Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controls shade avoidance syndrome in Arabidopsis.

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. In response to far-red (FR) light, expression of APOLO anti-correlates with that of its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. Accumulation of APOLO transcription fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves and in a late response to far-red light treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of auxin synthesis gene YUCCA2, and auxin efflux genes PID and WAG2. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant-environment interactions may therefore become a new tool for sustainable agriculture.

An atlas of the tomato epigenome reveals that KRYPTONITE shapes TAD-like boundaries through the control of H3K9ac distribution.

In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.

Predictable and stable epimutations induced during clonal plant propagation with embryonic transcription factor.

Clonal propagation is frequently used in commercial plant breeding and biotechnology programs because it minimizes genetic variation, yet it is not uncommon to observe clonal plants with stable phenotypic changes, a phenomenon known as somaclonal variation. Several studies have linked epigenetic modifications induced during regeneration with this newly acquired phenotypic variation. However, the factors that determine the extent of somaclonal variation and the molecular changes underpinning this process remain poorly understood. To address this gap in our knowledge, we compared clonally propagated Arabidopsis thaliana plants derived from somatic embryogenesis using two different embryonic transcription factors- RWP-RK DOMAIN-CONTAINING 4 (RKD4) or LEAFY COTYLEDON2 (LEC2) and from two epigenetically distinct founder tissues. We found that both the epi(genetic) status of the explant and the regeneration protocol employed play critical roles in shaping the molecular and phenotypic landscape of clonal plants. Phenotypic variation in regenerated plants can be largely explained by the inheritance of tissue-specific DNA methylation imprints, which are associated with specific transcriptional and metabolic changes in sexual progeny of clonal plants. For instance, regenerants were particularly affected by the inheritance of root-specific epigenetic imprints, which were associated with an increased accumulation of salicylic acid in leaves and accelerated plant senescence. Collectively, our data reveal specific pathways underpinning the phenotypic and molecular variation that arise and accumulate in clonal plant populations.

Polycomb-dependent differential chromatin compartmentalization determines gene coregulation in Arabidopsis.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

Individualized VDJ recombination predisposes the available Ig sequence space.

The process of recombination between variable (V), diversity (D), and joining (J) immunoglobulin (Ig) gene segments determines an individual's naive Ig repertoire and, consequently, (auto)antigen recognition. VDJ recombination follows probabilistic rules that can be modeled statistically. So far, it remains unknown whether VDJ recombination rules differ between individuals. If these rules differed, identical (auto)antigen-specific Ig sequences would be generated with individual-specific probabilities, signifying that the available Ig sequence space is individual specific. We devised a sensitivity-tested distance measure that enables inter-individual comparison of VDJ recombination models. We discovered, accounting for several sources of noise as well as allelic variation in Ig sequencing data, that not only unrelated individuals but also human monozygotic twins and even inbred mice possess statistically distinguishable immunoglobulin recombination models. This suggests that, in addition to genetic, there is also nongenetic modulation of VDJ recombination. We demonstrate that population-wide individualized VDJ recombination can result in orders of magnitude of difference in the probability to generate (auto)antigen-specific Ig sequences. Our findings have implications for immune receptor-based individualized medicine approaches relevant to vaccination, infection, and autoimmunity.

RWP-RK Domain 3 (OsRKD3) induces somatic embryogenesis in black rice.

