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Ferns and fern-allies, now known as Monylophytes, have received scant molecular attention, in relation to angiosperm group. The advent of high-throughput technologies allows to advance towards a greater knowledge of their elusive genome. In this work, samples of apogamous and sexual heart-shaped gametophytes from two ferns: the apomictic species Dryopteris affinis ssp. af-finisand its sexual relative Dryopteris oreades were extracted and identified. In total, a set of 218 proteins shared by these gametophytes were analysed by using the STRING database, and the proteome associated to metabolism, genetic information processing and responses to abitotic stress is discussed. Specifically, there are reported proteins involved in metabolism of carbohy-drates and lipids, biosynthesis of amino acids, metabolism of nucleotides, energy, and secondary compounds, oxido-reduction, transcription, translation, folding, sorting, and degradation, and response to abiotic stress. Some homologs of proteins found are MACCI-BOU (MAB1), MOSAIC DEATH 1 (MOD1), MAINTENANCE OF PHOTOSYSTEM II UNDER HIGH LIGHT 2 (MPH2), TRANSPARENT TESTA 5 (TT5), ALBINO OR GLASSY YELLOW 1 (AGY1), LEUCYL AMINOPEP-TIDASE 1 and 3 (LAP1 and LAP3), or LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (LOS1). The interactome of the set of proteins was also studied, being the most common interac-tions database and textmining. All these data about the interactions that exist between the stud-ied proteins of the ferns D. affinis and D. oreades, together with the description of their biological function, might contribute to better understand the functioning and development of ferns as well as to fulfil gaps of knowledge in plant evolution.

In multicellular organisms, sexual reproduction relies on the formation of highly specialized, differentiated cells, the gametes. At maturity, male and female gametes are quiescent, awaiting fertilization, with their cell cycle being arrested at a precise stage. Failure to establish quiescence leads to unwanted proliferation, abortion of the offspring, and a waste of resources. Upon fertilization, the cell cycle resumes, allowing the newly formed zygote to divide rapidly. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that enforce quiescence and reinstate cell division only after fertilization occurs are poorly understood. Here, we describe a sperm-derived signal that induces proliferation of theArabidopsiscentral cell precisely upon fertilization. We show that the mature central cell is arrested in S phase, caused by the activity of the conserved RETINOBLASTOMA RELATED1 (RBR1) protein. Paternal delivery of the core cell cycle component CYCD7;1 triggers RBR1 degradation, thereby stimulating S phase progression. Absence of CYCD7;1 delays RBR1 depletion, S phase reactivation, and central cell division, whereas its constitutive expression triggers proliferation of unfertilized central cells. In summary, we show that CYCD7;1 is a paternal signal that informs the central cell that fertilization occurred, thus unlocking quiescence and ensuring that cell division initiates just at the right time to ensure functional endosperm formation.

Historic yield advances in the major crops have, to a large extent, been achieved by selection for improved productivity of groups of plant individuals such as high-density stands. Research suggests that such improved group productivity depends on “cooperative” traits (e.g., erect leaves, short stems) that—while beneficial to the group—decrease individual fitness under competition. This poses a problem for some traditional breeding approaches, especially when selection occurs at the level of individuals, because “selfish” traits will be selected for and reduce yield in high-density monocultures. One approach, therefore, has been to select individuals based on ideotypes with traits expected to promote group productivity. However, this approach is limited to architectural and physiological traits whose effects on growth and competition are relatively easy to anticipate. Here, we developed a general and simple method for the discovery of alleles promoting cooperation in plant stands. Our method is based on the game-theoretical premise that alleles increasing cooperation benefit the monoculture group but are disadvantageous to the individual when facing noncooperative neighbors. Testing the approach using the model plant Arabidopsis thaliana, we found a major effect locus where the rarer allele was associated with increased cooperation and productivity in high-density stands. The allele likely affects a pleiotropic gene, since we find that it is also associated with reduced root competition but higher resistance against disease. Thus, even though cooperation is considered evolutionarily unstable except under special circumstances, conflicting selective forces acting on a pleiotropic gene might maintain latent genetic variation for cooperation in nature. Such variation, once identified in a crop, could rapidly be leveraged in modern breeding programs and provide efficient routes to increase yields.

SummaryIn plant communities, diversity often increases community productivity and functioning, but the specific underlying drivers are difficult to identify. Most ecological theories attribute the positive diversity effects to complementary niches occupied by different species or genotypes. However, the type of niche complementarity often remains unclear, including how complementarity is expressed in terms of trait differences between plants. Here, we use a gene-centred approach to identify differences associated with positive diversity effects in mixtures of naturalArabidopsis thalianagenotypes. Using two orthogonal genetic mapping approaches, we found that between-plant allelic differences at theAtSUC8locus contribute strongly to mixture overyielding. The corresponding gene encodes a proton-sucrose symporter and is expressed in root tissues. Genetic variation inAtSUC8affected the biochemical activities of protein variants and resulted in different sensitivities of root growth to changes in substrate pH. We thus speculate that - in the particular case studied here - evolutionary divergence along an edaphic gradient resulted in the niche complementarity between genotypes that now drives overyielding in mixtures. Identifying such genes important for ecosystem functioning may ultimately allow the linking of ecological processes to evolutionary drivers, help to identify the traits underlying positive diversity effects, and facilitate the development of high-performing crop variety mixtures in agriculture.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but crucial for intercellular communication and interactions with the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family are involved in a multitude of physiological and developmental processes, making it difficult to assess their primary or ancestral function. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e., rhizoid formation and cell expansion, and have reduced male fertility. In contrast, Mpfer null mutants and overexpression lines severely affect cell integrity and morphogenesis of the gametophyte. Thus, the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified to fulfil a multitude of specialized physiological and developmental and roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.Summary statementThe CrRLK1L family arose in land plants and the Marchantia polymorpha genome contains a single copy, MpFER, which is broadly expressed and regulates cell expansion and cell wall integrity.

