Candice C. Sheldon

The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation.

A MADS box gene, FLF (for FLOWERING LOCUS F), isolated from a late-flowering, T-DNA–tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis.

The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC)

In Arabidopsis, the MADS-box protein encoded by FLOWERING LOCUS C (FLC) is a repressor of flowering. Vernalization, which promotes flowering in the late-flowering ecotypes and many late-flowering mutants, decreases the level of FLC transcript and protein in the plant. This vernalization-induced reduction in FLC transcript levels is mitotically stable and occurs in all tissues. FLC activity is restored in each generation, as is the requirement of a low-temperature exposure for the promotion of flowering. The level of FLC determines the extent of the vernalization response in the promotion of flowering, and there is a quantitative relationship between the duration of cold treatment and the extent of down-regulation of FLC activity. We conclude that FLC is the central regulator of the induction of flowering by vernalization. Other vernalization-responsive late-flowering mutants, which are disrupted in genes that encode regulators of FLC, are late-flowering as a consequence of their elevated levels of FLC.

Different Regulatory Regions Are Required for the Vernalization-Induced Repression of FLOWERING LOCUS C and for the Epigenetic Maintenance of Repression

Vernalization, the promotion of flowering by a prolonged period of low temperature, results in repression of the floral repressor FLOWERING LOCUS C (FLC) and in early flowering. This repression bears the hallmark of an epigenetic event: the low expression state is maintained over many cell division cycles, but expression is derepressed in progeny. We show that the two stages of the response of FLC to vernalization, the repression of FLC and the maintenance of the repression during growth at normal temperatures after vernalization, are mediated through different regions of the FLC gene. Both promoter and intragenic regions are required for the responses. We also identify a 75-bp region in the FLC promoter that, in addition to intragenic sequences, is required for expression in nonvernalized plants.

Resetting of FLOWERING LOCUS C expression after epigenetic repression by vernalization

The epigenetic repression of FLOWERING LOCUS C (FLC) in winter-annual ecotypes of Arabidopsis by prolonged cold ensures that plants flower in spring and not during winter. Resetting of the FLC expression level in progeny is an important step in the life cycle of the plant. We show that both the paternally derived and the maternally derived FLC:GUS genes are reset to activity but that the timing of their first expression differs. The paternal FLC:GUS gene in vernalized plants is expressed in the male reproductive organs, the anthers, in both somatic tissue and in the sporogenous pollen mother cells, but there is no expression in mature pollen. In the progeny generation, the paternally derived FLC:GUS gene is expressed in the single-celled zygote (fertilized egg cell) and through embryo development, but not in the fertilized central cell, which generates the endosperm of the progeny seed. FLC:GUS is not expressed during female gametogenesis, with the maternally derived FLC:GUS being first expressed in the early multicellular embryo. We show that FLC activity during late embryo development is a prerequisite for the repressive action of FLC on flowering.

Short Vegetative Phase-Like MADS-Box Genes Inhibit Floral Meristem Identity in Barley

Analysis of the functions of Short Vegetative Phase (SVP)-like MADS-box genes in barley (Hordeum vulgare) indicated a role in determining meristem identity. Three SVP-like genes are expressed in vegetative tissues of barley: Barley MADS1 (BM1), BM10, and Vegetative to Reproductive Transition gene 2. These genes are induced by cold but are repressed during floral development. Ectopic expression of BM1 inhibited spike development and caused floral reversion in barley, with florets at the base of the spike replaced by tillers. Head emergence was delayed in plants that ectopically express BM1, primarily by delayed development after the floral transition, but expression levels of the barley VRN1 gene (HvVRN1) were not affected. Ectopic expression of BM10 inhibited spike development and caused partial floral reversion, where florets at the base of the spike were replaced by inflorescence-like structures, but did not affect heading date. Floral reversion occurred more frequently when BM1 and BM10 ectopic expression lines were grown in short-day conditions. BM1 and BM10 also inhibited floral development and caused floral reversion when expressed in Arabidopsis (Arabidopsis thaliana). We conclude that SVP-like genes function to suppress floral meristem identity in winter cereals.

The control of flowering by vernalization

The process by which vernalization, the exposure of a germinating seed or a juvenile plant to a prolonged period of low temperature, promotes flowering in the adult plant has remained a mystery for many years. The recent isolation of one of the key genes involved in vernalization, FLOWERING LOCUS C, has now provided an insight into the molecular mechanism involved, including the role of DNA methylation.

A Cluster of Arabidopsis Genes with a Coordinate Response to an Environmental Stimulus

Vernalization, the promotion of flowering after prolonged exposure to low temperatures, is an adaptive response of plants ensuring that flowering occurs at a propitious time in the annual seasonal cycle. In Arabidopsis, FLOWERING LOCUS C (FLC), which encodes a repressor of flowering, is a key gene in the vernalization response; plants with high-FLC expression respond to vernalization by downregulating FLC and thereby flowering at an earlier time. Vernalization has the hallmarks of an epigenetically regulated process. The downregulation of FLC by low temperatures is maintained throughout vegetative development but is reset at each generation. During our study of vernalization, we have found that a small gene cluster, including FLC and its two flanking genes, is coordinately regulated in response to genetic modifiers, to the environmental stimulus of vernalization, and in plants with low levels of DNA methylation. Genes encoded on foreign DNA inserted into the cluster also acquire the low-temperature response. At other chromosomal locations, FLC maintains its response to vernalization and imposes a parallel response on a flanking gene. This suggests that FLC contains sequences that confer changes in gene expression extending beyond FLC itself, perhaps through chromatin modification.

Mechanisms of gene repression by vernalization in Arabidopsis.

FLOWERING LOCUS C (FLC) is a major regulator of flowering time in Arabidopsis. Repression of FLC occurs in response to prolonged cold exposure (vernalization) and is associated with an enrichment of the repressive histone modification trimethylated H3 lysine 27 (H3K27me3) and a depletion of the active histone modification H3K4me3 at FLC chromatin. In two cases genes adjacent to FLC are also repressed by vernalization. NEOMYCIN PHOSPHOTRANSFERASE II (NPTII) adjacent to an FLC transgene is repressed by vernalization, and this is associated with an increase in H3K27me3, demonstrating that the epigenetic repression of FLC can confer a repressed epigenetic state to an adjacent transcription unit. The second case involves the two genes adjacent to the endogenous FLC gene, UPSTREAM OF FLC (UFC) and DOWNSTREAM OF FLC (DFC). Both genes are repressed by vernalization (Finnegan et al., 2004), but they require neither cis-acting nor trans-acting factors derived from the FLC gene nor the VERNALIZATION2 (VRN2) complex which trimethylates H3K27. This demonstrates that there are two different mechanisms of gene repression by vernalization. We further show that repression and H3K27 trimethylation of FLC still occurs in mutants of the VRN2 complex. In contrast, the VRN2 complex is essential for repression and H3K27 trimethylation of the FLC-related MADS AFFECTING FLOWERING (MAF) genes by vernalization. This suggest that other proteins are able to repress FLC, but not MAF, gene expression.