COP1 controls light-dependent chromatin remodeling.

Light is a crucial environmental factor that impacts various aspects of plant development. Phytochromes, as light sensors, regulate myriads of downstream genes to mediate developmental reprogramming in response to changes in environmental conditions. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is an E3 ligase for a number of substrates in light signaling, acting as a central repressor of photomorphogenesis. The interplay between phytochrome B (phyB) and COP1 forms an antagonistic regulatory module that triggers extensive gene expression reprogramming when exposed to light. Here, we uncover a role of COP1 in light-dependent chromatin remodeling through the regulation of VIL1 (VIN3-LIKE 1)/VERNALIZATION 5, a Polycomb protein. VIL1 directly interacts with phyB and regulates photomorphogenesis through the formation of repressive chromatin loops at downstream growth-promoting genes in response to light. Furthermore, we reveal that COP1 governs light-dependent formation of chromatin loop and limiting a repressive histone modification to fine-tune expressions of growth-promoting genes during photomorphogenesis through VIL1.

Warm temperature-triggered developmental reprogramming requires VIL1-mediated, genome-wide H3K27me3 accumulation in Arabidopsis.

Changes in ambient temperature immensely affect developmental programs in many species. Plants adapt to high ambient growth temperature in part by vegetative and reproductive developmental reprogramming, known as thermo-morphogenesis. Thermo-morphogenesis is accompanied by massive changes in the transcriptome upon temperature change. Here, we show that transcriptome changes induced by warm ambient temperature require VERNALIZATION INSENSITIVE 3-LIKE 1 (VIL1), a facultative component of the Polycomb repressive complex PRC2, in Arabidopsis. Warm growth temperature elicits genome-wide accumulation of H3K27me3 and VIL1 is necessary for the warm temperature-mediated accumulation of H3K27me3. Consistent with its role as a mediator of thermo-morphogenesis, loss of function of VIL1 results in hypo-responsiveness to warm ambient temperature. Our results show that VIL1 is a major chromatin regulator in responses to high ambient temperature.

Chromatin sensing: Integration of environmental signals to reprogram plant development through chromatin regulators.

Chromatin regulation in eukaryotes plays pivotal roles in controlling developmental regulatory gene network. This review explores the intricate interplay between chromatin regulators and environmental signals, elucidating their roles in shaping plant development. As sessile organisms, plants have evolved sophisticated mechanisms to perceive and respond to environmental cues, orchestrating developmental programs that ensure adaptability and survival. A central aspect of this dynamic response lies in the modulation of versatile gene regulatory networks, mediated in part by various chromatin regulators. Here, we summarized the current understanding of the molecular mechanisms through which chromatin regulators integrate environmental signals, influencing key aspects of plant development.

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.

Abscisic acid negatively regulates the Polycomb-mediated H3K27me3 through the PHD-finger protein, VIL1.

Polycomb dictates developmental programs in higher eukaryotes, including flowering plants. A phytohormone, abscisic acid (ABA), plays a pivotal role in seed and seedling development and mediates responses to multiple environmental stresses, such as salinity and drought. In this study, we show that ABA affects the Polycomb Repressive Complex 2 (PRC2)-mediated Histone H3 Lys 27 trimethylation (H3K27me3) through VIN3-LIKE1/VERNALIZATION 5 (VIL1/VRN5) to fine-tune the timely repression of ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABSCISIC ACID INSENSITIVE 4 (ABI4) in Arabidopsis thaliana. vil1 mutants exhibit hypersensitivity to ABA during early seed germination and show enhanced drought tolerance. Our study revealed that the ABA signaling pathway utilizes a facultative component of the chromatin remodeling complex to demarcate the level of expression of ABA-responsive genes.

Transcriptome and epigenome analyses of vernalization in Arabidopsis thaliana.

Vernalization accelerates flowering after prolonged winter cold. Transcriptional and epigenetic changes are known to be involved in the regulation of the vernalization response. Despite intensive applications of next-generation sequencing in diverse aspects of plant research, genome-wide transcriptome and epigenome profiling during the vernalization response has not been conducted. In this work, to our knowledge, we present the first comprehensive analyses of transcriptomic and epigenomic dynamics during the vernalization process in Arabidopsis thaliana. Six major clusters of genes exhibiting distinctive features were identified. Temporary changes in histone H3K4me3 levels were observed that likely coordinate photosynthesis and prevent oxidative damage during cold exposure. In addition, vernalization induced a stable accumulation of H3K27me3 over genes encoding many development-related transcription factors, which resulted in either inhibition of transcription or a bivalent status of the genes. Lastly, FLC-like and VIN3-like genes were identified that appear to be novel components of the vernalization pathway.

