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1.  The Arabidopsis SWI2/SNF2 Chromatin Remodeler BRAHMA Regulates Polycomb Function during Vegetative Development and Directly Activates the Flowering Repressor Gene SVP 
PLoS Genetics  2015;11(1):e1004944.
The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.
Author Summary
In flowering plants, the proper transition from vegetative growth to flowering is critical for their reproductive success and must be controlled precisely. Multiple genes have been shown to regulate the floral transition in response to environmental and endogenous cues. Among them is SHORT VEGETATIVE PHASE (SVP), a key flowering repressor gene in Arabidopsis. SVP is highly expressed during the vegetative phase to promote growth, but the mechanism by which the high expression level of SVP is maintained remains unknown. Here, we report a genome-wide study to examine the functional interplay between the BRM chromatin remodeler and the PcG proteins that catalyze trimethylation of lysine 27 on histone H3 (H3K27me3), a histone mark normally associated with transcriptionally repressed genes. We identify BRM as a direct upstream activator of SVP. BRM acts to keep the levels of H3K27me3 low at the SVP locus by inhibiting the binding and activities of the PcG proteins. Thus, our work identifies a previously unknown mechanism in regulation of flowering time and demonstrates the power of genome-wide approaches in dissecting regulatory networks controlling plant development.
PMCID: PMC4304717  PMID: 25615622
2.  Arabidopsis BREVIPEDICELLUS Interacts with the SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA to Regulate KNAT2 and KNAT6 Expression in Control of Inflorescence Architecture 
PLoS Genetics  2015;11(3):e1005125.
BREVIPEDICELLUS (BP or KNAT1), a class-I KNOTTED1-like homeobox (KNOX) transcription factor in Arabidopsis thaliana, contributes to shaping the normal inflorescence architecture through negatively regulating other two class-I KNOX genes, KNAT2 and KNAT6. However, the molecular mechanism of BP-mediated transcription regulation remains unclear. In this study, we showed that BP directly interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) both in vitro and in vivo. Loss-of-function BRM mutants displayed inflorescence architecture defects, with clustered inflorescences, horizontally orientated pedicels, and short pedicels and internodes, a phenotype similar to the bp mutants. Furthermore, the transcript levels of KNAT2 and KNAT6 were elevated in brm-3, bp-9 and brm-3 bp-9 double mutants. Increased histone H3 lysine 4 tri-methylation (H3K4me3) levels were detected in brm-3, bp-9 and brm-3 bp-9 double mutants. Moreover, BRM and BP co-target to KNAT2 and KNAT6 genes, and BP is required for the binding of BRM to KNAT2 and KNAT6. Taken together, our results indicate that BP interacts with the chromatin remodeling factor BRM to regulate the expression of KNAT2 and KNAT6 in control of inflorescence architecture.
Author Summary
BP is a class-I KNOX transcription factor that controls normal inflorescence architecture development by repressing the expression of two KNOX genes, KNAT2 and KNAT6. In this study, we showed that Arabidopsis BP directly interacts with the SWI2/SNF2 chromatin remodeling ATPase BRM. brm and bp mutants displayed similar inflorescence architecture defects, with clustered inflorescences, horizontally orientated pedicels, and short pedicels and internodes. Furthermore, BP and BRM co-target to KNAT2 and KNAT6 genes and repress their expression. This work reveals a new regulatory mechanism that BP associates with BRM in control of inflorescence architecture development.
PMCID: PMC4379049  PMID: 25822547
3.  BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis 
PLoS ONE  2013;8(3):e58588.
SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.
PMCID: PMC3594165  PMID: 23536800
4.  ARABIDOPSIS TRITHORAX-RELATED3/SET DOMAIN GROUP2 is Required for the Winter-Annual Habit of Arabidopsis thaliana 
Plant and Cell Physiology  2012;53(5):834-846.
The winter-annual habit of Arabidopsis thaliana requires active alleles of FLOWERING LOCUS C (FLC), which encodes a potent flowering repressor, and FRIGIDA (FRI), an activator of FLC. FLC activation by FRI is accompanied by an increase in specific histone modifications, such as tri-methylation of histone H3 at lysine 4 (H3K4me3), and requires three H3K4 methyltransferases, the Drosophila Trithorax-class ARABIDOPSIS TRITHORAX1 (ATX1) and ATX2, and yeast Set1-class ATX-RELATED7/SET DOMAIN GROUP25 (ATXR7/SDG25). However, lesions in all of these genes failed to suppress the enhanced FLC expression caused by FRI completely, suggesting that another H3K4 methyltransferase may participate in the FLC activation. Here, we show that ATXR3/SDG2, which is a member of a novel class of H3K4 methyltransferases, also contributes to FLC activation. An ATXR3 lesion suppressed the enhanced FLC expression and delayed flowering caused by an active allele of FRI in non-vernalized plants. The decrease in FLC expression in atxr3 mutants was accompanied by reduced H3K4me3 levels at FLC chromatin. We also found that the rapid flowering of atxr3 was epistatic to that of atxr7, suggesting that ATXR3 functions in FLC activation in sequence with ATXR7. Our results indicate that the novel-class H3K4 methyltransferase, ATXR3, is a transcriptional activator that plays a role in the FLC activation and establishing the winter-annual habit. In addition, ATXR3 also contributes to the activation of other FLC clade members, such as FLOWERING LOCUS M/MADS AFFECTING FLOWERING1 (FLM/MAF1) and MAF5, at least partially explaining the ATXR3 function in delayed flowering caused by non-inductive photoperiods.
