Natural variation in the plasticity of flowering time across Raphanus sativus var. raphanistroides characterized by different vernalization requirements

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.

Studies of natural genetic variation for the vernalization requirement in Arabidopsis have revealed two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), that are determinants of the vernalization-requiring, winter-annual habit. In this study, we show that FLC EXPRESSOR LIKE 4 (FLL4) is essential for up-regulation of FLC in winter-annual Arabidopsis accessions and establishment of a vernalization requirement. FLL4 is part of the FLC EXPRESSOR (FLX) gene family and both are non-redundantly involved in flowering-time control. Epistasis analysis among FRI, FLL4, FLX and autonomous-pathway genes reveals that FRI fve exhibits an extreme delay of flowering compared to fri fve, but mutants in other autonomous-pathway genes do not, indicating that FVE acts most antagonistically to FRI. FLL4 may represent a new member of a FRI-containing complex that activates FLC.

The requirement for vernalization, a need for prolonged cold to trigger flowering, aligns reproductive development with favorable spring conditions. In Arabidopsis thaliana vernalization depends on the cold-induced epigenetic silencing of the floral repressor locus FLC. Extensive natural variation in vernalization response is associated with A. thaliana accessions collected from different geographical regions. Here, we analyse natural variation for vernalization temperature requirement in accessions, including those from the northern limit of the A. thaliana range. Vernalization required temperatures above 0°C and was still relatively effective at 14°C in all the accessions. The different accessions had characteristic vernalization temperature profiles. One Northern Swedish accession showed maximum vernalization at 8°C, both at the level of flowering time and FLC chromatin silencing. Historical temperature records predicted all accessions would vernalize in autumn in N. Sweden, a prediction we validated in field transplantation experiments. The vernalization response of the different accessions was monitored over three intervals in the field and found to match that when the average field temperature was given as a constant condition. The vernalization temperature range of 0–14°C meant all accessions fully vernalized before snowfall in N. Sweden. These findings have important implications for understanding the molecular basis of adaptation and for predicting the consequences of climate change on flowering time.DOI:http://dx.doi.org/10.7554/eLife.06620.001

The requirement for vernalization, a need for prolonged cold to trigger flowering, aligns reproductive development with favorable spring conditions. In Arabidopsis thaliana vernalization depends on the cold-induced epigenetic silencing of the floral repressor locus FLC. Extensive natural variation in vernalization response is associated with A. thaliana accessions collected from different geographical regions. Here, we analyse natural variation for vernalization temperature requirement in accessions, including those from the northern limit of the A. thaliana range. Vernalization required temperatures above 0°C and was still relatively effective at 14°C in all the accessions. The different accessions had characteristic vernalization temperature profiles. One Northern Swedish accession showed maximum vernalization at 8°C, both at the level of flowering time and FLC chromatin silencing. Historical temperature records predicted all accessions would vernalize in autumn in N. Sweden, a prediction we validated in field transplantation experiments. The vernalization response of the different accessions was monitored over three intervals in the field and found to match that when the average field temperature was given as a constant condition. The vernalization temperature range of 0-14°C meant all accessions fully vernalized before snowfall in N. Sweden. These findings have important implications for understanding the molecular basis of adaptation and for predicting the consequences of climate change on flowering time.

Summary Flowering time is a key adaptive and agronomic trait. In Arabidopsis, natural variation in expression levels of the floral repressor FLOWERING LOCUS C (FLC) leads to differences in vernalization. In Brassica napus there are nine copies of FLC. Here, we study how these multiple FLC paralogues determine vernalization requirement as a system.We collected transcriptome time series for Brassica napus spring, winter, semi‐winter, and Siberian kale crop types. Modelling was used to link FLC expression dynamics to floral response following vernalization.We show that relaxed selection pressure has allowed expression of FLC paralogues to diverge, resulting in variation of FLC expression during cold treatment between paralogues and accessions. We find that total FLC expression dynamics best explains differences in cold requirement between cultivars, rather than expression of specific FLC paralogues.The combination of multiple FLC paralogues with different expression dynamics leads to rich behaviour in response to cold and a wide range of vernalization requirements in B. napus. We find evidence for different strategies to determine the response to cold in existing winter rapeseed accessions.