BACKGROUND: Plants have the unique capability to form embryos from both gametes and somatic cells, with the latter process known as somatic embryogenesis. Somatic embryogenesis (SE) can be induced by exposing plant tissues to exogenous growth regulators or by the ectopic activation of embryogenic transcription factors. Recent studies have revealed that a discrete group of RWP-RK DOMAIN-CONTAINING PROTEIN (RKD) transcription factors act as key regulators of germ cell differentiation and embryo development in land plants. The ectopic overexpression of reproductive RKDs is associated with increased cellular proliferation and the formation of somatic embryo-like structures that bypass the need for exogenous growth regulators. However, the precise molecular mechanisms implicated in the induction of somatic embryogenesis by RKD transcription factors remains unknown. RESULTS: In silico analyses have identified a rice RWP-RK transcription factor, named Oryza sativa RKD3 (OsRKD3), which is closely related to Arabidopsis thaliana RKD4 (AtRKD4) and Marchantia polymorpha RKD (MpRKD) proteins. Our study demonstrates that the ectopic overexpression of OsRKD3, which is expressed preferentially in reproductive tissues, can trigger the formation of somatic embryos in an Indonesian black rice landrace (Cempo Ireng) that is normally resistant to somatic embryogenesis. By analyzing the transcriptome of induced tissue, we identified 5,991 genes that exhibit differential expression in response to OsRKD3 induction. Among these genes, 50% were up-regulated while the other half were down-regulated. Notably, approximately 37.5% of the up-regulated genes contained a sequence motif in their promoter region, which was also observed in RKD targets from Arabidopsis. Furthermore, OsRKD3 was shown to mediate the transcriptional activation of a discrete gene network, which includes several transcription factors such as APETALA 2-like (AP2-like)/ETHYLENE RESPONSE FACTOR (ERF), MYB and CONSTANS-like (COL), and chromatin remodeling factors associated with hormone signal transduction, stress responses and post-embryonic pathways. CONCLUSIONS: Our data show that OsRKD3 modulates an extensive gene network and its activation is associated with the initiation of a somatic embryonic program that facilitates genetic transformation in black rice. These findings hold substantial promise for improving crop productivity and advancing agricultural practices in black rice.

A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere.

Partial maintenance of organ-specific epigenetic marks during plant asexual reproduction leads to heritable phenotypic variation.

Plants differ from animals in their capability to easily regenerate fertile adult individuals from terminally differentiated cells. This unique developmental plasticity is commonly observed in nature, where many species can reproduce asexually through the ectopic initiation of organogenic or embryogenic developmental programs. While organ-specific epigenetic marks are not passed on during sexual reproduction, the fate of epigenetic marks during asexual reproduction and the implications for clonal progeny remain unclear. Here we report that organ-specific epigenetic imprints in Arabidopsis thaliana can be partially maintained during asexual propagation from somatic cells in which a zygotic program is artificially induced. The altered marks are inherited even over multiple rounds of sexual reproduction, becoming fixed in hybrids and resulting in heritable molecular and physiological phenotypes that depend on the identity of the founder tissue. Consequently, clonal plants display distinct interactions with beneficial and pathogenic microorganisms. Our results demonstrate how novel phenotypic variation in plants can be unlocked through altered inheritance of epigenetic marks upon asexual propagation.

Specific chromatin changes mark lateral organ founder cells in the Arabidopsis inflorescence meristem.

Fluorescence-activated cell sorting (FACS) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were combined to analyse the chromatin state of lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis apetala1-1 cauliflower-1 double mutant inflorescence meristem. On a genome-wide level, we observed a striking correlation between transposase hypersensitive sites (THSs) detected by ATAC-seq and DNase I hypersensitive sites (DHSs). The mostly expanded DHSs were often substructured into several individual THSs, which correlated with phylogenetically conserved DNA sequences or enhancer elements. Comparing chromatin accessibility with available RNA-seq data, THS change configuration was reflected by gene activation or repression and chromatin regions acquired or lost transposase accessibility in direct correlation with gene expression levels in LOFCs. This was most pronounced immediately upstream of the transcription start, where genome-wide THSs were abundant in a complementary pattern to established H3K4me3 activation or H3K27me3 repression marks. At this resolution, the combined application of FACS/ATAC-seq is widely applicable to detect chromatin changes during cell-type specification and facilitates the detection of regulatory elements in plant promoters.

Peptide signalling during angiosperm seed development.

Cell-cell communication is pivotal for the coordination of various features of plant development. Recent studies in plants have revealed that, as in animals, secreted signal peptides play critical roles during reproduction. However, the precise signalling mechanisms in plants are not well understood. In this review, we discuss the known and putative roles of secreted peptides present in the seeds of angiosperms as key signalling factors involved in coordinating different aspects of seed development.

Epigenetic asymmetry of imprinted genes in plant gametes.

Plant imprinted genes show parent-of-origin expression in seed endosperm, but little is known about the nature of parental imprints in gametes before fertilization. We show here that single differentially methylated regions (DMRs) correlate with allele-specific expression of two maternally expressed genes in the seed and that one DMR is differentially methylated between gametes. Thus, plants seem to have developed similar strategies as mammals to epigenetically mark imprinted genes.

HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions.

