Flowering time is an important crop trait, as premature or delayed flowering can significantly reduce yields. Plants flower in response to a defined set of endogenous and environmental cues. While the effects of ambient temperature on flowering time are well-studied, the role of epigenetic modifiers in the floral thermosensory pathway remains largely unexplored. Here, we show that the histone demethylases EARLY FLOWERING 6 (ELF6) and JUMONJI 13 (JMJ13) act redundantly to fine-tune temperature-dependent flowering. We found that the elf6-3 jmj13-1 double mutant is hyposensitive to the flowering delay induced by low ambient temperature. Genetic analyses indicated that this response requires FLOWERING LOCUS M ( FLM ) and, to a lesser extent, its homolog FLOWERING LOCUS C ( FLC ). Consistent with these genetic requirements, transcript and chromatin immunoprecipitation (ChIP) analyses revealed that ELF6 and JMJ13 modulate FLM and FLC expression. ChIP assays showed that both proteins associate with FLM and FLC , but with distinct dynamics: JMJ13 exhibited low-level, constitutive occupancy, whereas ELF6 recruitment was temperature and development dependent. Remarkably, ELF6 binding was detected earlier in development under low ambient temperature, coincident with increased ELF6 protein accumulation in germinating seedlings. Together, our data support a model in which low ambient temperature promotes early ELF6 recruitment to limit repressive histone H3 lysine 27 trimethylation (H3K27me3) accumulation at thermoresponsive floral repressors, thereby enabling appropriate gene expression and flowering-time control. These findings identify H3K27me3 demethylases as key components of the floral thermosensory pathway and highlight epigenetic mechanisms linking ambient temperature to flowering.

BACKGROUND:The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with its precise expression controlled by complex transcriptional networks. While upstream regulatory regions are well-studied, the role of downstream cis-regulatory elements in modulating FT expression remains poorly characterized. RESULTS:Systematic dissection of the FT downstream region in its native chromosomal context using CRISPR/Cas9-mediated genome editing provides genetic evidence that a 2.3-kb sequence, encompassing the Block E enhancer immediately adjacent to the FT coding sequence, is essential for proper FT expression and timely flowering. Fine-scale deletions within Block E reveal that a 63-bp sequence containing one CCAAT-box and one G-box, both closely spaced, forms a core functional module, whereas other conserved motifs contribute modestly in a context-dependent manner. Strikingly, a cryptic CCAAT-box module downstream of Block E that becomes active when repositioned. This coincides with increased transcription factor occupancy and local chromatin accessibility. CONCLUSIONS:Our work reveals that quantitative FT expression and flowering time are governed by the spatial organization and chromatin context of downstream cis-regulatory elements. The positional sensitivity and modular logic of these elements provide framework for understanding and engineering quantitative gene regulation through targeted cis-regulatory design, a concept broadly applicable across diverse developmental systems.

Epigenetic regulators shape chromatin landscapes, allowing cells to express distinct gene sets depending on cell-type, developmental stage or environmental cues. These regulatory complexes rely on interactions with sequence-specific DNA binding proteins, such as the small family of TELOMERE REPEAT BINDING FACTORS (TRBs). TRBs are components of chromatin regulatory complexes with opposing functions, such as the epigenetic repressors Polycomb Repressive Complex 2 (PRC2) and a JMJ14/NAC complex that respectively add and removes the repressive H3K27me3 and positive H3K4me3 modification, but also with the plant-specific PEAT complex that is linked to histone acetylation and gene activation. We dissected the partial redundancy between TRB1, TRB2 and TRB3 in target gene selection and interaction with different chromatin regulatory complexes. High redundancy of TRBs is suggested by major phenotypic changes that are only observed trb triple mutants; however, we found different target site preference between TRB1-3 and preferred partnership with chromatin complexes. Furthermore, TRB paralogs interacted with the NuA4 histone acetylation complex, both together with and in absence of PEAT. Among the three paralogs, TRB1 had more unique binding sites and correlated stronger with PEAT and NuA4 functions. In contrast, TRB2 and TRB3 were more dependent on the presence of bona fide telo-box motifs and were more likely to be found at PRC2 associated sites. Overall, we provide insight into the diverse roles of TRBs in epigenetic gene regulation and how their diversification contributes to their apparent redundancy, as well as their observed activating and repressing effects on gene expression.

