Epigenomic and transcriptomic persistence of heat stress memory in strawberry (Fragaria vesca).

BACKGROUND: In plants, epigenetic stress memory has so far been found to be largely transient. Here, we wanted to assess the heritability of heat stress-induced epigenetic and transcriptomic changes following woodland strawberry (Fragaria vesca) reproduction. Strawberry is an ideal model to study epigenetic inheritance because it presents two modes of reproduction: sexual (self-pollinated plants) and asexual (clonally propagated plants named daughter plants). Taking advantage of this model, we investigated whether heat stress-induced DNA methylation changes can be transmitted via asexual reproduction. RESULTS: Our genome-wide study provides evidence for stress memory acquisition and maintenance in F. vesca. We found that specific DNA methylation marks or epimutations are stably transmitted over at least three asexual generations. Some of the epimutations were associated with transcriptional changes after heat stress. CONCLUSION: Our findings show that the strawberry methylome and transcriptome respond with a high level of flexibility to heat stress. Notably, independent plants acquired the same epimutations and those were inherited by their asexual progenies. Overall, the asexual progenies can retain some information in the genome of past stresses encountered by their progenitors. This molecular memory, also documented at the transcriptional level, might be involved in functional plasticity and stress adaptation. Finally, these findings may contribute to novel breeding approaches for climate-ready plants.

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.

Natural and induced epigenetic variation for crop improvement.

Maintaining global food security is a major challenge that requires novel strategies for crop improvement. Epigenetic regulation of plant responses to adverse environmental conditions provides a tunable mechanism to optimize plant growth, adaptation and ultimately yield. Epibreeding employs agricultural practices that rely on key epigenetic features as a means of engineering favorable phenotypic traits in target crops. This review summarizes recent findings on the role of epigenetic marks such as DNA methylation and histone modifications, in controlling phenotypic variation in crop species in response to environmental factors. The potential use of natural and induced epigenetic features as platforms for crop improvement via epibreeding, is discussed.

Extrachromosomal circular DNA: A neglected nucleic acid molecule in plants.

Throughout the years, most plant genomic studies were focused on nuclear chromosomes. Extrachromosomal circular DNA (eccDNA) has largely been neglected for decades since its discovery in 1965. While initial research showed that eccDNAs can originate from highly repetitive sequences, recent findings show that many regions of the genome can contribute to the eccDNA pool. Currently, the biological functions of eccDNAs, if any, are a mystery but recent studies have indicated that they can be regulated by different genomic loci and contribute to stress response and adaptation. In this review, we outline current relevant technological developments facilitating eccDNA identification and the latest discoveries about eccDNAs in plants. Finally, we explore the probable functions and future research directions that could be undertaken with respect to different eccDNA sources.

Experimentally heat-induced transposition increases drought tolerance in Arabidopsis thaliana.

Eukaryotic genomes contain a vast diversity of transposable elements (TEs). Formerly often described as selfish and parasitic DNA sequences, TEs are now recognised as a source of genetic diversity and powerful drivers of evolution. However, because their mobility is tightly controlled by the host, studies experimentally assessing how fast TEs may mediate the emergence of adaptive traits are scarce. We exposed Arabidopsis thaliana high-copy TE lines (hcLines) with up to c. eight-fold increased copy numbers of the heat-responsive ONSEN TE to drought as a straightforward and ecologically highly relevant selection pressure. We provide evidence for increased drought tolerance in five out of the 23 tested hcLines and further pinpoint one of the causative mutations to an exonic insertion of ONSEN in the ribose-5-phosphate-isomerase 2 gene. The resulting loss-of-function mutation caused a decreased rate of photosynthesis, plant size and water consumption. Overall, we show that the heat-induced transposition of a low-copy TE increases phenotypic diversity and leads to the emergence of drought-tolerant individuals in A. thaliana. This is one of the rare empirical examples substantiating the adaptive potential of mobilised stress-responsive TEs in eukaryotes. Our work demonstrates the potential of TE-mediated loss-of-function mutations in stress adaptation.

ecc_finder: A Robust and Accurate Tool for Detecting Extrachromosomal Circular DNA From Sequencing Data.

