- Email: [email protected]
Plant stress biology in epigenomic era
Journal Pre-proof Plant Stress Biology in Epigenomic Era Anna Perrone
PII:
S0168-9452(19)31549-3
DOI:
https://doi.org/10.1016/j.plantsci.2019.110376
Reference:
PSL 110376
To appear in:
Plant Science
Federico Martinelli
PII:
S0168-9452(19)31549-3
DOI:
https://doi.org/10.1016/j.plantsci.2019.110376
Reference:
PSL 110376
To appear in:
Plant Science
Received Date:
12 April 2019
Please cite this article as: Perrone A, Martinelli F, Plant Stress Biology in Epigenomic Era, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110376
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Plant Stress Biology in Epigenomic Era Anna Perronea, Federico Martinellib* a
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze, Palermo, 90128, Italy b Department of Biology, University of Firenze, Sesto Fiorentino, Florence, 50019, Italy
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*Corresponding authors: Federico Martinelli Email: [email protected] Anna Perrone Email: [email protected]
Highlights
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ABSTRACT
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Different DNA methylation enzymes are involved in different plant processes An integrated systems biology approach is needed in crop stress biology Priming drives a wide range of epigenetic mechanisms An epigenetic code is present in response to environmental stresses in plants
Recent progress in "omics" methodologies allow us to gain insight into the complex molecular
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regulatory networks underlying plant responses to environmental stresses. Among the different
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genome-wide analysis, epigenomics is the most under-investigated “omic” approach requiring more critical and speculative discussion about approaches, methods and experimental designs. Epigenomics allows us to gain insight into the molecular adaptation of plants in response to environmental stresses. The identification of epigenetic marks transmitted during filial generations enables new theories to be developed on the evolution of living organisms in relation to environmental changes. The molecular mechanisms driving the capacity of plants to memorize a stress and to generate stress-resistant progenies are still unclear and scarcely investigated. The 1
elucidation of these cryptic molecular switches will assist breeders in designing crops characterized by minimally compromised productivity in relation to stresses caused by climate change. The aim of this review is to briefly describe the most uptodate epigenomic approaches, update recent progresses in crop epigenomics in plant stress biology, and to stimulate the discussion of new epigenomic methods and approaches in the new era of “omic” sciences.
Keywords: epigenomics, omics, plant stress biology, priming, stress memory, transgenerational
1. Epigenetic mechanisms and techniques in plants
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modifications.
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Epigenetic mechanisms refer to all the modifications of gene expression that occur without altering the DNA sequence. The main epigenetic-driven modifications belong to three types of mechanisms:
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1) chromatin restructuring and histone post-translational modifications (PTMs), 2) DNA
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methylation changes, and 3) post-transcriptional modifications due to non-coding RNAs (microRNA or long non–coding RNAs).
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1.1 Chromatin restructuring and histone post-translational modifications Histone post-transcriptional modifications are key modifications significantly altering gene
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activities due to modifications at the N-terminal tail of core histones of the nucleosome. These
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chemical modifications that provoke chromatin restructuring consists of acetylation, methylation, phosphorylation and ubiquitination [1]. The first two modifications, histone acetylation and methylation, are generally due to the activity of large (HATs) and histone lysine methyltransferases (HKMTs) enzyme families. These epigenetic marks can be reversed by other enzymes such as histone deacetylases (HDACs) and histone demethylases (HDMs), respectively [2]. While phosphorylation of histone H3 Ser-10 has been linked with responses to salinity, cold stress and abscissic acid treatment [3], ubiquitination has been mainly associated with increases in 2
transcription [4]. Ubiquitination conjugation is a post-translational modification that is a key player in the signal transduction of oxidative stress responses inducing DNA damage repair, protein degradation, and trafficking [5]. Particularly changes in K63 ubiquitination in response to oxidative stresses have been shown to affect polysome stability and protein expression. These results implied that ubiquitination has a regulatory role in redox processes [5]. Moreover, the tails of the N-terminal histones modified through, for example, acetylation processes, have been shown to be crucial events both for defense mechanisms and for plant development [6].
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Studies on the pluripotency of plant cells demonstrate the existence of regulatory mechanisms that depend on important epigenetic factors in Arabidopsis. In this context, epigenetic modifications could be key players in reprogramming lineage-specified plant cells to pluripotency. Specifically,
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regeneration mechanisms [7] involve the histone acetyltransferase HAG1 / AtGCN5. Callus development causes the catalyzing of histone acetylation processes in different root-meristem gene
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loci, including WOX5, WOX14, SCR, PLT1 and PLT2, providing an epigenetic platform for their
1.1.1 Main techniques
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transcriptional activation.
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Chromatin Immuno-Precipitation combined with chip (DNA microarray) analysis (ChIP-CHIP) has been the most common technique used for the genome-wide analysis of chromatin epigenetic
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modifications before the arrival of the next-generation of sequencing technologies. While the ChIP-
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CHIP technique is effective in easily detecting histone positions with specific modifications of their N-terminal tails [8], ChIP-sequencing combines chromatin immunoprecipitation (ChIP) with genome-wide DNA sequencing providing a deeper level of identification of the DNA sequences bound with transcription factors and other regulating proteins. ChIP-seq consists of the selection of DNA fragments linked with the studied proteins and the analysis of the DNA fragments with deep sequencing platforms (Illumina, PacBio, Roche etc..). The combination of chromatin precipitation and DNA next-generation sequencing has highly advanced our understanding of the mechanism of 3
histone modifications in regulating gene expression [9]. ChIP-sequencing requires a known genomic sequence reference that might be represented by the studied species or a phylogenetically closely related one. Chip-seq is currently the most-desirable approach for studying histone modifications because it allows a better depth/costs ratio and the identification of new unknown SNPs responsible for chromatin restructuring, absent in arrays used in the ChIP-CHIP approach.
1.2 DNA methylation analysis
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One of the most commonly analyzed epigenetic modification is the methylation of cytosine occurring at the fifth carbon position. This epigenetic modification is frequently transient in response to the environment although DNA methylation can be inherited in the next generation
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[10]. DNA methylation plays a key role in transcriptional regulation activity during embryonic development, in key biological phenomenon such as genome stability (transposon silencing) and
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genomic imprinting during cell differentiation. Although DNA methylation has been frequently
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linked with the repression of gene expression, in some instances it has been responsible for transcription activation [11]. For example, in Arabidopsis thaliana Harris et al. found that DNA methylation promoted the recruitment of a transcriptional activation protein complex to chromatin
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sites inducing transposon silencing in neighbouring genes. It has been debated that DNA methylation is a mechanism that has occurred during evolution in
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order to silence transposons considered to be genomic parasites in the host genomes. This is
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corroborated by the fact that transposons are generally highly methylated and represent the main targets for epigenetic silencing. Unfortunately, the study of epigenetic modifications in genomic regions rich in transposon elements is not easy due to the difficulties in determining the link between DNA methylation and transposon polymorphism for their repetitive nature and their capacity to create large insertion/deletion polymorphisms. Methylation is site specific [12] and frequently occurs at pericentric heterochromatin regions driven by histone H3 lysine 9 (H3-K9) methylation [13]. Several findings support the idea that DNA methylation also plays a key role in 4
modifying genome size, structure and genetic variations. Frequently, cytosine methylation occurs when it is present in three sequences: CG, CHG and CHH (where H = A, T or C). DNA methylation has been clearly linked to abiotic stress responses in plants (Figure 1). Each of these methylation marks are catalyzed by different enzymes. Methyltransferase1 (MET1) is responsible for the CG methylation while Chromomethylase3 (CMT3) provokes CHG methylation [14]. Domains Rearranged Methyltransferases (DRM1 and DRM2) that are guided by small interfering RNAs (siRNAs) drive the methylation of CHH [15]. CHH methylation is due to RNA-dependent DNA
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methylation (RdDM) and requires plant-specific RNA polymerases IV and V. Pol IV produces precursor transcripts of 24-nt small RNAs (sRNAs) that are able to target scaffold transcripts using the complementary sequence. The maintaining of the cytosine methylation during replication
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depends on proteins such as Decrease in DNA Methylation 1 [DDM1; 14] while DNA methylations are generally deleted by specific classes of DNA glycosylases such as DEMETER (DME),
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REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 and DML3 [15]. In plant stress
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responses, DNA methylation is a key modulator of both pathogen and symbiont interactions with plants [16] and previous works have linked DNA methylation with host susceptibility [17]. Methylome analyses permitted a better clarification of epigenomic plasticity mechanisms. For
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example, studies of epigenetic analysis on the bean [18]
have facilitated the drawing up of
preliminary epigenomic maps based on chromatin accessibility, histone modifications and DNA
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methylation linked with important agronomic traits.
1.2.1 Main techniques
Next-generation sequencing technologies represent the most powerful method of identifying epigenetic modifications occurring at the DNA level. It has permitted new discoveries in the mechanisms of epigenetic regulation, through the understanding of the biological meaning of DNA methylation, chromatin modeling and the role of small RNA. NGS technologies are represented by various popular platforms, including the 454 FLX (Roche) [19], the Genome Analyzer / Hiseq 5
(Illumina Solexa) [20,21] and the SOLiD (Life Technologies) and new platforms such as Heliscope (Helicos) [22] and PacBio RS (Pacific Biosciences) [23] for single molecular sequencing, and Ion Torrent (Life Technologies), based on semiconductor chips [24]. The most common technique for genome-wide methylation analysis is the Whole Genome Bisulphite sequencing (WGBS) that consists in the treatment of DNA with sodium bisulphite followed by next-generation sequencing. Bisulphite treatment of DNA provokes the conversion of unmodified cytosines to uracil maintaining 5mC unchanged. The use of restriction enzymes sensitive to DNA methylation permits the
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enriching of DNA in methylated or un-methylated sequences. WGBS allows DNA methylation sites to be identified in a genome-wide manner. This approach has been used to generate different plant methylomes [25]. Bisulphite treatment of DNA sequences precedes the hybridization of DNA
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fragments to tilling arrays or their direct sequencing. In addition, antibodies specific for m5C can be used to analyze them before the analysis using arrays or sequencing platforms (MeDip-ChIP or
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MeDip-Seq). The bisulphite-treated DNA can be analyzed with PCR for target epigenetic analysis
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followed by sequencing [26]. However, the PCR amplification that is also included in the genomewide analysis technique is not exempt from bias. False positive and false negative results can also be produced due the incomplete cytosine conversion by sodium bisulphite or the over-conversion of
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5mC. Indeed, PacBio sequencing that does not require bisulphite treatment is able to eliminate this bias. Although methylome analysis through sequencing might be relatively expensive for large
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plant genomes [27], the reduction of the cost of sequencing is doubling every 18 months, rendering
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these analyses affordable for small projects (< 10000-20000 $). A method for DNA methylation is the identification of target regions with padlock-probe based targeting coupled with promoter tiling arrays [28]. An alternative is a reduced representation bisulphite sequencing (RRBS), which consists of the fractionation of the genome and the enrichment of fragments with high CpG content regions before sequencing [29]. In addition, pyrosequencing has been used without the use of methylation specific PCR [30]. This is employed to determine the specific CpG sites in the amplified product. Based on the amount of incorporated C and T, it is possible to determine the 6
ratio of C and T and individual sites of methylation. However, the costs for this technique are high. An application of this technique is represented by the analysis of genomic imprinting through the use of allele specific primers with single nucleotide polymorphisms. Another technique for the analysis of DNA methylation is the methylation-sensitive single-strand conformation analysis (MSSSCA). This method employs the single-strand conformation analysis to resolve sequence differences between methylated and unmethylated samples due to the conversion of bisulphite of cytosine to thiamine while the methylated cytosine remains unchanged. The efficiency of SSCA is
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70-95% [31]. High-resolution melting analysis (HRM) also permits the analysis of DNA methylation when quantitative real-time PCR analysis is completed. It consists of applying melting cycles that allow converted to be distinguished from unconverted bisulphite-treated DNA. The PCR
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products are analyzed by temperature ramping that will result in the liberation of intercalating fluorescent dye during the melting process. The rapidity of the melting and consequent release of
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the dye is due to the C to T content that represents the DNA methylation amount [32]. The
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weakness of this method is the ability to analyze only one or a few amplicons each time [34-35]. Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique suitable for a quick quantitation of methylation occurrence at individual CpG sites [35]. It uses the primer
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extension method that has been optimized for detecting single-nucleotide polymorphisms. In this technique, bisulphite treatment is used together with PCR amplifications with bisulphite-specific
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primers. They are designed to anneal to the sequence up to the base pair immediately before the site
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of DNA methylation. This is a simple method for screening DNA methylation in a large number of samples. This method also promotes genomic sequencing since the electrophoretic bands may be isolated and directly sequenced. Other methodologies employ matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry analysis to differentiate between the two polymorphic primer extension products based on the GOOD assay designed for SNP genotyping. Base-specific cleavage/MALDI-TOF consists in the in vitro transcription of the region under study using an RNA polymerase promoter site. RNAse A treatment follows retro7
transcription and the base specificity is obtained by adding incorporating cleavage resistant dTTP or dCTP if cytosine or uracil specific cleaves are desired, respectively. The fragments obtained by the cleavage
are
analyzed
with
MALDI-TOF.
The
bisulphite
treatment
provokes
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introduction/removal of cleavage sites by C to U conversions or a shift in fragment mass by G to A conversions in the amplified reverse strand. C-specific cleavage will only work at the methylated CpG sites. This technique has a great efficiency, allowing identification of CpG sites in multiple tissues with low costs. The EpiTYPER™ assay allows the detection and quantification of DNA
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methylation in a specific region through a high-resolution scan of selected regions. This method enables us to perform an epigenomic analysis of a region surrounding a single CpGs and can be employed to validate the CpGs detected by genome-wide approaches. This technique is considered
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a rapid method for the targeted quantification of individual CpGs in a high throughput manner through the base-specific cleavage after bisulphite-conversion of DNA coupled with MALDI-TOF
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MS [36].