Significance Plant reproduction relies on proper pollen tube growth to reach the female gametes. This process is highly regulated by a family of secreted signaling peptides that are recognized by cell-wall monitoring proteins to enable plant fertilization. Here, we report that these signaling peptides are sensed both by specific cell-wall and membrane-anchored proteins, which together enable cell-wall remodeling and, consequently, pollen tube growth., Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by Catharanthus roseus RLK1-like (CrRLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2–RALF4 and LRX8–RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX–RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue lrx infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION

Although land plant germ cells have received much attention, knowledge about their specification is still limited. We thus identified transcripts enriched in egg cells of the bryophyte model species Physcomitrium patens, compared the results with angiosperm egg cells, and selected important candidate genes for functional analysis. We used laser-assisted microdissection to perform a cell-type-specific transcriptome analysis on egg cells for comparison with available expression profiles of vegetative tissues and male reproductive organs. We made reporter lines and knockout mutants of the two BONOBO (PbBNB) genes and studied their role in reproduction. We observed an overlap in gene activity between bryophyte and angiosperm egg cells, but also clear differences. Strikingly, several processes that are male-germline specific in Arabidopsis are active in the P. patens egg cell. Among those were the moss PbBNB genes, which control proliferation and identity of both female and male germlines. Pathways shared between male and female germlines were most likely present in the common ancestors of land plants, besides sex-specifying factors. A set of genes may also be involved in the switches between the diploid and haploid moss generations. Nonangiosperm gene networks also contribute to the specification of the P. patens egg cell.

SUMMARYPollen tubes live a life on a razor’s edge. They must maintain cell wall integrity whilst growing towards the ovule at extraordinary speed but explosively burst at just the right moment to release the sperm cells—with fatal consequences for reproduction if things go wrong. The precisely controlled growth of the pollen tube depends on the fine-tuned balance between the expansive force of turgor pressure and the restraining effect of the cell wall. Currently, it is not well understood how the composition of the cell wall affects its mechanical properties. Using Arabidopsis mutants, we have investigated these interactions by combining experimental and simulation techniques to determine instantaneous and time-dependent mechanical parameters. This allowed, for the first time, the quantification of the effects of cell wall biochemistry on turgor pressure and cell wall elasticity and to predict their effects on growth rate. Our systems biology approach is widely applicable to study the implications of mechanical stress on growth.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. Then, after entering interphase, the positions of the scattered centromeres are stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWNs have little effect on chromatin organization involved in the control of gene expression, indicating the robustness of chromatin organization regardless of the type of centromere distribution.

Background Apomixis, the asexual reproduction through seeds, occurs in over 40 plant families and avoids the hidden cost of sex. Apomictic plants are thought to have an advantage in sparse populations and when colonizing new areas but may have a disadvantage in changing environments because they propagate via fixed genotypes. In this study, we separated the influences of different genetic backgrounds (potentially reflecting local adaptation) from those of the mode of reproduction, i.e., sexual vs. apomictic, on nine fitness-related traits in Hieracium pilosella L. We aimed to test whether apomixis per se may provide a fitness advantage in different competitive environments in a common garden setting. Results To separate the effects of genetic background from those of reproductive mode, we generated five families of apomictic and sexual full siblings by crossing two paternal with four maternal parents. Under competition, apomictic plants showed reproductive assurance (probability of seeding, fertility), while offspring of sexual plants with the same genetic background had a higher germination rate. Sexual plants grew better (biomass) than apomictic plants in the presence of grass as a competitor but apomictic plants spread further vegetatively (maximum stolon length) when their competitors were sexual plants of the same species. Furthermore, genetic background as represented by the five full-sibling families influenced maximum stolon length, the number of seeds, and total fitness. Under competition with grass, genetic background influenced fecundity, the number of seeds, and germination rate. Conclusions Our results suggest that both the mode of reproduction as well as the genetic background affect the success of H. pilosella in competitive environments. Total fitness, the most relevant trait for adaptation, was only affected by the genetic background. However, we also show for the first time that apomixis per se has effects on fitness-related traits that are not confounded by-and thus independent of-the genetic background., BMC Biology, 19 (1), ISSN:1741-7007