PMCID: PMC3345368  PMID: 22378382
ARABIDOPSIS TRITHORAX; Flowering; FLOWERING LOCUS C; Histone methylation; Winter-annual Arabidopsis
5.  A Companion Cell–Dominant and Developmentally Regulated H3K4 Demethylase Controls Flowering Time in Arabidopsis via the Repression of FLC Expression 
PLoS Genetics  2012;8(4):e1002664.
Flowering time relies on the integration of intrinsic developmental cues and environmental signals. FLC and its downstream target FT are key players in the floral transition in Arabidopsis. Here, we characterized the expression pattern and function of JMJ18, a novel JmjC domain-containing histone H3K4 demethylase gene in Arabidopsis. JMJ18 was dominantly expressed in companion cells; its temporal expression pattern was negatively and positively correlated with that of FLC and FT, respectively, during vegetative development. Mutations in JMJ18 resulted in a weak late-flowering phenotype, while JMJ18 overexpressors exhibited an obvious early-flowering phenotype. JMJ18 displayed demethylase activity toward H3K4me3 and H3K4me2, and bound FLC chromatin directly. The levels of H3K4me3 and H3K4me2 in chromatins of FLC clade genes and the expression of FLC clade genes were reduced, whereas FT expression was induced and the protein expression of FT increased in JMJ18 overexpressor lines. The early-flowering phenotype caused by the overexpression of JMJ18 was mainly dependent on the functional FT. Our findings suggest that the companion cell–dominant and developmentally regulated JMJ18 binds directly to the FLC locus, reducing the level of H3K4 methylation in FLC chromatin and repressing the expression of FLC, thereby promoting the expression of FT in companion cells to stimulate flowering.
Author Summary
Flowering is an important developmental transition during plant life cycle and the key process for production of the next generation. Flowering time is controlled by both intrinsic developmental and environmental signals. FLC and its target FT work as repressor and activator, respectively, to regulate flowering time in Arabidopsis; thus the regulation of FLC and FT expression is the key for the control of floral transition. Epigenetic modifications are critical for transcription regulation. Here, we show that a novel JmjC domain-containing histone H3K4 demethylase, JMJ18, is a key regulator for the expression of FLC and FT in companion cells and flowering time. JMJ18 is dominantly expressed in vascular tissue; its temporal expression pattern was developmentally regulated, and negatively and positively correlated with FLC and FT, respectively. JMJ18 mutation leads to weak late-flowering, while JMJ18 overexpressor exhibited an obvious early-flowering phenotype. JMJ18 binds to chromatin of FLC, represses its expression, and releases expression of FT in companion cells. Our results suggest that JMJ18 is a developmentally regulated companion cell–dominantly expressed signal to control flowering time by binding to FLC—reducing level of H3K4 methylation in FLC and repressing expression of FLC—thereby promoting expression of FT in companion cells during vegetative development in Arabidopsis.
PMCID: PMC3334889  PMID: 22536163
6.  Arabidopsis RRP6L1 and RRP6L2 Function in FLOWERING LOCUS C Silencing via Regulation of Antisense RNA Synthesis 
PLoS Genetics  2014;10(9):e1004612.
The exosome complex functions in RNA metabolism and transcriptional gene silencing. Here, we report that mutations of two Arabidopsis genes encoding nuclear exosome components AtRRP6L1 and AtRRP6L2, cause de-repression of the main flowering repressor FLOWERING LOCUS C (FLC) and thus delay flowering in early-flowering Arabidopsis ecotypes. AtRRP6L mutations affect the expression of known FLC regulatory antisense (AS) RNAs AS I and II, and cause an increase in Histone3 K4 trimethylation (H3K4me3) at FLC. AtRRP6L1 and AtRRP6L2 function redundantly in regulation of FLC and also act independently of the exosome core complex. Moreover, we discovered a novel, long non-coding, non-polyadenylated antisense transcript (ASL, for Antisense Long) originating from the FLC locus in wild type plants. The AtRRP6L proteins function as the main regulators of ASL synthesis, as these mutants show little or no ASL transcript. Unlike ASI/II, ASL associates with H3K27me3 regions of FLC, suggesting that it could function in the maintenance of H3K27 trimethylation during vegetative growth. AtRRP6L mutations also affect H3K27me3 levels and nucleosome density at the FLC locus. Furthermore, AtRRP6L1 physically associates with the ASL transcript and directly interacts with the FLC locus. We propose that AtRRP6L proteins participate in the maintenance of H3K27me3 at FLC via regulating ASL. Furthermore, AtRRP6Ls might participate in multiple FLC silencing pathways by regulating diverse antisense RNAs derived from the FLC locus.