1.3 Non-coding RNA-mediated epigenetic regulation It is known that 90% of the eukariotic genome is transcribed but only 1-2% of the genome is
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translated to proteins. This means that there is a large number of RNA molecules that are circulating in the cells whose functions are unknown but most probably having a key role in the post-
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transcriptional regulation [37]. These RNA molecules are noncoding RNAs (ncRNAs) that are the
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third main actors in epigenetic modifications. Noncoding RNA molecules can be divided in different groups based on their size, function and regulation mechanisms: microRNAs (miRNAs), short interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and long non-coding (lncRNAs). Another classification has divided them into two categories: short ncRNAs (<30 nts; including miRNAs, siRNAs, and piRNAs) and long ncRNAs (>200 nts). Small RNAs are important activators/inhibitors of plant environmental stresses. For example, it has been shown that the plant pathogen Botrytis cinerea delivers small RNAs into the host cells to repress plant defense genes and 8
thus deactivate host immunity [38]. Besides lncRNAs that clearly function in chromatin modifications, a few other ncRNAs also play roles in other biological processes in plants [38]. For example, the soybean early nodulin gene, Enod40, is involved in the regulation of symbiotic interactions between leguminous plants and soil bacteria [38]. Several stress-responsive miRNAs have been found in different crops leading to the possibility of using miRNA-driven RNAinterference (RNAi) to improve key traits and provide phenotypic plasticity in a climate change scenario [39]. RNA-mediated chromatin-level silencing is strongly involved in stress responses and
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natural epigenetic variation and consequently in phenotypic diversity, physiological plasticity and evolutionary change [40]. Epigenomic regulation of chromatin structure and genome stability is essential for genetic interpretation information and key roles have emerged for small RNAs,
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proteins with domains that bind methylated DNA and DNA glycosylases in these processes [41,42]. The analysis of cytosine methylome (methylC-seq), transcriptome (mRNA-seq) and small RNA
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transcriptome (small RNA-seq) in Arabidopsis inflorescences showed methylation patterns on a
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genomic scale and a direct relationship between the sRNA position and DNA methylation [43]. In addition, genomic analysis in Arabidopsisis showed that various methylated cytosines have been detected by bisulphite sequencing using NGS technologies, precisely through the so-called BS-seq
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[44-46].
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1.3.1 Main techniques
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The most potent method for analyzing ncRNA (including miRNA) is the use of next-generation sequencing because it permits us not only to conduct a quantitative analysis of their expression but also the identification of single nuclear polymorphisms in RNA sequences [47]. Novaseq 6000 (Illumina) is one of the most-desired platforms for studying rare transcripts and splice variants due to the higher number of reads with longer reads than previous platforms (i.e. Hiseq 2500) (250 bp instead of 75 bp). In silico methods are used since they are able to distinguish lncRNAs from protein-coding transcripts based on the absence of discernible open-reading frames. Full-length 9
targeted cDNA sequencing may be the best method to determine the coding potential of transcripts, although this approach is time-consuming and expensive. Alternatively, tiling DNA microarray for genome-wide transcriptome analysis can be employed to detect and determine the expression of lncRNAs in plants. Tiling DNA microarray analyses have been used to identify new stress-induced lncRNAs [48]. High-throughput deep RNA sequencing is able to identify missing or incomplete transcripts of lncRNA and to determine the levels of lncRNA expression. A similar approach was used to identify rare alternative splicing variants of a lncRNA, HOTAIR [49]. An approach with a
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combination of different kinds of techniques (genome-wide histone modification analysis and deep sequencing) may permit the identification of the number of long intergenic ncRNAs (lincRNAs) transcribed from a K4-K36 domain, which marks active promoters with trimethylation [49].
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A summary of all the mentioned techniques divided by the three main epigenetic mechanisms is
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provided in Table 1.
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1. What is the role of epigenomics in system biology?
An integrated systems biology approach is required to gain insight into the complex molecular regulatory networks that modulate the expression of plant traits. Epigenomics is one of the most
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unexplored “omic” approaches to understand the adaptation of living organisms to rapid local environment change, weather or other stresses. However, the analysis of epigenomic changes in
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relation to plant environmental stresses requires a strong integration of the different “omic”
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analyses (Figure 2). Transcriptome analysis is essential to validate the gene silencing that is predicted by DNA methylation data. The use of the other analyses (proteomics and metabolomics) are commonly used to gain insight into the post-transcriptional and post-translational regulation mechanisms linked to specific epigenetic changes. The epigenomic modification has a real effect at the phenotypic level if the consequent change in transcription is not reversed at the posttranscriptional level. Indeed, proteomic and metabolomic platforms are essential to determine if the epigenetic modification has effects on composition at protein and metabolite levels. The power of 10
epigenomic analysis can be better exploited for plant species whose genomes have been sequenced. Transcriptomic approaches permit analysis of the expression of many thousands of genes, identifying splice variants, rare genes, single nucleotide polymorphisms. However, transcriptomic results are usually inconclusive and not strictly correlated to phenotype changes since cells frequently activate multiple regulatory mechanisms for their adaptation to stress responses. Proteomic analysis permits us to understand how protein changes are modulated by rapid or chronic environmental stresses in crops [50]. A recent integrated approach has been proposed correlating
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genes and protein expression, named proteogenomics [51]. Proteomics can be used to validate the expression of the genes while transcriptomic data permits the construction of customized protein databases, avoiding the need for homology-based approaches [52-54]. Metabolomics is the
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untargeted high-throughput technique that can be linked to epigenetic modifications. Metabolomic approaches allow a comprehensive analysis of the cell metabolism and could validate epigenomics.
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This “omic” approach has been less employed compared with the other “omic” analyses especially
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for trait mapping and plant selections [55]. It allows for deep characterization of how plant metabolism is affected in response to environmental stimuli and changing climatic conditions. The recent progresses in metabolomics allow quantification of a wide range of metabolites from a small
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amount of analyzed samples (> 10 mg). Metabolomics has been used to understand cell dehydration, water stress responses and plant-microbe interactions [55], providing a great
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contribution to shedding light on the link between genotype and metabolic pathways leading to the
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phenotype. They help identify key points of molecular network regulation. Plants biosynthesize more than a hundred thousand metabolites with different chemical structures and quantity, each one with key roles in physiological, development, and stress response processes [56-57]. Integrated multi-omic approaches are highly effective in discovering epigenomic mechanisms of regulation of multi-factorial quantitative traits. The real challenge in analyzing both “omic” data is understanding the complex gene/protein regulatory networks identification due to gene-gene and protein-protein interactions. Nodes, molecular players (genes or proteins), are generally indicated in different colors 11
depending on their trend of expression (up, down or un-regulated between control and stress condition), different forms based on their functions, and different sizes proportional to the number of connections (Figure S1). Through the construction of these networks it is possible to identify not only the highly interactive nodes (hubs), but also low interactive proteins that play a key role in the networks because they connect hubs or cores of highly interconnected proteins/genes. In this regard, the interaction between key highly interactive proteins and non-coding RNA often plays an important role in plant stress responses [56]. For example, WRKY transcription factors are typically
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key players in modulating environmental stress responses in plants at the interactome level. Several targets of miRNAs encode WRKY transcription factors [57] implying that miRNAs are involved
in the regulation of the expression of WRKYs. On the other hand, some miRNA promoters are
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abundant in W box sequences, the cis-elements recognized by WRKYs [58]. For example, the response to powdery mildew has been shown to be modulated by WRKY-smRNA interactome
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[59]
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2. Approaches for investigating epigenomic transient and transgenerational mechanisms We propose a general workflow to investigate plant transgenerational epigenomic modifications in
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response to environmental stresses. First, the stress-epigenetic response should be analyzed in controlled conditions to reduce undesired environmental variability. Basing ourselves on the
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evidence that DNA methylation generally causes the silencing of the methylated gene, the decrease in transcript abundance of the mRNA should be observed. In fact, the most desired approach is to
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integrate epigenomics with transcriptomic and metabolomic approaches to observe the inhibition of expression of the methylated genes [60-63]. The use of proteomic approaches would allow the confirmation of the epigenetic-driven changes in transcript abundance although the magnitude of analysis is generally 10-fold less than RNA-seq. ITRAQ proteomic approaches could partially contribute to fill this gap through the analysis of more than 6000 proteins using small biological samples (> 50-100 mg). The gap that still exists between transcriptomics and proteomics (even with iTRAQ) precludes the conclusion that the absence of the protein targeted by the DNA methylation 12
is a consequence of a variation in its expression. It might be due that the protein was not present in the protein extract. The heterogenous chemical nature of the proteins renders the extraction complex and difficult, and does not allow the recovery of all types of proteins during the extraction processes. In this regard, Phenol-based protocols are commonly used to extract proteins from recalcitrant plant tissues [64]. Once DNA methylation analysis, transcriptomics and proteomics are conducted, a list of potential epigenetic marks can be delivered and associated with the considered stress response. Quantitative RT-PCRs may be conducted for the targeted list of methylated genes
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to confirm the trend of altered expression in relation to the detected DNA methylation. Seeds can be collected and DNA may be extracted from zygotic tissues to perform a genome-wide DNA methylation analysis in order to see if the methylation signal is transmitted in the filial generation.
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The epigenetic patterns of the seeds can be compared with the one related to the same tissue (leaf, fruit, shoots etc.) coming from the first generation to see if the DNA methylation is a transient
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stimulus that occurrs only when the stress is present, or if it is provided by inheritable stress
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memory. In addition, the epigenetic modification should be analyzed after the occurrence of the stress if the unstressed condition could be restored after the treatment (irrigation after drought etc.). The presence of the same epigenetic mark in parental and filial generations in absence of stress
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should prove the transgenerational inheritance excluding its transient nature. Natural epigenetic variation may originate from polymorphisms in transposon insertions and repeats. Epigenetic works
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in model plant species such as Arabidopsis thaliana have shown that epigenetic regulation is closely
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associated with functional aspects. However, epigenetic studies on Arabidopsis thaliana have limitations, such as the low value methylated cytosine in the genome (5%) while many crops contain more than 20% [63]. In addition, this plant contains very few transposable elements compared to other crops (review by Lee and Kim, 2014) [65]. Mutations in epigenetic regulators appear to have a greater impact in crops than Arabidopsis. Furthermore, while the distribution of genes along the Arabidopsis chromosomes is fairly homogeneous, this situation may differ in crops. For example, the genome of Vitis vinifera (L.) is characterized by the alternation of large regions 13
with high and low genetic density [66-67]. Several epigenetic studies have been performed in crops such as tomato fruits which constitute an important model for studies on grapes where DNA methylation and histone modifications should play a pivotal role in fruit veraison [68]. In woody crops, epigenetic modifications can be investigated using clonal propagation. The stress applied in the first vegetative generation can be analyzed in the second upcoming vegetation derived from the meristematic apex previously subjected to the studied stress. The propagation through cuttings is a desirable way to identify how the epigenetic modifications is
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stable or transient. Grape has recently been proposed as a model plant to study epigenomics in perennial plants [68]. This is due for the following reasons: 1) it is a model for non-climateric fruits and flower development, 2) it presents flower buds that occur one year after they open, and 3) it is
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clonally propagated and commonly grafted.
It is well-known that environmental conditions of the previous year have a significant impact on the
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flower and subsequently on the development of the fruit. Other specific requirements that affect
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flowering and fructification are represented by clonal grafting and propagation. In this context, the epigenetic variability added to the genetic diversity of the grape could shape the phenotypic variations of the plant. The clonal diversity within the grape varieties was identified using the
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methylation-sensitive amplified polymorphism technique, allowing the understanding of the utility of epigenetic markers in intra-varietal diversity studies [69]. Another important impact on the
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epigenetic state of a plant is represented by grafting. The understanding of these mechanisms is
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useful to improve grape tolerance to highly threatening environmental stresses enhancing the product quality. Interestingly, systemic acquired resistance to downy mildew pathogen has been shown to be due to DNA methylation inherited in offspring [70]. Another crop that might be considered as a model for epigenomic studies is tomato due to the easy availability of the mutants. Chemical treatments inducing DNA methylations may be useful for generating epimutations although they might not be stable as genetic mutants. Without the need of using genetic recombination, epimutagenesis may represent an important means of exploring allelic 14
variations and the new combinations of alleles. The analysis of DNA methylation in grape in relation to fruit qualitative traits could represent an important tool of identifying interesting epialleles usable in crop breeding. Specifically, DNA methylation can generate epialleles with various levels of expression, leading to a continuous quantitative variation of a crop trait. Herbaceous auto-gamous species are great candidates to understand if epigenetic modifications have a transient or transgenerational nature. Another model plant for cereals is Brachypodium since it has a rapid life cycle, belongs to the Graminaceae family, is easy to cultivate, and has a small
3. Epigenetic regulation of plant stress responses
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diploid high-quality genome that has been recently sequenced.
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The recent progresses in the genome-wide analysis of epigenetic marks have allowed gaining insights into the dynamics and plasticity of plant adaptation to altered environmental conditions.
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Plants are sessile living organisms and they are particularly characterized by a finely-tuned
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adaptation to changing environmental conditions through the trigger or repression of epigenetic mechanisms. Most of the epigenetic features are transiently activated in response to the stress occurrence and reverted when the environmental stimuli are no longer present. However, a certain
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part of these epigenetic marks is “memorized” by the plant determining stress priming. This phenomenon consists in an improved resistance/response to an environmental stress due to the
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activation of responses (priming fingerprinting) which remain latent but prompted to be activated
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again in the presence of a subsequent stress. This induced stress resistance could be inherited in offspring resulting in an improved response to a particular stress that has been previously occurred in parents. Priming is directly linked to the concept of plant stress memory. It is known that plants do not have the same group of germline cells for all their life span as animals do. New germ cells come from meristematic cells during their entire life so that any epigenetic modification that occurrs during vegetative growth may be transmitted to the offspring. Any stable epigenetic information acquired by the genetic structures of the somatic cells might be transmitted to the next generation. 15
The presence of a stress may induce epigenetic heritable traits that represents a ‘memory' inherited in the next generations which helps the colonization of different environments. This imprint, or stress memory, can be defined as the structural, genetic, and biochemical modifications occurred as a consequence of stress exposure rendering a plant more resistant (although it might also be more sensitive in some cases) to future exposure to the same stress factor. The most common histone modification associated with stress memory is H3K4me3 although also H3K4me2 and H3K27me3 have also been found in systemic acquired response and bacterial stress
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(Figure 3). Chromatin modifications, nucleosome positioning, and DNA methylation are playing a key role in plant stress memory and several epigenetic mechanisms have been shown to be responsible for priming of plants [71] (Figure 4). Although priming may compromise plant
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productivity in the short-term, for example through a reduction of photosynthesis, it usually induces tolerance to subsequent stress and therefore favors productivity in the long-term perspective [72].
