Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

Brassica rapa L., which includes Chinese cabbage, turnip, and pak choi, has more complex flowering time regulation than does Arabidopsis thaliana due to the presence of multiple paralogous flowering time genes. FLOWERING LOCUS C (FLC) is one of the key genes regulating the flowering time, and B. rapa has four FLC paralogs. BrFLC5 on the reference genome is deemed a pseudogene because of a mutation (from G to A) in the splice site of the third intron, but there are some accessions with a G nucleotide in the splice site. In this study, we genotyped 310 B. rapa accessions and found that 19 had homozygous and 81 had heterozygous putative functional BrFLC5 alleles. Accessions of turnip showed the highest proportion with a functional BrFLC5 allele. BrFLC5 acts as a floral repressor when overexpressed in A. thaliana. The BrFLC5 expression level varied in pre-vernalized plants, and this transcriptional variation was not associated with the G/A polymorphism in the third intron. Three accessions having a higher BrFLC5 expression in pre-vernalized plants had a 584-bp insertion in the promoter region. Many regions homologous to this 584-bp sequence are present in the B. rapa genome, and this 584-bp inserted region has tandem duplications of an AT-rich sequence in its central region. The possibility that a high expression of a functional BrFLC5 could contribute to producing premature bolting-resistant lines in B. rapa vegetables is discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01405-0.

Differences in the transcriptional immune response to Albugo candida between white rust resistant and susceptible cultivars in Brassica rapa L.

Albugo candida causing white rust disease decreases the yield of Brassica rapa vegetables greatly. Resistant and susceptible cultivars in B. rapa vegetables have different immune responses against A. candida inoculation, however, the mechanism of how host plants respond to A. candida is still unknown. Using RNA-sequencing, we identified differentially expressed genes (DEGs) between A. candida inoculated [48 and 72 h after inoculation (HAI)] and non-inoculated samples in resistant and susceptible cultivars of komatsuna (B. rapa var. perviridis). Functional DEGs differed between the resistant and susceptible cultivars in A. candida inoculated samples. Salicylic acid (SA) responsive genes tended to be changed in their expression levels by A. candida inoculation in both resistant and susceptible cultivars, but different genes were identified in the two cultivars. SA-dependent systemic acquired resistance (SAR) involving genes were upregulated following A. candida inoculation in the resistant cultivar. Particular genes categorized as SAR that changed expression levels overlapped between A. candida and Fusarium oxysporum f. sp. conglutinans inoculated samples in resistant cultivar, suggesting a role for SAR in defense response to both pathogens particularly in the effector-triggered immunity downstream pathway. These findings will be useful for understanding white rust resistance mechanisms in B. rapa.

Corrigendum: Will epigenetics be a key player in crop breeding?

[This corrects the article DOI: 10.3389/fpls.2022.958350.].

Will epigenetics be a key player in crop breeding?

If food and feed production are to keep up with world demand in the face of climate change, continued progress in understanding and utilizing both genetic and epigenetic sources of crop variation is necessary. Progress in plant breeding has traditionally been thought to be due to selection for spontaneous DNA sequence mutations that impart desirable phenotypes. These spontaneous mutations can expand phenotypic diversity, from which breeders can select agronomically useful traits. However, it has become clear that phenotypic diversity can be generated even when the genome sequence is unaltered. Epigenetic gene regulation is a mechanism by which genome expression is regulated without altering the DNA sequence. With the development of high throughput DNA sequencers, it has become possible to analyze the epigenetic state of the whole genome, which is termed the epigenome. These techniques enable us to identify spontaneous epigenetic mutations (epimutations) with high throughput and identify the epimutations that lead to increased phenotypic diversity. These epimutations can create new phenotypes and the causative epimutations can be inherited over generations. There is evidence of selected agronomic traits being conditioned by heritable epimutations, and breeders may have historically selected for epiallele-conditioned agronomic traits. These results imply that not only DNA sequence diversity, but the diversity of epigenetic states can contribute to increased phenotypic diversity. However, since the modes of induction and transmission of epialleles and their stability differ from that of genetic alleles, the importance of inheritance as classically defined also differs. For example, there may be a difference between the types of epigenetic inheritance important to crop breeding and crop production. The former may depend more on longer-term inheritance whereas the latter may simply take advantage of shorter-term phenomena. With the advances in our understanding of epigenetics, epigenetics may bring new perspectives for crop improvement, such as the use of epigenetic variation or epigenome editing in breeding. In this review, we will introduce the role of epigenetic variation in plant breeding, largely focusing on DNA methylation, and conclude by asking to what extent new knowledge of epigenetics in crop breeding has led to documented cases of its successful use.