Author Summary
Arabidopsis FLOWERING LOCUS C (FLC) delays flowering; therefore, repressing expression of FLC provides a critical mechanism to regulate flowering. This mechanism involves multiple levels of regulation, including genetic regulation by transcription factors, and epigenetic regulation by modifications of genomic DNA and histones at the FLC locus. This work examines the role of non-coding RNAs in the epigenetic regulation of FLC, finding that the different RNAs produced from the FLC locus may have different functions in altering the epigenetic landscape at the FLC locus, and revealing that AtRRP6L1 and AtRRP6L2, two components of the exosome, an RNA-processing complex, play roles in regulating these non-coding RNAs. Therefore, this work explores the complex ties between RNA processing, non-coding RNAs, and epigenetic regulation of FLC, a key repressor of flowering.
PMCID: PMC4161302  PMID: 25211139
7.  Arabidopsis COMPASS-Like Complexes Mediate Histone H3 Lysine-4 Trimethylation to Control Floral Transition and Plant Development 
PLoS Genetics  2011;7(3):e1001330.
Histone H3 lysine-4 (H3K4) methylation is associated with transcribed genes in eukaryotes. In Drosophila and mammals, both di- and tri-methylation of H3K4 are associated with gene activation. In contrast to animals, in Arabidopsis H3K4 trimethylation, but not mono- or di-methylation of H3K4, has been implicated in transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS or COMPASS-like in yeast and mammals. Here, we report that Arabidopsis homologs of the COMPASS and COMPASS-like complex core components known as Ash2, RbBP5, and WDR5 in humans form a nuclear subcomplex during vegetative and reproductive development, which can associate with multiple putative H3K4 methyltransferases. Loss of function of ARABIDOPSIS Ash2 RELATIVE (ASH2R) causes a great decrease in genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Knockdown of ASH2R or the RbBP5 homolog suppresses the expression of a crucial Arabidopsis floral repressor, FLOWERING LOCUS C (FLC), and FLC homologs resulting in accelerated floral transition. ASH2R binds to the chromatin of FLC and FLC homologs in vivo and is required for H3K4 trimethylation, but not for H3K4 dimethylation in these loci; overexpression of ASH2R causes elevated H3K4 trimethylation, but not H3K4 dimethylation, in its target genes FLC and FLC homologs, resulting in activation of these gene expression and consequent late flowering. These results strongly suggest that H3K4 trimethylation in FLC and its homologs can activate their expression, providing concrete evidence that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings suggest that there are multiple COMPASS-like complexes in Arabidopsis and that these complexes deposit trimethyl but not di- or mono-methyl H3K4 in target genes to promote their expression, providing a molecular explanation for the observed coupling of H3K4 trimethylation (but not H3K4 dimethylation) with active gene expression in Arabidopsis.
Author Summary
Histones can be covalently modified and histone modifications regulate chromatin structure and gene transcription. One such modification is histone H3 lysine-4 (H3K4) methylation, which can be mono-, di-, or tri-methylated. In animals such as fruitfly and mammals, both di- and tri-methylation of H3K4 are associated with active gene expression. In contrast to animals, in the flowering plant Arabidopsis only H3K4 trimethylation has been implicated in gene transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS-like in mammals. Here, we report that COMPASS-like H3K4 methyltransferase complexes exist in Arabidopsis. Loss of function of a core complex protein causes a great decrease in Arabidopsis genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Our analyses of several direct target genes of these COMPASS-like complexes show that they mediate deposition of trimethyl but not dimethyl H3K4 in these loci to activate their expression, providing concrete evidence for the notion that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings provide a molecular explanation for the observed coupling of trimethylation but not dimethylation of H3K4 with active gene expression in Arabidopsis. In addition, we found that H3K4 trimethylation regulates leaf growth and development, flowering, and embryo development.
PMCID: PMC3053346  PMID: 21423667
8.  The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure 
The Plant Journal   2008;53(3):530-540.
Loss-of-function siz1 mutations caused early flowering under short days. siz1 plants have elevated salicylic acid (SA) levels, which are restored to wild-type levels by expressing nahG, bacterial salicylate hydroxylase. The early flowering of siz1 was suppressed by expressing nahG, indicating that SIZ1 represses the transition to flowering mainly through suppressing SA-dependent floral promotion signaling under short days. Previous results have shown that exogenous SA treatment does not suppress late flowering of autonomous pathway mutants. However, the siz1 mutation accelerated flowering time of an autonomous pathway mutant, luminidependens, by reducing the expression of FLOWERING LOCUS C (FLC), a floral repressor. This result suggests that SIZ1 promotes FLC expression, possibly through an SA-independent pathway. Evidence indicates that SIZ1 is required for the full activation of FLC expression in the late-flowering FRIGIDA background. Interestingly, increased FLC expression and late flowering of an autonomous pathway mutant, flowering locus d (fld), was not suppressed by siz1, suggesting that SIZ1 promotes FLC expression by repressing FLD. Consistent with this, SIZ1 facilitates sumoylation of FLD that can be suppressed by mutations in three predicted sumoylation motifs in FLD (i.e. FLDK3R). Furthermore, expression of FLDK3R in fld protoplasts strongly reduced FLC transcription compared with expression of FLD, and this affect was linked to reduced acetylation of histone 4 in FLC chromatin. Taken together, the results suggest that SIZ1 is a floral repressor that not only represses the SA-dependent pathway, but also promotes FLC expression by repressing FLD activity through sumoylation, which is required for full FLC expression in a FRIGIDA background.