The life cycle of flowering plants is partially defined by environmental cues like day length and temperature. In the model plant Arabidopsis thaliana and temperate cereals, such as barley (Hordeum vulgare) and wheat (Triticum spp.), differences in life cycle control have been associated with a natural variation in FLOWERING LOCUS C (FLC) and VERNALIZATION 1-3 (VRN1-3). In sugar beet (Beta vulgaris L.), variation in vernalization requirement and life cycle is determined by a major gene at the B locus. This gene has recently been identified as a pseudo-response regulator (PRR) gene BOLTING TIME CONTROL 1 (BTC1). A second gene in beet with homology to BTC1 and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 7 (APRR7) in Arabidopsis was identified and termed Beta vulgaris PSEUDO RESPONSE REGULATOR 7 (BvPRR7). We functionally characterized BvPRR7 by transgenic analysis in Arabidopsis and expression profiling during development in beet. We show that BvPRR7 was diurnally regulated and responded to cold. Constitutive expression of BvPRR7 distorted diurnal rhythms and caused late flowering in Arabidopsis suggesting a conserved function of BvPRR7 in clock regulation. Conceivably, the retention of a functional role of BvPRR7 in clock regulation may have facilitated the evolution of a distinct role as major floral regulator of the second PRR7 homolog in beet, BTC1.

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.

The evolution of diverse life history strategies has allowed Arabidopsis thaliana to adapt to worldwide locations, spanning a range of latitudinal and environmental conditions. Arabidopsis thaliana accessions are either vernalization-requiring winter annuals or rapid cyclers, with extensive natural variation in vernalization requirement and response. Genetic and molecular analysis of this variation has enhanced our understanding of the mechanisms involved in life history determination, with translation to both natural and crop systems in the Brassicaceae and beyond.

Wheat varieties with a winter growth habit require long exposures to low temperatures (vernalization) to accelerate flowering. Natural variation in four vernalization genes regulating this requirement has favored wheat adaptation to different environments. The first three genes (VRN1-VRN3) have been cloned and characterized before. Here we show that the fourth gene, VRN-D4, originated by the insertion of a ∼290-kb region from chromosome arm 5AL into the proximal region of chromosome arm 5DS. The inserted 5AL region includes a copy of VRN-A1 that carries distinctive mutations in its coding and regulatory regions. Three lines of evidence confirmed that this gene is VRN-D4: it cosegregated with VRN-D4 in a high-density mapping population; it was expressed earlier than other VRN1 genes in the absence of vernalization; and induced mutations in this gene resulted in delayed flowering. VRN-D4 was found in most accessions of the ancient subspecies Triticum aestivum ssp. sphaerococcum from South Asia. This subspecies showed a significant reduction of genetic diversity and increased genetic differentiation in the centromeric region of chromosome 5D, suggesting that VRN-D4 likely contributed to local adaptation and was favored by positive selection. Three adjacent SNPs in a regulatory region of the VRN-D4 first intron disrupt the binding of GLYCINE-RICH RNA-BINDING PROTEIN 2 (TaGRP2), a known repressor of VRN1 expression. The same SNPs were identified in VRN-A1 alleles previously associated with reduced vernalization requirement. These alleles can be used to modulate vernalization requirements and to develop wheat varieties better adapted to different or changing environments.

The transition from vegetative to floral meristems in higher plants is determined by the coincidence of internal and environmental signals. Contrary to the photoperiod pathway, convergent evolution of the cold-dependent pathway has implicated different genes between dicots and monocots. Whereas no association between natural variation in vernalization requirement and Flowering time locus T (FT) gene polymorphism has been described in Arabidopsis, recent studies in Triticeae suggest implication of orthologous copies of FT in the cold response. In our study, we show that nucleotide polymorphisms on A and D copies of the wheat FT gene were associated with variations for heading date in a collection of 239 lines representing diverse geographical origins and status (landraces, old or recent cultivars). Interestingly, polymorphisms in the non-coding intronic region were strongly associated to flowering variation observed on plants grown without vernalization. But differently from VRN1, no epistatic interaction between FT homeologous copies was revealed. In agreement with the results of association study, the A and D copies of FT were mapped in regions including major QTLs for earliness traits in hexaploid wheat. This work, by identifying additional homeoalleles involved in wheat vernalization pathway, will contribute to a better understanding of the control of flowering, hence providing tools for the breeding of varieties with enhanced adaptation to changing environments.