The Transcription Factors TaTDRL and TaMYB103 Synergistically Activate the Expression of TAA1a in Wheat, Which Positively Regulates the Development of Microspore in Arabidopsis.

Pollen fertility plays an important role in the application of heterosis in wheat (Triticum aestivum L.). However, the key genes and mechanisms underlying pollen abortion in K-type male sterility remain unclear. TAA1a is an essential gene for pollen development in wheat. Here, we explored the mechanism involved in its transcriptional regulation during pollen development, focusing on a 1315-bp promoter region. Several cis-acting elements were identified in the TAA1a promoter, including binding motifs for Arabidopsis thaliana AtAMS and AtMYB103 (CANNTG and CCAACC, respectively). Evolutionary analysis indicated that TaTDRL and TaMYB103 were the T. aestivum homologs of AtAMS and AtMYB103, respectively, and encoded nucleus-localized transcription factors containing 557 and 352 amino acids, respectively. TaTDRL and TaMYB103 were specifically expressed in wheat anthers, and their expression levels were highest in the early uninucleate stage; this expression pattern was consistent with that of TAA1a. Meanwhile, we found that TaTDRL and TaMYB03 directly interacted, as evidenced by yeast two-hybrid and bimolecular fluorescence complementation assays, while yeast one-hybrid and dual-luciferase assays revealed that both TaTDRL and TaMYB103 could bind the TAA1a promoter and synergistically increase its transcriptional activity. Furthermore, TaTDRL-EAR and TaMYB103-EAR transgenic Arabidopsis plants displayed abnormal microspore morphology, reduced pollen viability, and lowered seed setting rates. Additionally, the expression of AtMS2, a TAA1a homolog, was significantly lower in the two repressor lines than in the corresponding overexpression lines or WT plants. In summary, we identified a potential transcriptional regulatory mechanism associated with wheat pollen development.

Rice hybrid mimics have stable yields equivalent to those of the F1 hybrid and suggest a basis for hybrid vigour.

MAIN CONCLUSION: We have developed long term stable high yielding rice lines, Hybrid Mimics, from commercial hybrids. The vigour of the Mimic and the hybrid are developmental changes. These Mimics could substitute for hybrid seed for planting. We have used two pre-existing high-yielding hybrid systems (FLY1 and DY527) to develop Hybrid Mimics. In the FLY1 hybrid system we selected, under field conditions, F6 lines which have high grain yields and biomass equivalent to the F1 hybrids, stable over subsequent F7, F8 and later generations. We have termed these lines Hybrid Mimics. The mimics are mostly homozygous as a consequence of selfing in each generation. We have repeated this selection procedure in the second independent hybrid system DY527, producing Mimics with similar characteristics to the F1 hybrid. In both hybrid systems the selection criterion, based on the phenotype of the F1 hybrid, results in the Mimics having grain yield and biomass similar to that of the F1 hybrid. In each generation of the breeding program the plant population has increased phenotypic homogeneity. The genomes of the Mimic plants do not contain any common heterozygous segments negating claims that the vigour of hybrids depends upon heterozygosity of particular loci. Both hybrids and Mimics have early germination and commence photosynthesis before the parents, providing enhanced growth which is maintained throughout the life cycle. The biochemical parameters of photosynthesis in the hybrids and Mimics do not differ from those of the parents. Grain quality and resistance to the two major diseases, bacterial blight and rice blast are similar in the Mimics and hybrids. The Mimics overcome the major disadvantage of hybrids where F2 phenotypic segregation prevents their use as a crop beyond the F1 generation.

Development of a New DNA Marker for Fusarium Yellows Resistance in Brassica rapa Vegetables.

In vegetables of Brassica rapa L., Fusarium oxysporum f. sp. rapae (For) or F. oxysporum f. sp. conglutinans (Foc) cause Fusarium yellows. A resistance gene against Foc (FocBr1) has been identified, and deletion of this gene results in susceptibility (focbr1-1). In contrast, a resistance gene against For has not been identified. Inoculation tests showed that lines resistant to Foc were also resistant to For, and lines susceptible to Foc were susceptible to For. However, prediction of disease resistance by a dominant DNA marker on FocBr1 (Bra012688m) was not associated with disease resistance of For in some komatsuna lines using an inoculation test. QTL-seq using four F2 populations derived from For susceptible and resistant lines showed one causative locus on chromosome A03, which covers FocBr1. Comparison of the amino acid sequence of FocBr1 between susceptible and resistant alleles (FocBr1 and FocBo1) showed that six amino acid differences were specific to susceptible lines. The presence and absence of FocBr1 is consistent with For resistance in F2 populations. These results indicate that FocBr1 is essential for For resistance, and changed amino acid sequences result in susceptibility to For. This susceptible allele is termed focbr1-2, and a new DNA marker (focbr1-2m) for detection of the focbr1-2 allele was developed.