Ratiometric time-lapse FRET analysis requires a robust and accurate processing pipeline to eliminate bias in intensity measurements on fluorescent images before further quantitative analysis can be conducted. This level of robustness can only be achieved by supplementing automated tools with built-in flexibility for manual ad-hoc adjustments. FRET-IBRA is a modular and fully parallelized configuration file-based tool written in Python. It simplifies the FRET processing pipeline to achieve accurate, registered, and unified ratio image stacks. The flexibility of this tool to handle discontinuous image frame sequences with tailored configuration parameters further streamlines the processing of outliers and time-varying effects in the original microscopy images. FRET-IBRA offers cluster-based channel background subtraction, photobleaching correction, and ratio image construction in an all-in-one solution without the need for multiple applications, image format conversions, and/or plug-ins. The package accepts a variety of input formats and outputs TIFF image stacks along with performance measures to detect both the quality and failure of the background subtraction algorithm on a per frame basis. Furthermore, FRET-IBRA outputs images with superior signal-to-noise ratio and accuracy in comparison to existing background subtraction solutions, whilst maintaining a fast runtime. The FRET-IBRA package has been extensively used in quantifying the spatial distribution of calcium ions during pollen tube growth under mechanical constraints. Benchmarks against existing tools clearly demonstrate the need for FRET-IBRA in extracting reliable insights from FRET microscopy images of dynamic physiological processes at high spatial and temporal resolution. The source code for Linux and Mac operating systems is released under the BSD license and, along with installation instructions, test images, example configuration files, and a step-by-step tutorial, is freely available at github.com/gmunglani/fret-ibra.Author SummaryFRET is a fundamental imaging technique used to generate fluorescence signals sensitive to molecular conformations and interactions. Despite its wide use and the large body of literature on the theoretical steps required to process images generated from this procedure, we were unable to locate a tool that contained the entire processing workflow, whilst allowing the user the flexibility to adjust parameters for maximum accuracy and runtime efficiency. FRET-IBRA was thus created to be an all-in-one, open-source, parallel solution to process FRET images, while eliminating complications arising from repeated image format conversions. Besides enhancing the background subtraction algorithm for FRET images, several additional options were implemented for the user to extract the cleanest signal possible for their specific use case. FRET-IBRA is primarily built for flexibility when handling large image stacks by supporting sequences of image frames to be treated independently, greatly reducing time spent on splitting and concatenating image stacks. In accuracy and speed benchmarks against more general background subtraction packages, FRET-IBRA was able to provide the cleanest results with a fast runtime, leading to reliable analysis without additional tuning.

Genomic imprinting leads to different expression levels of maternally and paternally derived alleles. Over the last years, major progress has been made in identifying novel imprinted candidate genes in plants, owing to affordable next-generation sequencing technologies. However, reports on sequencing the transcriptome of hybrid F1 seed tissues strongly disagree about how many and which genes are imprinted. This raises questions about the relative impact of biological, environmental, technical, and analytic differences or biases. Here, we adopt a statistical approach, frequently used in RNA-seq data analysis, which properly models count overdispersion and considers replicate information of reciprocal crosses. We show that our statistical pipeline outperforms other methods in identifying imprinted genes in simulated and real data. Accordingly, reanalysis of genome-wide imprinting studies in Arabidopsis and maize shows that, at least for the Arabidopsis dataset, an increased agreement across datasets can be observed. For maize, however, consistent reanalysis did not yield in a larger overlap between the datasets. This suggests that the discrepancy across publications might be partially due to different analysis pipelines but that technical, biological, and environmental factors underlie much of the discrepancy between datasets. Finally, we show that the set of genes that can be characterized regarding allelic bias by all studies with minimal confidence is small (~8,000/27,416 genes for Arabidopsis and ~12,000/39,469 for maize). In conclusion, we propose to use biologically replicated reciprocal crosses, high sequence coverage, and a generalized linear model approach to identify differentially expressed alleles in developing seeds.

The sensory hairs of the Venus flytrap (Dionaea muscipula Ellis) detect mechanical stimuli imparted by their prey and fire bursts of electrical signals called action potentials (APs). APs are elicited when the hairs are sufficiently stimulated and two consecutive APs can trigger closure of the trap. Earlier experiments have identified thresholds for the relevant stimulus parameters, namely the angular displacement $$\theta $$ θ and angular velocity $$\omega $$ ω . However, these experiments could not trace the deformation of the trigger hair’s sensory cells, which are known to transduce the mechanical stimulus. To understand the kinematics at the cellular level, we investigate the role of two relevant mechanical phenomena: viscoelasticity and intercellular fluid transport using a multi-scale numerical model of the sensory hair. We hypothesize that the combined influence of these two phenomena and $$\omega $$ ω contribute to the flytrap’s rate-dependent response to stimuli. In this study, we firstly perform sustained deflection tests on the hair to estimate the viscoelastic material properties of the tissue. Thereafter, through simulations of hair deflection tests at different loading rates, we were able to establish a multi-scale kinematic link between $$\omega $$ ω and the cell wall stretch $$\delta $$ δ . Furthermore, we find that the rate at which $$\delta $$ δ evolves during a stimulus is also proportional to $$\omega $$ ω . This suggests that mechanosensitive ion channels, expected to be stretch-activated and localized in the plasma membrane of the sensory cells, could be additionally sensitive to the rate at which stretch is applied.

Summary Establishing the embryonic body plan of multicellular organisms relies on precisely orchestrated cell divisions coupled with pattern formation, which, in animals, are regulated by Polycomb group (PcG) proteins. The conserved Polycomb Repressive Complex 2 (PRC2) mediates H3K27 trimethylation and comes in different flavors in Arabidopsis. The PRC2 catalytic subunit MEDEA is required for seed development; however, a role for PRC2 in embryonic patterning has been dismissed. Here, we demonstrate that embryos derived from medea eggs abort because MEDEA is required for patterning and cell lineage determination in the early embryo. Similar to PcG proteins in mammals, MEDEA regulates embryonic patterning and growth by controlling cell-cycle progression through repression of CYCD1;1, which encodes a core cell-cycle component. Thus, Arabidopsis embryogenesis is epigenetically regulated by PcG proteins, revealing that the PRC2-dependent modulation of cell-cycle progression was independently recruited to control embryonic cell proliferation and patterning in animals and plants., Graphical abstract, Highlights • MEDEA, a Polycomb Repressive Complex 2 (PRC2) subunit, is required in embryo and endosperm • MEDEA links cell proliferation and differentiation during embryonic pattern formation • The PRC2 protein MEDEA directly regulates core cell-cycle components, i.e., cyclin CYCD1;1 • Body plan and cell proliferation are epigenetically regulated in both animals and plants, In animals, Polycomb group (PcG) proteins are essential for embryogenesis. Simonini et al. show that growth and patterning of plant embryos are also epigenetically regulated by PcG proteins, with the PRC2 histone methyltransferase MEDEA directly repressing the core cell-cycle component CYCD1;1 in the Arabidopsis embryo.

