Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications.
ABSTRACT: Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.
Project description:The role in flowering time of the MADS-box transcription factor fruitfulL (FUL) has been proposed in many works. FUL has been connected to several flowering pathways as a target of the photoperiod, ambient temperature, and age pathways and it is has been shown to promote flowering in a partially redundant manner with suppressor of overexpression of constans 1 (SOC1). However, the position of FUL in these genetic networks, as well as the functional output of FUL activity during floral transition, remains unclear. In this work, a genetic approach has been undertaken to understand better the functional hierarchies involving FUL and other MADS-box factors with well established roles as floral integrators such as SOC1, short vegetative phase (svp) or flowering locus C (FLC). Our results suggest a prominent role of FUL in promoting reproductive transition when photoinductive signalling is suppressed by short-day conditions or by high levels of FLC expression, as in non-vernalized winter ecotypes. A model is proposed where the sequential formation of FUL-SVP and FUL-SOC1 heterodimers may mediate the vegetative and meristem identity transitions, counteracting the repressive effect of FLC and SVP on flowering.
Project description: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.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.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.
Project description:The turnip (Brassica rapa var. rapa) is a biennial crop that is planted in late summer/early fall and forms fleshy tubers for food in temperate regions. The harvested tubers then overwinter and are planted again the next spring for flowering and seeds. FLOWERING LOCUS C (FLC) is a MADS-box transcription factor that acts as a major repressor of floral transition by suppressing the flowering promoters FT and SOC1. Here we show that vernalization effectively represses tuber formation and promotes flowering in Tibetan turnip. We functionally characterized four FLC homologues (BrrFLC1, FLC2, FLC3, and FLC5), and found that BrrFLC2 and BrrFLC1 play a major role in repressing flowering in turnip and in transgenic Arabidopsis. In contrast, tuber formation was correlated with BrrFLC1 expression in the hypocotyl and was repressed under cold treatment following the quantitative downregulation of BrrFLC1. Grafting experiments of non-vernalized and vernalized turnips revealed that vernalization independently suppressed tuberization in the tuber or hypocotyl of the rootstock or scion, which occurred in parallel with the reduction in BrrFLC1 activity. Together, our results demonstrate that the Tibetan turnip is highly responsive to cold exposure, which is associated with the expression levels of BrrFLC genes.
Project description:FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.
Project description:The floral transition in Arabidopsis is tightly controlled by complex genetic regulatory networks in response to endogenous and environmental flowering signals. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SHORT VEGETATIVE PHASE (SVP), two key MADS-domain transcription factors, perceive these signals and function as antagonistic flowering regulators. To understand how they mediate the floral transition, we mapped in vivo binding sites of SOC1 and SVP using chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays (ChIP-chip). Genes encoding proteins with transcription regulator activity and transcription factor activity were the most enriched groups of genes bound by SOC1 and SVP, indicating their central roles in flowering regulatory networks. In combination with gene expression microarray studies, we further identified the genes whose expression was directly regulated by SOC1 or SVP. Among the common direct targets identified, APETALA2 (AP2)-like genes that repress FT and SOC1 expression were downregulated by SOC1, but upregulated by SVP, revealing a complex feedback regulation among key genes determining the integration of flowering signals. SOC1 regulatory regions were also accessed by SOC1 itself and SVP, suggesting that self-activation and repression by SVP contribute to the regulation of SOC1 expression. In addition, ChIP-chip analysis demonstrated that miR156e and miR172a, which are involved in the regulation of AP2-like genes, were direct targets of SOC1 and SVP, respectively. Taken together, these findings reveal that feedback regulatory loops mediated by SOC1 and SVP are essential components of the gene regulatory networks underpinning the integration of flowering signals during the floral transition. soc1-101D and 35S:SVP ChIPed with SOC1 or SVP polyclonal antibody respectively vs. soc1-2 or svp-41 in Arabidopsis 9-day-old whole seedlings
Project description:The adaptive success of flowering plants is largely due to their ability to align floral production with optimal conditions. In Arabidopsis thaliana, MADS-box repressors of the FLC/MAF-clade prevent flowering under non-inductive conditions, although the role of some members is not yet clearly defined. Using a genetic strategy, we identified the KH-domain gene HEN4, previously shown to be involved in MADS-box floral homeotic gene regulation, as a modulator of flowering time. Loss-of-function hen4 mutants are early-flowering, and their response to low growth-temperature (16?°C) and day-length is altered. Interestingly, hen4 plants showed dramatic reduction of FLC and MAF4 transcripts, whereas other flowering repressors of the same clade (FLM, MAF2, MAF3, MAF5) remained unaltered. We also determined that hen4, partly due to loss of FLC, accelerates the vegetative phase-change. This report provides insight into flowering time control and highlights the potential of versatile regulators such as HEN4 to coordinate the juvenile-to-adult transition and floral timing.
