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.     

Vernalization-induced trimethylation of histone H3 lysine 27 at FLC is not maintained in mitotically quiescent cells

Vernalization promotes flowering in Arabidopsis through epigenetic repression of the floral repressor, FLOWERING LOCUS C (FLC). Vernalization, like other polycomb-mediated repression events, occurs in two stages; FLC repression is established at low temperatures, then maintained during subsequent growth at 22 degrees C. Low temperatures induce VIN3 activity, which is required for changes in histone modifications and the associated FLC repression. Plant polycomb proteins FIE, VRN2, CLF, and SWN, together with VIN3, form a complex that adds histone H3 lysine 27 methylation at FLC in vernalized plants. VRN1 and LHP1 are required for maintenance of FLC repression. Tissue must be undergoing cell division during low-temperature treatments for acceleration of flowering to occur. We show that low-temperature treatments repress FLC in cells that are not mitotically active, but this repression is not fully maintained. Trimethyl-lysine 27 (K27me3), is enriched at the start of the FLC gene during the cold, before spreading across the locus after vernalization. In the absence of DNA replication, K27me3 is added to chromatin at the start of FLC but is removed on return to 22 degrees C. This suggests that DNA replication is essential for maintenance of vernalization-induced repression of FLC.     

Quantitative effects of vernalization on FLC and SOC1 expression

Prolonged exposure to cold results in early flowering in Arabidopsis winter annual ecotypes, with longer exposures resulting in a greater promotion of flowering than shorter exposures. The promotion of flowering is mediated through an epigenetic down-regulation of the floral repressor FLOWERING LOCUS C (FLC). We present results that provide an insight into the quantitative regulation of FLC by vernalization. Analysis of the effect of seed or plant cold treatment on FLC expression indicates that the time-dependent nature of vernalization on FLC expression is mediated through the extent of the initial repression of FLC and not by affecting the ability to maintain the repressed state. In the over-expression mutant flc-11, the time-dependent repression of FLC correlates with the proportional deacetylation of histone H3. Our results indicate that sequences within intron 1 and the activities of both VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2) are required for efficient establishment of FLC repression; however, VRN1 and VRN2 are not required for maintenance of the repressed state during growth after the cold exposure. SUPPRESSOR OF OVER-EXPRESSION OF CO 1 (SOC1), a downstream target of FLC, is quantitatively induced by vernalization in a reciprocal manner to FLC. In addition, we show that SOC1 undergoes an acute induction by both short and long cold exposures.     

The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis.

The acceleration of flowering by a long period of low temperature, vernalization, is an adaptation that ensures plants overwinter before flowering. Vernalization induces a developmental state that is mitotically stable, suggesting that it may have an epigenetic basis. The VERNALIZATION2 (VRN2) gene mediates vernalization and encodes a nuclear-localized zinc finger protein with similarity to Polycomb group (PcG) proteins of plants and animals. In wild-type Arabidopsis, vernalization results in the stable reduction of the levels of the floral repressor FLC. In vrn2 mutants, FLC expression is downregulated normally in response to vernalization, but instead of remaining low, FLC mRNA levels increase when plants are returned to normal temperatures. VRN2 function therefore stably maintains FLC repression after a cold treatment, serving as a mechanism for the cellular memory of vernalization.     

The downregulation of FLOWERING LOCUS C (FLC) expression in plants with low levels of DNA methylation and by vernalization occurs by distinct mechanisms