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Abscisic acid is known to be involved in plant responses to reiterated abiotic stresses and might be
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involved in priming [73. In some plant species, it was observed that ABA levels are higher under drought conditions if the plants have previously been subjected to water stress, implying that ABA may be linked to drought stress memory [74]. In this regard, Virlouvet and Fromm (2015) [75]
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showed that previously stressed plants have the capacity to maintain stomatal apertures that remain partially closed during a recovery period, which reduces transpiration during a subsequent drought
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episode. An increased expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) and
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ALDEHYDE OXIDASE 3 (AAO3), key modulators of ABA biosynthesis, was linked with drought memory.
3.1 Abiotic stresses Recent studies have demonstrated that chromatin and DNA methylation changes play an important role in resistance and tolerance mechanisms to different abiotic stresses such as cold, salt stress, drought, osmolality, or mineral changes (Figure 5), thereby pointing out the importance of 16
epigenetic regulations in these aspects [77-75]. In addition, genome-wide analyses of histone PTMs have revealed global epigenomic reprogramming in plants under abiotic stresses. Recently, a specialized histone H1 variant was shown to be required for DNA methylation changes linked with stress responses in Arabidopsis [75]. Two DEAD-box RNA helicases are responsible for epigenetic silencing of gene expression leading to inactivation of abiotic stress responses in Arabidopsis [76]. Arabidopsis mutants deficient in genes involved in the RdDM pathway or with defects in maintenance of CHG methylation showed a variable sensitivity of regulation of the stomatal index
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in conditions of low relative humidity [77]. In addition, they showed hypersensitivity to heat stress [78] or high sensitivity to phosphate starvation [79]. These results are consistent with an important
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function of the DNA methylation dynamics in the regulation of abiotic stress-responsive genes.
3.1.1 Drought and salt stress
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Drought causes extensive remodeling of DNA methylation patterns as it was observed in different
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plants species 4-81]. Modulation of DNA methylation at repetitive elements seems to be important to modulate the expression of adjacent genes. H3K4me3 modifications under drought stress have been observed [82]. It has been confirmed that H3K4me3 might be a marker for highly expressed
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genes in stress conditions. In populus, epigenetic modifications have been linked to drought responses, affecting the transcriptomic changes in response to stress [83]. In apple, a variation of
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approximately 54%, 38% and 8.5% of methylation respectively at CG, CHG and CHH sequences
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has been linked to changes in drought-sensitivity/tolerance. A part of these epigenetic changes occurred at genes encoding transcription factors (TFs) and transposable elements (TEs). It is worth noting that epigenetic effects of drought stress vary depending on many factors, such as the intensity and duration of the stress, plant genotype or growth phase, and also genomic imprinting left by previous stress episodes. In barley, drought stress-induced genome-wide DNA methylation changes were associated with water deficit [84]. Drought-stressed poplar has showed genome-wide changes in DNA methylation implying that epigenetic mechanisms are involved in adaption of trees 17
to environmental changes [85]. This has been suggested in corn where transposon elements could function as local enhancers for stress responsive genes [86]. Epigenetic responses to salinity are known to play an important role in plant resilience to this type of stress. Salt stress affected DNA methylation in key stress-related genes such as WRKYs in Arabidopsis thaliana [87], poplar [88], rice [89] forest species[90] and wheat [91]. Acetylation, methylation and phosphorylation of histones play an important role in plant responses to salt stress. In addition, salinity triggers the activation of cell wall related genes such as expansin β2 and
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xyloglucan endotransglucosylase in roots and these gene expression changes have been linked with enhanced acetylation of histones in proximity of the promoter and the open reading frames of these genes [91]. An interaction between salt and drought stress has been observed. Salinity promoted
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drought tolerance in Arabidopsis through the modification of H3K27me3. A prolonged absence of H3K27me3 induced a transient and quick transcription of high affinity K+transporter 1 [68].
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Another epigenetic mechanism involved in plant stress memory modulates the accumulation of high
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levels of proline, an aminoacid with antioxidant activities during abiotic stresses [50]. The intraspecific variability in salt tolerance in wheat has been associated with changes in differentially methylated regions (DMRs) containing genes closely associated with transposons [92]. The picture
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of the molecular regulatory mechanisms is complex since hypermethylated regions responsible for silencing of heterochromatin has also been linked to the activity of small RNAs [93]. Other
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evidences in different crops have confrimed the role of epigenomic modifications in drought and
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salt responses [94-97].
3.1.2 Metal-related stresses
Manganese oxide nanoparticles provoke DNA hypomethylation in the moss Physcomitrella patens [98]. Epigenetic studies have been conducted in Populus, a model plant for investigating molecular long-term responses under metal excess conditions. Chip-Seq and RNA-seq data were integrated in poplar showing that genes with a H3K4me3 modification were usually upregulated, while genes 18
with a H3K27me3 modification on the 5-UTR were repressed under excess of Zn [99]. The epigenome of leaves of poplar plants in response to AMF inoculation on multi-metal polluted or unpolluted soil was investigated using the MSAP approach. Few changes in cytosine methylation patterns were observed after 4 months after from planting, whereas increased hypomethylation was observed after 6 months from mycorrhizal treatment in the presence of heavy metals [100]. Transgenerational inheritance of changes in DNA methylation patterns in response to heavy metal stress was shown in rice (Oryza sativa L.) [101]. The DNA methylation changes were observed in
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leaves immediately after heavy metal treatment and they consist in CHG hypomethylation; CHGdemethylated states that were heritable to offsprings. MSAP technique was useful to determine the effect of metal contamination on cytosine residues in CCGG motifs in the genome of Red maple
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(Acer rubum), a common deciduous tree in northern America. Zinc stress has been associated with enhanced methylation linked to reduced levels of key hormones such as gibberellic acid, zeatin and
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indole acetic acid and increased levels of abscisic acid [102]. Finally, Aluminum stress altered DNA
retrotransposons [103].
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3.1.3 Cold stress
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methylation in corn and it was associated with polymorphisms in long terminal repeat
Non-coding RNAs are involved in epigenetic regulation of vernalization through the silencing of
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FLC even after winter cold. Molecular studies showed that chromatin-remodeling processes are
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responsible for the regulation of FLC expression. A high expression level of FLC has been associated with delayed flowering, whereas flowering is promoted when FLC is repressed by vernalization mediated by non-coding RNA regulation. VERNALIZATION INSENSITIVE 3 (VIN3) was identified as an essential gene to trigger repression of FLC by vernalization. VIN3 encodes a plant homeodomain (PHD) finger protein that is induced only during a cold period. The PHD finger motif in VIN3 is often found in various components of chromatin-remodeling complexes [104]. Vernalization provokes an increased association of PHD-PRC2 with FLC chromatin and an 19
enhanced deposition of H3K27me3 marks at the FLC chromatin [104]. Increased enrichments of PRC2 and H3K27me3 are hallmarks for the stable repression of FLC by vernalization. Another lncRNA involved in vernalization is a cold-inducible intronic lncRNA, COLDAIR [105]. Transcriptional silencing of MET1 gene in corn roots in response to cold causes demethylation of the Ac/Ds transposon system [106]. The Tam3 transposon methylation at CHH sites was observed in snapdragon during cold conditions [107]. Plants have developed mechanisms to survive in an ever-changing environment through the active regulation of chromatin. Particularly, it was shown
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that the interaction of HOS15 with histone deacetylase 2C (HD2C) modulates the action of promoters of cold-responsive COR genes. Epigenetic mechanisms have been observed to play a key role in modulating cold tolerance in several plants such as brassica rapa and sugar beet [108-
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109].
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3.2 Biotic stresses
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Plants are sessile organisms that have evolved different mechanisms compared to animals to escape the exposure of adverse environmental factors, including pathogens. Plants have evolved complex mechanisms of protection, among which the ability to establish symbiotic interactions to prevent
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infections is particularly relevant. Recent researches have evidenced several regulatory mechanisms controlling the plant-microbe interactions. In this context, epigenetic mechanisms are important key
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modulators of both pathogenic and symbiotic interactions between plants and microbes (Figure 6).
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DNA methylation is an epigenetic modification of DNA that prevents the loss of gene expression in plant defenses against pathogens and plays a crucial role in microbial-plant interactions. For example, several research reports confirmed the role played by recurrent DNA methylations in the induction of enhanced resistance to pathogens [110-111]. Responses to biotic and abiotic stresses are stored by intracellular molecules that allow plants to respond more efficiently to future environmental stresses. Systemic acquired resistance (SAR) or defense priming is a classic example of memory response to future stresses able to promote hypersensitivity and rapid expression of 20
defensive genes through the action of the NPR1 gene as it was found in Arabidopsis [112]. Regulation of the epigenetic expression of NPR1/NIM1 genes is an important example of H3 histone modifications affecting stress memory in response to biotic stresses [113]. Regulation of gene expression by DNA methylation is crucial to modulate the transcription of genes involved in the biosynthesis of the SA affecting plant defense mechanisms. DNA methylation could mediate the response to biotic stress through the increase of SA and the modulation of the downstream genes [1114]. These data suggested that methylation levels within TEs may dynamically control the
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expressions of transposon and the proximal gene in response to stress in Arabidopsis [115]. Chromatin remodeling plays a crucial role in plant immunity against both necrotrophic and biotrophic pathogens [110]. As previously reported, mutant bon1-1 with loss of- function leads to
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constitutive defense response mechanisms to the virulent pathogen Pst DC3000 through the activation of HUB1 and HUB2 and consequently the induction of autoimmune responses [116]. It
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was found that dimethylated or trimethylated histone H3 Lys 27 (H3K27me2/3) is involved in the
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expression of responsive genes to biotic stresses in rice. Jumonji C (jmjC) protein gene encodes a histone Lys demethylase that specifically reverses the activity of H3K27me2/3. During pathogenic infections, the expression of JMJ705 is induced. The overexpression of JMJ705 in transgenic plants
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removes levels of H3K27me2/3, inducing their expression and enhancing plant resistance to biotic stress through the involvement of jasmonic acid. JMJ704 positively regulates rice tolerance to
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bacterial blight through the epigenetic suppression of defense regulators, that is a distinct way from
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its homolog JMJ705 [116]. Furthermore, during plant infection processes, chromatin-based transcriptional regulation can also affect the expression of the effector gene in fungi [117]. Different studies have focused on the concept that pathogen infections induce histone modifications affecting key response genes. Changes in DNA methylation has been shown to affect plant resistance to diseases. For example, significant differences in cytosine methylation have been observed between resistant and sensitive genotypes to Fusarium wilt in chickpea [118]. DNA hypomethylation at specific pericentromeric regions affects priming and biotic stress resistance [119]. Responses to 21
geminivirus infection are modulated by cytosine DNA methylation. In this regard, the interaction between V2 protein of Tomato Yellow Leaf Curl Virus (TYLCV) and the host HDA6 affects the recruitment of MET1 by HDA6, causing hypomethylation of the viral DNA genome and the consequent enhancement in plant susceptibility to TYLCV infection [120]. DNA methylation and histone modifications are involved in priming since the modification of chromatin structure modifies the genetic responses to a subsequent attack. It has been shown that MeJA promotes histone modifications in the promotion of defense-related genes, as well as DNA methylation
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modifications in the genome. This supports the hypothesis that epigenetic regulation plays a key role in priming onset. Transposable elements are involved in plant immunity as shown by the transposon ATCOPIA93 long-terminal repeat (LTR) that is negatively regulated by DNA
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methylation and modifies immune-responsive genes during pathogen attacks [121]. RNA-directed DNA-methylation (RdDM) inhibits transcription through post-transcriptional gene silencing driven
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by Dicer-like (DCL) proteins that are responsible for the formation of small-interfering RNAs
expression,
while
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(siRNAs). Indeed, hypomethylation is linked to biotic stress, which is linked with enhanced DCL4 hypermethylation
is
associated
with
DCL4
down-regulation
[122].
Hypomethylation of centromeric regions and terminal heterochromatic blocks have been observed
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in response to infections of virulent and avirulent strains of the fungus Colletotrichum lindemuthianum [123]. Finally, it has been demonstrated that histone methyltransferase plays an
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important role in plant defenses against fungal pathogens through the modulation of genes involved
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in the jasmonic acid (JA) and/or ethylene signaling pathway. Methylation of histone H3 lysine 36 mediated by the histone methyltransferase SET DOMAIN GROUP8 (SDG8) may work as a memory of permissive expression of several defense genes promoting priming [124].
