The H3K4 demethylase JMJ1 is required for proper timing of flowering in Brachypodium distachyon.

Flowering is a key developmental transition in the plant life cycle. In temperate climates, flowering often occurs in response to the perception of seasonal cues such as changes in day-length and temperature. However, the mechanisms that have evolved to control the timing of flowering in temperate grasses are not fully understood. We identified a Brachypodium distachyon mutant whose flowering is delayed under inductive long-day conditions due to a mutation in the JMJ1 gene, which encodes a Jumonji domain-containing protein. JMJ1 is a histone demethylase that mainly demethylates H3K4me2 and H3K4me3 in vitro and in vivo. Analysis of the genome-wide distribution of H3K4me1, H3K4me2, and H3K4me3 in wild-type plants by chromatin immunoprecipitation and sequencing combined with RNA sequencing revealed that H3K4m1 and H3K4me3 are positively associated with gene transcript levels, whereas H3K4me2 is negatively correlated with transcript levels. Furthermore, JMJ1 directly binds to the chromatin of the flowering regulator genes VRN1 and ID1 and affects their transcription by modifying their H3K4me2 and H3K4me3 levels. Genetic analyses indicated that JMJ1 promotes flowering by activating VRN1 expression. Our study reveals a role for JMJ1-mediated chromatin modification in the proper timing of flowering in B. distachyon.

INDETERMINATE1-mediated expression of FT family genes is required for proper timing of flowering in Brachypodium distachyon.

The transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 (FT1) and FT-Like (FTL) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 (BdID1) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 (FT-LIKE9) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1. We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1, FT2, FTL12, and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses.

PHYTOCHROME C regulation of photoperiodic flowering via PHOTOPERIOD1 is mediated by EARLY FLOWERING 3 in Brachypodium distachyon.

Daylength sensing in many plants is critical for coordinating the timing of flowering with the appropriate season. Temperate climate-adapted grasses such as Brachypodium distachyon flower during the spring when days are becoming longer. The photoreceptor PHYTOCHROME C is essential for long-day (LD) flowering in B. distachyon. PHYC is required for the LD activation of a suite of genes in the photoperiod pathway including PHOTOPERIOD1 (PPD1) that, in turn, result in the activation of FLOWERING LOCUS T (FT1)/FLORIGEN, which causes flowering. Thus, B. distachyon phyC mutants are extremely delayed in flowering. Here we show that PHYC-mediated activation of PPD1 occurs via EARLY FLOWERING 3 (ELF3), a component of the evening complex in the circadian clock. The extreme delay of flowering of the phyC mutant disappears when combined with an elf3 loss-of-function mutation. Moreover, the dampened PPD1 expression in phyC mutant plants is elevated in phyC/elf3 mutant plants consistent with the rapid flowering of the double mutant. We show that loss of PPD1 function also results in reduced FT1 expression and extremely delayed flowering consistent with results from wheat and barley. Additionally, elf3 mutant plants have elevated expression levels of PPD1, and we show that overexpression of ELF3 results in delayed flowering associated with a reduction of PPD1 and FT1 expression, indicating that ELF3 represses PPD1 transcription consistent with previous studies showing that ELF3 binds to the PPD1 promoter. Indeed, PPD1 is the main target of ELF3-mediated flowering as elf3/ppd1 double mutant plants are delayed flowering. Our results indicate that ELF3 operates downstream from PHYC and acts as a repressor of PPD1 in the photoperiod flowering pathway of B. distachyon.

Vernalization: winter and the timing of flowering in plants.

Plants have evolved many systems to sense their environment and to modify their growth and development accordingly. One example is vernalization, the process by which flowering is promoted as plants sense exposure to the cold temperatures of winter. A requirement for vernalization is an adaptive trait that helps prevent flowering before winter and permits flowering in the favorable conditions of spring. In Arabidopsis and cereals, vernalization results in the suppression of genes that repress flowering. We describe recent progress in understanding the molecular basis of this suppression. In Arabidopsis, vernalization involves the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are silenced epigenetically via histone modifications. We also discuss the similarities and differences in vernalization between Arabidopsis and cereals.

Mutations in the predicted DNA polymerase subunit POLD3 result in more rapid flowering of Brachypodium distachyon.

