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.     

RFLP Analysis of the <Emphasis Type="Italic">FLOWERING LOCUS C</Emphasis> Gene in Six <Emphasis Type="Italic">Brassica</Emphasis> Species

The FLOWERING LOCUS C (FLC) gene controls the transition of arabidopsis plants to flowering following cold induction (vernalization). Time to flowering in annual and biennial species of Brassicaceae supposedly depends on the number of FLC copies. We analyzed DNA restriction fragment length polymorphism in six Brassica species with diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes using for a hybridization probe an FLC homolog previously cloned in our laboratory from B. juncea. The characteristic variations in the patterns of restriction fragments corresponded to the genomic composition of Brassica species and, in some cases, correlated with the timing of floral transition.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 399–405.Original Russian Text Copyright © 2005 by Martynov, Khavkin.     

Genome-wide identification of flowering time genes associated with vernalization and the regulatory flowering networks in Chinese cabbage

Flowering time (Ft) is the most important characteristic of Chinese cabbage with high leaf yields and late-flowering are favorable traits, while little knowledge on genes involved in Ft and the flowering mechanism in this crop. In this study, we conducted genome-wide RNA-seq analysis using an inbred Chinese cabbage ‘4004’ line in response to vernalization and compared the Ft gene expression with radish crop. A number of Ft genes which play roles in flowering pathways were performed quantitative RT-PCR analysis to verify the regulatory flowering gene network in Chinese cabbage. We found that a total of 223 Ft genes in Chinese cabbage, and 50 of these genes responded to vernalization. The majority of flowering enhancers were upregulated, whereas most flowering repressors were downregulated in response to vernalization as confirmed by RT-qPCR. Among the major Ft genes, the expression of BrCOL1-2, BrFT1/2, BrSOC1/2/3, BrFLC1/2/3/5, and BrMAF was strongly affected by vernalization. In reference to comparative RNA-seq profiling of Ft genes, Chinese cabbage and radish revealed substantially different vernalization response in particular GA flowering pathway. Thus, this study provides new insight into functional divergence in flowering pathways and the regulatory mechanisms in Brassicaceae crops. Further analysis of the major integrator genes between early and late-flowering inbred lines facilitates understanding flowering trait variation and molecular basis of flowering in Chinese cabbage.     

Mapping loci controlling vernalization requirement in Brassica rapa

Brassica cultivars are classified as biennial or annual based on their requirement for a period of cold treatment (vernalization) to induce flowering. Genes controlling the vernalization requirement were identified in a Brassica rapa F₂ population derived from a cross between an annual and a biennial oilseed cultivar by using an RFLP linkage map and quantitative trait locus (QTL) analysis of flowering time in F₃ lines. Two genomic regions were strongly associated with variation for flowering time of unvernalized plants and alleles from the biennial parent in these regions delayed flowering. These QTLs had no significant effect on flowering time after plants were vernalized for 6 weeks, suggesting that they control flowering time through the requirement for vernalization. The two B. rapa linkage groups containing these QTLs had RFLP loci in common with two B. napus linkage groups that were shown previously to contain QTLs for flowering time. An RFLP locus detected by the cold-induced gene COR6.6 cloned from Arabidopsis thaliana mapped very near to one of the B. rapa QTLs for flowering time.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

Reduction of the geomagnetic field delays Arabidopsis thaliana flowering time through downregulation of flowering‐related genes

