Local climate and vernalization requirements explain the latitudinal patterns of flowering initiation in the crop wild relative Linum bienne

Background and Aims: Days to flowering initiation in species with large geographic distributions often correlate with latitude. Latitude reflects climatic gradients, but it is unclear if large-scale differentiation in flowering results from adaptation to local climate, and whether adaptation to local climate could constrain shifts in distribution and colonization of new environments. Methods: In its Western range in Europe, L. bienne populations were surveyed to describe latitudinal patterns of flowering initiation and determine its correlation with the local climate of populations. This was measured under standardized greenhouse conditions, with a vernalization experiment to learn if chilling advances flowering, and with a reciprocal transplant experiment at three sites along the latitudinal gradient, recording flowering at the central site and plant survival in all sites. Also, genetic differentiation of populations along the latitudinal range was studied using microsatellite markers. Key Results: Flowering initiation varied with latitude, with southern populations flowering earlier than northern populations. Latitude also predicted population response to vernalization, with chilling inducing a greater advance of flowering initiation in northern than southern populations. In general, plant survival in the reciprocal transplant experiment decreased with the geographic distance of populations to the experimental site and, at the central site, flowering initiation varied with latitude of origin. However, across experiments, the local climate of populations better predicted the differentiation in flowering initiation and vernalization response than latitude of origin. Finally, the microsatellite data revealed genetic differentiation of populations forming two groups that agree with a Mediterranean and Atlantic lineage. Conclusions: The consistent result across experiments of a latitudinal cline in flowering initiation and in the vernalization response suggests that flowering is under genetic regulation and yet dependent on particular environmental and climatic cues at local scale. However, the genetic differentiation suggests that past population history might influenced the flowering initiation patterns detected.

Molecular labeling of Vrn, Ppd genes and vernalization response of the ultra-early lines of spring bread wheat Triticum aestivum L.

Background. The knowledge of genetic control of vernalization response in the ultra-early accessions can facilitate bread wheat breeding for a high adaptive capacity. Materials and methods. The study involved the ultra-early lines Rico (k-65588) and Rimax (k-67257) as the earliest maturing lines in the VIR bread wheat collection, as well as 10 Rifor lines (k-67120, k-67121, k-67250-67256) with a high rate of development before heading. A late ripening accession ‘Forlani Roberto’ (k-42641) and ‘Leningradskaya 6’ variety (k-64900), regionally adapted to Northwestern Russia, were also studied. The alleles of the Vrn and Ppd genes were identified by the PCR analysis using the allele-specific primers published in literature sources. The response to vernalization (30 days at 3°C) and a short 12-hour day were determined using a methodology accepted at VIR. Results. The ultra-early lines respond to a short 12-hour day and 30-day vernalization very poorly. The genotype of ultra-early wheat lines is mainly represented by three genes, Vrn-A1, Vrn-B1a, and Vrn-D1, which ensure insensitivity to vernalization alongside with the expression of Ppd-D1a, which controls the response to photoperiod. The ultra-early lines Rifor 4 and Rifor 5 have a recessive allele vrn-A1a, like the original ‘Forlani Roberto’ accession. The lines Rifor 4 and Rifor 5 are vernalization-insensitive under the long day and have a very weak response under the short day (3.5±0.42 days and 4.0±0.61 days, respectively). However, ‘Forlani Roberto’ with the vrn-A1a gene responds to vernalization in the same way under any photoperiod (12.3±1.58 days and 12.2±0.74 days). Conclusion The ultra-early lines of bread wheat Rifor 4 and Rifor 5 with the vrn-A1a gene can have no response to vernalization or have a low level response. This effect can be a reason for the formation of a complex of modifier genes along with the dominant gene Vrn-D1, which forms during the hybridization of F7-8 Rico × Forlani Roberto. The ultra-early lines of bread wheat Rico, Rimax and Rifor (k-67120, k-67121, k-67250-67256) can serve as effective sources of genes for earliness in common wheat breeding.

Vernalization-triggered expression of the antisense transcript COOLAIR is mediated by CBF genes

Many plants undergo vernalization, a long-term winter-triggered acceleration of flowering, to align their flowering time with spring. In Arabidopsis thaliana, this is achieved by silencing a floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, an antisense noncoding RNA expressed at the FLC locus, is induced during the early phase of vernalization, preceding FLC suppression. However, the mechanism by which long-term cold induces COOLAIR is not well understood. Here, we showed that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3′-end of FLC and CRT/DRE-binding factors (CBFs) are required for vernalization-induced COOLAIR activation. The CBFs bind to CRT/DREs at the 3′-end of FLC, both in vitro and in vivo, and the CBFs levels increased gradually during vernalization. Additionally, vernalization-induced COOLAIR expression was highly suppressed in the cbfs mutant, in which all CBFs were knocked-out. Contrastingly, CBF-overexpressing plants showed COOLAIR upregulation, even at warm temperatures. We propose that COOLAIR is induced by CBFs in the early phase of vernalization but is downregulated as CBFs are evicted from closed FLC chromatin during the late vernalization phase. We also demonstrated that cbfs and COOLAIR mutants have a normal vernalization response, although they show defects in vernalization-induced COOLAIR activation, indicating that COOLAIR is not necessary for this process.