Characterization of Histone H3 Lysine 4 and 36 Tri-methylation in Brassica rapa L.

Covalent modifications of histone proteins act as epigenetic regulators of gene expression. We report the distribution of two active histone marks (H3K4me3 and H3K36me3) in 14-day leaves in two lines of Brassica rapa L. by chromatin immunoprecipitation sequencing. Both lines were enriched with H3K4me3 and H3K36me3 marks at the transcription start site, and the transcription level of a gene was associated with the level of H3K4me3 and H3K36me3. H3K4me3- and H3K36me3-marked genes showed low tissue-specific gene expression, and genes with both H3K4me3 and H3K36me3 had a high level of expression and were constitutively expressed. Bivalent active and repressive histone modifications such as H3K4me3 and H3K27me3 marks or antagonistic coexistence of H3K36me3 and H3K27me3 marks were observed in some genes. Expression may be susceptible to changes by abiotic and biotic stresses in genes having both H3K4me3 and H3K27me3 marks. We showed that the presence of H3K36me3 marks was associated with different gene expression levels or tissue specificity between paralogous paired genes, suggesting that H3K36me3 might be involved in subfunctionalization of the subgenomes.

Genome-wide analysis of long noncoding RNAs, 24-nt siRNAs, DNA methylation and H3K27me3 marks in Brassica rapa.

Long noncoding RNAs (lncRNAs) are RNA fragments that generally do not code for a protein but are involved in epigenetic gene regulation. In this study, lncRNAs of Brassica rapa were classified into long intergenic noncoding RNAs, natural antisense RNAs, and intronic noncoding RNAs and their expression analyzed in relation to genome-wide 24-nt small interfering RNAs (siRNAs), DNA methylation, and histone H3 lysine 27 trimethylation marks (H3K27me3). More than 65% of the lncRNAs analyzed consisted of one exon, and more than 55% overlapped with inverted repeat regions (IRRs). Overlap of lncRNAs with IRRs or genomic regions encoding for 24-nt siRNAs resulted in increased DNA methylation levels when both were present. LncRNA did not overlap greatly with H3K27me3 marks, but the expression level of intronic noncoding RNAs that did coincide with H3K27me3 marks was higher than without H3K27me3 marks. The Brassica genus comprises important vegetables and oil seed crops grown across the world. B. rapa is a diploid (AA genome) thought to be one of the ancestral species of both B. juncea (AABB genome) and B. napus (AACC) through genome merging (allotetrapolyploidization). Complex genome restructuring and epigenetic alterations are thought to be involved in these allotetrapolyploidization events. Comparison of lncRNAs between B. rapa and B. nigra, B. oleracea, B. juncea, and B. napus showed the highest conservation with B. oleracea. This study presents a comprehensive analysis of the epigenome structure of B. rapa at multi-epigenetic levels (siRNAs, DNA methylation, H3K27me3, and lncRNAs) and identified a suite of candidate lncRNAs that may be epigenetically regulated in the Brassica genus.

The transcriptional response to salicylic acid plays a role in Fusarium yellows resistance in Brassica rapa L.

KEY MESSAGE: Fusarium yellows resistant and susceptible lines in Brassica rapa showed different salicylic acid responses; the resistant line showed a similar response to previous reports, but the susceptible line differed. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease. Previous studies showed that genes related to salicylic acid (SA) response were more highly induced following Foc infection in Brassica rapa Fusarium yellows resistant lines than susceptible lines. However, SA-induced genes have not been identified at the whole genome level and it was unclear whether they were up-regulated by Foc inoculation. Transcriptome analysis with and without SA treatment in the B. rapa Fusarium yellows susceptible line 'Misugi' and the resistant line 'Nanane' was performed to obtain insights into the relationship between SA sensitivity/response and Fusarium yellows resistance. 'Nanane's up-regulated genes were related to SA response and down-regulated genes were related to jasmonic acid (JA) or ethylene (ET) response, but differentially expressed genes in 'Misugi' were not. This result suggests that Fusarium yellows resistant and susceptible lines have a different SA response and that an antagonistic transcription between SA and JA/ET responses was found only in a Fusarium yellows resistant line. SA-responsive genes were induced by Foc inoculation in Fusarium yellows resistant (RJKB-T23) and susceptible lines (RJKB-T24). By contrast, 39 SA-induced genes specific to RJKB-T23 might function in the defense response to Foc. In this study, SA-induced genes were identified at the whole genome level, and the possibility, the defense response to Foc observed in a resistant line could be mediated by SA-induced genes, is suggested. These results will be useful for future research concerning the SA importance in Foc or other diseases resistance in B. rapa.