Extrachromosomal circular DNA (eccDNA) has been observed in different species for decades, and more and more evidence shows that this specific type of DNA molecules may play an important role in rapid adaptation. Therefore, characterizing the full landscape of eccDNA has become critical, and there are several protocols for enriching eccDNAs and performing short-read or long-read sequencing. However, there is currently no available bioinformatic tool to identify eccDNAs from Nanopore reads. More importantly, the current tools based on Illumina short reads lack an efficient standardized pipeline notably to identify eccDNA originating from repeated loci and cannot be applied to very large genomes. Here, we introduce a comprehensive tool to solve both of these two issues. Applying ecc_finder to eccDNA-seq data (either mobilome-seq, Circle-Seq and CIDER-seq) from Arabidopsis, human, and wheat (with genome sizes ranging from 120Mb to 17 Gb), we document the improvement of computational time, sensitivity, and accuracy and demonstrate ecc_finder wide applicability and functionality.

Genomic impact of stress-induced transposable element mobility in Arabidopsis.

Transposable elements (TEs) have long been known to be major contributors to plant evolution, adaptation and crop domestication. Stress-induced TE mobilization is of particular interest because it may result in novel gene regulatory pathways responding to stresses and thereby contribute to stress adaptation. Here, we investigated the genomic impacts of stress induced TE mobilization in wild type Arabidopsis plants. We find that the heat-stress responsive ONSEN TE displays an insertion site preference that is associated with specific chromatin states, especially those rich in H2A.Z histone variant and H3K27me3 histone mark. In order to better understand how novel ONSEN insertions affect the plant's response to heat stress, we carried out an in-depth transcriptomic analysis. We find that in addition to simple gene knockouts, ONSEN can produce a plethora of gene expression changes such as: constitutive activation of gene expression, alternative splicing, acquisition of heat-responsiveness, exonisation and genesis of novel non-coding and antisense RNAs. This report shows how the mobilization of a single TE-family can lead to a rapid rise of its copy number increasing the host's genome size and contribute to a broad range of transcriptomic novelty on which natural selection can then act.

The plant mobile domain proteins MAIN and MAIL1 interact with the phosphatase PP7L to regulate gene expression and silence transposable elements in Arabidopsis thaliana.

Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.

Divergent DNA Methylation Signatures of Juvenile Seedlings, Grafts and Adult Apple Trees.

The vast majority of previous studies on epigenetics in plants have centered on the study of inheritance of DNA methylation patterns in annual plants. In contrast, perennial plants may have the ability to accumulate changes in DNA methylation patterns over numerous years. However, currently little is known about long-lived perennial and clonally reproducing plants that may have evolved different DNA methylation inheritance mechanisms as compared to annual plants. To study the transmission of DNA methylation patterns in a perennial plant, we used apple (Malus domestica) as a model plant. First, we investigated the inheritance of DNA methylation patterns during sexual reproduction in apple by comparing DNA methylation patterns of mature trees to juvenile seedlings resulting from selfing. While we did not observe a drastic genome-wide change in DNA methylation levels, we found clear variations in DNA methylation patterns localized in regions enriched for genes involved in photosynthesis. Using transcriptomics, we also observed that genes involved in this pathway were overexpressed in seedlings. To assess how DNA methylation patterns are transmitted during clonal propagation we then compared global DNA methylation of a newly grafted tree to its mature donor tree. We identified significant, albeit weak DNA methylation changes resulting from grafting. Overall, we found that a majority of DNA methylation patterns from the mature donor tree are transmitted to newly grafted plants, however with detectable specific local differences. Both the epigenomic and transcriptomic data indicate that grafted plants are at an intermediate phase between an adult tree and seedling and inherit part of the epigenomic history of their donor tree.

Skin Color in Apple Fruit (Malus × domestica): Genetic and Epigenetic Insights.