Variations in magnetic field (MF) intensity are known to induce plant morphological and gene expression changes. In Arabidopsis thaliana Col‐0, near‐null magnetic field (NNMF, i.e., <100 nT MF) causes a delay in the transition to flowering, but the expression of genes involved in this response has been poorly studied. Here, we showed a time‐course quantitative analysis of the expression of both leaf (including clock genes, photoperiod pathway, GA20ox, SVP, and vernalization pathway) and floral meristem (including GA2ox, SOC1, AGL24, LFY, AP1, FD, and FLC) genes involved in the transition to flowering in A. thaliana under NNMF. NNMF induced a delayed flowering time and a significant reduction of leaf area index and flowering stem length, with respect to controls under geomagnetic field. Generation experiments (F₁‐ and F₂‐NNMF) showed retention of flowering delay. The quantitative expression (qPCR) of some A. thaliana genes expressed in leaves and floral meristem was studied during transition to flowering. In leaves and flowering meristem, NNMF caused an early downregulation of clock, photoperiod, gibberellin, and vernalization pathways and a later downregulation of TSF, AP1, and FLC. In the floral meristem, the downregulation of AP1, AGL24, FT, and FLC in early phases of floral development was accompanied by a downregulation of the gibberellin pathway. The progressive upregulation of AGL24 and AP1 was also correlated to the delayed flowering by NNMF. The flowering delay is associated with the strong downregulation of FT, FLC, and GA20ox in the floral meristem and FT, TSF, FLC, and GA20ox in leaves. Bioelectromagnetics. 39:361–374, 2018. © 2018 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.     

Environmental and genetic interactions reveal FLOWERING LOCUS C as a modulator of the natural variation for the plasticity of flowering in Arabidopsis

The timing of flowering initiation depends strongly on the environment, a property termed as the plasticity of flowering. Such plasticity determines the adaptive potential of plants because it provides phenotypic buffer against environmental changes, and its natural variation contributes to evolutionary adaptation. We addressed the genetic mechanisms of the natural variation for this plasticity in Arabidopsis thaliana by analysing a population of recombinant inbred lines derived from Don‐0 and Ler accessions collected from distinct climates. Quantitative trait locus (QTL) mapping in four environmental conditions differing in photoperiod, vernalization treatment and ambient temperature detected the folllowing: (i) FLOWERING LOCUS C (FLC) as a large effect QTL affecting flowering time differentially in all environments; (ii) numerous QTL displaying smaller effects specifically in some conditions; and (iii) significant genetic interactions between FLC and other loci. Hence, the variation for the plasticity of flowering is determined by a combination of environmentally sensitive and specific QTL, and epistasis. Analysis of FLC from Don identified a new and more active allele likely caused by a cis‐regulatory deletion covering the non‐coding RNA COLDAIR. Further characterization of four FLC natural alleles showed different environmental and genetic interactions. Thus, FLC appears as a major modulator of the natural variation for the plasticity of flowering to multiple environmental factors.     

Mapping and characterization of <Emphasis Type="Italic">FLC</Emphasis> homologs and QTL analysis of flowering time in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The FLC gene product is an inhibitor of flowering in Arabidopsis. FLC homologs in Brassica species are thought to control vernalization. We cloned four FLC homologs (BoFLCs) from Brassica oleracea. Three of these, BoFLC1, BoFLC3 and BoFLC5, have been previously characterized. The fourth novel sequence displayed 98% sequence homology to the previously identified gene BoFLC4, but also showed 91% homology to BrFLC2 from Brassica rapa. Phylogenetic analysis showed that this clone belongs to the FLC2 clade. Therefore, we designated this gene BoFLC2. Based on the segregation of RFLP, SRAP, CAPS, SSR and AFLP loci, a detailed linkage map of B. oleracea was constructed in the F₂ progeny obtained from a cross of B. oleracea cv. Green Comet (broccoli; non-vernalization type) and B. oleracea cv. Reiho (cabbage; vernalization type), which covered 540 cM, 9 major linkage groups. Six quantitative trait loci (QTL) controlling flowering time were detected. BoFLC1, BoFLC3 and BoFLC5 were not linked to the QTLs controlling flowering time. However, the largest QTL effect was located in the region where BoFLC2 was mapped. Genotyping of F₂ plants at the BoFLC2 locus showed that most of the early flowering plants were homozygotes of BoFLC-GC, whereas most of the late- and non-flowering plants were homozygotes of BoFLC-Rei. The BoFLC2 homologs present in plants of the non-vernalization type were non-functional, due to a frameshift in exon 4. Moreover, duplications and deletions of BoFLC2 were detected in broccoli and a rapid cycling line, respectively. These results suggest that BoFLC2 contributes to the control of flowering time in B. oleracea.