The timing of reproduction is a critical developmental decision in the life cycle of many plant species. Fine mapping of a rapid-flowering mutant was done using whole-genome sequence data from bulked DNA from a segregating F2 mapping populations. The causative mutation maps to a gene orthologous with the third subunit of DNA polymerase δ (POLD3), a previously uncharacterized gene in plants. Expression analyses of POLD3 were conducted via real time qPCR to determine when and in what tissues the gene is expressed. To better understand the molecular basis of the rapid-flowering phenotype, transcriptomic analyses were conducted in the mutant vs wild-type. Consistent with the rapid-flowering mutant phenotype, a range of genes involved in floral induction and flower development are upregulated in the mutant. Our results provide the first characterization of the developmental and gene expression phenotypes that result from a lesion in POLD3 in plants.

Establishment of a vernalization requirement in Brachypodium distachyon requires REPRESSOR OF VERNALIZATION1.

A requirement for vernalization, the process by which prolonged cold exposure provides competence to flower, is an important adaptation to temperate climates that ensures flowering does not occur before the onset of winter. In temperate grasses, vernalization results in the up-regulation of VERNALIZATION1 (VRN1) to establish competence to flower; however, little is known about the mechanism underlying repression of VRN1 in the fall season, which is necessary to establish a vernalization requirement. Here, we report that a plant-specific gene containing a bromo-adjacent homology and transcriptional elongation factor S-II domain, which we named REPRESSOR OF VERNALIZATION1 (RVR1), represses VRN1 before vernalization in Brachypodium distachyon That RVR1 is upstream of VRN1 is supported by the observations that VRN1 is precociously elevated in an rvr1 mutant, resulting in rapid flowering without cold exposure, and the rapid-flowering rvr1 phenotype is dependent on VRN1 The precocious VRN1 expression in rvr1 is associated with reduced levels of the repressive chromatin modification H3K27me3 at VRN1, which is similar to the reduced VRN1 H3K27me3 in vernalized plants. Furthermore, the transcriptome of vernalized wild-type plants overlaps with that of nonvernalized rvr1 plants, indicating loss of rvr1 is similar to the vernalized state at a molecular level. However, loss of rvr1 results in more differentially expressed genes than does vernalization, indicating that RVR1 may be involved in processes other than vernalization despite a lack of any obvious pleiotropy in the rvr1 mutant. This study provides an example of a role for this class of plant-specific genes.

An ortholog of CURLY LEAF/ENHANCER OF ZESTE like-1 is required for proper flowering in Brachypodium distachyon.

Many plants require prolonged exposure to cold to acquire the competence to flower. The process by which cold exposure results in competence is known as vernalization. In Arabidopsis thaliana, vernalization leads to the stable repression of the floral repressor FLOWERING LOCUS C via chromatin modification, including an increase of trimethylation on lysine 27 of histone H3 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2). Vernalization in pooids is associated with the stable induction of a floral promoter, VERNALIZATION 1 (VRN1). From a screen for mutants with a reduced vernalization requirement in the model grass Brachypodium distachyon, we identified two recessive alleles of ENHANCER OF ZESTE-LIKE 1 (EZL1). EZL1 is orthologous to A. thaliana CURLY LEAF 1, a gene that encodes the catalytic subunit of PRC2. B. distachyon ezl1 mutants flower rapidly without vernalization in long-day (LD) photoperiods; thus, EZL1 is required for the proper maintenance of the vegetative state prior to vernalization. Transcriptomic studies in ezl1 revealed mis-regulation of thousands of genes, including ectopic expression of several floral homeotic genes in leaves. Loss of EZL1 results in the global reduction of H3K27me3 and H3K27me2, consistent with this gene making a major contribution to PRC2 activity in B. distachyon. Furthermore, in ezl1 mutants, the flowering genes VRN1 and AGAMOUS (AG) are ectopically expressed and have reduced H3K27me3. Artificial microRNA knock-down of either VRN1 or AG in ezl1-1 mutants partially restores wild-type flowering behavior in non-vernalized plants, suggesting that ectopic expression in ezl1 mutants may contribute to the rapid-flowering phenotype.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon.

The transition to reproductive development is a crucial step in the plant life cycle, and the timing of this transition is an important factor in crop yields. Here, we report new insights into the genetic control of natural variation in flowering time in Brachypodium distachyon, a nondomesticated pooid grass closely related to cereals such as wheat (Triticum spp.) and barley (Hordeum vulgare L.). A recombinant inbred line population derived from a cross between the rapid-flowering accession Bd21 and the delayed-flowering accession Bd1-1 were grown in a variety of environmental conditions to enable exploration of the genetic architecture of flowering time. A genotyping-by-sequencing approach was used to develop SNP markers for genetic map construction, and quantitative trait loci (QTLs) that control differences in flowering time were identified. Many of the flowering-time QTLs are detected across a range of photoperiod and vernalization conditions, suggesting that the genetic control of flowering within this population is robust. The two major QTLs identified in undomesticated B. distachyon colocalize with VERNALIZATION1/PHYTOCHROME C and VERNALIZATION2, loci identified as flowering regulators in the domesticated crops wheat and barley. This suggests that variation in flowering time is controlled in part by a set of genes broadly conserved within pooid grasses.

