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.

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.

Genome-edited HEADING DATE 3a knockout enhances leaf production in Perilla frutescens.

Environmental cues regulate the transition of many plants from vegetative to flowering development. Day length, or photoperiod, is one cue that synchronizes flowering by changing seasons. Consequently, the molecular mechanism of flowering control is prominent in Arabidopsis and rice, where essential genes like FLOWERING LOCUS T (FT) homolog, HEADING DATE 3a (Hd3a), have been connected to flowering regulation. Perilla is a nutrient-rich leaf vegetable, and the flowering mechanism remains largely elusive. We identified flowering-related genes under short-day conditions using RNA sequencing to develop an enhanced leaf production trait using the flowering mechanism in the perilla. Initially, an Hd3a-like gene was cloned from the perilla and defined as PfHd3a. Furthermore, PfHd3a is highly rhythmically expressed in mature leaves under short-day and long-day conditions. Ectopic expression of PfHd3a in Atft-1 mutant plants has been shown to complement Arabidopsis FT function, resulting in early flowering. In addition, our genetic approaches revealed that overexpression of PfHd3a in perilla caused early flowering. In contrast, the CRISPR/Cas9 generated PfHd3a-mutant perilla showed significantly late flowering, resulting in approximately 50% leaf production enhancement compared to the control. Our results suggest that PfHd3a plays a vital role in regulating flowering in the perilla and is a potential target for molecular breeding in the perilla.

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.

Temperature-mediated regulation of flowering time in Arabidopsis thaliana.

Throughout a plant's life cycle, temperature plays a major role in development. Regulatory modules use temperature cues to control gene expression, facilitating physiological change from germination to flowering. These regulatory modules control morphological and molecular responses to temperature changes caused by seasonal changes or by temporary fluctuations, providing a versatile plasticity of plants. In this review, we outline how temperature changes affect the regulatory modules that induce and repress flowering, in addition to general temperature regulation. Recent studies have identified several regulatory modules by which floral transition and growth responses are controlled in a temperature-dependent manner. This review will report on recent studies related to floral transition and ambient temperature response.

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.

Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors.

BACKGROUND: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. RESULTS: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. CONCLUSIONS: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

Phytochrome B triggers light-dependent chromatin remodelling through the PRC2-associated PHD finger protein VIL1.

To compensate for a sessile nature, plants have developed sophisticated mechanisms to sense varying environmental conditions. Phytochromes (phys) are light and temperature sensors that regulate downstream genes to render plants responsive to environmental stimuli1-4. Here, we show that phyB directly triggers the formation of a repressive chromatin loop by physically interacting with VERNALIZATION INSENSITIVE 3-LIKE1/VERNALIZATION 5 (VIL1/VRN5), a component of Polycomb Repressive Complex 2 (PRC2)5,6, in a light-dependent manner. VIL1 and phyB cooperatively contribute to the repression of growth-promoting genes through the enrichment of Histone H3 Lys27 trimethylation (H3K27me3), a repressive histone modification. In addition, phyB and VIL1 mediate the formation of a chromatin loop to facilitate the repression of ATHB2. Our findings show that phyB directly utilizes chromatin remodelling to regulate the expression of target genes in a light-dependent manner.

Chromatin architectural proteins regulate flowering time by precluding gene looping.

Chromatin structure is critical for gene expression and many other cellular processes. In Arabidopsis thaliana, the floral repressor FLC adopts a self-loop chromatin structure via bridging of its flanking regions. This local gene loop is necessary for active FLC expression. However, the molecular mechanism underlying the formation of this class of gene loops is unknown. Here, we report the characterization of a group of linker histone-like proteins, named the GH1-HMGA family in Arabidopsis, which act as chromatin architecture modulators. We demonstrate that these family members redundantly promote the floral transition through the repression of FLC A genome-wide study revealed that this family preferentially binds to the 5' and 3' ends of gene bodies. The loss of this binding increases FLC expression by stabilizing the FLC 5' to 3' gene looping. Our study provides mechanistic insights into how a family of evolutionarily conserved proteins regulates the formation of local gene loops.