Plants utilize environmental information to modify their developmental trajectories for optimal survival and reproduction. Over a century ago, day length (photoperiod) was identified as a major factor influencing developmental transitions, particularly the shift from vegetative to reproductive growth. In rice (Oryza sativa), exposure to day lengths shorter than a critical threshold accelerates flowering, while longer days inhibit this process. This response is mediated by HEADING DATE 1 (Hd1), a zinc finger transcription factor that is central in the photoperiodic flowering network. Hd1 acts as a repressor of flowering under long days but functions as a promoter of flowering under short days. However, how global transcription of genes downstream of Hd1 changes in response to the photoperiod is still not fully understood. Furthermore, it is unclear whether Hd1 target genes are solely involved in flowering time control or mediate additional functions. In this study, we utilized RNA-Seq to analyze the transcriptome of hd1 mutants under both long and short day conditions. We identified genes involved in the phenylpropanoid pathway that are deregulated under long days in the mutant. Quantitative profiling of cell wall components and abiotic stress assays suggested that Hd1 is involved in processes considered unrelated to flowering control. This indicates that day length perception and responses are intertwined with physiological processes beyond flowering.

In many species, FLOWERING LOCUS T (FT)-like genes promote the floral transition by integrating environmental signals, in particular photoperiod, and internal cues. Here we show that Brassica napus contains six FT-like genes and two pseudogenes belonging to three orthogroups. All B. napus FT-like genes induce early flowering when expressed at the shoot apical meristems of Arabidopsis thaliana ft mutants; however, BnaFT.C6 and non-orthologous FT-like genes do not encode fully functional mobile florigens. In the case of BnFT.C6, the functional change is associated with a T to C amino acid change that is restricted to semi-winter accessions. Expression of orthologs of FT is photoperiod-dependent, and two distal enhancers are conserved; however, the homeologs BnaFT.A7 and BnaFT.C6 show rearrangements of DNA motifs binding NF-Y/CO and NF-Y transcriptional activator complexes between the promoter and downstream enhancers. Motif rearrangements correlate with differences in tissue-specific expression. Furthermore, homeologs with rearranged motifs could not be transactivated by B. napus CO in transient assays, although they show LD photoperiod-dependent expression. We propose that differential diurnal expression of NF-Y genes contributes to the photoperiod-dependent regulation of B. napus FT genes.

Photosynthesis is an essential process in plants that synthesizes sugars used for growth and development, highlighting the importance of establishing robust methods to monitor photosynthetic activity. Infrared gas analysis (IRGA) can be used to track photosynthetic rates by measuring plant CO2 assimilation and release. Although much progress has been made in the development of IRGA technologies, challenges remain when using this technique on small herbaceous plants such as Arabidopsis thaliana. The use of whole plant chambers can overcome the difficulties associated with applying bulky leaf clamps to small delicate leaves. However, respiration from the roots and from soil-based microorganisms may skew these gas exchange measurements. Here, we present a simple method to efficiently perform IRGA on A. thaliana plants using a whole plant chamber that removes the confounding effects of respiration from roots and soil-based microorganisms from the measurements. We show that this method can be used to detect subtle changes in photosynthetic rates measured at different times of day, under different growth conditions, and between wild-type and plants with deficiencies in the photosynthetic machinery. Furthermore, we show that this method can be used to detect changes in photosynthetic rates even at very young developmental stages such as 10 d-old seedlings. This method contributes to the array of techniques currently used to perform IRGA on A. thaliana and can allow for the monitoring of photosynthetic rates of whole plants from young ages.