Arabidopsis Col/Ler and Ws/Ler hybrids and Hybrid Mimics produce seed yield heterosis through increased height, inflorescence branch and silique number.

MAIN CONCLUSION: The seed yield increase of the hybrids and their derived Mimics compared to parents is associated with increased plant height and inflorescence branch number which are correlated with decreased expression of FT, SOC1 and FUL. In Arabidopsis, plant size has been extensively investigated, but few studies have been carried out on seed yield heterosis. In hybrids between Columbia (Col) and Landsberg erecta (Ler), and Wassilewskija (Ws) and Ler, there was significant seed yield heterosis. F6/F7 Hybrid Mimics derived from hybrids of each of the two systems had seed yield increases similar to that of the F1 hybrid (approximately 50-70% greater than the average of the parents). Increased seed yield of the Hybrid Mimics was accompanied by changes of plant architecture with increased plant height and increased inflorescence branch number relative to the parents. Three of the Hybrid Mimic lines derived from the Ws/Ler system had 20% increase in seed yield relative to the F1 hybrid. Genes which repress flowering were up-regulated and the expression levels of flowering -promoting genes including FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FRUITFULL (FUL) were negatively correlated with the increase in seed yield in both hybrids and F7 Mimics of both systems.

Trichomes at the Base of the Petal Are Regulated by the Same Transcription Factors as Cotton Seed Fibers.

Many polypetalous plants have a constriction at the base of the petal that leaves a small gap that can provide entry into the young flower bud before the reproductive organs are fully developed. In cotton (Gossypium hirsutum L.), this gap is occluded by tufts of short unicellular trichomes superficially resembling the fibers found on cotton seeds. We are just beginning to understand the developmental regulation of the seed fibers and have previously characterized several MIXTA-like MYB transcription factors (TFs) that are critical for correct seed fiber development but know little about the molecular regulation of other types of cotton trichomes. Here, using RNAi or dominant suppression transgenic cotton lines and natural fiber mutants, we investigated the development and regulation of the petal base trichomes. Petal base trichomes and seed trichomes were also examined across several different species within and outside of the Malvoideae. We found that the petal base trichomes are regulated by the same MYB TFs as cotton seed fibers and, since they are more widely distributed across different taxa than the seed fibers, could have preceded them in the evolution of these important textile fibers produced by some cotton species.

In Arabidopsis thaliana Heterosis Level Varies among Individuals in an F1 Hybrid Population.

Heterosis or hybrid vigour is a phenomenon in which hybrid progeny exhibit superior yield and biomass to parental lines and has been used to breed F1 hybrid cultivars in many crops. A similar level of heterosis in all F1 individuals is expected as they are genetically identical. However, we found variation in rosette size in individual F1 plants from a cross between C24 and Columbia-0 accessions of Arabidopsis thaliana. Big-sized F1 plants had 26.1% larger leaf area in the first and second leaves than medium-sized F1 plants at 14 days after sowing in spite of the identical genetic background. We identified differentially expressed genes between big- and medium-sized F1 plants by microarray; genes involved in the category of stress response were overrepresented. We made transgenic plants overexpressing 21 genes, which were differentially expressed between the two size classes, and some lines had increased plant size at 14 or 21 days after sowing but not at all time points during development. Change of expression levels in stress-responsive genes among individual F1 plants could generate the variation in plant size of individual F1 plants in A. thaliana.

Early Establishment of Photosynthesis and Auxin Biosynthesis Plays a Key Role in Early Biomass Heterosis in Brassica napus (Canola) Hybrids.

Heterosis or hybrid vigor has been used widely for more than a decade in Canola (Brassica napus) production. Canola hybrids show heterosis in a variety of traits compared to parents, including increased biomass at the early stages of seedling establishment, which is a critical developmental step that impacts future plant growth and seed yield. In this study, we examined transcriptomes of two parental lines, Garnet (Gar) and NX0052 (0052), and their reciprocal hybrids, Gar/0052, at 4 and 8 days after sowing (DAS). In hybrids, early seedling biomass heterosis is correlated with earlier expression of genes in photosynthesis pathways relative to parents. The hybrids also showed early expression of genes in the auxin biosynthesis pathway, consistent with the higher auxin concentrations detected in hybrid seedlings at 4 DAS. Auxin is a key phytohormone that regulates plant development promoting cell expansion and cell proliferation. Consistent with the increased levels of auxin, hybrids have larger and more palisade cells than the parents at the same time point. We propose a possible mechanism of early biomass heterosis through the early establishment of photosynthesis and auxin biosynthesis, providing insights into how transcriptional changes in hybrids are translated into phenotypical heterosis. This finding could be utilized in future Canola breeding to identify hybrid combinations with the superior early seedling establishment and strong levels of hybrid vigor in later plant development.

