Establishment of the Winter-Annual Growth Habit via FRIGIDA-Mediated Histone Methylation at FLOWERING LOCUS C in Arabidopsis

In Arabidopsis thaliana, flowering-time variation exists among accessions, and the winter-annual (late-flowering without vernalization) versus rapid-cycling (early flowering) growth habit is typically determined by allelic variation at FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FRI upregulates the expression of FLC, a central floral repressor, to levels that inhibit flowering, resulting in the winter-annual habit. Here, we show that FRI promotes histone H3 lysine-4 trimethylation (H3K4me3) in FLC to upregulate its expression. We identified an Arabidopsis homolog of the human WDR5, namely, WDR5a, which is a conserved core component of the human H3K4 methyltransferase complexes called COMPASS-like. We found that recombinant WDR5a binds H3K4-methylated peptides and that WDR5a also directly interacts with an H3K4 methyltransferase, ARABIDOPSIS TRITHORAX1. FRI mediates WDR5a enrichment at the FLC locus, leading to increased H3K4me3 and FLC upregulation. WDR5a enrichment is not required for elevated H3K4me3 in FLC upon loss of function of an FLC repressor, suggesting that two distinct mechanisms underlie elevated H3K4me3 in FLC. Our findings suggest that FRI is involved in the enrichment of a WDR5a-containing COMPASS-like complex at FLC chromatin that methylates H3K4, leading to FLC upregulation and thus the establishment of the winter-annual growth habit.     

Independent FLC Mutations as Causes of Flowering-Time Variation in Arabidopsis thaliana and Capsella rubella

Capsella rubella is an inbreeding annual forb closely related to Arabidopsis thaliana, a model species widely used for studying natural variation in adaptive traits such as flowering time. Although mutations in dozens of genes can affect flowering of A. thaliana in the laboratory, only a handful of such genes vary in natural populations. Chief among these are FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). Common and rare FRI mutations along with rare FLC mutations explain a large fraction of flowering-time variation in A. thaliana. Here we document flowering time under different conditions in 20 C. rubella accessions from across the species’ range. Similar to A. thaliana, vernalization, long photoperiods and elevated ambient temperature generally promote flowering. In this collection of C. rubella accessions, we did not find any obvious loss-of-function FRI alleles. Using mapping-by-sequencing with two strains that have contrasting flowering behaviors, we identified a splice-site mutation in FLC as the likely cause of early flowering in accession 1408. However, other similarly early C. rubella accessions did not share this mutation. We conclude that the genetic basis of flowering-time variation in C. rubella is complex, despite this very young species having undergone an extreme genetic bottleneck when it split from C. grandiflora a few tens of thousands of years ago.     

Growth habit determination by the balance of histone methylation activities in Arabidopsis

In Arabidopsis, the rapid‐flowering summer‐annual versus the vernalization‐requiring winter‐annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter‐annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)‐specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36‐ and H3K4‐specific histone demethylase (HDM) activities of autonomous‐pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter‐annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36‐specific HMT and HDM activities.     

Genetic base of Arabidopsis thaliana (L.) Heynh.: Fitness of plants for extreme conditions in northern margins of species range

Flowering time and vernalization requirement were studied in eight natural Karelian populations (KPs) of Arabidopsis thaliana. These KPs consisted of late-flowering plants with elevated expression of flowering repressor FLC and a reduced expression level of flowering activator SOC1 compared to the early-flowering ecotypes Dijon-M and Cvi-0. Despite variations in flowering time and the vernalization requirement among the KPs, two-week-old seedlings showed no changes in either the nucleotide sequence of the FRI gene or the relative expression levels of FRI and its target gene FLC that would be responsible for this variation. An analysis of abscisic acid (ABA) biosynthesis and catabolism genes (NCED3 and CYP707A2) did not show significant differences between late-flowering KPs and the early-flowering ecotypes Dijon-M and Cvi-0. Cold treatment (4°C for 24 h) induced the expression of not only NCED3, but also RD29B, a gene involved in the ABA-dependent cold-response pathway. The relative levels of cold activation of these genes were nearly equal in all genotypes under study. Thus, the ABA-dependent cold response pathway does not depend on FLC expression. The lack of significant differences between northern populations, as well as the ecotypes Dijon- M (Europe) and Cvi-0 (Cape Verde Islands), indicates that this pathway is not crucial for fitness to the northern environment.     

Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, two MYST histone acetyltransferases HAM1 and HAM2 work redundantly to acetylate histone H4 lysine 5 (H4K5ace) in vitro. The double mutant ham1/ham2 is lethal, which suggests the critical role of HAM1 and HAM2 in development. Here, we used an artificial microRNA (amiRNA) strategy in Arabidopsis to uncover a novel function of HAM1 and HAM2. The amiRNA-HAM1/2 transgenic plants showed early flowering and reduced fertility. In addition, they responded normally to photoperiod, gibberellic acid treatment, and vernalization. The expression of flowering-repressor FLOWERING LOCUS C (FLC) and its homologues, MADS-box Affecting Flowering genes 3/4 (MAF3/4), were decreased in amiRNA-HAM1/2 lines. HAM1 overexpression caused late flowering and elevated expression of FLC and MAF3/4. Mutation of FLC almost rescued the late flowering with HAM1 overexpression, which suggests that HAM1 regulation of flowering time depended on FLC. Global H4 acetylation was decreased in amiRNA-HAM1/2 lines, but increased in HAM1-OE lines, which further confirmed the acetyltransferase activity of HAM1 in vivo. Chromatin immunoprecipitation revealed that H4 hyperacetylation and H4K5ace at FLC and MAF3/4 were less abundant in amiRNA-HAM1/2 lines than the wild type, but were enriched in HAM1-OE lines. Thus, HAM1 and HAM2 may affect flowering time by epigenetic modification of FLC and MAF3/4 chromatins at H4K5 acetylation.     

Vernalization-Mediated VIN3 Induction Overcomes the LIKE-HETEROCHROMATIN PROTEIN1/POLYCOMB REPRESSION COMPLEX2-Mediated Epigenetic Repression