The FLOWERING LOCUS T ( FT ) gene is a central integrator of floral induction in Arabidopsis thaliana , with expression tightly regulated by complex transcriptional networks. Using CRISPR/Cas9 genome editing, we dissected the functional architecture of the FT downstream region and reveal that a 2.3-kb region immediately downstream of the FT coding sequence containing the Block E enhancer is essential for proper FT expression and flowering. Fine-scale deletions revealed a 63-bp core module with adjacent CCAAT- and G-boxes, whereas other conserved motifs had minor, context-dependent effects. We also uncovered a cryptic CCAAT-box module that becomes active when repositioned, coinciding with increased transcription factor binding and local chromatin accessibility, indicating that enhancer function is governed by local chromatin and motif context. The cis -regulatory logic revealed here provides insights into manipulating gene expression through the architecture and spatial arrangement of enhancer elements, potentially applicable beyond flowering genes or plant species.

Transcriptional enhancers participate in cell and tissue differentiation in all multicellular organisms. Here, we characterized the candidate enhancer Vegetative to generative1 (Vgt1), a major QTL for flowering time in maize. Transgenic lines containing an inverted repeat that induces DNA methylation at Vgt1 showed early flowering and an accelerated growth rate during early development. DNA methylation of Vgt1 was associated with the downregulation of the AP2-like floral repressor ZmRap2.7 in specific leaf tissues at the early stages of maize development. In line with Vgt1 regulating ZmRap2.7, chromosome conformation capture data showed that Vgt1 physically interacts with the ZmRap2.7 transcription start site. Finally, chromatin immunoprecipitation of transiently expressed ZmRap2.7 in protoplasts indicated that this transcription factor binds to the promoters of several hundred genes. These genes include many genes that are differentially expressed in maize lines with and without extra DNA methylation at Vgt1. Altogether, we show that ZmRap2.7 is transcriptionally controlled by Vgt1 and is involved in regulating flowering time and other biological pathways, such as jasmonate signaling.

Flowering time is an important crop trait, as premature or delayed flowering can significantly reduce yields. Plants flower in response to a defined set of endogenous and environmental cues. The effect of ambient temperature on flowering time is extensively studied, but the role of epigenetic modifiers on the floral thermosensory pathway remains largely unexplored. Here we describe that the histone demethylases EARLY FLOWERING 6 (ELF6) and JUMONJI 13 (JMJ13) fine-tune temperature-dependent flowering. We found that the elf6-3 jmj13-1 double mutant is hyposensitive to the effect of low ambient temperature on flowering time. Genetic analyses demonstrate that ELF6 and JMJ13 function requires the cold-responsive floral repressors FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM). ELF6 and JMJ13 bind to the chromatin of FLC and FLM loci at both temperature regimes, with increased binding to FLM observed at low ambient temperatures. Interestingly, we also detected increased levels of H3 lysine 27 trimethylation (H3K27me3) in the elf6-3 jmj13-1 double mutant at low ambient temperature in FLC and FLM. Our work demonstrates that ELF6 and JMJ13 are required to modulate FLC and FLM gene expression in response to ambient temperature fluctuations, revealing H3K27me3 demethylases as novel players in the floral thermosensory pathway.

Plants utilize environmental information to modify their developmental trajectories for optimal survival and reproduction. Over a century ago, day length (photoperiod) was identified as a major factor influencing developmental transitions, particularly the shift from vegetative to reproductive growth. In rice, exposure to day lengths shorter than a critical threshold accelerates flowering, while longer days inhibit this process. This response is mediated by HEADING DATE 1 (Hd1), a zinc finger transcription factor that is central in the photoperiodic flowering network. Hd1 acts as a repressor of flowering under long days but functions as a promoter of flowering under short days. However, the transcriptional organization of this dual function is still not fully understood. In this study, we utilized RNA-Seq to analyze the transcriptome of hd1 mutants under both long and short day conditions. We identified genes involved in the phenylpropanoid pathway that are deregulated under long days in the mutant. Quantitative profiling of cell wall components and abiotic stress assays suggest that Hd1 is involved in processes considered unrelated to flowering control. This indicates that day length perception and responses are intertwined with physiological processes beyond flowering.