Nuclear import of LIKE HETEROCHROMATIN PROTEIN1 is redundantly mediated by importins α-1, α-2 and α-3.

LIKE HETEROCHROMATIN PROTEIN1 (LHP1) encodes the only plant homologue of the metazoan HETEROCHROMATIN PROTEIN1 (HP1) protein family. The LHP1 protein is necessary for proper epigenetic regulation of a range of developmental processes in plants. LHP1 is a transcriptional repressor of flowering-related genes, such as FLOWERING LOCUS T (FT), FLOWERING LOCUS C (FLC), AGAMOUS (AG) and APETALA 3 (AP3). We found that LHP1 interacts with importin α-1 (IMPα-1), importin α-2 (IMPα-2) and importin α-3 (IMPα-3) both in vitro and in vivo. A genetic approach revealed that triple mutation of impα-1, impα-2 and impα-3 resulted in Arabidopsis plants with a rapid flowering phenotype similar to that of plants with mutations in lhp1 due to the upregulation of FT expression. Nuclear targeting of LHP1 was severely impaired in the impα triple mutant, resulting in the de-repression of LHP1 target genes AG, AP3 and SHATTERPROOF 1 as well as FT. Therefore, the importin proteins IMPα-1, -2 and -3 are necessary for the nuclear import of LHP1.

Direct phosphorylation of HY5 by SPA kinases to regulate photomorphogenesis in Arabidopsis.

Elongated hypocotyl5 (HY5) is a key transcription factor that promotes photomorphogenesis. Constitutive photomorphogenic1 (COP1)-Suppressor of phytochrome A-105 (SPA) E3 ubiquitin ligase complex promotes ubiquitination and degradation of HY5 to repress photomorphogenesis in darkness. HY5 is also regulated by phosphorylation at serine 36 residue. However, the kinase responsible for phosphorylation of HY5 remains unknown. Here, using extensive in vitro and in vivo biochemical, genetic, and photobiological techniques, we have identified a new kinase that phosphorylates HY5 and demonstrated the significance of phosphorylation of HY5 in Arabidopsis thaliana. We show that SPA proteins are the missing kinases necessary for HY5 phosphorylation. SPAs can directly phosphorylate HY5 in vitro, and the phosphorylated HY5 is absent in the spaQ background in vivo. We also demonstrate that the unphosphorylated HY5 interacts strongly with both COP1 and SPA1 and is the preferred substrate for degradation, whereas the phosphorylated HY5 is more stable in the dark. In addition, the unphosphorylated HY5 actively binds to the target promoters and is the physiologically more active form. Consistently, the transgenic plants expressing the unphosphorylated form of HY5 display enhanced photomorphogenesis. Collectively, our study revealed the missing kinase responsible for direct phosphorylation of HY5 that fine-tunes its stability and activity to regulate photomorphogenesis.

DEK domain-containing proteins control flowering time in Arabidopsis.

Evolutionarily conserved DEK domain-containing proteins have been implicated in multiple chromatin-related processes, mRNA splicing and transcriptional regulation in eukaryotes. Here, we show that two DEK proteins, DEK3 and DEK4, control the floral transition in Arabidopsis. DEK3 and DEK4 directly associate with chromatin of related flowering repressors, FLOWERING LOCUS C (FLC), and its two homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, to promote their expression. The binding of DEK3 and DEK4 to a histone octamer in vivo affects histone modifications at FLC, MAF4 and MAF5 loci. In addition, DEK3 and DEK4 interact with RNA polymerase II and promote the association of RNA polymerase II with FLC, MAF4 and MAF5 chromatin to promote their expression. Our results show that DEK3 and DEK4 directly interact with chromatin to facilitate the transcription of key flowering repressors and thus prevent precocious flowering in Arabidopsis.

Modular function of long noncoding RNA, COLDAIR, in the vernalization response.