VERNALIZATION INSENSITIVE3 (VIN3) induction by vernalization is one of the earliest events in the vernalization response of Arabidopsis (Arabidopsis thaliana). However, the mechanism responsible for vernalization-mediated VIN3 induction is poorly understood. Here, we show that the constitutive repression of VIN3 in the absence of the cold is due to multiple repressive components, including a transposable element-derived sequence, LIKE-HETEROCHROMATIN PROTEIN1 and POLYCOMB REPRESSION COMPLEX2. Furthermore, the full extent of VIN3 induction by vernalization requires activating complex components, including EARLY FLOWERING7 and EARLY FLOWERING IN SHORT DAYS. In addition, we observed dynamic changes in the histone modifications present at VIN3 chromatin during the course of vernalization. Our results show that the induction of VIN3 includes dynamic changes at the level of chromatin triggered by long-term cold exposure.The transition from vegetative growth to reproductive growth is one of major developmental transitions in the life cycle of plants. Flowering plants have evolved to maximize the reproductive success by optimizing the timing of flowering. The onset of floral transition in flowering plants is affected by various environmental cues, including changing daylength and temperature. Plants use such environment cues to monitor seasonal changes and determine the timing of flowering. In temperate climates, the winter season imposes a prolonged period of cold to plants. In many plant species, exposure to prolonged period of cold provides competence to flower in the following spring through the process known as vernalization (for review, see Sung and Amasino, 2005; Dennis and Peacock, 2007).While most lab strains of Arabidopsis (Arabidopsis thaliana) do not require vernalization treatment to flower rapidly, many naturally occurring accessions of Arabidopsis flower very late unless vernalized (Clarke and Dean, 1994; Lee and Amasino, 1995; Michaels and Amasino, 1999; Gazzani et al., 2003). In Arabidopsis, the vernalization requirement is conferred by two dominant genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC; Lee et al., 1993; Clarke and Dean, 1994; Michaels and Amasino, 1999; Sheldon et al., 1999; Johanson et al., 2000). FLC encodes a MADS box DNA binding protein that functions as a repressor of the floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (Michaels and Amasino, 1999; Sheldon et al., 1999; Lee et al., 2000; Samach et al., 2000; Hepworth et al., 2002; Helliwell et al., 2006; Searle et al., 2006). FLC antagonizes the effect of CONSTANS (CO) by directly binding to regulatory regions within FT and SOC1. It appears that FRI contributes to the vernalization requirement solely by activating FLC. FRI encodes a protein with unknown biochemical function (Johanson et al., 2000). Vernalization results in the stable repression of FLC (Michaels and Amasino, 1999; Bastow et al., 2004; Sung and Amasino, 2004) so that floral integrators can be activated when the photoperiod pathway activates CO (Parcy, 2005). Thus, vernalization renders plants to be competent to flower upon exposure to inductive photoperiods in winter annuals and biennials.Other than FRI, another group of genes involved in FLC activation have been identified from screens for early flowering in certain genotypes or photoperiod conditions. They include EARLY FLOWERING5 (ELF5), ELF7, ELF8, VERNALIZATION INDEPENDENCE3 (VIP3), VIP4, EARLY FLOWERING IN SHORT DAYS4 (ESD4), PHOTOPERIOD INDEPENDENT EARLY1 (PIE1), EARLY FLOWERING IN SHORT DAYS (EFS), and ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1)/ATX2/ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7; Reeves et al., 2002; Zhang and van Nocker, 2002; Noh and Amasino, 2003; He et al., 2004; Noh et al., 2004; Oh et al., 2004; He and Amasino, 2005; Kim et al., 2005; Zhao et al., 2005; Choi et al., 2007; Saleh et al., 2008; Tamada et al., 2009). Some of these genes encode proteins with chromatin modification functions, including components of RNA Polymerase II-associated factor 1 (PAF1) complex (VIP3, VIP4, ELF7, and ELF8), a Histone H3 Lys-36 methyltransferase (EFS), a Histone H3 Lys-4 methyltransferase (ATX1, ATX2, and ATXR7), and a SWR1-related nucleosome remodeling factor (PIE1).Mitotically stable repression of FLC by vernalization is also achieved by chromatin modifications (Michaels and Amasino, 1999; Bastow et al., 2004; Sung and Amasino, 2004). FLC mRNA expression is repressed during the course of cold exposure, and several repressive histone marks accumulate at FLC chromatin, including methylations at Histone H3 Lys-9 (H3K9) and Histone H3 Lys-27 (H3K27). The accumulation of histone modifications at FLC chromatin depends on the activity of chromatin remodeling complexes. During the course of cold exposure, POLYCOMB REPRESSION COMPLEX2 (PRC2), which has H3K27 methyltransferase activity, is enriched at FLC chromatin (Wood et al., 2006; De Lucia et al., 2008) and establishes the stable repression of FLC through H3K27 methylation. PRC2 biochemically copurifies with members of the VERNALIZATION INSENSITIVE3 (VIN3) family of proteins, including VIN3, VIN3-LIKE1 (VIL1)/VERNALIZATION5 (VRN5), and VIL2/VERNALIZATION LIKE1 (VEL1; Wood et al., 2006; De Lucia et al., 2008).The vernalization response involves two phases. The first is a cold perception that measures the cumulative time of exposure to cold. Vernalization requires cold exposure over the course of weeks rather than minutes or hours. The second phase is essentially the output of the cold perception. When a sufficient duration of cold has been perceived, a series of changes of gene expression ensue, ultimately leading to the epigenetic repression of FLC. VIN3, which is a repressive chromatin-remodeling component, is induced only after a sufficient duration of cold has been perceived. One of the early molecular events in the vernalization response is the induction of VIN3 by prolonged cold exposure. Upstream of VIN3, there must be a biochemical mechanism to sense cold. However, nothing is known about the upstream event. The induction of VIN3 by cold is unique in that VIN3 induction takes several days of cold, unlike many cold-induced genes, which are induced within hours of cold exposure (Thomashow, 2001). Furthermore, VIN3 mRNA expression is quickly rerepressed once plants are moved to warm temperature.Interestingly, the induction of VIN3 also involves changes in active histone marks at VIN3 chromatin, including Histone H3 acetylation, Histone H4 acetylation, and Histone H3 Lys-4 trimethylation (H3K4me3; Finnegan et al., 2005; Bond et al., 2009). However, no chromatin remodeling complexes have been identified to have roles in those changes at VIN3 chromatin.Here, we show that VIN3 is in a constitutively silenced state, which is mediated by the presence of a transposable element (TE)-derived sequence in its promoter region and by the components of repressive complexes, including PRC2 and LHP1. In addition, the full extent of VIN3 induction by vernalization requires components of activating complexes, including PAF1 and EFS. Thus, VIN3 expression is under the influence of chromatin level regulators. Furthermore, VIN3 chromatin is in a transiently bivalent state when VIN3 mRNA is induced, having both a repressive histone mark and an active histone mark at VIN3 chromatin. Our results show that VIN3 is under a constitutively repressed state, which is transiently relieved from repression only when sufficient cold is provided.     

Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

VASCULAR PLANT ONE-ZINC FINGER1 and VOZ2 repress the FLOWERING LOCUS C clade members to control flowering time in Arabidopsis

Floral transition is regulated by environmental and endogenous signals. Previously, we identified VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2 as phytochrome B-interacting factors. VOZ1 and VOZ2 redundantly promote flowering and have pivotal roles in the downregulation of FLOWERING LOCUS C (FLC), a central repressor of flowering in Arabidopsis. Here, we showed that the late-flowering phenotypes of the voz1 voz2 mutant were suppressed by vernalization in the Columbia and FRIGIDA (FRI)-containing accessions, which indicates that the late-flowering phenotype of voz1 voz2 mutants was caused by upregulation of FLC. We also showed that the other FLC clade members, MADS AFFECTING FLOWERING (MAF) genes, were also a downstream target of VOZ1 and VOZ2 as their expression levels were also increased in the voz1 voz2 mutant. Our results suggest that the FLC clade genes integrate signals from VOZ1/VOZ2 and vernalization to regulate flowering.     

A Conserved Interaction between the SDI Domain of Bre2 and the Dpy-30 Domain of Sdc1 Is Required for Histone Methylation and Gene Expression

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.     

The vernalization gene FRIGIDA in cultivated Brassica species

The repressor FLOWERING LOCUS C (FLC) holds a key position among the genes, which drive Arabidopsis floral transition along the vernalization pathway. The FRIGIDA (FRI) gene activates FLC expression, and the interplay of strong and weak alleles of FLC and FRI in many cases explains the variations in Arabidopsis requirement for cold induction. In annual and biennial life forms of Brassica, the variations in time to flower have been also related to FLC; whereas the place of FRI in the vernalization process has not been sufficiently elucidated. In contrast to Arabidopsis, FRI in Brassica genomes A and C and presumably B is represented by two expressible loci, FRI.a and FRI.b, each of them manifesting genome-specific polymorphisms. FRI.a and FRI.b sequences from diploid species B. rapa (genome A) and B. oleracea (genome C) are conserved (96–99% similarity) in subgenomes A and C of tetraploid species B. carinata (genome BC), B. juncea (genome AB), and B. napus (genome AC). Phylogenetic analysis of FRI sequences in the genus Brassica clearly discerns the lineages A/C and B, while in the family Brassicaceae, two FRI clusters discriminated by such analysis correspond to the lineages I (including the genus Arabidopsis) and II (including the genus Brassica). The origin of two FRI loci is discussed in the context of the Brassicaceae evolution via paleopolyploidy and subsequent genome reorganization.