AbstractPhotosynthesis is an essential process in plants that synthesizes sugars used for growth and development, highlighting the importance of establishing robust methods to monitor photosynthetic activity. Infrared gas analysis (IRGA) can be used to track photosynthetic rates by measuring the CO2assimilation/release from a plant. Although much progress has been made in the development of IRGA technologies, challenges remain when using this technique on small herbaceous plants such asArabidopsis thaliana. The use of whole plant chambers can overcome the difficulties associated with applying bulky leaf clamps to small delicate leaves, however this introduces the risk of soil-based microorganisms skewing gas exchange measurements. Here, we present a simple method to efficiently perform IRGA onA. thalianaplants using a whole plant chamber that removes soil-borne effects from the measurements. We show that this method can be used to detect subtle changes in photosynthetic rates measured at different times of day, under different growth conditions, and between wild-type and plants with deficiencies in the photosynthetic machinery. Furthermore, we show that this method can be used to detect changes in photosynthetic rates even at very young developmental stages such as 10 d-old seedlings. This method contributes to the array of techniques currently used to perform IRGA onA. thalianaand can allow for the monitoring of photosynthetic rates of whole plants from young ages.

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species.

Photoperiod plays a key role in controlling the phase transition from vegetative to reproductive growth in flowering plants. Leaves are the major organs perceiving day-length signals, but how specific leaf cell types respond to photoperiod remains unknown. We integrated photoperiod-responsive chromatin accessibility and transcriptome data in leaf epidermis and vascular companion cells of Arabidopsis thaliana by combining isolation of nuclei tagged in specific cell/tissue types with assay for transposase-accessible chromatin using sequencing and RNA-sequencing. Despite a large overlap, vasculature and epidermis cells responded differently. Long-day predominantly induced accessible chromatin regions (ACRs); in the vasculature, more ACRs were induced and these were located at more distal gene regions, compared with the epidermis. Vascular ACRs induced by long days were highly enriched in binding sites for flowering-related transcription factors. Among the highly ranked genes (based on chromatin and expression signatures in the vasculature), we identified TREHALOSE-PHOSPHATASE/SYNTHASE 9 (TPS9) as a flowering activator, as shown by the late flowering phenotypes of T-DNA insertion mutants and transgenic lines with phloem-specific knockdown of TPS9. Our cell-type-specific analysis sheds light on how the long-day photoperiod stimulus impacts chromatin accessibility in a tissue-specific manner to regulate plant development.

Flowering transition is pivotal and tightly regulated by complex gene-regulatory-networks, in which AGL16 plays important roles. But the molecular function and binding property of AGL16 is not fully explored in vivo . With ChIP-seq and comparative transcriptomics approaches, we characterized the AGL16 targets spectrum and tested its close molecular and genetic interactions with SOC1 , the key flowering integrator. AGL16 bound to promoters of more than 2000 genes via CArG-box motifs that were highly similar to that of SOC1. Being consistent with this, AGL16 formed protein complex and shared a common set of targets with SOC1. However, only very few genes showed differential expression in the agl16-1 loss-of-function mutant, whereas in the soc1-2 knockout background, AGL16 repressed and activated the expression of 375 and 182 genes, respectively, with more than a quarter of the DEGs were also bound by AGL16. AGL16 targeted potentially to about seventy flowering time genes involved in multiple pathways. Corroborating with these, AGL16 repressed the flowering time stronger in soc1-2 than in Col-0 background. These data reveals that AGL16 regulates gene expression and flowering time with a partial dependency on SOC1 activity. Moreover, AGL16 participated in the regulation of water loss and seed dormancy. Our study thus defines the AGL16 molecular spectrum and provides insights underlining the molecular coordination of flowering and environmental adaptation.