4
A proposed experimental plan for epigenomics in crop stress biology
A typical experimental plan to identify epigenetic marks linked with plant abiotic stress responses might be the one shown in Figure S2. Thanks to the agronomic evaluations, we should be able to 22
select a resistant and a susceptible genotype to a particular stress (i.e. drought, salinity etc..) in a well-known studied crop such as bean. Both DNA methylations and chromatin remodeling marks might be identified at genome-wide levels using techniques such as whole genome bisulphite sequencing (WGBS) and ChIP-seq. A typical experimental design might provide the use of two genotypes for three stress condition (control, drought, salinity). If we analyze two tissues (roots and leaves), one time point (early stress) and three biological replicates we would have a total of 36 samples. Transcriptomic analysis will provide the list of the differentially regulated genes and will
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guide the epigenomic analysis focusing only on key genes affecting resistance/susceptibility to the studied stress. The integration of epigenomic and transcriptomic allows filtering of this huge preliminary list of epigenetic patterns focusing on those genes that have been previously known to
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play a key role in resistance to the studied stress. A reasonable number of genes (i.e. 25-50) might be analyzed with a targeted epigenetic approach. RNA-seq using the same samples analysed at
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epigenomic level (one resistant and one susceptible for each stress) should be employed. The
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analysis of more than one time point is preferred for transcriptomics due to the low cost of analysis compared to epigenomics. After read trimming, mapping, annotation, and differential expression analysis, the list of the genes differentially regulated by each stress will be determined for each
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analyzed genotype. A possible targeted approach might to perform PCRs coupled with bisulphite sequencing on the filial generation comparing progenies of stressed and unstressed parents. It is
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expected that most of the epigenetic marks should be linked to environmental variability. The
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identification of those epigenetic marks strictly linked to stress resistance is hard. An intensive bibliographic research is required in order to identify DNA methylation patterns involved in the modulation of stress responses. Particular attention should be paid to several gene categories typically affected by abiotic stresses such as transcription factors (WRKYs, MYBs etc.), biosynthesis of water soluble molecules (proline, sugars, etc.), hormone biosynthesis, signaling and response, secondary metabolism, protein folding, stress signaling receptors, and antioxidant pathways. The use of the same samples for both epigenomic and transcriptomic should be allowed 23
to reduce environmental variability external to the studied stress. Proteomics analysis would be another “omic” approach that is desirable to include when trying to identifying genome-wide epigenetic marks. Isobaric tags for relative and absolute quantification systems (iTRAQ) would allow analysis of at least 4000 proteins/sample. Proteins should be extracted by powdered tissues using procedures that allow extraction of a high amount of high-quality proteins. The digested peptides are analyzed using a QExactive mass spectrometer coupled with an Easy-LC and a nanospray ionization source. Data are acquired using a data-dependent ms/ms method. Raw data
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might be analyzed using X!Tandem peptide sequences, which are annotated when searching for them against Uniprot databases. The list of differentially abundant proteins between genotypic and control/stress comparisons are compared with the list of genes identified from transcriptomic
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analysis. Functional data mining tools for proteomic data analysis might be the same used for RNAseq data (i.e. Mapman, Pageman, DAVID, STRING etc..). Considering that the resolution power of
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proteomics is lower than RNA-seq, some genes found in RNA-seq analysis will not show
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correspondent proteins in the proteomic data. In addition, protein extraction procedures do not allow us to obtain sufficient amounts of proteins to be analyzed by iTRAQ. Indeed, it is advisable to not exclude epigenetic marks related to those genes that do not show a correspondent change in
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protein abundance. This is due to the inability of proteomic technologies to analyze the entire
Crop epigenetic adaptation to environment
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5
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proteome.
Epigenetic systems should play an important role in sensing any environmental perturbation modulating gene expression. Rapid epigenetic changes (transient or stably inherited) should be the key for the high flexibility of plant responses to the environmental changes [125]. Epigenetic mechanisms control genomic structure, particularly the heterochromatic state of centromeric telomeric regions. Epigenetics together with genetic variation are probably the most involved events in phenotypic diversity and in the plasticity of plants. Epigenetic variation depends strictly 24
on environmental inputs. Dynamic environmental or experimental conditions induce the generation of epialleles that may a have a different phenotype from the one subjected to alleles generated by genetic variation. The epigenetic variants can be inherited and lead to better adaptation to the environment. Examples of experimentally induced epialleles were observed in Arabidopsis by generating Epigenetic Recombinant Inbred Line populations (EpiRIL) derived from the decrease in DNA methylation 1-2 (ddm1-2) [126]. These EpiRILs have been shown to exhibit changes in growth capacity and are markedly susceptible to salinity stress compared to the Col0 parent line,
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demonstrating that the epigenotypes derived from decrease in DNA methylation influence the ability to adapt to salt stress [127]. Methylation processes have also been observed in natural plants and may strongly depend on specific environmental traits. Therefore, the induced or natural
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epigenetic diversity could represent an unexplored resource of phenotypic variation and constitute a useful means in plant breeding programs. Polyploidy plays a prominent role in plant evolution,
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especially in flowering plants. It is an important evolutionary process in plants where hybridization
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and chromosome doubling produce epigenetic modifications related to retrotransposons. In allopolyploids, interspecific hybridization may promote epigenetic modifications that may be stabilized and transmitted as epialleles into the filial generation progeny, which are subject to
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natural selection and adaptation. LTR retrotransposons are the most abundant type of transposable elements (TEs) in plants. The presence of cis-regulatory motifs in the LTRs plays a pivotal role in
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stress responses and in the complex evolutionary mechanisms occurring in plants. The insertions of
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LTR retrotransposons close to genes have been linked to alternative splicing and epigenetic control. These characteristics can become an active part of the evolution of gene families actively involved in the evolution such as resistance genes. Epigenetic modifications are induced by stresses causing genome modification through the action of retrotransposons that are moving in the genome generating rearrangements that can be stably inherited. Climate change due to increased emission of greenhouse gases should negatively affect plant diversity and productivity. This adverse effect might be reduced through plant adaptive mechanisms to abiotic stresses such as changes in the 25
production of reactive oxygen species triggering signal transduction networks that provide protection against environmental stresses [127].
6
Conclusions
A greater elucidation of the epigenetic modifications in both perennial and autogamous species would allow to decipher the complex interactions between genotype and environment, identifying the real impact of global climate changes in the adaptation and consequently in the quality and
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productivity of commercial crop species. In addition, the construction of crop methylomes would help to develop new molecular markers associated with epigenetic changes that will assist breeding activities. The next-generation markers might be integrated in current methods of genetic
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characterization such as those using DNA barcoding [128]. It will allow the identification of transient stress conditions to manage plant stresses in a cautious and sustainable way. Indeed, we
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propose that an “epigenetic code” should exist in addition to the well-known genetic code
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composed by 4 bases. The existence of an “epigenetic alphabeth” might explain the low correlation observed between genetic and phenotypic variance. Key players in the epigenetic code should be represented by DNA methylation in CG, CH, and CHH sequences. Other epigenetic mechanisms
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driven by chromatin restructuring should be driven by histone methylations such as H3K27me2/3 occurring in response to bacterial stress. The deciphering of this epigenetic alphabet is one of the
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most fascinating fields of modern genetics. This may greatly help to gain insight into complex
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mechanisms of plant epigenomic responses to environmental stresses in a climate change scenario.
AUTHOR CONTRIBUTIONS Both FM and AP contributed in writing the manuscript. Both authors have made substantial, direct and intellectual contribution to the work.
26
Conflict of Interest None
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N. Pumplin, A. Sarazin, P.E. Jullien, N.G. Bologna, S. Oberlin, O. Voinnet, DNA
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Figure legends Figure 1. Main DNA methylation mechanisms at cytosine position in CG, CHH, CHG sequences
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and their role in plants.
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Figure 2. Integration of different “omic” approaches to develop a systems-based approaches to
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investigate plant responses to environmental stresses.
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environmental stresses.
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Figure 3. Main epigenetic modifications linked to stress memory in relation to different
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Figure 4. Epigenetic mechanisms linked to priming in plants.
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Figure 5. Main epigenetic mechanisms involved in abiotic stress responses in plants.
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Figure 6. Main epigenetic mechanisms involved in plant biotic stress responses.
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Figure 7
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Figure 8
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Tables
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Table 1. A selected list of references dealing with epigenetic/epigenomic studies in plants, the type of mechanism, major findings or article’s subject, and technique were indicated.
49
Epigenetic Mechanisms
Main Findings/ Subjects
Used/Studied Techniques
Histone modification
ChIP-CHIP
Chen et al., 2018
DNA methylation
Zhong et al., 2013
DNA methylation
Zilberman and Henikoff, 2007
DNA methylation me-wide methylation analysis
The technique can be used to identify target sequences of transcription factors, and positions of histones with modified N-terminal tails Description of the ChIP-Seq workflow and its various applications in plants Plant epigenome is dynamic during fruit development and play a key role in processes such as ripening Combining different techniques with DNA microarrays and highthroughput sequencing is possible to map DNA methylation at genomewide scale Wholegenome arrays can be used for epigenetic analysis such as chromatinimmunoprecipitationchip studies, methylome characterization Opportunities and limitations of RRBS iwere described for nonmodel plant species
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Mockler et al., 2005
Paun et al., 2019
DNA methylation
Zhang et al., 2015
DNA methylation
Kunze, 2018
DNA methylation
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References dealing with Epigenetic/Epigenomic Technique Pellegrini and Ferrari, 2012
ChIP-Seq
Whole Genome Bisulphite sequencing
Different techniques
Padlock-probe combined with promoter tiling arrays
Reduced representation bisulphite sequencing (RRBS) Differentially Methylationmethylated genomic sensitive singlefragments were linked strand with sexual dimorphism conformation of pests in rice analysis The EpiTYPER™ assay EpiTYPER™ allows a fast and assay reproducible targeted quantification of 50 individual CpGs in a high throughput manner
DNA methylation
Agorio et al., 2017
DNA methylation
Rodriguez-Lopez et al., 2010
DNA methylation
Gonzalgo and Liang, 2007
DNA methylation
Van den Boom and Ehrich, 2009
Methylation levels in WRKY genes were negatively correlated with gene expression in response to NaCl treatments Data revealed a main role of this loop in maintaining a natural epiallele
Bisulphite coupled with PCR
High-resolution melting analysis allows adjusting thermal parameters for DNA hybridization and PCRbased techniques analyzing the impact of DNA methylation marks on the thermostability of regulatory sequences Methylation-sensitive single-nucleotide primer extension can be used to quantify DNA methylation
High-resolution melting analysis
Bisulphite treatment coupled with pyrosequencing
Methylationsensitive singlenucleotide primer extension ((MsSNuPE) Matrix-assisted laser desorption ionization/timeof-flight
Martinelli et al., 2018
Non-coding RNA
Next-generation sequencing
Matsui et al. 2010
Non-coding RNA
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Celik et al., 2019
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DNA methylation
Matrix-assisted laser desorption ionization/time-of-flight combines candidate gene amplification with base-specific cleavage or primer extension methods and MALDITOF mass spectrometric analysis to overcome high dynamicity of DNA methylation patterns MiRNAs may be included in nextgeneration breeding programs Tiling array can identify the stress-responsive genes in Arabidopsis thaliana
Tiling DNA microarray
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Table 2. A selected list of main epigenetic modifications in response to different environmental stresses. Levels of analysis (target or genome-wide analysis) and references were indicated.
DNA methylation Chromatin restructuring DNA methylation Histone modification
Level of Environmental Analysis Stresses (Genome-wide or target epigenetic specific) Abiotic Stress Responses Genome-wide Osmotic Genome-wide Cold
References
Sani et al., 2013 Kim et al., 2015
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Epigenetic modifications
Genome-wide Target epigenetic specific Target epigenetic specific Target epigenetic specific
Cold Drought, cold,
CHG methylation
Genome-wide
Phosphate starvation
DNA methylation
Genome-wide
Histone modification
Genome-wide
Drought
H3K4me3
Genome-wide
Drought
Target epigenetic specific
Salinity
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Low humidity Heat
Drought
Target epigenetic specific
Salinity
DNA methylation
Genome-wide
Salinity
DNA methylation
Genome-wide
Salinity
H3K27me3 H3K27me3 Differentially methylated regions Hypermethylated regions; heterochromatin
Genome-wide Genome-wide Genome-wide
Salinity Drought Salinity
Genome-wide
Salinity
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DNA methylation
General abiotic stresses
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DNA methylation (WRKY, GAPC1)
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Non coding RNA (RNA helicases) CHG methylation
Liu et al., 2017 Rutowicz et al., 2015 Khan et al., 2014 Tricker et al., 2012; Popova et al., 2013; Yong-Villalobos et al., 2015 Liang et al., 2014; Bräutigam et al., 2013 Gourcilleau et al., 2010; van Dijk et al., 2010; Çelik et al., 2019; Fei et al., 2017; Liu et al., 2018; Ferreira et al., 2019 Yaish et al., 2018 Al-Lawati et al., 2016 Sani et al., 2013 Garg et al., 2015 Banerjee and Roychoudhury, 2017 52
silencing DNA methylation
Target epigenetic specific
Aluminum stress
H3K27me3
Genome-wide
Zinc stress
CHG hypomethylation
Genome-wide
Heavy metals
Chromatin-remodeling processes; Non-coding RNAs MET1 transcriptional silencing Tam3 transposon methylation DNA methylation
Genome-wide
Cold (vernalization)
Agnieszka, 2018; Ghosh et al., 2018 Ariani et al., 2016; Erturk et al., 2015 Cicatelli et al., 2014 Kim and Sung 2013 Steward et al., 2000 Hashida et al., 2006 Liu et al., 2017
Cytosine methylation
Target epigenetic specific Genome-wide
Responses to pathogen infections Responses to Fusarium wilt
DNA hypomethylation
Genome-wide
General biotic stress
Cytosine DNA methylation DNA methylation
Genome-wide
Responses to geminivirus infection Responses to pathogen infections General biotic stress
Jaskiewicz et al., 2011 Mohammadi, et al., 2015 Furci et al., 2019 Wang et al., 2018 Zervudacki et al., 2018 Pumplin et al., 2016 Fonsêca et al.,
DNA methylation
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DNA methylation
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Target epigenetic Cold specific Target epigenetic Cold specific Target epigenetic Cold specific Biotic Stress Responses Target epigenetic General biotic stress specific Target epigenetic Stress memory specific Target epigenetic Stress memory specific Target epigenetic General biotic stress specific Target epigenetic Responses to necrotrophic specific and biotrophic pathogens Target epigenetic Response to pathogen Pst specific DC3000 Target epigenetic Response to pathogen Pst specific DC3000 Target epigenetic General biotic stress specific Target epigenetic Responses to fungal attacks specific
Chromatin remodeling
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Activation of HUB1 and HUB2 Histone deacetylase (HDA19) H3K27me2/3; Jumonji C Chromatin-based transcriptional regulation Histone modifications
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H3 histone modifications DNA methylation
Target epigenetic specific DNA methylation and Target epigenetic small-interfering RNAs specific Hypomethylation of Genome-wide
Responses to fungus C.