PMCID: PMC2254019  PMID: 18069938
SIZ1; SA; flowering; SUMO; FLD; FLC
9.  FVE, an Arabidopsis Homologue of the Retinoblastoma-Associated Protein That Regulates Flowering Time and Cold Response, Binds to Chromatin as a Large Multiprotein Complex 
Molecules and Cells  2011;32(3):227-234.
Some genetic studies indicate that plant homologues of proteins involved in chromatin modification and remodeling in other organisms may regulate plant development. Previously, we described an Arabidopsis mutant with altered cold-responsive gene expression (acg1) displaying a late flowering phenotype, a null allele of fve. FVE is a homologue of the mammalian retinoblastoma-associated protein (RbAp), one component of a histone deacetylase (HDAC) complex involved in transcriptional repression, and has been shown to be involved in the deacetylation of the FLOWERING LOCUS C (FLC) chromatin encoding for a repressor of flowering. In an effort to gain insight into the biochemical functions of FVE, we overexpressed FVE tagged with the hemagglutinin (HA) and FLAG epitope at the N-terminus in acg1 mutants. The results of physiological and molecular analyses demonstrated that FVE overexpression in acg1 rescued the mutant phenotypes, including late flowering and alterations in floral pathway gene expression such as FLC, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), and FLOWERING LOCUS T (FT), and also super-induced cold-responsive reporter gene expression. The chromatin immunoprecipitation experiments revealed the amplification of specific DNA regions of FLC and COLD-REGULATED 15A (COR15A), indicating that FVE may bind to the FLC and COR15A chromatin. Gel-filtration chromatography and the immunoprecipitation of putative FVE complexes showed that FVE forms a protein complex of approximately 1.0 MDa. These results demonstrate that FVE may exist as a multiprotein complex, similar to the mammalian HDAC complex harboring RbAp, to regulate flowering time and cold response by associating with the FLC and COR chromatin.
PMCID: PMC3887629  PMID: 21710206
chromatin; flowering; FVE; histone deacetylase; retinoblastoma-associated protein
10.  Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb Repressive Complex 2 Components 
PLoS ONE  2008;3(10):e3404.
Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.
PMCID: PMC2561057  PMID: 18852898
11.  Hypoxia 
Plant Signaling & Behavior  2009;4(8):773-776.
VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.1 Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,1 and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.4,7–10 In our recent paper11 we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.
PMCID: PMC2801397  PMID: 19820304
arabidopsis; VIN3; FLC; hypoxia; vernalization; chromatin remodelling; survival
12.  Arabidopsis MSI1 functions in photoperiodic flowering time control 
Appropriate timing of flowering is crucial for crop yield and the reproductive success of plants. Flowering can be induced by a number of molecular pathways that respond to internal and external signals such as photoperiod, vernalization or light quality, ambient temperature and biotic as well as abiotic stresses. The key florigenic signal FLOWERING LOCUS T (FT) is regulated by several flowering activators, such as CONSTANS (CO), and repressors, such as FLOWERING LOCUS C (FLC). Chromatin modifications are essential for regulated gene expression, which often involves the well conserved MULTICOPY SUPRESSOR OF IRA 1 (MSI1)-like protein family. MSI1-like proteins are ubiquitous partners of various complexes, such as POLYCOMB REPRESSIVE COMPLEX2 or CHROMATIN ASSEMBLY FACTOR 1. In Arabidopsis, one of the functions of MSI1 is to control the switch to flowering. Arabidopsis MSI1 is needed for the correct expression of the floral integrator gene SUPPRESSOR OF CO 1 (SOC1). Here, we show that the histone-binding protein MSI1 acts in the photoperiod pathway to regulate normal expression of CO in long day (LD) photoperiods. Reduced expression of CO in msi1-mutants leads to failure of FT and SOC1 activation and to delayed flowering. MSI1 is needed for normal sensitivity of Arabidopsis to photoperiod, because msi1-mutants responded less than wild type to an intermittent LD treatment of plants grown in short days. Finally, genetic analysis demonstrated that MSI1 acts upstream of the CO-FT pathway to enable an efficient photoperiodic response and to induce flowering.
PMCID: PMC3945484  PMID: 24639681
Arabidopsis; flowering time; chromatin; MSI1; photoperiod; FLOWERING LOCUS T (FT); CONSTANS (CO)
13.  The inhibitory effect of ABA on floral transition is mediated by ABI5 in Arabidopsis  
Journal of Experimental Botany  2013;64(2):675-684.
