Transposon dynamics in the emerging oilseed crop Thlaspi arvense.

Genome evolution is partly driven by the mobility of transposable elements (TEs) which often leads to deleterious effects, but their activity can also facilitate genetic novelty and catalyze local adaptation. We explored how the intraspecific diversity of TE polymorphisms might contribute to the broad geographic success and adaptive capacity of the emerging oil crop Thlaspi arvense (field pennycress). We classified the TE inventory based on a high-quality genome assembly, estimated the age of retrotransposon TE families and comprehensively assessed their mobilization potential. A survey of 280 accessions from 12 regions across the Northern hemisphere allowed us to quantify over 90,000 TE insertion polymorphisms (TIPs). Their distribution mirrored the genetic differentiation as measured by single nucleotide polymorphisms (SNPs). The number and types of mobile TE families vary substantially across populations, but there are also shared patterns common to all accessions. Ty3/Athila elements are the main drivers of TE diversity in T. arvense populations, while a single Ty1/Alesia lineage might be particularly important for transcriptome divergence. The number of retrotransposon TIPs is associated with variation at genes related to epigenetic regulation, including an apparent knockout mutation in BROMODOMAIN AND ATPase DOMAIN-CONTAINING PROTEIN 1 (BRAT1), while DNA transposons are associated with variation at the HSP19 heat shock protein gene. We propose that the high rate of mobilization activity can be harnessed for targeted gene expression diversification, which may ultimately present a toolbox for the potential use of transposition in breeding and domestication of T. arvense.

DNA methylation in the wild: epigenetic transgenerational inheritance can mediate adaptation in clones of wild strawberry (Fragaria vesca).

Due to the accelerating climate change, it is crucial to understand how plants adapt to rapid environmental changes. Such adaptation may be mediated by epigenetic mechanisms like DNA methylation, which could heritably alter phenotypes without changing the DNA sequence, especially across clonal generations. However, we are still missing robust evidence of the adaptive potential of DNA methylation in wild clonal populations. Here, we studied genetic, epigenetic and transcriptomic variation of Fragaria vesca, a predominantly clonally reproducing herb. We examined samples from 21 natural populations across three climatically distinct geographic regions, as well as clones of the same individuals grown in a common garden. We found that epigenetic variation was partly associated with climate of origin, particularly in non-CG contexts. Importantly, a large proportion of this variation was heritable across clonal generations. Additionally, a subset of these epigenetic changes affected the expression of genes mainly involved in plant growth and responses to pathogen and abiotic stress. These findings highlight the potential influence of epigenetic changes on phenotypic traits. Our findings indicate that variation in DNA methylation, which can be environmentally inducible and heritable, may enable clonal plant populations to adjust to their environmental conditions even in the absence of genetic adaptation.

Histone Deacetylases HD2A and HD2B Undergo Feedback Regulation by ABA and Modulate Drought Tolerance via Mediating ABA-Induced Transcriptional Repression.

Histone deacetylation catalyzed by histone deacetylase plays a critical role in gene silencing and subsequently controls many important biological processes. It was reported that the expression of the plant-specific histone deacetylase subfamily HD2s is repressed by ABA in Arabidopsis. However, little is known about the molecular relationship between HD2A/HD2B and ABA during the vegetative phase. Here, we describe that the hd2ahd2b mutant shows hypersensitivity to exogenous ABA during the germination and post-germination period. Additionally, transcriptome analyses revealed that the transcription of ABA-responsive genes was reprogrammed and the global H4K5ac level is specifically up-regulated in hd2ahd2b plants. ChIP-Seq and ChIP-qPCR results further verified that both HD2A and HD2B could directly and specifically bind to certain ABA-responsive genes. As a consequence, Arabidopsis hd2ahd2b plants displayed enhanced drought resistance in comparison to WT, which is consistent with increased ROS content, reduced stomatal aperture, and up-regulated drought-resistance-related genes. Moreover, HD2A and HD2B repressed ABA biosynthesis via the deacetylation of H4K5ac at NCED9. Taken together, our results indicate that HD2A and HD2B partly function through ABA signaling and act as negative regulators during the drought resistance response via the regulation of ABA biosynthesis and response genes.

Arabidopsis histone deacetylase HD2A and HD2B regulate seed dormancy by repressing DELAY OF GERMINATION 1.

Seed dormancy is a crucial developmental transition that affects the adaption and survival of plants. Arabidopsis DELAY OF GERMINATION 1 (DOG1) is known as a master regulator of seed dormancy. However, although several upstream factors of DOG1 have been reported, the exact regulation of DOG1 is not fully understood. Histone acetylation is an important regulatory layer, controlled by histone acetyltransferases and histone deacetylases. Histone acetylation strongly correlates with transcriptionally active chromatin, whereas heterochromatin is generally characterized by hypoacetylated histones. Here we describe that loss of function of two plant-specific histone deacetylases, HD2A and HD2B, resulted in enhanced seed dormancy in Arabidopsis. Interestingly, the silencing of HD2A and HD2B caused hyperacetylation of the DOG1 locus and promoted the expression of DOG1 during seed maturation and imbibition. Knockout of DOG1 could rescue the seed dormancy and partly rescue the disturbed development phenotype of hd2ahd2b. Transcriptomic analysis of the hd2ahd2b line shows that many genes involved in seed development were impaired. Moreover, we demonstrated that HSI2 and HSL1 interact with HD2A and HD2B. In sum, these results suggest that HSI2 and HSL1 might recruit HD2A and HD2B to DOG1 to negatively regulate DOG1 expression and to reduce seed dormancy, consequently, affecting seed development during seed maturation and promoting seed germination during imbibition.