Alonso et al., 2019 Alves et al., 2013 Jin et al., 2018 Dowen et al., 2012 Ding and Wang, 2015 Zou et al., 2014 Choi et al., 2012 Hou et al., 2015 Soyer et al., 2014
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Target epigenetic specific
lindemuthianum Responses to fungal pathogens
2014 Berr et al., 2010
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centromeric regions Methylation of histone H3 lysine 36
54
PII:
S0168-9452(19)31549-3
DOI:
https://doi.org/10.1016/j.plantsci.2019.110376
Reference:
PSL 110376
To appear in:
Plant Science
Federico Martinelli
PII:
S0168-9452(19)31549-3
DOI:
https://doi.org/10.1016/j.plantsci.2019.110376
Reference:
PSL 110376
To appear in:
Plant Science
Received Date:
12 April 2019
Please cite this article as: Perrone A, Martinelli F, Plant Stress Biology in Epigenomic Era, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110376
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Plant Stress Biology in Epigenomic Era Anna Perronea, Federico Martinellib* a
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze, Palermo, 90128, Italy b Department of Biology, University of Firenze, Sesto Fiorentino, Florence, 50019, Italy
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*Corresponding authors: Federico Martinelli Email: [email protected] Anna Perrone Email: [email protected]
Highlights
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ABSTRACT
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Different DNA methylation enzymes are involved in different plant processes An integrated systems biology approach is needed in crop stress biology Priming drives a wide range of epigenetic mechanisms An epigenetic code is present in response to environmental stresses in plants
Recent progress in "omics" methodologies allow us to gain insight into the complex molecular
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regulatory networks underlying plant responses to environmental stresses. Among the different
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genome-wide analysis, epigenomics is the most under-investigated “omic” approach requiring more critical and speculative discussion about approaches, methods and experimental designs. Epigenomics allows us to gain insight into the molecular adaptation of plants in response to environmental stresses. The identification of epigenetic marks transmitted during filial generations enables new theories to be developed on the evolution of living organisms in relation to environmental changes. The molecular mechanisms driving the capacity of plants to memorize a stress and to generate stress-resistant progenies are still unclear and scarcely investigated. The 1
elucidation of these cryptic molecular switches will assist breeders in designing crops characterized by minimally compromised productivity in relation to stresses caused by climate change. The aim of this review is to briefly describe the most uptodate epigenomic approaches, update recent progresses in crop epigenomics in plant stress biology, and to stimulate the discussion of new epigenomic methods and approaches in the new era of “omic” sciences.
Keywords: epigenomics, omics, plant stress biology, priming, stress memory, transgenerational
1. Epigenetic mechanisms and techniques in plants
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modifications.
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Epigenetic mechanisms refer to all the modifications of gene expression that occur without altering the DNA sequence. The main epigenetic-driven modifications belong to three types of mechanisms:
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1) chromatin restructuring and histone post-translational modifications (PTMs), 2) DNA
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methylation changes, and 3) post-transcriptional modifications due to non-coding RNAs (microRNA or long non–coding RNAs).
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1.1 Chromatin restructuring and histone post-translational modifications Histone post-transcriptional modifications are key modifications significantly altering gene
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activities due to modifications at the N-terminal tail of core histones of the nucleosome. These
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chemical modifications that provoke chromatin restructuring consists of acetylation, methylation, phosphorylation and ubiquitination [1]. The first two modifications, histone acetylation and methylation, are generally due to the activity of large (HATs) and histone lysine methyltransferases (HKMTs) enzyme families. These epigenetic marks can be reversed by other enzymes such as histone deacetylases (HDACs) and histone demethylases (HDMs), respectively [2]. While phosphorylation of histone H3 Ser-10 has been linked with responses to salinity, cold stress and abscissic acid treatment [3], ubiquitination has been mainly associated with increases in 2
transcription [4]. Ubiquitination conjugation is a post-translational modification that is a key player in the signal transduction of oxidative stress responses inducing DNA damage repair, protein degradation, and trafficking [5]. Particularly changes in K63 ubiquitination in response to oxidative stresses have been shown to affect polysome stability and protein expression. These results implied that ubiquitination has a regulatory role in redox processes [5]. Moreover, the tails of the N-terminal histones modified through, for example, acetylation processes, have been shown to be crucial events both for defense mechanisms and for plant development [6].
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Studies on the pluripotency of plant cells demonstrate the existence of regulatory mechanisms that depend on important epigenetic factors in Arabidopsis. In this context, epigenetic modifications could be key players in reprogramming lineage-specified plant cells to pluripotency. Specifically,
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regeneration mechanisms [7] involve the histone acetyltransferase HAG1 / AtGCN5. Callus development causes the catalyzing of histone acetylation processes in different root-meristem gene
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loci, including WOX5, WOX14, SCR, PLT1 and PLT2, providing an epigenetic platform for their
1.1.1 Main techniques
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transcriptional activation.
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Chromatin Immuno-Precipitation combined with chip (DNA microarray) analysis (ChIP-CHIP) has been the most common technique used for the genome-wide analysis of chromatin epigenetic
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modifications before the arrival of the next-generation of sequencing technologies. While the ChIP-
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CHIP technique is effective in easily detecting histone positions with specific modifications of their N-terminal tails [8], ChIP-sequencing combines chromatin immunoprecipitation (ChIP) with genome-wide DNA sequencing providing a deeper level of identification of the DNA sequences bound with transcription factors and other regulating proteins. ChIP-seq consists of the selection of DNA fragments linked with the studied proteins and the analysis of the DNA fragments with deep sequencing platforms (Illumina, PacBio, Roche etc..). The combination of chromatin precipitation and DNA next-generation sequencing has highly advanced our understanding of the mechanism of 3
histone modifications in regulating gene expression [9]. ChIP-sequencing requires a known genomic sequence reference that might be represented by the studied species or a phylogenetically closely related one. Chip-seq is currently the most-desirable approach for studying histone modifications because it allows a better depth/costs ratio and the identification of new unknown SNPs responsible for chromatin restructuring, absent in arrays used in the ChIP-CHIP approach.
1.2 DNA methylation analysis
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One of the most commonly analyzed epigenetic modification is the methylation of cytosine occurring at the fifth carbon position. This epigenetic modification is frequently transient in response to the environment although DNA methylation can be inherited in the next generation
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[10]. DNA methylation plays a key role in transcriptional regulation activity during embryonic development, in key biological phenomenon such as genome stability (transposon silencing) and
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genomic imprinting during cell differentiation. Although DNA methylation has been frequently
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linked with the repression of gene expression, in some instances it has been responsible for transcription activation [11]. For example, in Arabidopsis thaliana Harris et al. found that DNA methylation promoted the recruitment of a transcriptional activation protein complex to chromatin
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sites inducing transposon silencing in neighbouring genes. It has been debated that DNA methylation is a mechanism that has occurred during evolution in
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order to silence transposons considered to be genomic parasites in the host genomes. This is
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corroborated by the fact that transposons are generally highly methylated and represent the main targets for epigenetic silencing. Unfortunately, the study of epigenetic modifications in genomic regions rich in transposon elements is not easy due to the difficulties in determining the link between DNA methylation and transposon polymorphism for their repetitive nature and their capacity to create large insertion/deletion polymorphisms. Methylation is site specific [12] and frequently occurs at pericentric heterochromatin regions driven by histone H3 lysine 9 (H3-K9) methylation [13]. Several findings support the idea that DNA methylation also plays a key role in 4
modifying genome size, structure and genetic variations. Frequently, cytosine methylation occurs when it is present in three sequences: CG, CHG and CHH (where H = A, T or C). DNA methylation has been clearly linked to abiotic stress responses in plants (Figure 1). Each of these methylation marks are catalyzed by different enzymes. Methyltransferase1 (MET1) is responsible for the CG methylation while Chromomethylase3 (CMT3) provokes CHG methylation [14]. Domains Rearranged Methyltransferases (DRM1 and DRM2) that are guided by small interfering RNAs (siRNAs) drive the methylation of CHH [15]. CHH methylation is due to RNA-dependent DNA
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methylation (RdDM) and requires plant-specific RNA polymerases IV and V. Pol IV produces precursor transcripts of 24-nt small RNAs (sRNAs) that are able to target scaffold transcripts using the complementary sequence. The maintaining of the cytosine methylation during replication
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depends on proteins such as Decrease in DNA Methylation 1 [DDM1; 14] while DNA methylations are generally deleted by specific classes of DNA glycosylases such as DEMETER (DME),
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REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 and DML3 [15]. In plant stress
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responses, DNA methylation is a key modulator of both pathogen and symbiont interactions with plants [16] and previous works have linked DNA methylation with host susceptibility [17]. Methylome analyses permitted a better clarification of epigenomic plasticity mechanisms. For
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example, studies of epigenetic analysis on the bean [18]
have facilitated the drawing up of
preliminary epigenomic maps based on chromatin accessibility, histone modifications and DNA
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methylation linked with important agronomic traits.
1.2.1 Main techniques
Next-generation sequencing technologies represent the most powerful method of identifying epigenetic modifications occurring at the DNA level. It has permitted new discoveries in the mechanisms of epigenetic regulation, through the understanding of the biological meaning of DNA methylation, chromatin modeling and the role of small RNA. NGS technologies are represented by various popular platforms, including the 454 FLX (Roche) [19], the Genome Analyzer / Hiseq 5
(Illumina Solexa) [20,21] and the SOLiD (Life Technologies) and new platforms such as Heliscope (Helicos) [22] and PacBio RS (Pacific Biosciences) [23] for single molecular sequencing, and Ion Torrent (Life Technologies), based on semiconductor chips [24]. The most common technique for genome-wide methylation analysis is the Whole Genome Bisulphite sequencing (WGBS) that consists in the treatment of DNA with sodium bisulphite followed by next-generation sequencing. Bisulphite treatment of DNA provokes the conversion of unmodified cytosines to uracil maintaining 5mC unchanged. The use of restriction enzymes sensitive to DNA methylation permits the
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enriching of DNA in methylated or un-methylated sequences. WGBS allows DNA methylation sites to be identified in a genome-wide manner. This approach has been used to generate different plant methylomes [25]. Bisulphite treatment of DNA sequences precedes the hybridization of DNA
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fragments to tilling arrays or their direct sequencing. In addition, antibodies specific for m5C can be used to analyze them before the analysis using arrays or sequencing platforms (MeDip-ChIP or
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MeDip-Seq). The bisulphite-treated DNA can be analyzed with PCR for target epigenetic analysis
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followed by sequencing [26]. However, the PCR amplification that is also included in the genomewide analysis technique is not exempt from bias. False positive and false negative results can also be produced due the incomplete cytosine conversion by sodium bisulphite or the over-conversion of
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5mC. Indeed, PacBio sequencing that does not require bisulphite treatment is able to eliminate this bias. Although methylome analysis through sequencing might be relatively expensive for large
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plant genomes [27], the reduction of the cost of sequencing is doubling every 18 months, rendering
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these analyses affordable for small projects (< 10000-20000 $). A method for DNA methylation is the identification of target regions with padlock-probe based targeting coupled with promoter tiling arrays [28]. An alternative is a reduced representation bisulphite sequencing (RRBS), which consists of the fractionation of the genome and the enrichment of fragments with high CpG content regions before sequencing [29]. In addition, pyrosequencing has been used without the use of methylation specific PCR [30]. This is employed to determine the specific CpG sites in the amplified product. Based on the amount of incorporated C and T, it is possible to determine the 6
ratio of C and T and individual sites of methylation. However, the costs for this technique are high. An application of this technique is represented by the analysis of genomic imprinting through the use of allele specific primers with single nucleotide polymorphisms. Another technique for the analysis of DNA methylation is the methylation-sensitive single-strand conformation analysis (MSSSCA). This method employs the single-strand conformation analysis to resolve sequence differences between methylated and unmethylated samples due to the conversion of bisulphite of cytosine to thiamine while the methylated cytosine remains unchanged. The efficiency of SSCA is
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70-95% [31]. High-resolution melting analysis (HRM) also permits the analysis of DNA methylation when quantitative real-time PCR analysis is completed. It consists of applying melting cycles that allow converted to be distinguished from unconverted bisulphite-treated DNA. The PCR
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products are analyzed by temperature ramping that will result in the liberation of intercalating fluorescent dye during the melting process. The rapidity of the melting and consequent release of
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the dye is due to the C to T content that represents the DNA methylation amount [32]. The
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weakness of this method is the ability to analyze only one or a few amplicons each time [34-35]. Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique suitable for a quick quantitation of methylation occurrence at individual CpG sites [35]. It uses the primer
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extension method that has been optimized for detecting single-nucleotide polymorphisms. In this technique, bisulphite treatment is used together with PCR amplifications with bisulphite-specific
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primers. They are designed to anneal to the sequence up to the base pair immediately before the site
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of DNA methylation. This is a simple method for screening DNA methylation in a large number of samples. This method also promotes genomic sequencing since the electrophoretic bands may be isolated and directly sequenced. Other methodologies employ matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry analysis to differentiate between the two polymorphic primer extension products based on the GOOD assay designed for SNP genotyping. Base-specific cleavage/MALDI-TOF consists in the in vitro transcription of the region under study using an RNA polymerase promoter site. RNAse A treatment follows retro7
transcription and the base specificity is obtained by adding incorporating cleavage resistant dTTP or dCTP if cytosine or uracil specific cleaves are desired, respectively. The fragments obtained by the cleavage
are
analyzed
with
MALDI-TOF.
The
bisulphite
treatment
provokes
the
introduction/removal of cleavage sites by C to U conversions or a shift in fragment mass by G to A conversions in the amplified reverse strand. C-specific cleavage will only work at the methylated CpG sites. This technique has a great efficiency, allowing identification of CpG sites in multiple tissues with low costs. The EpiTYPER™ assay allows the detection and quantification of DNA
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methylation in a specific region through a high-resolution scan of selected regions. This method enables us to perform an epigenomic analysis of a region surrounding a single CpGs and can be employed to validate the CpGs detected by genome-wide approaches. This technique is considered
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a rapid method for the targeted quantification of individual CpGs in a high throughput manner through the base-specific cleavage after bisulphite-conversion of DNA coupled with MALDI-TOF
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MS [36].