Seed germination and flowering initiation are both transitions responding to similar seasonal cues. This study shows that ABSCISIC ACID-INSENSITIVE MUTANT 5 (ABI5), a bZIP transcription factor, which plays an important role in the abscisic acid (ABA)-arrested seed germination, is robustly associated with the floral transition in Arabidopsis. Under long-day conditions, overexpression of ABI5 could delay floral transition through upregulating FLOWERING LOCUS C (FLC) expression. In contrast, ectopically overexpressing FLC in an abi5 mutant reversed the earlier flowering phenotype. Further analysis indicated that transactivation of FLC could be promoted by ABI5 and/or other abscisic acid-responsive element (ABRE)-binding factors (ABFs). The expression of FLC that was promoted by ABI5 and/or other ABFs could be blocked in a triple SNF1-related protein kinase (SnRK) mutant, snrk2.2/2.3/2.6, despite the presence of ABA. In sharp contrast, when SnRK2.6 was coexpressed, the reduction of transactivity of FLC was reverted in mesophyll protoplasts of snrk2.2/2.3/2.6. Additional results from analysing transgenic plants carrying mutations of phosphoamino acids (ABI5 S42AS145AT201A), which are conserved in ABI5, suggested that SnRK2-mediated ABI5 and/or ABF phosphorylation may be crucial for promoting FLC expression. The transgenic plants ABI5 S42AS145AT201A were insensitive to ABA in seed germination, in addition to having an earlier flowering phenotype. Direct binding of ABI5 to the ABRE/G-box promoter elements existing in FLC was demonstrated by chromatin immunoprecipitation. Mutations at the ABRE/G-box regions in FLC promoter sequences abolished the ABI5-promoted transactivation of FLC. In summary, these results may decipher the inhibitory effect of ABA on floral transition in Arabidopsis.
PMCID: PMC3542054  PMID: 23307919
ABA; ABFs; ABI5; chromatin immunoprecipitation; FLC; flowering time; SnRK2s.
14.  Brm Inhibits the Proliferative Response of Keratinocytes and Corneal Epithelial Cells to Ultraviolet Radiation-Induced Damage 
PLoS ONE  2014;9(9):e107931.
Ultraviolet radiation (UV) from sunlight is the primary cause of skin and ocular neoplasia. Brahma (BRM) is part of the SWI/SNF chromatin remodeling complex. It provides energy for rearrangement of chromatin structure. Previously we have found that human skin tumours have a hotspot mutation in BRM and that protein levels are substantially reduced. Brm−/− mice have enhanced susceptibility to photocarcinogenesis. In these experiments, Brm−/− mice, with both or a single Trp53 allele were exposed to UV for 2 or 25 weeks. In wild type mice the central cornea and stroma became atrophic with increasing time of exposure while the peripheral regions became hyperplastic, presumably as a reparative process. Brm−/−, Trp53+/−, and particularly the Brm−/− Trp53+/− mice had an exaggerated hyperplastic regeneration response in the corneal epithelium and stroma so that the central epithelial atrophy or stromal loss was reduced. UV induced hyperplasia of the epidermis and corneal epithelium, with an increase in the number of dividing cells as determined by Ki-67 expression. This response was considerably greater in both the Brm−/− Trp53+/+ and Brm−/− Trp53+/− mice indicating that Brm protects from UV-induced enhancement of cell division, even with loss of one Trp53 allele. Cell division was disorganized in Brm−/− mice. Rather than being restricted to the basement membrane region, dividing cells were also present in the suprabasal regions of both tissues. Brm appears to be a tumour suppressor gene that protects from skin and ocular photocarcinogenesis. These studies indicate that Brm protects from UV-induced hyperplastic growth in both cutaneous and corneal keratinocytes, which may contribute to the ability of Brm to protect from photocarcinogenesis.
PMCID: PMC4177874  PMID: 25254962
15.  BRR2a Affects Flowering Time via FLC Splicing 
PLoS Genetics  2016;12(4):e1005924.
Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects.
Author Summary
Timing of flowering has a great effect on reproductive success and fitness. It is controlled by many external signals and internal states involving a large set of genes. Here we report that the Arabidopsis thaliana BRR2a gene is needed for normal flowering. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. BRR2a is needed for splicing of a subset of introns, most noticeably in the transcript of the flowering repressor FLC. Reduced FLC expression increased transcription of key floral activators, leading to early flowering in both short and long days. Genome-wide experiments established that full BRR2a activity was required only for a small group of introns. We propose that uncompromised BRR2a activity is most important for efficient splicing of a subset of introns of particular size, sequence and chromatin composition, and that FLC is most sensitive to splicing defects.
PMCID: PMC4839602  PMID: 27100965
16.  Local chromatin environment of a Polycomb target gene instructs its own epigenetic inheritance 
eLife  null;4:e07205.