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.

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.

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.

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.

Identification of Long Noncoding RNAs in the Developing Endosperm of Maize.

Maize endosperm consists of three distinct types of tissues, including the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Compartmentalization of these tissues during endosperm differentiation makes the endosperm development an excellent model to study changes in gene expression during development. By utilizing cryo-dissection of developing endosperm, morphologically distinct samples can be obtained for transcriptome and epigenome analysis. Here, we describe methods for the isolation of tissues from developing maize endosperm and for the transcriptome analysis to identify novel long noncoding RNAs. The transcriptome data can be further analyzed to illustrate spatiotemporal changes in both coding and noncoding transcripts during the endosperm development.

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.

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.

Spatio-temporal analysis of coding and long noncoding transcripts during maize endosperm development.

The maize endosperm consists of three major compartmentalized cell types: the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Differential genetic programs are activated in each cell type to construct functionally and structurally distinct cells. To compare gene expression patterns involved in maize endosperm cell differentiation, we isolated transcripts from cryo-dissected endosperm specimens enriched with BETL, AL, or SE at 8, 12, and 16 days after pollination (DAP). We performed transcriptome profiling of coding and long noncoding transcripts in the three cell types during differentiation and identified clusters of the transcripts exhibiting spatio-temporal specificities. Our analysis uncovered that the BETL at 12 DAP undergoes the most dynamic transcriptional regulation for both coding and long noncoding transcripts. In addition, our transcriptome analysis revealed spatio-temporal regulatory networks of transcription factors, imprinted genes, and loci marked with histone H3 trimethylated at lysine 27. Our study suggests that various regulatory mechanisms contribute to the genetic networks specific to the functions and structures of the cell types of the endosperm.

Accelerated vernalization response by an altered PHD-finger protein in Arabidopsis.

Vernalization is a response to the winter cold to acquire the competence to flower in next spring. VERNALIZATION INSENSITIVE 3 (VIN3) is a PHD-finger protein that binds to modified histones in vitro. VIN3 is induced by long-term cold and is necessary for Polycomb Repression Complex 2 (PRC2)-mediated tri-methylation of Histone H3 Lysine 27 (H3K27me3) at the FLC locus in Arabidopsis. An alteration in the PHD-finger domain of VIN3 changes the binding specificity of the PHD-finger domain of VIN3 in vitro and results in an accelerated vernalization response in vivo. The acceleration in vernalization response is achieved by increased enrichments of VIN3 and tri-methylation of Histone H3 Lysine 27 (H3K27me3) at the FLC locus without invoking the increased enrichment of Polycomb Repressive Complex 2. This result indicates that the binding specificity of the PHD-finger domain of VIN3 plays a role in mediating a proper vernalization response in Arabidopsis. Furthermore, this work shows a potential that the alteration of PHD-finger domains could be applied to alter various developmental processes in plants.

Vernalization-Triggered Intragenic Chromatin Loop Formation by Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) affect gene regulation through structural and regulatory interactions with associated proteins. The Polycomb complex often binds to lncRNAs in eukaryotes, and an lncRNA, COLDAIR, associates with Polycomb to mediate silencing of the floral repressor FLOWERING LOCUS C (FLC) during the process of vernalization in Arabidopsis. Here, we identified an additional Polycomb-binding lncRNA, COLDWRAP. COLDWRAP is derived from the repressed promoter of FLC and is necessary for the establishment of the stable repressed state of FLC by vernalization. Both COLDAIR and COLDWRAP are required to form a repressive intragenic chromatin loop at the FLC locus by vernalization. Our results indicate that vernalization-mediated Polycomb silencing is coordinated by lncRNAs in a cooperative manner to form a stable repressive chromatin structure.