1.3 Non-coding RNA-mediated epigenetic regulation It is known that 90% of the eukariotic genome is transcribed but only 1-2% of the genome is
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translated to proteins. This means that there is a large number of RNA molecules that are circulating in the cells whose functions are unknown but most probably having a key role in the post-
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transcriptional regulation [37]. These RNA molecules are noncoding RNAs (ncRNAs) that are the
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third main actors in epigenetic modifications. Noncoding RNA molecules can be divided in different groups based on their size, function and regulation mechanisms: microRNAs (miRNAs), short interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and long non-coding (lncRNAs). Another classification has divided them into two categories: short ncRNAs (<30 nts; including miRNAs, siRNAs, and piRNAs) and long ncRNAs (>200 nts). Small RNAs are important activators/inhibitors of plant environmental stresses. For example, it has been shown that the plant pathogen Botrytis cinerea delivers small RNAs into the host cells to repress plant defense genes and 8
thus deactivate host immunity [38]. Besides lncRNAs that clearly function in chromatin modifications, a few other ncRNAs also play roles in other biological processes in plants [38]. For example, the soybean early nodulin gene, Enod40, is involved in the regulation of symbiotic interactions between leguminous plants and soil bacteria [38]. Several stress-responsive miRNAs have been found in different crops leading to the possibility of using miRNA-driven RNAinterference (RNAi) to improve key traits and provide phenotypic plasticity in a climate change scenario [39]. RNA-mediated chromatin-level silencing is strongly involved in stress responses and
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natural epigenetic variation and consequently in phenotypic diversity, physiological plasticity and evolutionary change [40]. Epigenomic regulation of chromatin structure and genome stability is essential for genetic interpretation information and key roles have emerged for small RNAs,
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proteins with domains that bind methylated DNA and DNA glycosylases in these processes [41,42]. The analysis of cytosine methylome (methylC-seq), transcriptome (mRNA-seq) and small RNA
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transcriptome (small RNA-seq) in Arabidopsis inflorescences showed methylation patterns on a
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genomic scale and a direct relationship between the sRNA position and DNA methylation [43]. In addition, genomic analysis in Arabidopsisis showed that various methylated cytosines have been detected by bisulphite sequencing using NGS technologies, precisely through the so-called BS-seq
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[44-46].
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1.3.1 Main techniques
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The most potent method for analyzing ncRNA (including miRNA) is the use of next-generation sequencing because it permits us not only to conduct a quantitative analysis of their expression but also the identification of single nuclear polymorphisms in RNA sequences [47]. Novaseq 6000 (Illumina) is one of the most-desired platforms for studying rare transcripts and splice variants due to the higher number of reads with longer reads than previous platforms (i.e. Hiseq 2500) (250 bp instead of 75 bp). In silico methods are used since they are able to distinguish lncRNAs from protein-coding transcripts based on the absence of discernible open-reading frames. Full-length 9
targeted cDNA sequencing may be the best method to determine the coding potential of transcripts, although this approach is time-consuming and expensive. Alternatively, tiling DNA microarray for genome-wide transcriptome analysis can be employed to detect and determine the expression of lncRNAs in plants. Tiling DNA microarray analyses have been used to identify new stress-induced lncRNAs [48]. High-throughput deep RNA sequencing is able to identify missing or incomplete transcripts of lncRNA and to determine the levels of lncRNA expression. A similar approach was used to identify rare alternative splicing variants of a lncRNA, HOTAIR [49]. An approach with a
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combination of different kinds of techniques (genome-wide histone modification analysis and deep sequencing) may permit the identification of the number of long intergenic ncRNAs (lincRNAs) transcribed from a K4-K36 domain, which marks active promoters with trimethylation [49].
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A summary of all the mentioned techniques divided by the three main epigenetic mechanisms is
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provided in Table 1.
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1. What is the role of epigenomics in system biology?
An integrated systems biology approach is required to gain insight into the complex molecular regulatory networks that modulate the expression of plant traits. Epigenomics is one of the most
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unexplored “omic” approaches to understand the adaptation of living organisms to rapid local environment change, weather or other stresses. However, the analysis of epigenomic changes in
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relation to plant environmental stresses requires a strong integration of the different “omic”
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analyses (Figure 2). Transcriptome analysis is essential to validate the gene silencing that is predicted by DNA methylation data. The use of the other analyses (proteomics and metabolomics) are commonly used to gain insight into the post-transcriptional and post-translational regulation mechanisms linked to specific epigenetic changes. The epigenomic modification has a real effect at the phenotypic level if the consequent change in transcription is not reversed at the posttranscriptional level. Indeed, proteomic and metabolomic platforms are essential to determine if the epigenetic modification has effects on composition at protein and metabolite levels. The power of 10
epigenomic analysis can be better exploited for plant species whose genomes have been sequenced. Transcriptomic approaches permit analysis of the expression of many thousands of genes, identifying splice variants, rare genes, single nucleotide polymorphisms. However, transcriptomic results are usually inconclusive and not strictly correlated to phenotype changes since cells frequently activate multiple regulatory mechanisms for their adaptation to stress responses. Proteomic analysis permits us to understand how protein changes are modulated by rapid or chronic environmental stresses in crops [50]. A recent integrated approach has been proposed correlating
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genes and protein expression, named proteogenomics [51]. Proteomics can be used to validate the expression of the genes while transcriptomic data permits the construction of customized protein databases, avoiding the need for homology-based approaches [52-54]. Metabolomics is the
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untargeted high-throughput technique that can be linked to epigenetic modifications. Metabolomic approaches allow a comprehensive analysis of the cell metabolism and could validate epigenomics.
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This “omic” approach has been less employed compared with the other “omic” analyses especially
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for trait mapping and plant selections [55]. It allows for deep characterization of how plant metabolism is affected in response to environmental stimuli and changing climatic conditions. The recent progresses in metabolomics allow quantification of a wide range of metabolites from a small
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amount of analyzed samples (> 10 mg). Metabolomics has been used to understand cell dehydration, water stress responses and plant-microbe interactions [55], providing a great
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contribution to shedding light on the link between genotype and metabolic pathways leading to the
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phenotype. They help identify key points of molecular network regulation. Plants biosynthesize more than a hundred thousand metabolites with different chemical structures and quantity, each one with key roles in physiological, development, and stress response processes [56-57]. Integrated multi-omic approaches are highly effective in discovering epigenomic mechanisms of regulation of multi-factorial quantitative traits. The real challenge in analyzing both “omic” data is understanding the complex gene/protein regulatory networks identification due to gene-gene and protein-protein interactions. Nodes, molecular players (genes or proteins), are generally indicated in different colors 11
depending on their trend of expression (up, down or un-regulated between control and stress condition), different forms based on their functions, and different sizes proportional to the number of connections (Figure S1). Through the construction of these networks it is possible to identify not only the highly interactive nodes (hubs), but also low interactive proteins that play a key role in the networks because they connect hubs or cores of highly interconnected proteins/genes. In this regard, the interaction between key highly interactive proteins and non-coding RNA often plays an important role in plant stress responses [56]. For example, WRKY transcription factors are typically
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key players in modulating environmental stress responses in plants at the interactome level. Several targets of miRNAs encode WRKY transcription factors [57] implying that miRNAs are involved
in the regulation of the expression of WRKYs. On the other hand, some miRNA promoters are
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abundant in W box sequences, the cis-elements recognized by WRKYs [58]. For example, the response to powdery mildew has been shown to be modulated by WRKY-smRNA interactome
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[59]
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2. Approaches for investigating epigenomic transient and transgenerational mechanisms We propose a general workflow to investigate plant transgenerational epigenomic modifications in
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response to environmental stresses. First, the stress-epigenetic response should be analyzed in controlled conditions to reduce undesired environmental variability. Basing ourselves on the
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evidence that DNA methylation generally causes the silencing of the methylated gene, the decrease in transcript abundance of the mRNA should be observed. In fact, the most desired approach is to
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integrate epigenomics with transcriptomic and metabolomic approaches to observe the inhibition of expression of the methylated genes [60-63]. The use of proteomic approaches would allow the confirmation of the epigenetic-driven changes in transcript abundance although the magnitude of analysis is generally 10-fold less than RNA-seq. ITRAQ proteomic approaches could partially contribute to fill this gap through the analysis of more than 6000 proteins using small biological samples (> 50-100 mg). The gap that still exists between transcriptomics and proteomics (even with iTRAQ) precludes the conclusion that the absence of the protein targeted by the DNA methylation 12
is a consequence of a variation in its expression. It might be due that the protein was not present in the protein extract. The heterogenous chemical nature of the proteins renders the extraction complex and difficult, and does not allow the recovery of all types of proteins during the extraction processes. In this regard, Phenol-based protocols are commonly used to extract proteins from recalcitrant plant tissues [64]. Once DNA methylation analysis, transcriptomics and proteomics are conducted, a list of potential epigenetic marks can be delivered and associated with the considered stress response. Quantitative RT-PCRs may be conducted for the targeted list of methylated genes
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to confirm the trend of altered expression in relation to the detected DNA methylation. Seeds can be collected and DNA may be extracted from zygotic tissues to perform a genome-wide DNA methylation analysis in order to see if the methylation signal is transmitted in the filial generation.
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The epigenetic patterns of the seeds can be compared with the one related to the same tissue (leaf, fruit, shoots etc.) coming from the first generation to see if the DNA methylation is a transient
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stimulus that occurrs only when the stress is present, or if it is provided by inheritable stress
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memory. In addition, the epigenetic modification should be analyzed after the occurrence of the stress if the unstressed condition could be restored after the treatment (irrigation after drought etc.). The presence of the same epigenetic mark in parental and filial generations in absence of stress
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should prove the transgenerational inheritance excluding its transient nature. Natural epigenetic variation may originate from polymorphisms in transposon insertions and repeats. Epigenetic works
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in model plant species such as Arabidopsis thaliana have shown that epigenetic regulation is closely
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associated with functional aspects. However, epigenetic studies on Arabidopsis thaliana have limitations, such as the low value methylated cytosine in the genome (5%) while many crops contain more than 20% [63]. In addition, this plant contains very few transposable elements compared to other crops (review by Lee and Kim, 2014) [65]. Mutations in epigenetic regulators appear to have a greater impact in crops than Arabidopsis. Furthermore, while the distribution of genes along the Arabidopsis chromosomes is fairly homogeneous, this situation may differ in crops. For example, the genome of Vitis vinifera (L.) is characterized by the alternation of large regions 13
with high and low genetic density [66-67]. Several epigenetic studies have been performed in crops such as tomato fruits which constitute an important model for studies on grapes where DNA methylation and histone modifications should play a pivotal role in fruit veraison [68]. In woody crops, epigenetic modifications can be investigated using clonal propagation. The stress applied in the first vegetative generation can be analyzed in the second upcoming vegetation derived from the meristematic apex previously subjected to the studied stress. The propagation through cuttings is a desirable way to identify how the epigenetic modifications is
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stable or transient. Grape has recently been proposed as a model plant to study epigenomics in perennial plants [68]. This is due for the following reasons: 1) it is a model for non-climateric fruits and flower development, 2) it presents flower buds that occur one year after they open, and 3) it is
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clonally propagated and commonly grafted.
It is well-known that environmental conditions of the previous year have a significant impact on the
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flower and subsequently on the development of the fruit. Other specific requirements that affect
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flowering and fructification are represented by clonal grafting and propagation. In this context, the epigenetic variability added to the genetic diversity of the grape could shape the phenotypic variations of the plant. The clonal diversity within the grape varieties was identified using the
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methylation-sensitive amplified polymorphism technique, allowing the understanding of the utility of epigenetic markers in intra-varietal diversity studies [69]. Another important impact on the
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epigenetic state of a plant is represented by grafting. The understanding of these mechanisms is
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useful to improve grape tolerance to highly threatening environmental stresses enhancing the product quality. Interestingly, systemic acquired resistance to downy mildew pathogen has been shown to be due to DNA methylation inherited in offspring [70]. Another crop that might be considered as a model for epigenomic studies is tomato due to the easy availability of the mutants. Chemical treatments inducing DNA methylations may be useful for generating epimutations although they might not be stable as genetic mutants. Without the need of using genetic recombination, epimutagenesis may represent an important means of exploring allelic 14
variations and the new combinations of alleles. The analysis of DNA methylation in grape in relation to fruit qualitative traits could represent an important tool of identifying interesting epialleles usable in crop breeding. Specifically, DNA methylation can generate epialleles with various levels of expression, leading to a continuous quantitative variation of a crop trait. Herbaceous auto-gamous species are great candidates to understand if epigenetic modifications have a transient or transgenerational nature. Another model plant for cereals is Brachypodium since it has a rapid life cycle, belongs to the Graminaceae family, is easy to cultivate, and has a small
3. Epigenetic regulation of plant stress responses
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diploid high-quality genome that has been recently sequenced.
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The recent progresses in the genome-wide analysis of epigenetic marks have allowed gaining insights into the dynamics and plasticity of plant adaptation to altered environmental conditions.
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Plants are sessile living organisms and they are particularly characterized by a finely-tuned
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adaptation to changing environmental conditions through the trigger or repression of epigenetic mechanisms. Most of the epigenetic features are transiently activated in response to the stress occurrence and reverted when the environmental stimuli are no longer present. However, a certain
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part of these epigenetic marks is “memorized” by the plant determining stress priming. This phenomenon consists in an improved resistance/response to an environmental stress due to the
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activation of responses (priming fingerprinting) which remain latent but prompted to be activated
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again in the presence of a subsequent stress. This induced stress resistance could be inherited in offspring resulting in an improved response to a particular stress that has been previously occurred in parents. Priming is directly linked to the concept of plant stress memory. It is known that plants do not have the same group of germline cells for all their life span as animals do. New germ cells come from meristematic cells during their entire life so that any epigenetic modification that occurrs during vegetative growth may be transmitted to the offspring. Any stable epigenetic information acquired by the genetic structures of the somatic cells might be transmitted to the next generation. 15
The presence of a stress may induce epigenetic heritable traits that represents a ‘memory' inherited in the next generations which helps the colonization of different environments. This imprint, or stress memory, can be defined as the structural, genetic, and biochemical modifications occurred as a consequence of stress exposure rendering a plant more resistant (although it might also be more sensitive in some cases) to future exposure to the same stress factor. The most common histone modification associated with stress memory is H3K4me3 although also H3K4me2 and H3K27me3 have also been found in systemic acquired response and bacterial stress
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(Figure 3). Chromatin modifications, nucleosome positioning, and DNA methylation are playing a key role in plant stress memory and several epigenetic mechanisms have been shown to be responsible for priming of plants [71] (Figure 4). Although priming may compromise plant
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productivity in the short-term, for example through a reduction of photosynthesis, it usually induces tolerance to subsequent stress and therefore favors productivity in the long-term perspective [72].