Apple skin color is an important trait for organoleptic quality. In fact, it has a major influence on consumer choice. Skin color is, thus, one of the most important criteria taken into account by breeders. For apples, most novel varieties are so-called "mutants" or "sports" that have been identified in clonal populations. Indeed, many "sports" exist that show distinct phenotypic differences compared to the varieties from which they originated. These differences affect a limited number of traits of economic importance, including skin color. Until recently, the detailed genetic or epigenetic changes resulting in heritable phenotypic changes in sports was largely unknown. Recent technological advances and the availability of several high-quality apple genomes now provide the bases to understand the exact nature of the underlying molecular changes that are responsible for the observed phenotypic changes observed in sports. The present review investigates the molecular nature of sports affected in apple skin color giving arguments in favor of the genetic or epigenetic explanatory models.

The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis.

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV's largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.

Apple whole genome sequences: recent advances and new prospects.

In 2010, a major scientific milestone was achieved for tree fruit crops: publication of the first draft whole genome sequence (WGS) for apple (Malus domestica). This WGS, v1.0, was valuable as the initial reference for sequence information, fine mapping, gene discovery, variant discovery, and tool development. A new, high quality apple WGS, GDDH13 v1.1, was released in 2017 and now serves as the reference genome for apple. Over the past decade, these apple WGSs have had an enormous impact on our understanding of apple biological functioning, trait physiology and inheritance, leading to practical applications for improving this highly valued crop. Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly. Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees. High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders. We understand the species, geographical, and genomic origins of domesticated apple more precisely, as well as its relationship to wild relatives. The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable, environmentally sound, productive, and consumer-desirable apple production. This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs. Recommendations for "what's next" focus on necessary upgrades to the genome sequence data pool, as well as for use of the data, to reach new frontiers in genomics-based scientific understanding of apple.

Functional and molecular characterization of the conserved Arabidopsis PUMILIO protein, APUM9.

KEY MESSAGE: Here we demonstrate that the APUM9 RNA-binding protein and its co-factors play a role in mRNA destabilization and how this activity might regulate early plant development. APUM9 is a conserved PUF RNA-binding protein (RBP) under complex transcriptional control mediated by a transposable element (TE) that restricts its expression in Arabidopsis. Currently, little is known about the functional and mechanistic details of the plant PUF regulatory system and the biological relevance of the TE-mediated repression of APUM9 in plant development and stress responses. By combining a range of transient assays, we show here, that APUM9 binding to target transcripts can trigger their rapid decay via its conserved C-terminal RNA-binding domain. APUM9 directly interacts with DCP2, the catalytic subunit of the decapping complex and DCP2 overexpression induces rapid decay of APUM9 targeted mRNAs. We show that APUM9 negatively regulates the expression of ABA signaling genes during seed imbibition, and thereby might contribute to the switch from dormant stage to seed germination. By contrast, strong TE-mediated repression of APUM9 is important for normal plant growth in the later developmental stages. Finally, APUM9 overexpression plants show slightly enhanced heat tolerance suggesting that TE-mediated control of APUM9, might have a role not only in embryonic development, but also in plant adaptation to heat stress conditions.

A large-scale circular RNA profiling reveals universal molecular mechanisms responsive to drought stress in maize and Arabidopsis.

Drought is a major abiotic stress that threatens global food security. Circular RNAs (circRNAs) are endogenous RNAs. How these molecules influence plant stress responses remains elusive. Here, a large-scale circRNA profiling identified 2174 and 1354 high-confidence circRNAs in maize and Arabidopsis, respectively, and most were differentially expressed in response to drought. A substantial number of drought-associated circRNA-hosting genes were involved in conserved or species-specific pathways in drought responses. Comparative analysis revealed that circRNA biogenesis was more complex in maize than in Arabidopsis. In most cases, maize circRNAs were negatively correlated with sRNA accumulation. In 368 maize inbred lines, the circRNA-hosting genes were enriched for single nucleotide polymorphisms (SNPs) associated with circRNA expression and drought tolerance, implying either important roles of circRNAs in maize drought responses or their potential use as biomarkers for breeding drought-tolerant maize. Additionally, the expression levels of circRNAs derived from drought-responsible genes encoding calcium-dependent protein kinase and cytokinin oxidase/dehydrogenase were significantly associated with drought tolerance of maize seedlings. Specifically, Arabidopsis plants overexpressing circGORK (Guard cell outward-rectifying K+ -channel) were hypersensitive to abscisic acid, but insensitive to drought, suggesting a positive role of circGORK in drought tolerance. We report the transcriptomic profiling and transgenic studies of circRNAs in plant drought responses, and provide evidence highlighting the universal molecular mechanisms involved in plant drought tolerance.