A Vernalization Response in a Winter Safflower (Carthamus tinctorius) Involves the Upregulation of Homologs of FT, FUL, and MAF

Safflower (Carthamus tinctorius) is a member of the Asteraceae family that is grown in temperate climates as an oil seed crop. Most commercially grown safflower varieties can be sown in late winter or early spring and flower rapidly in the absence of overwintering. There are winter-hardy safflower accessions that can be sown in autumn and survive over-wintering. Here, we show that a winter-hardy safflower possesses a vernalization response, whereby flowering is accelerated by exposing germinating seeds to prolonged cold. The impact of vernalization was quantitative, such that increasing the duration of cold treatment accelerated flowering to a greater extent, until the response was saturated after 2 weeks exposure to low-temperatures. To investigate the molecular-basis of the vernalization-response in safflower, transcriptome activity was compared and contrasted between vernalized versus non-vernalized plants, in both ‘winter hardy’ and ‘spring’ cultivars. These genome-wide expression analyses identified a small set of transcripts that are both differentially expressed following vernalization and that also have different expression levels in the spring versus winter safflowers. Four of these transcripts were quantitatively induced by vernalization in a winter hardy safflower but show high basal levels in spring safflower. Phylogenetic analyses confidently assigned that the nucleotide sequences of the four differentially expressed transcripts are related to FLOWERING LOCUS T (FT), FRUITFUL (FUL), and two genes within the MADS-like clade genes. Gene models were built for each of these sequences by assembling an improved safflower reference genome using PacBio-based long-read sequencing, covering 85% of the genome, with N50 at 594,000 bp in 3000 contigs. Possible evolutionary relationships between the vernalization response of safflower and those of other plants are discussed.

Identification and Characterization of Perennial Ryegrass (Lolium perenne) Vernalization Genes

Perennial ryegrass (Lolium perenne) is a temperate grass species commonly used as pasture for livestock. Flowering (heading) of ryegrass impacts metabolizable energy content and seed yield, therefore this trait is important for both farmers and seed producers. In related grass species, the VRN genes (VRN1-3) have been largely implicated in the determination of vernalization response and are responsible for much of the intra-species variation in this trait. Many other important flowering-time regulators have been cataloged in the model grass Brachypodium distachyon; however, in several cases, such as VRN2, their ryegrass homologs have not been well-characterized. Here, ryegrass homologs of important flowering time genes from B. distachyon were identified through available synteny data and sequence similarity. Phylogenetic analysis of VRN3/FT-like and VRN2-like genes was performed to elucidate these families further. The expression patterns of these genes were assessed during vernalization. This confirmed the key roles played by LpVRN1 and LpFT3 in the promotion of flowering. Furthermore, two orthologs of VRN2 identified here, as well as an ortholog of CO9, were expressed prior to vernalization, and were repressed in flowering plants, suggesting a role in floral repression. Significant variability in expression of these flowering pathway genes in diverse genotypes was detected and may underlie variation in flowering time and vernalization response.

Genome Triplication Leads to Transcriptional Divergence of FLOWERING LOCUS C Genes During Vernalization in the Genus Brassica

The genus Brassica includes oil crops, vegetables, condiments, fodder crops, and ornamental plants. Brassica species underwent a whole genome triplication event after speciation between ancestral species of Brassica and closely related genera including Arabidopsis thaliana. Diploid species such as Brassica rapa and Brassica oleracea have three copies of genes orthologous to each A. thaliana gene, although deletion in one or two of the three homologs has occurred in some genes. The floral transition is one of the crucial events in a plant’s life history, and time of flowering is an important agricultural trait. There is a variation in flowering time within species of the genus Brassica, and this variation is largely dependent on a difference in vernalization requirements. In Brassica, like in A. thaliana, the key gene of vernalization is FLOWERING LOCUS C (FLC). In Brassica species, the vernalization response including the repression of FLC expression by cold treatment and the enrichment of the repressive histone modification tri-methylated histone H3 lysine 27 (H3K27me3) at the FLC locus is similar to A. thaliana. B. rapa and B. oleracea each have four paralogs of FLC, and the allotetraploid species, Brassica napus, has nine paralogs. The increased number of paralogs makes the role of FLC in vernalization more complicated; in a single plant, paralogs vary in the expression level of FLC before and after vernalization. There is also variation in FLC expression levels between accessions. In this review, we focus on the regulatory circuits of the vernalization response of FLC expression in the genus Brassica.

Natural variation in autumn FLC levels, rather than epigenetic silencing, aligns vernalization to different climates

Plants monitor temperatures over long timescales to assess seasons and time developmental transitions. In Arabidopsis thaliana, winter is registered during vernalization through the temperature-dependent repression and epigenetic silencing of floral repressor FLOWERING LOCUS C (FLC). Natural Arabidopsis accessions show considerable variation in vernalization, however which aspect of the FLC repression mechanism is most important for adaptation to different climates is not clear. By analyzing FLC silencing in natural variants throughout winter in three field sites, we find that FLC starting levels and early phases of silencing are the major variables underlying vernalization response, rather than establishment of epigenetic silencing. This results in an intricate interplay between promotion and delay of flowering to balance survival, and through a post-vernalization effect of FLC, reproductive effort via branch production. These data reveal how non-coding FLC variation aligns vernalization response to different climatic conditions and year-on-year fluctuations in natural temperature profiles.Impact StatementAlleles of the major floral repressor vary in their initial expression to underpin the ability of Arabidopsis to survive year-on-year climatic fluctuations.