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Abscisic acid is known to be involved in plant responses to reiterated abiotic stresses and might be
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involved in priming [73. In some plant species, it was observed that ABA levels are higher under drought conditions if the plants have previously been subjected to water stress, implying that ABA may be linked to drought stress memory [74]. In this regard, Virlouvet and Fromm (2015) [75]
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showed that previously stressed plants have the capacity to maintain stomatal apertures that remain partially closed during a recovery period, which reduces transpiration during a subsequent drought
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episode. An increased expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) and
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ALDEHYDE OXIDASE 3 (AAO3), key modulators of ABA biosynthesis, was linked with drought memory.
3.1 Abiotic stresses Recent studies have demonstrated that chromatin and DNA methylation changes play an important role in resistance and tolerance mechanisms to different abiotic stresses such as cold, salt stress, drought, osmolality, or mineral changes (Figure 5), thereby pointing out the importance of 16
epigenetic regulations in these aspects [77-75]. In addition, genome-wide analyses of histone PTMs have revealed global epigenomic reprogramming in plants under abiotic stresses. Recently, a specialized histone H1 variant was shown to be required for DNA methylation changes linked with stress responses in Arabidopsis [75]. Two DEAD-box RNA helicases are responsible for epigenetic silencing of gene expression leading to inactivation of abiotic stress responses in Arabidopsis [76]. Arabidopsis mutants deficient in genes involved in the RdDM pathway or with defects in maintenance of CHG methylation showed a variable sensitivity of regulation of the stomatal index
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in conditions of low relative humidity [77]. In addition, they showed hypersensitivity to heat stress [78] or high sensitivity to phosphate starvation [79]. These results are consistent with an important
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function of the DNA methylation dynamics in the regulation of abiotic stress-responsive genes.
3.1.1 Drought and salt stress
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Drought causes extensive remodeling of DNA methylation patterns as it was observed in different
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plants species 4-81]. Modulation of DNA methylation at repetitive elements seems to be important to modulate the expression of adjacent genes. H3K4me3 modifications under drought stress have been observed [82]. It has been confirmed that H3K4me3 might be a marker for highly expressed
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genes in stress conditions. In populus, epigenetic modifications have been linked to drought responses, affecting the transcriptomic changes in response to stress [83]. In apple, a variation of
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approximately 54%, 38% and 8.5% of methylation respectively at CG, CHG and CHH sequences
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has been linked to changes in drought-sensitivity/tolerance. A part of these epigenetic changes occurred at genes encoding transcription factors (TFs) and transposable elements (TEs). It is worth noting that epigenetic effects of drought stress vary depending on many factors, such as the intensity and duration of the stress, plant genotype or growth phase, and also genomic imprinting left by previous stress episodes. In barley, drought stress-induced genome-wide DNA methylation changes were associated with water deficit [84]. Drought-stressed poplar has showed genome-wide changes in DNA methylation implying that epigenetic mechanisms are involved in adaption of trees 17
to environmental changes [85]. This has been suggested in corn where transposon elements could function as local enhancers for stress responsive genes [86]. Epigenetic responses to salinity are known to play an important role in plant resilience to this type of stress. Salt stress affected DNA methylation in key stress-related genes such as WRKYs in Arabidopsis thaliana [87], poplar [88], rice [89] forest species[90] and wheat [91]. Acetylation, methylation and phosphorylation of histones play an important role in plant responses to salt stress. In addition, salinity triggers the activation of cell wall related genes such as expansin β2 and
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xyloglucan endotransglucosylase in roots and these gene expression changes have been linked with enhanced acetylation of histones in proximity of the promoter and the open reading frames of these genes [91]. An interaction between salt and drought stress has been observed. Salinity promoted
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drought tolerance in Arabidopsis through the modification of H3K27me3. A prolonged absence of H3K27me3 induced a transient and quick transcription of high affinity K+transporter 1 [68].
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Another epigenetic mechanism involved in plant stress memory modulates the accumulation of high
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levels of proline, an aminoacid with antioxidant activities during abiotic stresses [50]. The intraspecific variability in salt tolerance in wheat has been associated with changes in differentially methylated regions (DMRs) containing genes closely associated with transposons [92]. The picture
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of the molecular regulatory mechanisms is complex since hypermethylated regions responsible for silencing of heterochromatin has also been linked to the activity of small RNAs [93]. Other
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evidences in different crops have confrimed the role of epigenomic modifications in drought and
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salt responses [94-97].
3.1.2 Metal-related stresses
Manganese oxide nanoparticles provoke DNA hypomethylation in the moss Physcomitrella patens [98]. Epigenetic studies have been conducted in Populus, a model plant for investigating molecular long-term responses under metal excess conditions. Chip-Seq and RNA-seq data were integrated in poplar showing that genes with a H3K4me3 modification were usually upregulated, while genes 18
with a H3K27me3 modification on the 5-UTR were repressed under excess of Zn [99]. The epigenome of leaves of poplar plants in response to AMF inoculation on multi-metal polluted or unpolluted soil was investigated using the MSAP approach. Few changes in cytosine methylation patterns were observed after 4 months after from planting, whereas increased hypomethylation was observed after 6 months from mycorrhizal treatment in the presence of heavy metals [100]. Transgenerational inheritance of changes in DNA methylation patterns in response to heavy metal stress was shown in rice (Oryza sativa L.) [101]. The DNA methylation changes were observed in
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leaves immediately after heavy metal treatment and they consist in CHG hypomethylation; CHGdemethylated states that were heritable to offsprings. MSAP technique was useful to determine the effect of metal contamination on cytosine residues in CCGG motifs in the genome of Red maple
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(Acer rubum), a common deciduous tree in northern America. Zinc stress has been associated with enhanced methylation linked to reduced levels of key hormones such as gibberellic acid, zeatin and
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indole acetic acid and increased levels of abscisic acid [102]. Finally, Aluminum stress altered DNA
retrotransposons [103].
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3.1.3 Cold stress
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methylation in corn and it was associated with polymorphisms in long terminal repeat
Non-coding RNAs are involved in epigenetic regulation of vernalization through the silencing of
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FLC even after winter cold. Molecular studies showed that chromatin-remodeling processes are
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responsible for the regulation of FLC expression. A high expression level of FLC has been associated with delayed flowering, whereas flowering is promoted when FLC is repressed by vernalization mediated by non-coding RNA regulation. VERNALIZATION INSENSITIVE 3 (VIN3) was identified as an essential gene to trigger repression of FLC by vernalization. VIN3 encodes a plant homeodomain (PHD) finger protein that is induced only during a cold period. The PHD finger motif in VIN3 is often found in various components of chromatin-remodeling complexes [104]. Vernalization provokes an increased association of PHD-PRC2 with FLC chromatin and an 19
enhanced deposition of H3K27me3 marks at the FLC chromatin [104]. Increased enrichments of PRC2 and H3K27me3 are hallmarks for the stable repression of FLC by vernalization. Another lncRNA involved in vernalization is a cold-inducible intronic lncRNA, COLDAIR [105]. Transcriptional silencing of MET1 gene in corn roots in response to cold causes demethylation of the Ac/Ds transposon system [106]. The Tam3 transposon methylation at CHH sites was observed in snapdragon during cold conditions [107]. Plants have developed mechanisms to survive in an ever-changing environment through the active regulation of chromatin. Particularly, it was shown
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that the interaction of HOS15 with histone deacetylase 2C (HD2C) modulates the action of promoters of cold-responsive COR genes. Epigenetic mechanisms have been observed to play a key role in modulating cold tolerance in several plants such as brassica rapa and sugar beet [108-
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109].
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3.2 Biotic stresses
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Plants are sessile organisms that have evolved different mechanisms compared to animals to escape the exposure of adverse environmental factors, including pathogens. Plants have evolved complex mechanisms of protection, among which the ability to establish symbiotic interactions to prevent
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infections is particularly relevant. Recent researches have evidenced several regulatory mechanisms controlling the plant-microbe interactions. In this context, epigenetic mechanisms are important key
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modulators of both pathogenic and symbiotic interactions between plants and microbes (Figure 6).
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DNA methylation is an epigenetic modification of DNA that prevents the loss of gene expression in plant defenses against pathogens and plays a crucial role in microbial-plant interactions. For example, several research reports confirmed the role played by recurrent DNA methylations in the induction of enhanced resistance to pathogens [110-111]. Responses to biotic and abiotic stresses are stored by intracellular molecules that allow plants to respond more efficiently to future environmental stresses. Systemic acquired resistance (SAR) or defense priming is a classic example of memory response to future stresses able to promote hypersensitivity and rapid expression of 20
defensive genes through the action of the NPR1 gene as it was found in Arabidopsis [112]. Regulation of the epigenetic expression of NPR1/NIM1 genes is an important example of H3 histone modifications affecting stress memory in response to biotic stresses [113]. Regulation of gene expression by DNA methylation is crucial to modulate the transcription of genes involved in the biosynthesis of the SA affecting plant defense mechanisms. DNA methylation could mediate the response to biotic stress through the increase of SA and the modulation of the downstream genes [1114]. These data suggested that methylation levels within TEs may dynamically control the
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expressions of transposon and the proximal gene in response to stress in Arabidopsis [115]. Chromatin remodeling plays a crucial role in plant immunity against both necrotrophic and biotrophic pathogens [110]. As previously reported, mutant bon1-1 with loss of- function leads to
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constitutive defense response mechanisms to the virulent pathogen Pst DC3000 through the activation of HUB1 and HUB2 and consequently the induction of autoimmune responses [116]. It
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was found that dimethylated or trimethylated histone H3 Lys 27 (H3K27me2/3) is involved in the
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expression of responsive genes to biotic stresses in rice. Jumonji C (jmjC) protein gene encodes a histone Lys demethylase that specifically reverses the activity of H3K27me2/3. During pathogenic infections, the expression of JMJ705 is induced. The overexpression of JMJ705 in transgenic plants
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removes levels of H3K27me2/3, inducing their expression and enhancing plant resistance to biotic stress through the involvement of jasmonic acid. JMJ704 positively regulates rice tolerance to
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bacterial blight through the epigenetic suppression of defense regulators, that is a distinct way from
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its homolog JMJ705 [116]. Furthermore, during plant infection processes, chromatin-based transcriptional regulation can also affect the expression of the effector gene in fungi [117]. Different studies have focused on the concept that pathogen infections induce histone modifications affecting key response genes. Changes in DNA methylation has been shown to affect plant resistance to diseases. For example, significant differences in cytosine methylation have been observed between resistant and sensitive genotypes to Fusarium wilt in chickpea [118]. DNA hypomethylation at specific pericentromeric regions affects priming and biotic stress resistance [119]. Responses to 21
geminivirus infection are modulated by cytosine DNA methylation. In this regard, the interaction between V2 protein of Tomato Yellow Leaf Curl Virus (TYLCV) and the host HDA6 affects the recruitment of MET1 by HDA6, causing hypomethylation of the viral DNA genome and the consequent enhancement in plant susceptibility to TYLCV infection [120]. DNA methylation and histone modifications are involved in priming since the modification of chromatin structure modifies the genetic responses to a subsequent attack. It has been shown that MeJA promotes histone modifications in the promotion of defense-related genes, as well as DNA methylation
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modifications in the genome. This supports the hypothesis that epigenetic regulation plays a key role in priming onset. Transposable elements are involved in plant immunity as shown by the transposon ATCOPIA93 long-terminal repeat (LTR) that is negatively regulated by DNA
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methylation and modifies immune-responsive genes during pathogen attacks [121]. RNA-directed DNA-methylation (RdDM) inhibits transcription through post-transcriptional gene silencing driven
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by Dicer-like (DCL) proteins that are responsible for the formation of small-interfering RNAs
expression,
while
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(siRNAs). Indeed, hypomethylation is linked to biotic stress, which is linked with enhanced DCL4 hypermethylation
is
associated
with
DCL4
down-regulation
[122].
Hypomethylation of centromeric regions and terminal heterochromatic blocks have been observed
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in response to infections of virulent and avirulent strains of the fungus Colletotrichum lindemuthianum [123]. Finally, it has been demonstrated that histone methyltransferase plays an
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important role in plant defenses against fungal pathogens through the modulation of genes involved
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in the jasmonic acid (JA) and/or ethylene signaling pathway. Methylation of histone H3 lysine 36 mediated by the histone methyltransferase SET DOMAIN GROUP8 (SDG8) may work as a memory of permissive expression of several defense genes promoting priming [124].