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts.

Epigenetic reprogramming in plant reproductive lineages.

Monoecious flowering plants produce both microgametophytes (pollen) and megagametophytes (embryo sacs) containing the male and female gametes, respectively, which participate in double fertilization. Much is known about cellular and developmental processes giving rise to these reproductive structures and the formation of gametes. However, little is known about the role played by changes in the epigenome in dynamically shaping these defining events during plant sexual reproduction. This has in part been hampered by the inaccessibility of these structures-especially the female gametes, which are embedded within the female reproductive tissues of the plant sporophyte. However, with the recent development of new cellular isolation technologies that can be coupled to next-generation sequencing, a new wave of epigenomic studies indicate that an intricate epigenetic regulation takes place during the formation of male and female reproductive lineages. In this mini review, we assess the fast growing body of evidence for the epigenetic regulation of the developmental fate and function of plant gametes. We describe how small interfereing RNAs and DNA methylation machinery play a part in setting up unique epigenetic landscapes in different gametes, which may be responsible for their different fates and functions during fertilization. Collectively these studies will shed light on the dynamic epigenomic landscape of plant gametes or 'epigametes' and help to answer important unresolved questions on the sexual reproduction of flowering plants, especially those underpinning the formation of two products of fertilization, the embryo and the endosperm.

The lncRNA MARS modulates the epigenetic reprogramming of the marneral cluster in response to ABA.

Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. In this study, we identified the long non-coding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, which controls the local epigenetic activation of its surrounding region in response to abscisic acid (ABA). MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel non-coding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways.

Progress and challenges for the machine learning-based design of fit-for-purpose monoclonal antibodies.

Although the therapeutic efficacy and commercial success of monoclonal antibodies (mAbs) are tremendous, the design and discovery of new candidates remain a time and cost-intensive endeavor. In this regard, progress in the generation of data describing antigen binding and developability, computational methodology, and artificial intelligence may pave the way for a new era of in silico on-demand immunotherapeutics design and discovery. Here, we argue that the main necessary machine learning (ML) components for an in silico mAb sequence generator are: understanding of the rules of mAb-antigen binding, capacity to modularly combine mAb design parameters, and algorithms for unconstrained parameter-driven in silico mAb sequence synthesis. We review the current progress toward the realization of these necessary components and discuss the challenges that must be overcome to allow the on-demand ML-based discovery and design of fit-for-purpose mAb therapeutic candidates.

Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development.

Cell-cell communication in plants is essential for the correct co-ordination of reproduction, growth, and development. Studies to dissect this mode of communication have previously focussed primarily on the action of plant hormones as mediators of intercellular signalling. In animals, peptide signalling is a well-documented intercellular communication system, however, relatively little is known about this system in plants. In recent years, numerous reports have emerged about small, secreted peptides controlling different aspects of plant reproduction. Interestingly, most of these peptides are cysteine-rich, and there is convincing evidence suggesting multiple roles for related cysteine-rich peptides (CRPs) as signalling factors in developmental patterning as well as during plant pathogen responses and symbiosis. In this review, we discuss how CRPs are emerging as key signalling factors in regulating multiple aspects of vegetative growth and reproductive development in plants.

A transposon surveillance mechanism that safeguards plant male fertility during stress.

Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses1,2. Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production3. Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells.

Heterosis in early seed development: a comparative study of F1 embryo and endosperm tissues 6 days after fertilization.

Heterosis specifies the superior performance of heterozygous individuals and although used in plant breeding the underlying molecular mechanisms still remain largely elusive. In this study, we demonstrate the manifestation of heterosis in hybrid maize embryo and endosperm tissue 6 days after fertilization in crosses of several inbred lines. We provide a comparative analysis of heterosis-associated gene expression in these tissues by a combined approach of suppression subtractive hybridization and microarray hybridizations. Non-additive expression pattern indicated a trans-regulatory mechanism to act early after fertilization in hybrid embryo and endosperm although the majority of genes showed mid-parental expression levels in embryo and dosage dependent expression levels in endosperm. The consistent expression pattern within both tissues and both inbred line genotype combinations of genes coding for chromatin related proteins pointed to heterosis-related epigenetic processes. These and genes involved in other biological processes, identified in this study, might provide entry points for the investigation of regulatory networks associated with the specification of heterosis.