Quantitative surface free energy with micro-colloid probe pairs.

Measurement of the surface free energy (SFE) of a material allows the prediction of its adhesion properties. Materials can have microscale or sub-microscale surface inhomogeneities, engineered or random, which affect the surface macroscopic behaviour. However, quantitative characterization of the SFE at such length scales remains challenging in view of the variety of instruments and techniques available, the poor knowledge of critical variables and parameters during measurements and the need for appropriate contact models to derive the SFE from the measurements. Failure to characterize adhesion correctly may result in defective components or lengthy process optimization costing billions to industry. Conversely, for planar and homogeneous surfaces, contact angle (CA) measurements are standardized and allow for calculating the SFE using for example the Owen-Wendt model, relying only on the properties of the probing liquids. As such, we assessed and report here a method to correlate quantitative measurements of force-distance curves made with an atomic force microscope (AFM) and with silica and polystyrene (PS) colloidal probe pairs, with quantitative CA measurements and CA-derived SFE values. We measured five surfaces (mica, highly oriented pyrolytic graphite, thermally grown silica on silicon, silicon, and silicon with a super-hydrophobic coating), ranging from hydrophilic to super-hydrophobic, and found an excellent classification of the AFM measurements when these are represented by a set of principal components (PCs), and when both silica and PS colloidal probes are considered together. A regression of the PCs onto the CA measurements allows recovery of the SFE at the length scale of the colloidal probes, which is here ca. 1 micron. We found that once the PC-regression model is built, test sets of only ten AFM force-distance curves are sufficient to predict the local SFE with the calibrated silica and PS colloidal probes.

The ecologically relevant genetics of plant-plant interactions.

Interactions among plants have been long recognized as a major force driving plant community dynamics and crop yield. Surprisingly, our knowledge of the ecological genetics associated with variation of plant-plant interactions remains limited. In this opinion article by scientists from complementary disciplines, the international PLANTCOM network identified four timely questions to foster a better understanding of the mechanisms mediating plant assemblages. We propose that by identifying the key relationships among phenotypic traits involved in plant-plant interactions and the underlying adaptive genetic and molecular pathways, while considering environmental fluctuations at diverse spatial and time scales, we can improve predictions of genotype-by-genotype-by-environment interactions and modeling of productive and stable plant assemblages in wild habitats and crop fields.

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.

DNA methylation dynamics during stress response in woodland strawberry (Fragaria vesca).

Environmental stresses can result in a wide range of physiological and molecular responses in plants. These responses can also impact epigenetic information in genomes, especially at the level of DNA methylation (5-methylcytosine). DNA methylation is the hallmark heritable epigenetic modification and plays a key role in silencing transposable elements (TEs). Although DNA methylation is an essential epigenetic mechanism, fundamental aspects of its contribution to stress responses and adaptation remain obscure. We investigated epigenome dynamics of wild strawberry (Fragaria vesca) in response to variable ecologically relevant environmental conditions at the DNA methylation level. F. vesca methylome responded with great plasticity to ecologically relevant abiotic and hormonal stresses. Thermal stress resulted in substantial genome-wide loss of DNA methylation. Notably, all tested stress conditions resulted in marked hot spots of differential DNA methylation near centromeric or pericentromeric regions, particularly in the non-symmetrical DNA methylation context. Additionally, we identified differentially methylated regions (DMRs) within promoter regions of transcription factor (TF) superfamilies involved in plant stress-response and assessed the effects of these changes on gene expression. These findings improve our understanding on stress-response at the epigenome level by highlighting the correlation between DNA methylation, TEs and gene expression regulation in plants subjected to a broad range of environmental stresses.

Genetic and environmental drivers of large-scale epigenetic variation in Thlaspi arvense.

Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being lower in colder regions and in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylated regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.

Transcriptional response of a target plant to benzoxazinoid and diterpene allelochemicals highlights commonalities in detoxification.

