vernalization sensu stricto is crucial for understanding the quantitative effects of the environmental signal on wheat flowering. By submitting near isogenic lines of wheat differing in their allelic composition at the VRN1 locus to various temperature and photoperiod treatments, it is shown that, at the whole-plant level, the vernalization process has a positive response to temperature with complex interactions with photoperiod. In addition, the phenotypic variation associated with the presence of different spring homoeoalleles of VRN1 is not induced by a residual vernalization requirement. The results demonstrate that a precise definition of vernalization is necessary to understand and model temperature and photoperiod effects on wheat flowering. It is suggested that this definition should be used as the basis for gene expression studies and assessment of functioning of the wheat flowering gene network, including an explicit account of the quantitative effect of environmental variables.     

The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis.

The acceleration of flowering by a long period of low temperature, vernalization, is an adaptation that ensures plants overwinter before flowering. Vernalization induces a developmental state that is mitotically stable, suggesting that it may have an epigenetic basis. The VERNALIZATION2 (VRN2) gene mediates vernalization and encodes a nuclear-localized zinc finger protein with similarity to Polycomb group (PcG) proteins of plants and animals. In wild-type Arabidopsis, vernalization results in the stable reduction of the levels of the floral repressor FLC. In vrn2 mutants, FLC expression is downregulated normally in response to vernalization, but instead of remaining low, FLC mRNA levels increase when plants are returned to normal temperatures. VRN2 function therefore stably maintains FLC repression after a cold treatment, serving as a mechanism for the cellular memory of vernalization.