The long noncoding RNA COLDAIR is necessary for the repression of a floral repressor FLOWERING LOCUS C (FLC) during vernalization in Arabidopsis thaliana. The repression of FLC is mediated by increased enrichment of Polycomb Repressive Complex 2 (PRC2) and subsequent trimethylation of Histone H3 Lysine 27 (H3K27me3) at FLC chromatin. In this study we found that the association of COLDAIR with chromatin occurs only at the FLC locus and that the central region of the COLDAIR transcript is critical for this interaction. A modular motif in COLDAIR is responsible for the association with PRC2 in vitro, and the mutations within the motif that reduced the association of COLDAIR with PRC2 resulted in vernalization insensitivity. The vernalization insensitivity caused by mutant COLDAIR was rescued by the ectopic expression of the wild-type COLDAIR. Our study reveals the molecular framework in which COLDAIR lncRNA mediates the PRC2-mediated repression of FLC during vernalization.

NO FLOWERING IN SHORT DAY (NFL) is a bHLH transcription factor that promotes flowering specifically under short-day conditions in Arabidopsis.

Flowering in plants is a dynamic and synchronized process where various cues including age, day length, temperature and endogenous hormones fine-tune the timing of flowering for reproductive success. Arabidopsis thaliana is a facultative long day (LD) plant where LD photoperiod promotes flowering. Arabidopsis still flowers under short-day (SD) conditions, albeit much later than in LD conditions. Although factors regulating the inductive LD pathway have been extensively investigated, the non-inductive SD pathway is much less understood. Here, we identified a key basic helix-loop-helix transcription factor called NFL (NO FLOWERING IN SHORT DAY) that is essential to induce flowering specifically under SD conditions in Arabidopsis. nfl mutants do not flower under SD conditions, but flower similar to the wild type under LD conditions. The no-flowering phenotype in SD is rescued either by exogenous application of gibberellin (GA) or by introducing della quadruple mutants in the nfl background, suggesting that NFL acts upstream of GA to promote flowering. NFL is expressed at the meristematic regions and NFL is localized to the nucleus. Quantitative RT-PCR assays using apical tissues showed that GA biosynthetic genes are downregulated and the GA catabolic and receptor genes are upregulated in the nfl mutant compared with the wild type, consistent with the perturbation of the endogenous GA biosynthetic and catabolic intermediates in the mutant. Taken together, these data suggest that NFL is a key transcription factor necessary for promotion of flowering under non-inductive SD conditions through the GA signaling pathway.

Phosphorylation of CBP20 Links MicroRNA to Root Growth in the Ethylene Response.

Ethylene is one of the most important hormones for plant developmental processes and stress responses. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of cap binding protein 20 (CBP20) at Ser245 is regulated by ethylene, and the phosphorylation is involved in root growth. The constitutive phosphorylation mimic form of CBP20 (CBP20S245E or CBP20S245D), while not the constitutive de-phosphorylation form of CBP20 (CBP20S245A) is able to rescue the root ethylene responsive phenotype of cbp20. By genome wide study with ethylene regulated gene expression and microRNA (miRNA) expression in the roots and shoots of both Col-0 and cbp20, we found miR319b is up regulated in roots while not in shoots, and its target MYB33 is specifically down regulated in roots with ethylene treatment. We described both the phenotypic and molecular consequences of transgenic over-expression of miR319b. Increased levels of miR319b (miR319bOE) leads to enhanced ethylene responsive root phenotype and reduction of MYB33 transcription level in roots; over expression of MYB33, which carrying mutated miR319b target site (mMYB33) in miR319bOE is able to recover both the root phenotype and the expression level of MYB33. Taken together, we proposed that ethylene regulated phosphorylation of CBP20 is involved in the root growth and one pathway is through the regulation of miR319b and its target MYB33 in roots.

RNA-seq assistant: machine learning based methods to identify more transcriptional regulated genes.