While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we first report that inArabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2cisrecruitment. This study unveils a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.Abstract FigureTeano et al. report that that linker histone H1 and a group of H1-related telomeric proteins interplay to selectively influence thePolycombrepressive landscape at genes and telomeric repeats inArabidopsis. These findings provide a mechanistic framework by which H1 influences the epigenome and nuclear organization in a sequence-specific manner.HighlightsH1 promotes PRC2 activity and limits accessibility at a majority of genesH1 prevents PRC2 activity at telomeric DNA sequencesPRC2 repression is achieved by restricting accessibility to TRB proteinsH1 orchestrates the spatial organization of telomeres and interstitial telomeres (ITRs)

The identity of cells in an organism is largely defined by their specific transcriptional profile. During cell division, these profiles need to be faithfully inherited to the daughter cells to ensure the maintenance of cell structure and function in a cell lineage. Here, you will learn how two groups of chromatin regulators, the Polycomb group (PcG) and the Trithorax group (TrxG), act in an antagonistic manner to maintain differential gene expression states. Members of the PcG cooperate in large multiprotein complexes to modify histones with repressive marks, resulting in condensed chromatin domains. Conversely, the TrxG proteins counteract the repressed domains by opening nucleosomal structures and establishing activating histone modifications. PcG and TrxG proteins are evolutionary highly conserved and control diverse processes, such as the identity of stem cells in mammalian development to the process of vernalization in plants.

Alterations in chromatin function and epigenetic mechanisms are a hallmark of cancer. The disruption of epigenetic processes has been linked to altered gene expression and to cancer initiation and progression. Recent cancer genome sequencing projects revealed that numerous epigenetic regulators are frequently mutated in various cancers. This information has not only started to be utilized as prognostic and predictive markers to guide treatment decisions but also provided important information for the understanding of the molecular mechanisms of epigenetic regulation in both physiological and pathological conditions. Furthermore, the reversible nature of epigenetic aberrations has led to the emergence of the promising field of epigenetic therapy that has already provided new therapeutic options for patients with malignancies characterized by epigenetic alterations, laying the basis for new and personalized medicine.

During regenerative processes, cells are required to restructure parts of a damaged or worn-out organ and tissue. Here, you will become acquainted with the strategies that organisms developed to provide the material for tissue and organ repair. On the one hand, somatic cells can become dedifferentiated to increase their developmental potential and produce the plasticity required to replace the entire cellular complexity of a damaged part. On the other hand, organisms retain organ-specific stem cells with a restricted developmental potency and use these to provide the “spare parts” for replacing damaged cells. In all cases, a substantial reprogramming of the epigenome of these cells accompanies the restructuring process. In vitro strategies have been developed to drive cells back to a pluripotent state, allowing a better understanding of the underlying chromatin adjustments and providing a rich source for cellular therapies.

Most chromatin-modifying enzymes use metabolites as cofactors. Consequently, the cellular metabolism can influence the capacity of the cell to write or erase chromatin marks. This points to an intimate relationship between metabolic and epigenetic regulation. In this chapter, we describe the biosynthetic pathways of cofactors that are implicated in epigenetic and chromatin regulation and provide examples of how metabolic pathways can influence chromatin and epigenetic processes as well as their interplay in developmental and cancer biology.

The nucleus of a eukaryotic cell is a very busy place. Not only during replication of the DNA, but at any time in the cell cycle specific enzymes need access to genetic information to process reactions such as transcription and DNA repair. Yet, the nucleosomal structure of chromatin is primarily inhibitory to these processes and needs to be resolved in a highly orchestrated manner to allow developmental, organismal, and cell type-specific nuclear activities. This chapter explains how nucleosomes organize and structure the genome by interacting with specific DNA sequences. Variants of canonical histones can change the stability of the nucleosomal structure and also provide additional epigenetic layers of information. Chromatin remodeling complexes work locally to alter the regular beads-on-a-string organization and provide access to transcription and other DNA processing factors. Conversely, factors like histone chaperones and highly precise templating and copying mechanisms are required for the reassembly of nucleosomes and reestablishment of the epigenetic landscape after passage of activities processing DNA sequence information. A very intricate molecular machinery ensures a highly dynamic yet heritable chromatin template.