Inheritance of gene expression states is fundamental for cells to ‘remember’ past events, such as environmental or developmental cues. The conserved Polycomb Repressive Complex 2 (PRC2) maintains epigenetic repression of many genes in animals and plants and modifies chromatin at its targets. Histones modified by PRC2 can be inherited through cell division. However, it remains unclear whether this inheritance can direct long-term memory of individual gene expression states (cis memory) or instead if local chromatin states are dictated by the concentrations of diffusible factors (trans memory). By monitoring the expression of two copies of the Arabidopsis Polycomb target gene FLOWERING LOCUS C (FLC) in the same plants, we show that one copy can be repressed while the other is active. Furthermore, this ‘mixed’ expression state is inherited through many cell divisions as plants develop. These data demonstrate that epigenetic memory of FLC expression is stored not in trans but in cis.
eLife digest
Genetic material is contained within molecules of DNA. In plants and many other organisms, these DNA molecules are packaged around proteins called histones to make a structure known as chromatin. Altering how the DNA is packaged in chromatin can control the activity of genes. For example, a group of proteins called the Polycomb Repressive Complex 2 (PRC2) adds methyl tags to histones, which can alter the packaging of chromatin to lower the activity of particular genes.
When a cell divides, it is sometimes important that genes in the daughter cells have similar levels of activity as the parent cell. This allows individual cells to ‘remember’ past events, such as exposure to cold temperatures or other environmental conditions. The pattern of methyl tags on histones can be passed onto the daughter cells, but it is not clear if this is actually responsible for providing the memory.
One gene that PRC2 regulates is called FLC, which influences when a plant called Arabidopsis produces flowers. If the plants are exposed to cold temperatures, the activity of FLC is repressed. FLC activity remains low after the period of cold has ended to ensure that the plants produce flowers at an appropriate time. If this 'memory of cold' is held locally in the chromatin of the FLC gene, then it should be possible for two copies of the FLC gene in the same cell to show different gene activities. However, if the memory is stored more globally inside each cell by other proteins, then the two copies should have identical gene activities.
To distinguish between these two possibilities, Berry et al. added different fluorescent tags to two copies of the FLC gene in Arabidopsis plants, which allowed the activity of each gene copy to be tracked in individual cells under a microscope. The experiments show that one copy of FLC may be switched off, while the other remains switched on inside the same cell. Furthermore, it was found that this pattern of gene activity is passed onto the daughter cells when the original cell divides.
Berry et al.'s findings show that the memory of FLC gene activity is stored locally in the chromatin of the FLC gene itself. The alteration of histones by PRC2 is one important aspect of the packaging of chromatin. The next challenge is to identify what other features of chromatin are required for a gene to be able to store this memory locally.
PMCID: PMC4450441  PMID: 25955967
epigenetics; cis memory; Polycomb; FLOWERING LOCUS C; vernalization; Arabidopsis
17.  Antagonistic Roles for BRM and BRG1 SWI/SNF Complexes in Differentiation*♦ 
The Journal of Biological Chemistry  2009;284(15):10067-10075.
The mammalian SWI/SNF chromatin-remodeling complex is essential for the multiple changes in gene expression that occur during differentiation. However, the basis within the complex for specificity in effecting positive versus negative changes in gene expression has only begun to be elucidated. The catalytic core of the complex can be either of two closely related ATPases, BRM or BRG1, with the potential that the choice of alternative subunits is a key determinant of specificity. Short hairpin RNA-mediated depletion of the ATPases was used to explore their respective roles in the well characterized multistage process of osteoblast differentiation. The results reveal an unexpected role for BRM-specific complexes. Instead of impeding differentiation as was seen with BRG1 depletion, depletion of BRM caused accelerated progression to the differentiation phenotype. Multiple tissue-specific differentiation markers, including the tightly regulated late stage marker osteocalcin, become constitutively up-regulated in BRM-depleted cells. Chromatin immunoprecipitation analysis of the osteocalcin promoter as a model for the behavior of the complexes indicates that the promoter is a direct target of both BRM- and BRG1-containing complexes. BRG1 complexes, which are required for activation, are associated with the promoter well before induction, but the concurrent presence of BRM-specific complexes overrides their activation function. BRM-specific complexes are present only on the repressed promoter and are required for association of the co-repressor HDAC1. These findings reveal an unanticipated degree of specialization of function linked with the choice of ATPase and suggest a new paradigm for the roles of the alternative subunits during differentiation.
PMCID: PMC2665061  PMID: 19144648
18.  Arabidopsis TFL2/LHP1 Specifically Associates with Genes Marked by Trimethylation of Histone H3 Lysine 27 
PLoS Genetics  2007;3(6):e86.
TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) is the only Arabidopsis protein with overall sequence similarity to the HETEROCHROMATIN PROTEIN 1 (HP1) family of metazoans and S. pombe. TFL2/LHP1 represses transcription of numerous genes, including the flowering-time genes FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC), as well as the floral organ identity genes AGAMOUS (AG) and APETALA 3 (AP3). These genes are also regulated by proteins of the Polycomb repressive complex 2 (PRC2), and it has been proposed that TFL2/LHP1 represents a potential stabilizing factor of PRC2 activity. Here we show by chromatin immunoprecipitation and hybridization to an Arabidopsis Chromosome 4 tiling array (ChIP-chip) that TFL2/LHP1 associates with hundreds of small domains, almost all of which correspond to genes located within euchromatin. We investigated the chromatin marks to which TFL2/LHP1 binds and show that, in vitro, TFL2/LHP1 binds to histone H3 di- or tri-methylated at lysine 9 (H3K9me2 or H3K9me3), the marks recognized by HP1, and to histone H3 trimethylated at lysine 27 (H3K27me3), the mark deposited by PRC2. However, in vivo TFL2/LHP1 association with chromatin occurs almost exclusively and co-extensively with domains marked by H3K27me3, but not H3K9me2 or -3. Moreover, the distribution of H3K27me3 is unaffected in lhp1 mutant plants, indicating that unlike PRC2 components, TFL2/LHP1 is not involved in the deposition of this mark. Rather, our data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2.