A methyltransferase required for proper timing of the vernalization response in Arabidopsis.

Prolonged exposure to winter cold enables flowering in many plant species through a process called vernalization. In Arabidopsis, vernalization results from the epigenetic silencing of the floral repressor flowering locus C (FLC) via a Polycomb Repressive Complex 2 (PRC2)-mediated increase in the density of the epigenetic silencing mark H3K27me3 at FLC chromatin. During cold exposure, a gene encoding a unique, cold-specific PRC2 component, vernalization insensitive 3 (VIN3), which is necessary for PRC2-mediated silencing of FLC, is induced. Here we show that set domain group 7 (SDG7) is required for proper timing of VIN3 induction and of the vernalization process. Loss of SDG7 results in a vernalization-hypersensitive phenotype, as well as more rapid cold-mediated up-regulation of VIN3. In the absence of cold, loss of SDG7 results in elevated levels of long noncoding RNAs, which are thought to participate in epigenetic repression of FLC. Furthermore, loss of SDG7 results in increased H3K27me3 deposition on FLC chromatin in the absence of cold exposure and enhanced H3K27me3 spreading during cold treatment. Thus, SDG7 is a negative regulator of vernalization, and loss of SDG7 creates a partially vernalized state without cold exposure.

Evolution of VRN2/Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering.

Flowering of many plant species is coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides competence to flower after prolonged cold exposure, and a vernalization requirement prevents flowering from occurring prior to winter. In winter wheat (Triticum aestivum) and barley (Hordeum vulgare), three genes VRN1, VRN2, and FT form a regulatory loop that regulates the initiation of flowering. Prior to cold exposure, VRN2 represses FT. During cold, VRN1 expression increases, resulting in the repression of VRN2, which in turn allows activation of FT during long days to induce flowering. Here, we test whether the circuitry of this regulatory loop is conserved across Pooideae, consistent with their niche transition from the tropics to the temperate zone. Our phylogenetic analyses of VRN2-like genes reveal a duplication event occurred before the diversification of the grasses that gave rise to a CO9 and VRN2/Ghd7 clade and support orthology between wheat/barley VRN2 and rice (Oryza sativa) Ghd7 Our Brachypodium distachyon VRN1 and VRN2 knockdown and overexpression experiments demonstrate functional conservation of grass VRN1 and VRN2 in the promotion and repression of flowering, respectively. However, expression analyses in a range of pooids demonstrate that the cold repression of VRN2 is unique to core Pooideae such as wheat and barley. Furthermore, VRN1 knockdown in B. distachyon demonstrates that the VRN1-mediated suppression of VRN2 is not conserved. Thus, the VRN1-VRN2 feature of the regulatory loop appears to have evolved late in the diversification of temperate grasses.

Interaction of photoperiod and vernalization determines flowering time of Brachypodium distachyon.

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1.

Vernalization is the process by which sensing a prolonged exposure to winter cold leads to competence to flower in the spring. In winter annual Arabidopsis thaliana accessions, flowering is suppressed in the fall by expression of the potent floral repressor FLOWERING LOCUS C (FLC). Vernalization promotes flowering via epigenetic repression of FLC. Repression is accompanied by a series of histone modifications of FLC chromatin that include dimethylation of histone H3 at Lys9 (H3K9) and Lys27 (H3K27). Here, we report that A. thaliana LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) is necessary to maintain the epigenetically repressed state of FLC upon return to warm conditions typical of spring. LHP1 is enriched at FLC chromatin after prolonged exposure to cold, and LHP1 activity is needed to maintain the increased levels of H3K9 dimethylation at FLC chromatin that are characteristic of the vernalized state.

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.

My favourite flowering image: Maryland Mammoth tobacco.

Almost 100 years ago, the study of Maryland Mammoth tobacco by Garner and Allard was one in a long series of studies that have led to a better understanding of how plants "decide" when to flower. deciphering how plants "decide" when to flower. The extreme phenotype of Maryland Mammoth tobacco, in which a single recessive mutation changes a day-neutral to a strictly photoperiod-requiring plant, impressively illustrates the action of the photoperiodic pathway of flowering.