Crosses between the wild tomato species Solanum peruvianum and Solanum chilense result in hybrid seed failure (HSF), characterized by endosperm misdevelopment and embryo arrest. We previously showed that genomic imprinting, the parent-of-origin dependent expression of alleles, is perturbed in the hybrid endosperm, with many of the normally paternally expressed genes losing their imprinted status. Here, we report transcriptome-based analyses of gene and small RNA (sRNA) expression levels. We identified 2,295 genes and 387 sRNA clusters as differentially expressed when comparing reciprocal hybrid seed to seeds and endosperms from the two within-species crosses. Our analyses uncovered a pattern of overdominance in endosperm gene expression in both hybrid cross directions, in marked contrast to the patterns of sRNA expression in whole seeds. Intriguingly, patterns of increased gene expression resemble the previously reported increased maternal expression proportions in hybrid endosperms. We identified physical clusters of sRNAs; differentially expressed sRNAs exhibit reduced transcript abundance in hybrid seeds of both cross directions. Moreover, sRNAs map to genes coding for key proteins involved in epigenetic regulation of gene expression, suggesting a regulatory feedback mechanism. We describe examples of genes that appear to be targets of sRNA-mediated gene silencing; in these cases, reduced sRNA abundance is concomitant with increased gene expression in hybrid seeds. Our analyses also show that S. peruvianum dominance impacts gene and sRNA expression in hybrid seeds. Overall, our study indicates roles for sRNA-mediated epigenetic regulation in HSF between closely related wild tomato species., Genome Biology and Evolution, 13 (8), ISSN:1759-6653

This chapter provides an introduction to chromosome-wide dosage compensation systems. We will examine the evolution of dosage compensation, which is thought to be driven by the appearance of differentiated sex chromosomes. In a subset of species with X chromosomal sex determination or XY sex chromosome systems, expression of X-linked genes is regulated by chromosome-wide modifications that equalize gene expression differences between males and females. The molecular mechanisms of X chromosome-wide dosage compensation have been studied in flies, worms, and mammals. Each of these species uses a distinct dosage compensation strategy with a different molecular mechanism. In the wormCaenorhabditis elegans, gene expression on the two X chromosomes of hermaphrodites is reduced to a level that approximates a single X chromosome in males. The fruit flyDrosophila melanogasterachieves dosage compensation by increased transcription of the single X chromosome in males to a level that is similar to the two X chromosomes in females. Lastly, in mammals, one of the two X chromosomes in female cells is transcriptionally inactive and a single X chromosome is transcribed in both sexes. Studies of dosage compensation systems provide insights into how epigenetic regulation controls gene expression and chromatin organization differentially within a cell.

Frontiers in Plant Science, 12, ISSN:1664-462X

Although epigenetic states are typically associated with DNA-methylation and posttranslational histone modifications, RNAs often play an important role in their regulation. Specific examples have already been discussed in the context of dosage compensation (see book ► Chap. 10.1007/978-3-030-68670-3_4 of Wutz) and genomic imprinting (see book ► Chap. 10.1007/978-3-030-68670-3_5 of Grossniklaus). In this Chapter, we will take a closer look at a particular class of RNAs implicated in gene silencing. Although the focus will lie on RNA-based silencing mechanisms in plants, many of its components, such as RNase III-related DICERLIKE endonucleases or small RNA-binding ARGONAUTE proteins, are conserved in animals, plants, and fungi. On the one hand, small RNAs are involved in post-transcriptional silencing by targeting mRNAs for degradation or inhibiting their translation, a feature that has been exploited for large-scale genetic screens. On the other hand, they also play a central role in transcriptional gene silencing, for instance in the repression of transposable elements across a wide variety of organisms. In plants, this involves a complex system whereby small RNAs derived from transposons and repeats direct DNA-methylation and repressive histone modifications in a sequence-specific manner. Recent results link this so-called RNA-dependent DNA-methylation to paramutation, a classical epigenetic phenomenon where one allele directs a heritable epigenetic change in another.

A typical cell contains two sets of chromosomes: one that was inherited from the mother, the other from the father. Usually, autosomal alleles are expressed at similar levels from the maternally and paternally inherited chromosomes. This chapter is dedicated to an exception of this rule: the expression of genes that are regulated by genomic imprinting depends on the parental origin of the allele, leading to the non-equivalence of maternal and paternal genomes. Genomic imprinting is a paradigm of epigenetic gene regulation as genetically identical alleles can exist in two expression states within the same nucleus. The imprints marking the parental alleles are established in the parental germline, maintained during the development of the offspring, but reset before they are passed on to the next generation. In mammals, the primary imprint is usually a differentially methylated region at the locus but there are also examples where histone modifications mark the parental alleles. Many imprinted genes play important roles for development and are associated with human disease. Interestingly, genomic imprinting evolved independently in mammals and seed plants and similar mechanisms have been recruited to regulate imprinted expression in the two kingdoms. We will discuss evolutionary constraints that could have led to the evolution of genomic imprinting in these seemingly disparate lineages.

BackgroundApomixis, the asexual reproduction through seeds, occurs in over 40 plant families and avoids the hidden cost of sex. Apomictic plants are thought to have an advantage in sparse populations and when colonizing new areas but may have a disadvantage in changing environments because they propagate via fixed genotypes.In this study, we separated the influences of different genetic backgrounds (potentially reflecting local adaptation) from those of the mode of reproduction, i.e., sexual vs. apomictic, on nine fitness-related traits in Hieracium pilosella L. We aimed to test whether apomixis per se may provide a fitness advantage in different competition environments in a common garden setting.ResultsTo separate the effects of genetic background from those of reproductive mode, we generated five families of apomictic and sexual full siblings by crossing two paternal with four maternal parents. Under competition, apomictic plants showed reproductive assurance (probability of seeding, fertility), while offspring of sexual plants with the same genetic background had a higher germination rate. Sexual plants grew better (biomass) than apomictic plants in the presence of grass as a competitor but apomictic plants spread further vegetatively (maximum stolon length) when their competitors were sexual plants of the same species. Furthermore, genetic background as represented by the five full-sibling families influenced maximum stolon length, the number of seeds, and total fitness. Under competition with grass, genetic background influenced fecundity, the number of seeds, and germination rate.ConclusionsOur results suggest that both the mode of reproduction as well as the genetic background affect the success of H. pilosella in competitive environments. Total fitness, the most relevant trait for adaptation, was only affected by the genetic background. However, we also show for the first time that apomixis per se has effects on fitness-related traits that are not confounded by — and thus independent of — the genetic background.