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

In order to successfully reproduce, plants must sense changes in their environment and flower at the correct time. Many plants utilize day length and vernalization, a mechanism for verifying that winter has occurred, to determine when to flower. Our study used available temperature and day length data from different climates to provide a general understanding how this information processing of environmental signals could have evolved in plants. For climates where temperature fluctuation correlations decayed exponentially, a simple stochastic model characterizing vernalization was able to reconstruct the switch-like behavior of the core flowering regulatory genes. For these and other climates, artificial neural networks were used to predict flowering gene expression patterns. For temperate plants, long-term cold temperature and short-term day length measurements were sufficient to produce robust flowering time decisions from the neural networks. Additionally, evolutionary simulations on neural networks confirmed that the combined signal of temperature and day length achieved the highest fitness relative to neural networks with access to only one of those inputs. We suggest that winter temperature memory is a well-adapted strategy for plants' detection of seasonal changes, and absolute day length is useful for the subsequent triggering of flowering.

The multicellular embryo, and ultimately the entire organism, is a derivative of the fertilized egg cell. Unlike in animals, transcription factor networks orchestrating faithful egg development are still largely unknown in plants. We have identified that egg cell differentiation in Arabidopsis require interplay between evolutionarily conserved onco-protein homologs RETINOBLASTOMA-RELATED (RBR) and redundant MYB proteins MYB64/MYB119. RBR physically interacts with the MYBs; and with plant-specific transcription factors belonging to the RWP-RK-domain (RKD) family and LEAFY COTYLEDON1 (LEC1), which participate in development of egg cells and inherent stress response. RBR binds to most of these egg cell-expressed loci at the DNA level, partially overlapping with sites of histone methylation H3K27me3. Since deregulation of RKD s phenocopies mutants of RBR and the MYB s in terms of cell proliferation in the egg cell spatial domain, all the corresponding proteins are likely required to restrict parthenogenetic cell divisions of the egg cells. Cross-talk among these transcription factors, and direct regulation by RBR, govern egg cell development and expression of egg-to-zygotic polarity factors of the WUSCHEL RELATED HOMEOBOX family. Together, a network of RBR-centric transcription factors underlies egg cell development and stress response, possibly, in combination with several other predicted nodes. Author summary The RETINOBLASTOMA protein is one of the core components of the Eukaryotic cell cycle, and corresponding evolutionary homologs have been implicated not only to repress cell division but also to control differentiation and development. How RETINOBLASTOMA RELATED (RBR) associate with other higher order regulators to control faithful egg cell development in sexual plants is pivotal for manipulation of successful reproduction in general, and engineering of parthenogenesis when asexual or apomictic seed progeny are desirable over sexual plants. Using a suite of molecular methods, we show that a RBR-associated transcription factor network operates to specify egg cells in Arabidopsis . Complex cross-regulation within these transcription factors seems to be necessary for successful maternal egg cell to zygotic transition and reproductive stress response. Detailed genetic analysis implicate that RBR and its interactive partners belonging to MYB and RWP-RK transcription factor families are possibly required to prevent parthenogenesis of the sexual egg cells. Novel RBR networks and stress nodes explained in this study might help to improve our understanding of sexual and asexual reproduction.

FLOWERING LOCUS T ( FT ) plays a major role in regulating the floral transition in response to an inductive long day photoperiod in Arabidopsis thaliana . Expression of FT in leaves is dependent on the distal transcriptional enhancer Block C , located 5-kilobases (kb) upstream of the transcriptional start site (TSS). We expressed an inverted repeat of Block C to induce local DNA methylation and heterochromatin formation, which lead to FT downregulation in an inductive photoperiod. Using targeted DNA methylation as a tool to uncover further regulatory regions at the FT locus, we identified Block E , located 1 kb downstream of the gene, as a novel enhancer of FT . As Block C , Block E is conserved across Brassicaceae and located in accessible chromatin. Block C and E act as additive transcriptional enhancers that, in combination with the proximal FT promoter, control expression of FT in response to photoperiod in the leaf phloem.

Two new studies show that a plant-specific complex composed of EBS, or its homolog SHL, and EMF1 acts as a chromatin reader within the Polycomb pathway and effects gene repression. Two domains of EBS and SHL bind distinct chromatin modifications that are associated with active and repressed chromatin.