Project description:The MADS-box transcription factors FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) are major transcriptional repressors controlling flowering time. They enhance responses to environmental cues such as winter temperatures, high ambient temperatures and photoperiod, acting at least in part by blocking transcription of the floral pathway integrators. As other MADS-box transcription factors, FLC and SVP can interact in vivo forming multimeric complexes. Mutations in either FLC or SVP lead to similar early flowering, suggesting that FLC-SVP interaction might be the major control unit. Here we have analyzed the coordinated regulatory modes of these two key transcription factors at a genome-wide level through ChIP-seq and gene expression microarrays. Genome-wide identification of SVP and FLC DNA-binding occupancy events revealed that their binding scenarios are strongly yet differently affected by the presence of the cognate partner both at a quantitative as well as a qualitative level. Also, we identified a subgroup of genes whose regulation exclusively depends on the combinatorial binding of these two proteins, strengthening the role of the SVP-FLC complex. Some of these genes are involved in the control of flowering through direct and indirect regulation of Gibberellin-related genes such as GA2ox8, DDF1 and TEM1. Interestingly, we identified cis-regulatory elements enriched uniquely at complex-bound sites. This study decoded the regulatory code mediated by the major flowering repressors SVP and FLC. Overall design: ChIP followed by high-throughput sequencing for two MADS-box transcription factors in Arabidopsis seedlings (ecotype: Col-0 with active FRI)
Project description:Several pathways conferring environmental flowering responses in Arabidopsis converge on developmental processes that act in the shoot apical meristem to mediate the floral transition. Many characterized mutations impair environmental flowering responses, however downstream developmental processes have been more refractory to mutagenesis. We constructed a quintuple mutant in which several environmental pathways are impaired and showed that its flowering responses to changes in photoperiod and ambient temperature are almost abolished. Analysis of the quintuple mutant by RNA-seq showed that expression of gibberellin biosynthetic genes and transcription factors that contribute to the age pathway correlate with flowering. Mutagenesis of the quintuple mutant recovered two late-flowering mutants, quintuple ems 1 (qem1) and qem2. The causal genes were identified by isogenic mapping and transgenic complementation. The qem1 mutation was an allele of ga20ox2, confirming the importance of gibberellin for flowering in the absence of environmental responses. By contrast, qem2 is in CHROMATIN REMODELING 4 (CHR4), which was not previously genetically implicated in floral induction. Using co-immunoprecipitation, RNA-seq and ChIP-seq, we show that CHR4 interacts with transcription factors involved in floral meristem identity and affects expression of key floral regulators. We conclude that CHR4 plays important roles in the inflorescence meristem to promote floral identity. Overall design: Examination of differentially expressed genes in betweeen WT and svp flc ft tsf soc1, svp flc ft tsf soc1 and qem2 and col and chr4-2 apices in a short day time course.
Project description:BACKGROUND: MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. RESULTS: To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. CONCLUSIONS: Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them.