Polycomb proteins regulate the quantitative induction of VERNALIZATION INSENSITIVE 3 in response to low temperatures.

Vernalization, the promotion of flowering in response to low temperatures, is one of the best characterized examples of epigenetic regulation in plants. The promotion of flowering is proportional to the duration of the cold period, but the mechanism by which plants measure time at low temperatures has been a long-standing mystery. We show that the quantitative induction of the first gene in the Arabidopsis vernalization pathway, VERNALIZATION INSENSITIVE 3 (VIN3), is regulated by the components of Polycomb Response Complex 2, which trimethylates histone H3 lysine 27 (H3K27me3). In differentiated animal cells, H3K27me3 is mostly associated with long-term gene repression, whereas, in pluripotent embyonic stem cells, many cell lineage-specific genes are inactive but exist in bivalent chromatin that carries both active (H3K4me3) and repressive (H3K27me3) marks on the same molecule. During differentiation, bivalent domains are generally resolved to an active or silent state. We found that H3K27me3 maintains VIN3 in a repressed state prior to cold exposure; this mark is not removed during VIN3 induction. Instead, active VIN3 is associated with bivalently marked chromatin. The continued presence of H3K27me3 ensures that induction of VIN3 is proportional to the duration of the cold, and that plants require prolonged cold to promote the transition to flowering. The observation that Polycomb proteins control VIN3 activity defines a new role for Polycomb proteins in regulating the rate of gene induction.

Arabidopsis trithorax-related3/SET domain GROUP2 is required for the winter-annual habit of Arabidopsis thaliana.

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.

Natural variation in the temperature range permissive for vernalization in accessions of Arabidopsis thaliana.

Vernalization is an acceleration of flowering in response to chilling, and is normally studied in the laboratory at near-freezing (2-4 °C) temperatures. Many vernalization-requiring species, such as Arabidopsis thaliana, are found in a range of habitats with varying winter temperatures. Natural variation in the temperature range that elicits a vernalization response in Arabidopsis has not been fully explored. We characterized the effect of intermediate temperatures (7-19 °C) on 15 accessions and the well-studied reference line Col-FRI. Although progressively warmer temperatures are gradually less effective at activating expression of the vernalization-specific gene VERNALIZATION-INSENSITIVE 3 (VIN3) and in accelerating flowering, there is substantial natural variation in the upper threshold (T(max) ) of the flowering-time response. VIN3 is required for the T(max) (13 °C) response of Col-FRI. Surprisingly, even 16 °C treatment caused induction of VIN3 in six tested lines, despite the ineffectiveness of this temperature in accelerating flowering for two of them. Finally, we present evidence that mild acceleration of flowering by 19 °C exposure may counterbalance the flowering time delay caused by non-inductive photoperiods in at least one accession, creating an appearance of photoperiod insensitivity.

ARABIDOPSIS TRITHORAX-RELATED7 is required for methylation of lysine 4 of histone H3 and for transcriptional activation of FLOWERING LOCUS C.

In the winter-annual accessions of Arabidopsis thaliana, presence of an active allele of FRIGIDA (FRI) elevates expression of FLOWERING LOCUS C (FLC), a repressor of flowering, and thus confers a vernalization requirement. FLC activation by FRI involves methylation of Lys 4 of histone H3 (H3K4) at FLC chromatin. Many multicellular organisms that have been examined contain two classes of H3K4 methylases, a yeast (Saccharomyces cerevisiae) Set1 class and a class related to Drosophila melanogaster Trithorax. In this work, we demonstrate that ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7), a putative Set1 class H3K4 methylase, is required for proper FLC expression. The atxr7 mutation partially suppresses the delayed flowering of a FRI-containing line. The rapid flowering of atxr7 is associated with reduced FLC expression and is accompanied by decreased H3K4 methylation and increased H3K27 methylation at FLC. Thus, ATXR7 is required for the proper levels of these histone modifications that set the level of FLC expression to create a vernalization requirement in winter-annual accessions. Previously, it has been reported that lesions in ATX1, which encodes a Trithorax class H3K4 methylase, partially suppress the delayed flowering of winter-annual Arabidopsis. We show that the flowering phenotype of atx1 atxr7 double mutants is additive relative to those of single mutants. Therefore, both classes of H3K4 methylases appear to be required for proper regulation of FLC expression.