BACKGROUND: Although different quality controls have been applied at different stages of the sample preparation and data analysis to ensure both reproducibility and reliability of RNA-seq results, there are still limitations and bias on the detectability for certain differentially expressed genes (DEGs). Whether the transcriptional dynamics of a gene can be captured accurately depends on experimental design/operation and the following data analysis processes. The workflow of subsequent data processing, such as reads alignment, transcript quantification, normalization, and statistical methods for ultimate identification of DEGs can influence the accuracy and sensitivity of DEGs analysis, producing a certain number of false-positivity or false-negativity. Machine learning (ML) is a multidisciplinary field that employs computer science, artificial intelligence, computational statistics and information theory to construct algorithms that can learn from existing data sets and to make predictions on new data set. ML-based differential network analysis has been applied to predict stress-responsive genes through learning the patterns of 32 expression characteristics of known stress-related genes. In addition, the epigenetic regulation plays critical roles in gene expression, therefore, DNA and histone methylation data has been shown to be powerful for ML-based model for prediction of gene expression in many systems, including lung cancer cells. Therefore, it is promising that ML-based methods could help to identify the DEGs that are not identified by traditional RNA-seq method. RESULTS: We identified the top 23 most informative features through assessing the performance of three different feature selection algorithms combined with five different classification methods on training and testing data sets. By comprehensive comparison, we found that the model based on InfoGain feature selection and Logistic Regression classification is powerful for DEGs prediction. Moreover, the power and performance of ML-based prediction was validated by the prediction on ethylene regulated gene expression and the following qRT-PCR. CONCLUSIONS: Our study shows that the combination of ML-based method with RNA-seq greatly improves the sensitivity of DEGs identification.

The Binding Specificity of the PHD-Finger Domain of VIN3 Moderates Vernalization Response.

Vernalization is a response to winter cold to initiate flowering in spring. VERNALIZATION INSENSITIVE3 (VIN3) is induced by winter cold and is essential to vernalization response in Arabidopsis (Arabidopsis thaliana). VIN3 encodes a PHD-finger domain that binds to modified histones in vitro. An alteration in the binding specificity of the PHD-finger domain of VIN3 results in a hypervernalization response. The hypervernalization response is achieved by increased enrichments of VIN3 and trimethylation of Histone H3 Lys 27 at the FLC locus without invoking the increased enrichment of Polycomb Repressive Complex 2. Our result shows that the binding specificity of the PHD-finger domain of VIN3 plays a role in mediating a proper vernalization response in Arabidopsis.

The catalytic subunit of Arabidopsis DNA polymerase α ensures stable maintenance of histone modification.

Mitotic inheritance of identical cellular memory is crucial for development in multicellular organisms. The cell type-specific epigenetic state should be correctly duplicated upon DNA replication to maintain cellular memory during tissue and organ development. Although a role of DNA replication machinery in maintenance of epigenetic memory has been proposed, technical limitations have prevented characterization of the process in detail. Here, we show that INCURVATA2 (ICU2), the catalytic subunit of DNA polymerase α in Arabidopsis, ensures the stable maintenance of repressive histone modifications. The missense mutant allele icu2-1 caused a defect in the mitotic maintenance of vernalization memory. Although neither the recruitment of CURLY LEAF (CLF), a SET-domain component of Polycomb Repressive Complex 2 (PRC2), nor the resultant deposition of the histone mark H3K27me3 required for vernalization-induced FLOWERING LOCUS C (FLC) repression were affected, icu2-1 mutants exhibited unstable maintenance of the H3K27me3 level at the FLC region, which resulted in mosaic FLC de-repression after vernalization. ICU2 maintains the repressive chromatin state at additional PRC2 targets as well as at heterochromatic retroelements. In icu2-1 mutants, the subsequent binding of LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a functional homolog of PRC1, at PRC2 targets was also reduced. We demonstrated that ICU2 facilitates histone assembly in dividing cells, suggesting a possible mechanism for ICU2-mediated epigenetic maintenance.

Coordination of the vernalization response through a VIN3 and FLC gene family regulatory network in Arabidopsis.

Vernalization is an environmentally induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. Vernalization triggers the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are epigenetically silenced via chromatin modifications. In Arabidopsis thaliana, VERNALIZATION INSENSITIVE3 (VIN3) and its related plant homeodomain finger proteins act together with Polycomb Repressive Complex 2 to increase repressive histone marks at floral repressor loci, including FLOWERING LOCUS C (FLC) and its related genes, by vernalization. Here, we show that VIN3 family of proteins nonredundantly functions to repress different subsets of the FLC gene family during the course of vernalization. Each VIN3 family protein binds to modified histone peptides in vitro and directly associates with specific sets of FLC gene family chromatins in vivo to mediate epigenetic silencing. In addition, members of the FLC gene family are also differentially regulated during the course of vernalization to mediate proper vernalization response. Our results show that these two gene families cooperated during the course of evolution to ensure proper vernalization response through epigenetic changes.