This chapter provides an introduction to chromatin. We will examine the organization of the genome into a nucleosomal structure. DNA is wrapped around a globular complex of 8 core histone proteins, two of each histone H2A, H2B, H3, and H4. This nucleosomal arrangement is the context in which information can be established along the sequence of the DNA for regulating different aspects of the chromosome, including transcription, DNA replication and repair processes, recombination, kinetochore function, and telomere function. Posttranslational modifications of histone proteins and modifications of DNA bases underlie chromatin-based epigenetic regulation. Enzymes that catalyze histone modifications are considered writers. Conceptually, erasers remove these modifications, and readers are proteins binding these modifications and can target specific functions. On a larger scale, the 3-dimensional (3D) organization of chromatin in the nucleus also contributes to gene regulation. Whereas chromosomes are condensed during mitosis and segregated during cell division, they occupy discrete volumes called chromosome territories during interphase. Looping or folding of DNA can bring regulatory elements including enhancers close to gene promoters. Recent techniques facilitate understanding of 3D contacts at high resolution. Lastly, chromatin is dynamic and changes in histone occupancy, histone modifications, and accessibility of DNA contribute to epigenetic regulation.

Insects fall prey to the Venus flytrap (Dionaea muscipula) when they touch the sensory hairs located on the flytrap lobes, causing sudden trap closure. The mechanical stimulus imparted by the touch produces an electrical response in the sensory cells of the trigger hair. These cells are found in a constriction near the hair base, where a notch appears around the hair’s periphery. There are mechanosensitive ion channels (MSCs) in the sensory cells that open due to a change in membrane tension; however, the kinematics behind this process is unclear. In this study, we investigate how the stimulus acts on the sensory cells by building a multi-scale hair model, using morphometric data obtained from μ-CT scans. We simulated a single-touch stimulus and evaluated the resulting cell wall stretch. Interestingly, the model showed that high stretch values are diverted away from the notch periphery and, instead, localized in the interior regions of the cell wall. We repeated our simulations for different cell shape variants to elucidate how the morphology influences the location of these high-stretch regions. Our results suggest that there is likely a higher mechanotransduction activity in these ’hotspots’, which may provide new insights into the arrangement and functioning of MSCs in the flytrap. Dataset: 10.3929/ethz-b-000448954

In population genomics, genetic diversity measures play an important role in genome scans for divergent sites. In population epigenomics, comparable tools are rare although the epigenome can vary at several levels of organization. We propose a model-free, information-theoretic approach, the Jensen-Shannon divergence (JSD), as a flexible diversity index for epigenomic diversity. Here, we demonstrate how JSD uncovers the relationship between genomic features and cell type-specific methylome diversity in Arabidopsis thaliana. However, JSD is applicable to any epigenetic mark and any collection of individuals, tissues, or cells, for example to assess the heterogeneity in healthy organs and tumors.

Establishing the body plan of a multicellular organism relies on precisely orchestrated cell divisions coupled with pattern formation. In animals, cell proliferation and embryonic patterning are regulated byPolycombgroup (PcG) proteins that form various multisubunit complexes (Grossniklaus and Paro, 2014). The evolutionary conservedPolycombRepressive Complex 2 (PRC2) trimethylates histone H3 at lysine 27 (H3K27me3) and comes in different flavors in the model plantArabidopsis thaliana(Förderer et al., 2016; Grossniklaus and Paro, 2014). The histone methyltransferase MEDEA (MEA) is part of the FERTILIZATION INDEPENDENT SEED (FIS)-PRC2 required for seed development4. Although embryos derived frommeamutant egg cells show morphological abnormalities (Grossniklaus et al., 1998), defects in the development of the placenta-like endosperm are considered the main cause of seed abortion (Kinoshita et al., 1999; Scott et al., 1998), and a role of FIS-PRC2 in embryonic patterning was dismissed (Bouyer et al., 2011; Leroy et al., 2007). Here, we demonstrate that endosperm lackingMEAactivity sustains normal embryo development and that embryos derived frommeamutant eggs abort even in presence of a wild-type endosperm becauseMEAis required for embryonic patterning and cell lineage determination. We show that, similar to PcG proteins in mammals, MEA regulates embryonic growth by repressing the transcription of core cell cycle components. Our work demonstrates thatArabidopsisembryogenesis is under epigenetic control of maternally expressed PcG proteins, revealing that PRC2 was independently recruited to control embryonic cell proliferation and patterning in animals and plants.