4
A proposed experimental plan for epigenomics in crop stress biology
A typical experimental plan to identify epigenetic marks linked with plant abiotic stress responses might be the one shown in Figure S2. Thanks to the agronomic evaluations, we should be able to 22
select a resistant and a susceptible genotype to a particular stress (i.e. drought, salinity etc..) in a well-known studied crop such as bean. Both DNA methylations and chromatin remodeling marks might be identified at genome-wide levels using techniques such as whole genome bisulphite sequencing (WGBS) and ChIP-seq. A typical experimental design might provide the use of two genotypes for three stress condition (control, drought, salinity). If we analyze two tissues (roots and leaves), one time point (early stress) and three biological replicates we would have a total of 36 samples. Transcriptomic analysis will provide the list of the differentially regulated genes and will
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guide the epigenomic analysis focusing only on key genes affecting resistance/susceptibility to the studied stress. The integration of epigenomic and transcriptomic allows filtering of this huge preliminary list of epigenetic patterns focusing on those genes that have been previously known to
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play a key role in resistance to the studied stress. A reasonable number of genes (i.e. 25-50) might be analyzed with a targeted epigenetic approach. RNA-seq using the same samples analysed at
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epigenomic level (one resistant and one susceptible for each stress) should be employed. The
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analysis of more than one time point is preferred for transcriptomics due to the low cost of analysis compared to epigenomics. After read trimming, mapping, annotation, and differential expression analysis, the list of the genes differentially regulated by each stress will be determined for each
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analyzed genotype. A possible targeted approach might to perform PCRs coupled with bisulphite sequencing on the filial generation comparing progenies of stressed and unstressed parents. It is
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expected that most of the epigenetic marks should be linked to environmental variability. The
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identification of those epigenetic marks strictly linked to stress resistance is hard. An intensive bibliographic research is required in order to identify DNA methylation patterns involved in the modulation of stress responses. Particular attention should be paid to several gene categories typically affected by abiotic stresses such as transcription factors (WRKYs, MYBs etc.), biosynthesis of water soluble molecules (proline, sugars, etc.), hormone biosynthesis, signaling and response, secondary metabolism, protein folding, stress signaling receptors, and antioxidant pathways. The use of the same samples for both epigenomic and transcriptomic should be allowed 23
to reduce environmental variability external to the studied stress. Proteomics analysis would be another “omic” approach that is desirable to include when trying to identifying genome-wide epigenetic marks. Isobaric tags for relative and absolute quantification systems (iTRAQ) would allow analysis of at least 4000 proteins/sample. Proteins should be extracted by powdered tissues using procedures that allow extraction of a high amount of high-quality proteins. The digested peptides are analyzed using a QExactive mass spectrometer coupled with an Easy-LC and a nanospray ionization source. Data are acquired using a data-dependent ms/ms method. Raw data
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might be analyzed using X!Tandem peptide sequences, which are annotated when searching for them against Uniprot databases. The list of differentially abundant proteins between genotypic and control/stress comparisons are compared with the list of genes identified from transcriptomic
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analysis. Functional data mining tools for proteomic data analysis might be the same used for RNAseq data (i.e. Mapman, Pageman, DAVID, STRING etc..). Considering that the resolution power of
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proteomics is lower than RNA-seq, some genes found in RNA-seq analysis will not show
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correspondent proteins in the proteomic data. In addition, protein extraction procedures do not allow us to obtain sufficient amounts of proteins to be analyzed by iTRAQ. Indeed, it is advisable to not exclude epigenetic marks related to those genes that do not show a correspondent change in
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protein abundance. This is due to the inability of proteomic technologies to analyze the entire
Crop epigenetic adaptation to environment
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5
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proteome.
Epigenetic systems should play an important role in sensing any environmental perturbation modulating gene expression. Rapid epigenetic changes (transient or stably inherited) should be the key for the high flexibility of plant responses to the environmental changes [125]. Epigenetic mechanisms control genomic structure, particularly the heterochromatic state of centromeric telomeric regions. Epigenetics together with genetic variation are probably the most involved events in phenotypic diversity and in the plasticity of plants. Epigenetic variation depends strictly 24
on environmental inputs. Dynamic environmental or experimental conditions induce the generation of epialleles that may a have a different phenotype from the one subjected to alleles generated by genetic variation. The epigenetic variants can be inherited and lead to better adaptation to the environment. Examples of experimentally induced epialleles were observed in Arabidopsis by generating Epigenetic Recombinant Inbred Line populations (EpiRIL) derived from the decrease in DNA methylation 1-2 (ddm1-2) [126]. These EpiRILs have been shown to exhibit changes in growth capacity and are markedly susceptible to salinity stress compared to the Col0 parent line,
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demonstrating that the epigenotypes derived from decrease in DNA methylation influence the ability to adapt to salt stress [127]. Methylation processes have also been observed in natural plants and may strongly depend on specific environmental traits. Therefore, the induced or natural
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epigenetic diversity could represent an unexplored resource of phenotypic variation and constitute a useful means in plant breeding programs. Polyploidy plays a prominent role in plant evolution,
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especially in flowering plants. It is an important evolutionary process in plants where hybridization
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and chromosome doubling produce epigenetic modifications related to retrotransposons. In allopolyploids, interspecific hybridization may promote epigenetic modifications that may be stabilized and transmitted as epialleles into the filial generation progeny, which are subject to
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natural selection and adaptation. LTR retrotransposons are the most abundant type of transposable elements (TEs) in plants. The presence of cis-regulatory motifs in the LTRs plays a pivotal role in
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stress responses and in the complex evolutionary mechanisms occurring in plants. The insertions of
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LTR retrotransposons close to genes have been linked to alternative splicing and epigenetic control. These characteristics can become an active part of the evolution of gene families actively involved in the evolution such as resistance genes. Epigenetic modifications are induced by stresses causing genome modification through the action of retrotransposons that are moving in the genome generating rearrangements that can be stably inherited. Climate change due to increased emission of greenhouse gases should negatively affect plant diversity and productivity. This adverse effect might be reduced through plant adaptive mechanisms to abiotic stresses such as changes in the 25
production of reactive oxygen species triggering signal transduction networks that provide protection against environmental stresses [127].
6
Conclusions
A greater elucidation of the epigenetic modifications in both perennial and autogamous species would allow to decipher the complex interactions between genotype and environment, identifying the real impact of global climate changes in the adaptation and consequently in the quality and
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productivity of commercial crop species. In addition, the construction of crop methylomes would help to develop new molecular markers associated with epigenetic changes that will assist breeding activities. The next-generation markers might be integrated in current methods of genetic
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characterization such as those using DNA barcoding [128]. It will allow the identification of transient stress conditions to manage plant stresses in a cautious and sustainable way. Indeed, we
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propose that an “epigenetic code” should exist in addition to the well-known genetic code
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composed by 4 bases. The existence of an “epigenetic alphabeth” might explain the low correlation observed between genetic and phenotypic variance. Key players in the epigenetic code should be represented by DNA methylation in CG, CH, and CHH sequences. Other epigenetic mechanisms
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driven by chromatin restructuring should be driven by histone methylations such as H3K27me2/3 occurring in response to bacterial stress. The deciphering of this epigenetic alphabet is one of the
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most fascinating fields of modern genetics. This may greatly help to gain insight into complex
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mechanisms of plant epigenomic responses to environmental stresses in a climate change scenario.
AUTHOR CONTRIBUTIONS Both FM and AP contributed in writing the manuscript. Both authors have made substantial, direct and intellectual contribution to the work.
26
Conflict of Interest None
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2. C.S. Pikaard, O.M. Scheid, Epigenetic regulation in plants, Cold Spring Harb. Perspect. Biol. 6 (2015) a019315. doi: 10.1101/cshperspect. a019315.
3. A. Sokol, A. Kwiatkowska, A. Jerzmanowski, M. Prymakowska Bosak, Up-regulation of
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stress-inducible genes in tobacco and Arabidopsis cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modifications,
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Figure legends Figure 1. Main DNA methylation mechanisms at cytosine position in CG, CHH, CHG sequences
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and their role in plants.
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Figure 2. Integration of different “omic” approaches to develop a systems-based approaches to
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investigate plant responses to environmental stresses.
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environmental stresses.
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Figure 3. Main epigenetic modifications linked to stress memory in relation to different
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Figure 4. Epigenetic mechanisms linked to priming in plants.
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Figure 5. Main epigenetic mechanisms involved in abiotic stress responses in plants.
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Figure 6. Main epigenetic mechanisms involved in plant biotic stress responses.
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Figure 7
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Figure 8
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Tables
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Table 1. A selected list of references dealing with epigenetic/epigenomic studies in plants, the type of mechanism, major findings or article’s subject, and technique were indicated.
49
Epigenetic Mechanisms
Main Findings/ Subjects
Used/Studied Techniques
Histone modification
ChIP-CHIP
Chen et al., 2018
DNA methylation
Zhong et al., 2013
DNA methylation
Zilberman and Henikoff, 2007
DNA methylation me-wide methylation analysis
The technique can be used to identify target sequences of transcription factors, and positions of histones with modified N-terminal tails Description of the ChIP-Seq workflow and its various applications in plants Plant epigenome is dynamic during fruit development and play a key role in processes such as ripening Combining different techniques with DNA microarrays and highthroughput sequencing is possible to map DNA methylation at genomewide scale Wholegenome arrays can be used for epigenetic analysis such as chromatinimmunoprecipitationchip studies, methylome characterization Opportunities and limitations of RRBS iwere described for nonmodel plant species
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Mockler et al., 2005
Paun et al., 2019
DNA methylation
Zhang et al., 2015
DNA methylation
Kunze, 2018
DNA methylation
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References dealing with Epigenetic/Epigenomic Technique Pellegrini and Ferrari, 2012
ChIP-Seq
Whole Genome Bisulphite sequencing
Different techniques
Padlock-probe combined with promoter tiling arrays
Reduced representation bisulphite sequencing (RRBS) Differentially Methylationmethylated genomic sensitive singlefragments were linked strand with sexual dimorphism conformation of pests in rice analysis The EpiTYPER™ assay EpiTYPER™ allows a fast and assay reproducible targeted quantification of 50 individual CpGs in a high throughput manner
DNA methylation
Agorio et al., 2017
DNA methylation
Rodriguez-Lopez et al., 2010
DNA methylation
Gonzalgo and Liang, 2007
DNA methylation
Van den Boom and Ehrich, 2009
Methylation levels in WRKY genes were negatively correlated with gene expression in response to NaCl treatments Data revealed a main role of this loop in maintaining a natural epiallele
Bisulphite coupled with PCR
High-resolution melting analysis allows adjusting thermal parameters for DNA hybridization and PCRbased techniques analyzing the impact of DNA methylation marks on the thermostability of regulatory sequences Methylation-sensitive single-nucleotide primer extension can be used to quantify DNA methylation
High-resolution melting analysis
Bisulphite treatment coupled with pyrosequencing
Methylationsensitive singlenucleotide primer extension ((MsSNuPE) Matrix-assisted laser desorption ionization/timeof-flight
Martinelli et al., 2018
Non-coding RNA
Next-generation sequencing
Matsui et al. 2010
Non-coding RNA
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Celik et al., 2019
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DNA methylation
Matrix-assisted laser desorption ionization/time-of-flight combines candidate gene amplification with base-specific cleavage or primer extension methods and MALDITOF mass spectrometric analysis to overcome high dynamicity of DNA methylation patterns MiRNAs may be included in nextgeneration breeding programs Tiling array can identify the stress-responsive genes in Arabidopsis thaliana
Tiling DNA microarray
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Table 2. A selected list of main epigenetic modifications in response to different environmental stresses. Levels of analysis (target or genome-wide analysis) and references were indicated.
DNA methylation Chromatin restructuring DNA methylation Histone modification
Level of Environmental Analysis Stresses (Genome-wide or target epigenetic specific) Abiotic Stress Responses Genome-wide Osmotic Genome-wide Cold
References
Sani et al., 2013 Kim et al., 2015
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Epigenetic modifications
Genome-wide Target epigenetic specific Target epigenetic specific Target epigenetic specific
Cold Drought, cold,
CHG methylation
Genome-wide
Phosphate starvation
DNA methylation
Genome-wide
Histone modification
Genome-wide
Drought
H3K4me3
Genome-wide
Drought
Target epigenetic specific
Salinity
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Low humidity Heat
Drought
Target epigenetic specific
Salinity
DNA methylation
Genome-wide
Salinity
DNA methylation
Genome-wide
Salinity
H3K27me3 H3K27me3 Differentially methylated regions Hypermethylated regions; heterochromatin
Genome-wide Genome-wide Genome-wide
Salinity Drought Salinity
Genome-wide
Salinity
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DNA methylation
General abiotic stresses
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DNA methylation (WRKY, GAPC1)
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Non coding RNA (RNA helicases) CHG methylation
Liu et al., 2017 Rutowicz et al., 2015 Khan et al., 2014 Tricker et al., 2012; Popova et al., 2013; Yong-Villalobos et al., 2015 Liang et al., 2014; Bräutigam et al., 2013 Gourcilleau et al., 2010; van Dijk et al., 2010; Çelik et al., 2019; Fei et al., 2017; Liu et al., 2018; Ferreira et al., 2019 Yaish et al., 2018 Al-Lawati et al., 2016 Sani et al., 2013 Garg et al., 2015 Banerjee and Roychoudhury, 2017 52
silencing DNA methylation
Target epigenetic specific
Aluminum stress
H3K27me3
Genome-wide
Zinc stress
CHG hypomethylation
Genome-wide
Heavy metals
Chromatin-remodeling processes; Non-coding RNAs MET1 transcriptional silencing Tam3 transposon methylation DNA methylation
Genome-wide
Cold (vernalization)
Agnieszka, 2018; Ghosh et al., 2018 Ariani et al., 2016; Erturk et al., 2015 Cicatelli et al., 2014 Kim and Sung 2013 Steward et al., 2000 Hashida et al., 2006 Liu et al., 2017
Cytosine methylation
Target epigenetic specific Genome-wide
Responses to pathogen infections Responses to Fusarium wilt
DNA hypomethylation
Genome-wide
General biotic stress
Cytosine DNA methylation DNA methylation
Genome-wide
Responses to geminivirus infection Responses to pathogen infections General biotic stress
Jaskiewicz et al., 2011 Mohammadi, et al., 2015 Furci et al., 2019 Wang et al., 2018 Zervudacki et al., 2018 Pumplin et al., 2016 Fonsêca et al.,
DNA methylation
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DNA methylation
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Target epigenetic Cold specific Target epigenetic Cold specific Target epigenetic Cold specific Biotic Stress Responses Target epigenetic General biotic stress specific Target epigenetic Stress memory specific Target epigenetic Stress memory specific Target epigenetic General biotic stress specific Target epigenetic Responses to necrotrophic specific and biotrophic pathogens Target epigenetic Response to pathogen Pst specific DC3000 Target epigenetic Response to pathogen Pst specific DC3000 Target epigenetic General biotic stress specific Target epigenetic Responses to fungal attacks specific
Chromatin remodeling
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Activation of HUB1 and HUB2 Histone deacetylase (HDA19) H3K27me2/3; Jumonji C Chromatin-based transcriptional regulation Histone modifications
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H3 histone modifications DNA methylation
Target epigenetic specific DNA methylation and Target epigenetic small-interfering RNAs specific Hypomethylation of Genome-wide
Responses to fungus C.
Alonso et al., 2019 Alves et al., 2013 Jin et al., 2018 Dowen et al., 2012 Ding and Wang, 2015 Zou et al., 2014 Choi et al., 2012 Hou et al., 2015 Soyer et al., 2014
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Target epigenetic specific
lindemuthianum Responses to fungal pathogens
2014 Berr et al., 2010
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centromeric regions Methylation of histone H3 lysine 36
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