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.

Adaptive expansion of the maize maternally expressed gene (Meg) family involves changes in expression patterns and protein secondary structures of its members.

BACKGROUND: The Maternally expressed gene (Meg) family is a locally-duplicated gene family of maize which encodes cysteine-rich proteins (CRPs). The founding member of the family, Meg1, is required for normal development of the basal endosperm transfer cell layer (BETL) and is involved in the allocation of maternal nutrients to growing seeds. Despite the important roles of Meg1 in maize seed development, the evolutionary history of the Meg cluster and the activities of the duplicate genes are not understood. RESULTS: In maize, the Meg gene cluster resides in a 2.3 Mb-long genomic region that exhibits many features of non-centromeric heterochromatin. Using phylogenetic reconstruction and syntenic alignments, we identified the pedigree of the Meg family, in which 11 of its 13 members arose in maize after allotetraploidization ~4.8 mya. Phylogenetic and population-genetic analyses identified possible signatures suggesting recent positive selection in Meg homologs. Structural analyses of the Meg proteins indicated potentially adaptive changes in secondary structure from α-helix to ß-strand during the expansion. Transcriptomic analysis of the maize endosperm indicated that 6 Meg genes are selectively activated in the BETL, and younger Meg genes are more active than older ones. In endosperms from B73 by Mo17 reciprocal crosses, most Meg genes did not display parent-specific expression patterns. CONCLUSIONS: Recently-duplicated Meg genes have different protein secondary structures, and their expressions in the BETL dominate over those of older members. Together with the signs of positive selections in the young Meg genes, these results suggest that the expansion of the Meg family involves potentially adaptive transitions in which new members with novel functions prevailed over older members.

Epigenetic regulation by long noncoding RNAs in plants.

Many eukaryotes, including plants, produce a large number of long noncoding RNAs (lncRNAs).Growing number of lncRNAs are being reported to have regulatory roles in various developmental processes.Emerging mechanisms underlying the function of lncRNAs indicate that lncRNAs are versatile regulatory molecules. They function as potent cis- and trans-regulators of gene expression, including the formation of modular scaffolds that recruit chromatin-modifying complexes to target chromatin. LncRNAs have also been reported in plants. Here, we describe our current understanding on potential roles of lncRNA in plants.

Ca2+-dependent GTPase, extra-large G protein 2 (XLG2), promotes activation of DNA-binding protein related to vernalization 1 (RTV1), leading to activation of floral integrator genes and early flowering in Arabidopsis.

Heterotrimeric G proteins, consisting of Gα, Gß, and Gγ subunits, play important roles in plant development and cell signaling. In Arabidopsis, in addition to one prototypical G protein α subunit, GPA1, there are three extra-large G proteins, XLG1, XLG2, and XLG3, of largely unknown function. Each extra-large G (XLG) protein has a C-terminal Gα-like region and a ∼400 amino acid N-terminal extension. Here we show that the three XLG proteins specifically bind and hydrolyze GTP, despite the fact that these plant-specific proteins lack key conserved amino acid residues important for GTP binding and hydrolysis of GTP in mammalian Gα proteins. Moreover, unlike other known Gα proteins, these activities require Ca(2+) instead of Mg(2+) as a cofactor. Yeast two-hybrid library screening and in vitro protein pull-down assays revealed that XLG2 interacts with the nuclear protein RTV1 (related to vernalization 1). Electrophoretic mobility shift assays show that RTV1 binds to DNA in vitro in a non-sequence-specific manner and that GTP-bound XLG2 promotes the DNA binding activity of RTV1. Overexpression of RTV1 results in early flowering. Combined overexpression of XLG2 and RTV1 enhances this early flowering phenotype and elevates expression of the floral pathway integrator genes, FT and SOC1, but does not repress expression of the floral repressor, FLC. Chromatin immunoprecipitation assays show that XLG2 increases RTV1 binding to FT and SOC1 promoters. Thus, a Ca(2+)-dependent G protein, XLG2, promotes RTV1 DNA binding activity for a subset of floral integrator genes and contributes to floral transition.