Plant growth and morphogenesis are tightly controlled processes of division and expansion of individual cells. To fully describe the factors that influence cell expansion, it is necessary to quantify the counteracting forces of turgor pressure and cell wall stiffness, which together determine whether and how a cell expands. Several methods have been developed to measure these parameters, but most of them provide only values for one or the other, and thus require complex models to derive the missing quantity. Furthermore, available methods for turgor measurement are either accurate but invasive, like the pressure probe; or they lack accuracy, such as incipient plasmolysis or indentation-based methods that rely on information about the mechanical properties of the cell wall. Here, we describe a system that overcomes many of the above-mentioned disadvantages using growing pollen tubes of Lilium longiflorum as a model. By combining non-invasive microindentation and cell compression experiments, we separately measure turgor pressure and cell wall elasticity on the same pollen tube in parallel. Due to the modularity of the setup and the large range of the micro-positioning system, our method is not limited to pollen tubes but could be used to investigate the biomechanical properties of many other cell types or tissues.

SummaryIn multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the first cells of the germline, so-called spore mother cells (SMCs), differentiate as the reproductive organs form. Here, we explored how organ growth influences and contributes to SMC differentiation. We generated a collection of 92 annotated 3D images capturing ovule primordium ontogeny at cellular resolution in Arabidopsis. We identified a spatio-temporal pattern of cell divisions that acts in a domain-specific manner as the primordium forms, which is coupled with the emergence of a single SMC. Using tissue growth models, we uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis also reveals that SMC characteristics first arise in more than one cell but SMC fate becomes progressively restricted to a single cell during organ growth. Altered primordium geometry coincided with a delay in this fate restriction process inkataninmutants. Altogether, our study suggests that tissue geometry canalizes and modulates reproductive cell fate in the Arabidopsis ovule primordium.

Pollen tubes face many obstacles on their way to the ovule. They have to decide whether to navigate around cells or penetrate the cell wall and grow through it or even within it. Besides chemical sensing, which directs the pollen tubes on their path to the ovule, this involves mechanosensing to determine the optimal strategy in specific situations. Mechanical cues then need to be translated into physiological signals, which eventually lead to changes in the growth behavior of the pollen tube. To study these events, we have developed a system to directly quantify the forces involved in pollen tube navigation. We combined a lab-on-a-chip device with a microelectromechanical systems-based force sensor to mimic the pollen tube's journey from stigma to ovary in vitro. A force-sensing plate creates a mechanical obstacle for the pollen tube to either circumvent or attempt to penetrate while measuring the involved forces in real time. The change of growth behavior and intracellular signaling activities can be observed with a fluorescence microscope.

Flowers, from their initiation in the inflorescence meristem until their opening at anthesis, are particularly sensitive to subtle changes in environmental conditions. Short episodes of either cold or heat stress experienced during stamen development may lead to varying degrees of male sterility and can affect the ploidy of the gametes. In an attempt to understand the developmental origins of these and other phenotypes that are caused by harsh environmental conditions, an acute heat stress was simultaneously applied to all stages of anther development, and its consequences on pollen viability at anthesis and seed set were traced back to heat stress at specific developmental stages. We identified a critical stage during microsporogenesis that is highly sensitive to heat stress. The acute heat stress experienced during these stages completely blocked stamen and pollen development, likely mediated by its effects on both anther wall development and the formation of polyads in the germline. Interestingly, as we investigated heat shocks at earlier developmental stages, a gradual recovery of male fertility was observed, characterized by some abnormalities in locule development and by fusions of maturing pollen grains. Overall, our findings establish a developmental framework for the diverse effects of heat stress on stamen and pollen development, which is relevant to different fields of research. Acute heat stresses are expected to increase in frequency with global warming and might negatively affect plant fitness and crop yields in the short term by affecting male fertility and seed set. Moreover, the formation of polyads and the fusion of pollen grains offer insights into the developmental origins of evolutionary relevant processes, such as polyploidization, which might offer opportunities for environmental adaptations in the long term.

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.

Plant growth and morphogenesis are tightly controlled processes of division and expansion of individual cells. To fully describe the factors that influence cell expansion, it is necessary to quantify the counteracting forces of turgor pressure and cell wall stiffness, which together determine whether and how a cell expands. Several methods have been developed to measure these parameters, but most of them provide only values for one or the other, and thus require complex models to derive the missing quantity. Furthermore, available methods for turgor measurement are either accurate but invasive, like the pressure probe; or they lack accuracy, such as incipient plasmolysis or indentation-based methods that rely on information about the mechanical properties of the cell wall. Here, we describe a system that overcomes many of the above-mentioned disadvantages using growing pollen tubes of Lilium longiflorum as a model. By combining non-invasive microindentation and cell compression experiments, we separately measure turgor pressure and cell wall elasticity on the same pollen tube in parallel. Due to the modularity of the setup and the large range of the micro-positioning system, our method is not limited to pollen tubes but could be used to investigate the biomechanical properties of many other cell types or tissues.