BACKGROUND: Plants growing in proximity to other plants are exposed to a variety of metabolites that these neighbors release into the environment. Some species produce allelochemicals to inhibit growth of neighboring plants, which in turn have evolved ways to detoxify these compounds. RESULTS: In order to understand how the allelochemical-receiving target plants respond to chemically diverse compounds, we performed whole-genome transcriptome analysis of Arabidopsis thaliana exposed to either the benzoxazinoid derivative 2-amino- 3H-phenoxazin-3-one (APO) or momilactone B. These two allelochemicals belong to two very different compound classes, benzoxazinoids and diterpenes, respectively, produced by different Poaceae crop species. CONCLUSIONS: Despite their distinct chemical nature, we observed similar molecular responses of A. thaliana to these allelochemicals. In particular, many of the same or closely related genes belonging to the three-phase detoxification pathway were upregulated in both treatments. Further, we observed an overlap between genes upregulated by allelochemicals and those involved in herbicide detoxification. Our findings highlight the overlap in the transcriptional response of a target plant to natural and synthetic phytotoxic compounds and illustrate how herbicide resistance could arise via pathways involved in plant-plant interaction.

Mutation bias reflects natural selection in Arabidopsis thaliana.

Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences1. Here we test this assumption with large surveys of de novo mutations in the plant Arabidopsis thaliana. In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome-mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions (r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias2 reduces the occurrence of deleterious mutations in Arabidopsis, challenging the prevailing paradigm that mutation is a directionless force in evolution.

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates.

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high-quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe non-coding RNAs, pseudogenes and transposable elements, and highlight tissue-specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome-wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

MethylScore, a pipeline for accurate and context-aware identification of differentially methylated regions from population-scale plant whole-genome bisulfite sequencing data.

Whole-genome bisulfite sequencing (WGBS) is the standard method for profiling DNA methylation at single-nucleotide resolution. Different tools have been developed to extract differentially methylated regions (DMRs), often built upon assumptions from mammalian data. Here, we present MethylScore, a pipeline to analyse WGBS data and to account for the substantially more complex and variable nature of plant DNA methylation. MethylScore uses an unsupervised machine learning approach to segment the genome by classification into states of high and low methylation. It processes data from genomic alignments to DMR output and is designed to be usable by novice and expert users alike. We show how MethylScore can identify DMRs from hundreds of samples and how its data-driven approach can stratify associated samples without prior information. We identify DMRs in the A. thaliana 1,001 Genomes dataset to unveil known and unknown genotype-epigenotype associations .

The EpiDiverse Plant Epigenome-Wide Association Studies (EWAS) Pipeline.

Bisulfite sequencing is a widely used technique for determining DNA methylation and its relationship with epigenetics, genetics, and environmental parameters. Various techniques were implemented for epigenome-wide association studies (EWAS) to reveal meaningful associations; however, there are only very few plant studies available to date. Here, we developed the EpiDiverse EWAS pipeline and tested it using two plant datasets, from P. abies (Norway spruce) and Q. lobata (valley oak). Hence, we present an EWAS implementation tested for non-model plant species and describe its use.

GSNOR Contributes to Demethylation and Expression of Transposable Elements and Stress-Responsive Genes.

In the past, reactive nitrogen species (RNS) were supposed to be stress-induced by-products of disturbed metabolism that cause oxidative damage to biomolecules. However, emerging evidence demonstrates a substantial role of RNS as endogenous signals in eukaryotes. In plants, S-nitrosoglutathione (GSNO) is the dominant RNS and serves as the •NO donor for S-nitrosation of diverse effector proteins. Remarkably, the endogenous GSNO level is tightly controlled by S-nitrosoglutathione reductase (GSNOR) that irreversibly inactivates the glutathione-bound NO to ammonium. Exogenous feeding of diverse RNS, including GSNO, affected chromatin accessibility and transcription of stress-related genes, but the triggering function of RNS on these regulatory processes remained elusive. Here, we show that GSNO reductase-deficient plants (gsnor1-3) accumulate S-adenosylmethionine (SAM), the principal methyl donor for methylation of DNA and histones. This SAM accumulation triggered a substantial increase in the methylation index (MI = [SAM]/[S-adenosylhomocysteine]), indicating the transmethylation activity and histone methylation status in higher eukaryotes. Indeed, a mass spectrometry-based global histone profiling approach demonstrated a significant global increase in H3K9me2, which was independently verified by immunological detection using a selective antibody. Since H3K9me2-modified regions tightly correlate with methylated DNA regions, we also determined the DNA methylation status of gsnor1-3 plants by whole-genome bisulfite sequencing. DNA methylation in the CG, CHG, and CHH contexts in gsnor1-3 was significantly enhanced compared to the wild type. We propose that GSNOR1 activity affects chromatin accessibility by controlling the transmethylation activity (MI) required for maintaining DNA methylation and the level of the repressive chromatin mark H3K9me2.

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

Epigenetics in plant organismic interactions.

Plants are hubs of organismic interactions. They constantly engage in beneficial or competitive interactions with fungi, oomycetes, bacteria, insects, nematodes, and other plants. To adjust the molecular processes necessary for the establishment and maintenance of beneficial interactions and for the defense against pathogens and herbivores, plants have evolved intricate regulatory mechanisms. Besides the canonical plant immune system that acts as the primary defense, epigenetic mechanisms have started to emerge as another regulatory entity and as a target of pathogens trying to overcome the plant's defenses. In this review, we highlight recent advances in understanding the contribution of various epigenetic components and of epigenetic diversity to plant-organismic interactions.