The SCOOP12 peptide regulates defense response and root elongation in Arabidopsis thaliana.

Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner.

Biotic Stress-Induced Priming and De-Priming of Transcriptional Memory in Arabidopsis and Apple.

Under natural growth conditions, plants experience various and repetitive biotic and abiotic stresses. Salicylic acid (SA) is a key phytohormone involved in the response to biotic challenges. Application of synthetic SA analogues can efficiently prime defense responses, and leads to improved pathogen resistance. Because SA analogues can result in long-term priming and memory, we identified genes for which expression was affected by the SA analogue and explored the role of DNA methylation in this memorization process. We show that treatments with an SA analogue can lead to long-term transcriptional memory of particular genes in Arabidopsis. We found that subsequent challenging of such plants with a bacterial elicitor reverted this transcriptional memory, bringing their expression back to the original pre-treatment level. We also made very similar observations in apple (Malus domestica), suggesting that this expression pattern is highly conserved in plants. Finally, we found a potential role for DNA methylation in the observed transcriptional memory behavior. We show that plants defective in DNA methylation pathways displayed a different memory behavior. Our work improves our understanding of the role of transcriptional memory in priming, and has important implication concerning the application of SA analogues in agricultural settings.

The 5'-3' Exoribonuclease XRN4 Regulates Auxin Response via the Degradation of Auxin Receptor Transcripts.

Auxin is a major hormone which plays crucial roles in instructing virtually all developmental programs of plants. Its signaling depends primarily on its perception by four partially redundant receptors of the TIR1/AFB2 clade (TAARs), which subsequently mediate the specific degradation of AUX/IAA transcriptional repressors to modulate the expression of primary auxin-responsive genes. Auxin homeostasis depends on complex regulations at the level of synthesis, conjugation, and transport. However, the mechanisms and principles involved in the homeostasis of its signaling are just starting to emerge. We report that xrn4 mutants exhibit pleiotropic developmental defects and strong auxin hypersensitivity phenotypes. We provide compelling evidences that these phenotypes are directly caused by improper regulation of TAAR transcript degradation. We show that the cytoplasmic 5'-3' exoribonuclease XRN4 is required for auxin response. Thus, our work identifies new targets of XRN4 and a new level of regulation for TAAR transcripts important for auxin response and for plant development.

Ecological plant epigenetics: Evidence from model and non-model species, and the way forward.

Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.

Inhibition of RNA polymerase II allows controlled mobilisation of retrotransposons for plant breeding.

BACKGROUND: Retrotransposons play a central role in plant evolution and could be a powerful endogenous source of genetic and epigenetic variability for crop breeding. To ensure genome integrity several silencing mechanisms have evolved to repress retrotransposon mobility. Even though retrotransposons fully depend on transcriptional activity of the host RNA polymerase II (Pol II) for their mobility, it was so far unclear whether Pol II is directly involved in repressing their activity. RESULTS: Here we show that plants defective in Pol II activity lose DNA methylation at repeat sequences and produce more extrachromosomal retrotransposon DNA upon stress in Arabidopsis and rice. We demonstrate that combined inhibition of both DNA methylation and Pol II activity leads to a strong stress-dependent mobilization of the heat responsive ONSEN retrotransposon in Arabidopsis seedlings. The progenies of these treated plants contain up to 75 new ONSEN insertions in their genome which are stably inherited over three generations of selfing. Repeated application of heat stress in progeny plants containing increased numbers of ONSEN copies does not result in increased activation of this transposon compared to control lines. Progenies with additional ONSEN copies show a broad panel of environment-dependent phenotypic diversity. CONCLUSIONS: We demonstrate that Pol II acts at the root of transposon silencing. This is important because it suggests that Pol II can regulate the speed of plant evolution by fine-tuning the amplitude of transposon mobility. Our findings show that it is now possible to study induced transposon bursts in plants and unlock their use to induce epigenetic and genetic diversity for crop breeding.