Apomixis, the asexual reproduction through seeds, is thought to provide reproductive assurance when ploidy is not even and/or when population density is low. Therefore, apomicts are expected to be more abundant, and the frequency of apomictic offspring higher, at early stages of primary succession when mates are rare. To test this hypothesis, we sampled facultative apomictic Hieracium pilosella L. along the successional gradient on a glacier forefield and determined their ploidy, the level of apomixis in their offspring, and the genetic diversity of the entire meta-population and within subpopulations. We found that apomixis is more common in odd- and aneuploid cytotypes, which are more frequent at early stages of primary succession. However, apomixis was uncommon at all successional stages and sexual hexaploids were dominating throughout. Reproductive assurance was reflected in the higher fertility of all odd-ploid apomictic plants (3×, 5×) by avoiding meiosis, illustrating that apomixis provides an escape from sterility, as proposed by Darlington. Odd-ploid plants are supposedly better colonizers (Baker’s law), which is supported by their higher occurrence close to the glacier snout. Independent of succession, we found gene flow between apomicts and sexuals, which allows for the continuous creation of new apomictic and sexual genotypes. We conclude that apomixis in H. pilosella does indeed provide an escape from sterility, and therefore reproductive assurance, in aneuploid cytotypes. We further propose that apomixis preserves beneficial combinations of unlinked alleles in every generation for as long as apomictic genotypes persist in the population., Scientific Reports, 10 (1), ISSN:2045-2322

Pollen tubes face many obstacles on their way to the ovule. They have to decide whether to navigate around cells or penetrate the cell wall and grow through it or even within it. Besides chemical sensing, which directs the pollen tubes on their path to the ovule, this involves mechanosensing to determine the optimal strategy in specific situations. Mechanical cues then need to be translated into physiological signals, which eventually lead to changes in the growth behavior of the pollen tube. To study these events, we have developed a system to directly quantify the forces involved in pollen tube navigation. We combined a lab-on-a-chip device with a microelectromechanical systems-based force sensor to mimic the pollen tube's journey from stigma to ovary in vitro. A force-sensing plate creates a mechanical obstacle for the pollen tube to either circumvent or attempt to penetrate while measuring the involved forces in real time. The change of growth behavior and intracellular signaling activities can be observed with a fluorescence microscope.

Marchantia polymorpha has recently become a prime model for cellular, evo‐devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule‐scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high‐density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map‐based cloning, feasible.

Leu-rich repeat extensins (LRXs) are chimeric proteins containing an N-terminal Leu-rich repeat (LRR) and a C-terminal extensin domain. LRXs are involved in cell wall formation in vegetative tissues and required for plant growth. However, the nature of their role in these cellular processes remains to be elucidated. Here, we used a combination of molecular techniques, light microscopy, and transmission electron microscopy to characterize mutants of pollen-expressed LRXs in Arabidopsis (Arabidopsisthaliana). Mutations in multiple pollen-expressed lrx genes cause severe defects in pollen germination and pollen tube growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Ca2+ availability partially suppresses the pollen tube growth defects, suggesting that LRX proteins influence Ca2+-related processes. Furthermore, we show that LRX protein localizes to the cell wall, and its LRR-domain (which likely mediates protein-protein interactions) is associated with the plasma membrane. Mechanical analyses by cellular force microscopy and finite element method-based modeling revealed significant changes in the material properties of the cell wall and the fine-tuning of cellular biophysical parameters in the mutants compared to the wild type. The results indicate that LRX proteins might play a role in cell wall-plasma membrane communication, influencing cell wall formation and cellular mechanics.

Pollen tubes are tip-growing plant cells that deliver the sperm cells to the ovules for double fertilization of the egg cell and the endosperm. Various directional cues can trigger the reorientation of pollen tube growth direction on their passage through the female tissues. Among the external stimuli, protons serve an important, regulatory role in the control of pollen tube growth. The generation of local guidance cues has been challenging when investigating the mechanisms of perception and processing of such directional triggers in pollen tubes. Here, we developed and characterized a microelectrode device to generate a local proton gradient and proton flux through water electrolysis. We confirmed that the cytoplasmic pH of pollen tubes varied with environmental pH change. Depending on the position of the pollen tube tip relative to the proton gradient, we observed alterations in the growth behavior, such as bursting at the tip, change in growth direction, or complete growth arrest. Bursting and growth arrest support the hypothesis that changes in the extracellular H+ concentration may interfere with cell wall integrity and actin polymerization at the growing tip. A change in growth direction for some pollen tubes implies that they can perceive the local proton gradient and respond to it. We also showed that the growth rate is directly correlated with the extracellular pH in the tip region. Our microelectrode approach provides a simple method to generate protons and investigate their effect on plant cell growth.

Background The three-dimensional (3D) organization of chromosomes is linked to epigenetic regulation and transcriptional activity. However, only few functional features of 3D chromatin architecture have been described to date. The KNOT is a 3D chromatin structure in Arabidopsis, comprising 10 interacting genomic regions termed KNOT ENGAGED ELEMENTs (KEEs). KEEs are enriched in transposable elements and associated small RNAs, suggesting a function in transposon biology. Results Here, we report the KNOT’s involvement in regulating invasive DNA elements. Transgenes can specifically interact with the KNOT, leading to perturbations of 3D nuclear organization, which correlates with the transgene’s expression: high KNOT interaction frequencies are associated with transgene silencing. KNOT-linked silencing (KLS) cannot readily be connected to canonical silencing mechanisms, such as RNA-directed DNA methylation and post-transcriptional gene silencing, as both cytosine methylation and small RNA abundance do not correlate with KLS. Furthermore, KLS exhibits paramutation-like behavior, as silenced transgenes can lead to the silencing of active transgenes in trans. Conclusion Transgene silencing can be connected to a specific feature of Arabidopsis 3D nuclear organization, namely the KNOT. KLS likely acts either independent of or prior to canonical silencing mechanisms, such that its characterization not only contributes to our understanding of chromosome folding but also provides valuable insights into how genomes are defended against invasive DNA elements. Electronic supplementary material The online version of this article (10.1186/s13059-019-1722-3) contains supplementary material, which is available to authorized users.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.