Author Summary
Stable repression of gene expression is an important aspect of the developmental programs of higher organisms. In plants and animals, DNA is organized within chromatin, which contains at its core a set of evolutionarily conserved proteins called histones. These proteins can be modified for example by methylation or acetylation of lysines or phosphorylation of serines. Specific combinations of these histone modifications are interpreted by other chromatin proteins and thereby play essential roles in gene regulation. One such potential effector of the histone code in the flowering plant Arabidopsis is TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1). Here we present highly detailed “epigenomic” maps that establish that TFL2/LHP1 associates with a subset of Arabidopsis genes that are marked by tri-methylation of Lysine 27 of histone H3. In plants and animals, an evolutionarily conserved complex called PRC2 deposits this mark. In Drosophila and mammals this modified histone is then read by another complex, called PRC1, to maintain the stable repression of genes. In Arabidopsis however, no PRC1 complex exists, and our results provide evidence that TFL2/LHP1 may fulfill a related function.
PMCID: PMC1885283  PMID: 17542647
19.  Modulation of BRAHMA expression by the mitogen-activated protein kinase / extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation 
Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK) / extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.
PMCID: PMC4221548  PMID: 25026375
SWI/SNF Chromatin remodeling enzymes; BRG1/BRM; melanoma; BRAF(V600E); mitogen-activated protein kinase / extracellular signal regulated kinase (ERK1/2) pathway; vemurafenib
20.  ABSCISIC ACID-INSENSITIVE 4 negatively regulates flowering through directly promoting Arabidopsis FLOWERING LOCUS C transcription 
Journal of Experimental Botany  2015;67(1):195-205.
FLC is the direct target of both of the transcription factors ABI4 and ABI5, and ABA inhibits floral transition by activating FLC transcription through ABI4.
During the life cycle of a plant, one of the major biological processes is the transition from the vegetative to the reproductive stage. In Arabidopsis, flowering time is precisely controlled by extensive environmental and internal cues. Gibberellins (GAs) promote flowering, while abscisic acid (ABA) is considered as a flowering suppressor. However, the detailed mechanism through which ABA inhibits the floral transition is poorly understood. Here, we report that ABSCISIC ACID-INSENSITIVE 4 (ABI4), a key component in the ABA signalling pathway, negatively regulates floral transition by directly promoting FLOWERING LOCUS C (FLC) transcription. The abi4 mutant showed the early flowering phenotype whereas ABI4-overexpressing (OE-ABI4) plants had delayed floral transition. Consistently, quantitative reverse transcription–PCR (qRT–PCR) assay revealed that the FLC transcription level was down-regulated in abi4, but up-regulated in OE-ABI4. The change in FT level was consistent with the pattern of FLC expression. Chromatin immunoprecipitation-qPCR (ChIP-qPCR), electrophoretic mobility shift assay (EMSA), and tobacco transient expression analysis showed that ABI4 promotes FLC expression by directly binding to its promoter. Genetic analysis demonstrated that OE-ABI4::flc-3 could not alter the flc-3 phenotype. OE-FLC::abi4 showed a markedly delayed flowering phenotype, which mimicked OE-FLC::WT, and suggested that ABI4 acts upstream of FLC in the same genetic pathway. Taken together, these findings suggest that ABA inhibits the floral transition by activating FLC transcription through ABI4.
PMCID: PMC4682436  PMID: 26507894
ABA; ABI4; chromatin immunoprecipitation; FLC; flowering; transcription factor
The BRMS1 metastasis suppressor interacts with the protein AT rich interactive domain 4A (ARID4A, retinoblastoma-binding protein 1, RBBP1) as part of SIN3:histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1mut) and mapped ARID4A interactions. BRMS1L174D disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens (Y2H) but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation (co-IP). Deletion of the first coiled-coil domain (BRMS1ΔCC1) did not disrupt direct (Y2H) interaction, but did prevent association by co-IP. These results suggest altered complex composition with BRMS1mut. Although basal transcription repression was impaired and the pro-metastatic protein osteopontin (OPN) was differentially down-regulated by BRMS1L174D and BRMS1ΔCC1, both down-regulated epidermal growth factor receptor (EGFR) and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1mut that modify the composition of a SIN3:HDAC chromatin remodeling complex leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as EGFR, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/cellular functions may prove important for future strategies targeting metastasis.
PMCID: PMC2293288  PMID: 18211900
22.  Concerted genomic targeting of H3K27 demethylase REF6 and chromatin-remodeling ATPase BRM in Arabidopsis 
Nature genetics  2016;48(6):687-693.
SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level1–5, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase6, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM–REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.
PMCID: PMC5134324  PMID: 27111034
23.  Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription 
Molecular Cell  2014;54(1):156-165.
Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.
Graphical Abstract
•Alternative splicing of noncoding antisense transcripts affects flowering•Arabidopsis FLC gene transcription modulated by lncRNA isoforms•Positive feedback links chromatin state and antisense transcript splicing•Quantitative gene regulation by coupling antisense splicing to chromatin states
Marquardt et al. demonstrate that noncoding transcripts antisense to the Arabidopsis floral repressor gene FLC are alternatively spliced. Interfering with this alternative splicing alters FLC transcription quantitatively and affects the timing of flowering. Thus, this work reveals a function for a long noncoding antisense transcript in Arabidopsis.
PMCID: PMC3988885  PMID: 24725596
24.  Polycomb repression 
Plant Signaling & Behavior  2008;3(6):412-414.
In our recent paper1 we suggested a molecular explanation for the long standing observation that plants need to be mitotically active to respond to a prolonged period of low temperatures by flowering early (vernalization).2 In Arabidopsis, vernalization is associated with the epigenetic repression of the floral repressor, FLC.3–5 FLC repression is established during the low temperature treatment and is marked by the loss of chromatin marks associated with active genes and the gain of histone H3 trimethyl-lysine 27 (K27me3) at the start of transcription/translation.1 After the end of the cold treatment, this repressive modification spreads across FLC chromatin to mark the entire locus.1 In cells not undergoing mitosis, we found that FLC is repressed by low temperatures, but that this repression is only partially maintained. We concluded that DNA replication is not required for the initial response to low temperatures, but rather for the maintenance of this response. Here we discuss the implications of our observations in terms of the plasticity of chromatin modifications in plants.
PMCID: PMC2634319  PMID: 19704583
trimethyl lysine 27; FLC; VIN3; bivalent domain; histone replacement
25.  Seasonal shift in timing of vernalization as an adaptation to extreme winter 
eLife  null;4:e06620.
The requirement for vernalization, a need for prolonged cold to trigger flowering, aligns reproductive development with favorable spring conditions. In Arabidopsis thaliana vernalization depends on the cold-induced epigenetic silencing of the floral repressor locus FLC. Extensive natural variation in vernalization response is associated with A. thaliana accessions collected from different geographical regions. Here, we analyse natural variation for vernalization temperature requirement in accessions, including those from the northern limit of the A. thaliana range. Vernalization required temperatures above 0°C and was still relatively effective at 14°C in all the accessions. The different accessions had characteristic vernalization temperature profiles. One Northern Swedish accession showed maximum vernalization at 8°C, both at the level of flowering time and FLC chromatin silencing. Historical temperature records predicted all accessions would vernalize in autumn in N. Sweden, a prediction we validated in field transplantation experiments. The vernalization response of the different accessions was monitored over three intervals in the field and found to match that when the average field temperature was given as a constant condition. The vernalization temperature range of 0–14°C meant all accessions fully vernalized before snowfall in N. Sweden. These findings have important implications for understanding the molecular basis of adaptation and for predicting the consequences of climate change on flowering time.
eLife digest
Plants are not able to move around and so they need to be able to adapt their growth and development to seasonal changes in their environment. For example, prolonged exposure to cold temperatures during winter can prime some plants to flower when temperatures increase in the spring—a process called vernalization. In these plants, extended periods of cold temperatures lead to lower activity of a gene called FLC, which normally inhibits flowering.
In the plant Arabidopsis thaliana, vernalization requires several months of exposure to temperatures between 0–6°C. Recently, A. thaliana plants from southern Europe were found to vary in the temperature requirements for vernalization, responding to temperatures higher than 6°C. This suggested that plants from northern Europe might vernalize preferentially at lower temperatures. Here, Duncan et al. compared vernalization in a collection of A. thaliana plants (or ‘accessions’) sampled from different regions of Sweden and the UK.
The experiments show that all the accessions needed temperatures above 0°C to vernalize and that vernalization still worked relatively well at temperatures as high as 14°C. The optimal temperature range for vernalization differed between the accessions, but plants from more northern areas did not necessarily vernalize at lower temperatures. For example, for one particular accession from northern Sweden, the temperature that is optimum for vernalization was 8°C, a notably higher temperature than expected.
Historical local climate records suggested that this accession would vernalize before the first snowfall of the winter in North Sweden. Duncan et al. confirmed this proposal with field experiments. Plants were grown in natural field sites in September and then moved into a greenhouse. The experiments show that the plants complete vernalization by November, which strongly suggests that FLC is silenced during autumn rather than during winter, as previously thought. This changed temperature response is due, in part, to a small number of tiny genetic differences in regions of the FLC gene that do not code for protein.
These findings have important implications for future studies of vernalization and flowering time, and for understanding how plants will adapt to on going and future climate change. The next step is to understand what causes these changed temperature responses at a molecular level, which should enable selective breeding for flowering and harvest date in a range of crops.
PMCID: PMC4532801  PMID: 26203563
vernalization; epigenetics; adaptation; Arabidopsis

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