The appropriate timing of flowering is crucial for plant reproductive success. Studies of the molecular mechanism of flower induction in the model plant Arabidopsis thaliana showed long days and vernalization as major environmental promotive factors. Noccaea caerulescens has an obligate vernalization requirement that has not been studied at the molecular genetics level. Here, we characterize the vernalization requirement and response of four geographically diverse biennial/perennial N. caerulescens accessions: Ganges (GA), Lellingen (LE), La Calamine (LC), and St. Felix de Pallières (SF). Differences in vernalization responsiveness among accessions suggest that natural variation for this trait exists within N. caerulescens. Mutants which fully abolish the vernalization requirement were identified and were shown to contain mutations in the FLOWERING LOCUS C (NcFLC) and SHORT VEGETATIVE PHASE (NcSVP) genes, two key floral repressors in this species. At high temperatures, the non-vernalization requiring flc-1 mutant reverts from flowering to vegetative growth, which is accompanied with a reduced expression of LFY and AP1. This suggested there is “crosstalk” between vernalization and ambient temperature, which might be a strategy to cope with fluctuations in temperature or adopt a more perennial flowering attitude and thus facilitate a flexible evolutionary response to the changing environment across the species range.

Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate flowering (vernalization), whereas spring varieties do not have this requirement. In this study, we show that in these species, the vernalization gene VRN3 is linked completely to a gene similar to Arabidopsis FLOWERING LOCUS T (FT). FT induction in the leaves results in a transmissible signal that promotes flowering. Transcript levels of the barley and wheat orthologues, designated as HvFT and TaFT, respectively, are significantly higher in plants homozygous for the dominant Vrn3 alleles (early flowering) than in plants homozygous for the recessive vrn3 alleles (late flowering). In wheat, the dominant Vrn3 allele is associated with the insertion of a retroelement in the TaFT promoter, whereas in barley, mutations in the HvFT first intron differentiate plants with dominant and recessive VRN3 alleles. Winter wheat plants transformed with the TaFT allele carrying the promoter retroelement insertion flowered significantly earlier than nontransgenic plants, supporting the identity between TaFT and VRN-B3. Statistical analyses of flowering times confirmed the presence of significant interactions between vernalization and FT allelic classes in both wheat and barley (P < 0.0001). These interactions were supported further by the observed up-regulation of HvFT transcript levels by vernalization in barley winter plants (P = 0.002). These results confirmed that the wheat and barley FT genes are responsible for natural allelic variation in vernalization requirement, providing additional sources of adaptive diversity to these economically important crops.

FLOWERING LOCUS C (FLC) is one of the main genes influencing the vernalization requirement and natural flowering time variation in the annual Arabidopsis thaliana. Here we studied the effects of vernalization on flowering and its genetic basis in the perennial Arabidopsis lyrata. Two tandemly duplicated FLC genes (FLC1 and FLC2) were compared with respect to expression and DNA sequence. The effect of vernalization on flowering and on the expression of FLC1 was studied in three European populations. The genetic basis of the FLC1 expression difference between two of the populations was further studied by expression quantitative trait locus (eQTL) mapping and sequence analysis. FLC1 was shown to have a likely role in the vernalization requirement for flowering in A.lyrata. Vernalization decreased its expression and the northern study populations showed higher FLC1 expression than the southern one. eQTL mapping between two of the populations revealed one eQTL affecting FLC1 expression in the genomic region containing the FLC genes. Most FLC1 sequence differences between the study populations were found in the promoter region and in the first intron. Variation in the FLC1 sequence may cause differences in FLC1 expression between late- and early-flowering A.lyrata populations.

Interactions among factors regulating phenological development and acclimation rate determine low-temperature tolerance in wheat.

Analysis of how seasonal cues influence the timing of the floral transition has revealed many important principles for how epigenetic regulation can integrate a variety of environmental cues with developmental signals. The study of the pathways that necessitate overwintering in plants and their ability to respond to prolonged cold (the vernalization requirement and response pathways) has elaborated different chromatin regulatory pathways and the involvement of noncoding RNAs. The major target of these vernalization pathways in Arabidopsis (Arabidopsis thaliana) is Flowering Locus C (FLC). A relatively simple picture of FLC regulation is emerging of a few core complexes and mechanisms that antagonize each other's actions. This balance provides a fine degree of control that has nevertheless permitted evolution of a wide range of natural variation in vernalization in Arabidopsis. Similar simple routes of adaptation may underlie life history variation between species.