FLOWERING LOCUS C (FLC), a repressor of flowering, is a major determinant of flowering time in Arabidopsis. FLC expression is repressed by vernalization and in plants with low levels of DNA methylation, resulting in early flowering. This repression is not associated with changes of DNA methylation within the FLC locus in either vernalized plants or plants with low levels of DNA methylation. In both cases, there is a reduction of histone H3 trimethyl-lysine 4 (K4) and acetylation of both histones H3 and H4 around the promoter-translation start of FLC. The expression of the two genes flanking FLC is also repressed in both conditions and repression is associated with decreased histone H3 acetylation. The changes in histone modifications at the FLC gene cluster, which are similar in vernalized plants and in plants with reduced DNA methylation, must arise by different mechanisms. VERNALIZATION 1, VERNALIZATION 2 and VERNALIZATION INSENSITIVE 3 modulate FLC expression in vernalized plants; these proteins play no role in the downregulation of FLC in plants with low levels of DNA methylation. Chimeric FLC::GUS transgenes respond to vernalization but these same transgenes show a position-dependent response to low levels of DNA methylation. In plants with reduced DNA methylation, expression of the five MADS AFFECTING FLOWERING (MAF) genes is repressed, suggesting that DNA methylation alters the expression of a trans-acting regulator common to FLC and members of the related MAF gene family. Our observations suggest that DNA methylation is not part of the vernalization pathway.     

Encoding memory of winter by noncoding RNAs

In some plant species, prolonged exposure to low temperature during the winter season is necessary to acquire the competence to flower in the following spring. This process, known as vernalization, is an epigenetic change in that a mitotically stable change of the developmental potential of the meristem (competence to flower) is maintained even in the absence of the inducing signal (prolonged cold exposure). In Arabidopsis, vernalization results in stable epigenetic repression of a potent floral repressor, FLOWERING LOCUS C (FLC). Increased enrichment of Polycomb Repressive Complex 2 (PRC2) and trimethylated Histone H3 Lys 27 (H3K27me3) at FLC chromatin is necessary for the stable maintenance of FLC repression by vernalization. Recent recognition of long noncoding RNAs (ncRNAs) in vernalization response indicates that long ncRNAs are evolutionarily conserved components for PRC2-mediated repression in eukaryotes.     

植物非编码RNA调控春化作用的表观遗传

在自然界中许多高等植物需要通过冬季的低温阶段实现从营养生长到生殖生长的时期转化,这一生物学过程称作春化作用。小麦(Triticum aestivum L.)和油菜(Brassica napus L.)等作物以种子为产品器官,生产上往往通过茬口安排和栽培措施使植株尽早通过春化作用,以促进花芽形成和花器官发育,而大白菜(B rapa ssp.pekinenesis)和甘蓝(B.oleracea)等作物以叶球等营养器官作为产品器官,生产上则设法避免低温引起的春化作用,以保证产品器官的充分生长。FLOWERING LOCUS C(FLC)作为一种重要的开花抑制蛋白负调控春化作用,参与植株从营养生长向生殖生长的转化过程。文章综述了春化中FLC表达受抑制主要通过低温诱导表达FLC基因区域的非编码RNA以及VRN1、VRN2、VIN3等蛋白参与介导组蛋白甲基化,从而在表观遗传上控制春化作用的进程和产品器官的正常发育。     

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.     

Vernalization: a model for investigating epigenetics and eukaryotic gene regulation in plants

The transition from vegetative to reproductive development is a highly regulated process that, in many plant species, is sensitive to environmental cues that provide seasonal information to initiate flowering during optimal times of the year. One environmental cue is the cold of winter. Winter annuals and biennials typically require prolonged exposure to the cold of winter to flower rapidly in the spring. This process by which flowering is promoted by cold exposure is known as vernalization. The winter-annual habit of Arabidopsis thaliana is established by the ability of FRIGIDA to promote high levels of expression of the potent floral repressor FLOWERING LOCUS C (FLC). In Arabidopsis, vernalization results in the silencing of FLC in a mitotically stable (i.e., epigenetic) manner that is maintained for the remainder of the plant life cycle. The repressed "off" state of FLC has features characteristic of facultative heterochromatin. Upon passing to the next generation, the "off" state of FLC is reset to the "on" state. The environmental induction and mitotic stability of vernalization-mediated FLC repression as well as the subsequent resetting in the next generation provides a system for studying several aspects of epigenetic control of gene expression.