Leaf growth in early development is key to biomass heterosis in Arabidopsis.

Arabidopsis thaliana hybrids have similar properties to hybrid crops, with greater biomass relative to the parents. We asked whether the greater biomass was due to increased photosynthetic efficiency per unit leaf area or to overall increased leaf area and increased total photosynthate per plant. We found that photosynthetic parameters (electron transport rate, CO2 assimilation rate, chlorophyll content, and chloroplast number) were unchanged on a leaf unit area and unit fresh weight basis between parents and hybrids, indicating that heterosis is not a result of increased photosynthetic efficiency. To investigate the possibility of increased leaf area producing more photosynthate per plant, we studied C24×Landsberg erecta (Ler) hybrids in detail. These hybrids have earlier germination and leaf growth than the parents, leading to a larger leaf area at any point in development of the plant. The developing leaves of the hybrids are significantly larger than those of the parents, with consequent greater production of photosynthate and an increased contribution to heterosis. The set of leaves contributing to heterosis changes as the plant develops; the four most recently emerged leaves make the greatest contribution. As a leaf matures, its contribution to heterosis attenuates. While photosynthesis per unit leaf area is unchanged at any stage of development in the hybrid, leaf area is greater and the amount of photosynthate per plant is increased.

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.

In Arabidopsis hybrids and Hybrid Mimics, up-regulation of cell wall biogenesis is associated with the increased plant size.

Hybrid breeding is of economic importance in agriculture for increasing yield, yet the basis of heterosis is not well understood. In Arabidopsis, crosses between different accessions produce hybrids with different levels of heterosis relative to parental phenotypes in biomass. In all hybrids, the advantage of the F1 hybrid in both phenotypic uniformity and yield gain is lost in the heterogeneous F2. F5/F6 Hybrid Mimics generated from a cross between C24 and Landsberg erecta (Ler) ecotypes demonstrated that the large plant phenotype of the F1 hybrids can be stabilized. Hybrid Mimic selection was applied to Wassilewskija (Ws)/Ler and Col/Ler hybrids. The two hybrids show different levels of heterosis. The Col/Ler hybrid generated F7 Hybrid Mimics with rosette diameter and fresh weight equivalent to the F1 hybrid at 30 DAS; F7 Ws/Ler Hybrid Mimics outperformed the F1 hybrid in both the rosette size and biomass. Transcriptome analysis revealed up-regulation of cell wall biosynthesis, and cell wall expansion genes could be a common pathway in increased size in the Arabidopsis hybrids and Hybrid Mimics. Intercross of two independent Hybrid Mimic lines can further increase the biomass gain. Our results encourage the use of Hybrid Mimics for breeding and for investigating the molecular basis of heterosis.

The histone modification H3 lysine 27 tri-methylation has conserved gene regulatory roles in the triplicated genome of Brassica rapa L.

Brassica rapa L. is an important vegetable and oilseed crop. We investigated the distribution of the histone mark tri-methylation of H3K27 (H3K27me3) in B. rapa and its role in the control of gene expression at two stages of development (2-day cotyledons and 14-day leaves) and among paralogs in the triplicated genome. H3K27me3 has a similar distribution in two inbred lines, while there was variation of H3K27me3 sites between tissues. Sites that are specific to 2-day cotyledons have increased transcriptional activity, and low levels of H3K27me3 in the gene body region. In 14-day leaves, levels of H3K27me3 were associated with decreased gene expression. In the triplicated genome, H3K27me3 is associated with paralogs that have tissue-specific expression. Even though B. rapa and Arabidopsis thaliana are not closely related within the Brassicaceae, there is conservation of H3K27me3-marked sites in the two species. Both B. rapa and A. thaliana require vernalization for floral initiation with FLC being the major controlling locus. In all four BrFLC paralogs, low-temperature treatment increases H3K27me3 at the proximal nucleation site reducing BrFLC expression. Following return to normal temperature growth conditions, H3K27me3 spreads along all four BrFLC paralogs providing stable repression of the gene.

The role of FRIGIDA and FLOWERING LOCUS C genes in flowering time of Brassica rapa leafy vegetables.

There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower.