Drought-induced circular RNAs in maize roots: separating signal from noise.

Circular RNAs (CircRNAs) play an important role in diverse biological processes; however, their origin and functions, especially in plants, remain largely unclear. Here, we used two maize (Zea mays) inbred lines, as well as 14 of their derivative RILs with different drought sensitivity, to systematically characterize 8,790 circRNAs in maize roots under well-watered (WW) and water-stress (WS) conditions. We found that a diverse set of circRNAs expressed at significantly higher levels under WS. Enhanced expression of circRNAs was associated with longer flanking introns and an enrichment of long interspersed nuclear element (LINE) retrotransposable elements. The epigenetic marks found at the back-splicing junctions of circRNA-producing genes were markedly different from canonical splicing, characterized by increased levels of H3K36me3/H3K4me1, as well as decreased levels of H3K9Ac/H3K27Ac. We found that genes expressing circRNAs are subject to relaxed selection. The significant enrichment of trait-associated sites along their genic regions suggested that genes giving rise to circRNAs were associated with plant survival rate under drought stress, implying that circRNAs play roles in plant drought responses. Furthermore, we found that overexpression of circMED16, one of the drought-responsive circRNAs, enhances drought tolerance in Arabidopsis (Arabidopsis thaliana). Our results provide a framework for understanding the intricate interplay of epigenetic modifications and how they contribute to the fine-tuning of circRNA expression under drought stress.

Heritable changes of epialleles near genes in maize can be triggered in the absence of CHH methylation.

Trans-chromosomal interactions resulting in changes in DNA methylation during hybridization have been observed in several plant species. However, little is known about the causes or consequences of these interactions. Here, we compared DNA methylomes of F1 hybrids that are mutant for a small RNA biogenesis gene, Mop1 (Mediator of paramutation1), with that of their parents, wild-type siblings, and backcrossed progeny in maize (Zea mays). Our data show that hybridization triggers global changes in both trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM), most of which involved changes in CHH methylation. In more than 60% of these TCM differentially methylated regions (DMRs) in which small RNAs are available, no significant changes in the quantity of small RNAs were observed. Methylation at the CHH TCM DMRs was largely lost in the mop1 mutant, although the effects of this mutant varied depending on the location of these DMRs. Interestingly, an increase in CHH at TCM DMRs was associated with enhanced expression of a subset of highly expressed genes and suppressed expression of a small number of lowly expressed genes. Examination of the methylation levels in backcrossed plants demonstrates that both TCM and TCdM can be maintained in the subsequent generation, but that TCdM is more stable than TCM. Surprisingly, although increased CHH methylation in most TCM DMRs in F1 plants required Mop1, initiation of a new epigenetic state of these DMRs did not require a functional copy of this gene, suggesting that initiation of these changes is independent of RNA-directed DNA methylation.

Heritable changes of epialleles in maize can be triggered in the absence of DNA methylation.

Trans-chromosomal interactions resulting in changes in DNA methylation during hybridization have been observed in several plant species. However, very little is known about the causes or consequences of these interactions. Here, we compared DNA methylomes of F1 hybrids that are mutant for a small RNA biogenesis gene, Mop1 (mediator of paramutation1) with that of their parents, wild type siblings, and backcrossed progeny in maize. Our data show that hybridization triggers global changes in both trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM), most of which involved changes in CHH methylation. In more than 60% of these TCM differentially methylated regions (DMRs) in which small RNAs are available, no significant changes in the quantity of small RNAs were observed. Methylation at the CHH TCM DMRs was largely lost in the mop1 mutant, although the effects of this mutant varied depending on the location of the CHH DMRs. Interestingly, an increase in CHH at TCM DMRs was associated with enhanced expression of a subset of highly expressed genes and suppressed expression of a small number of lowly expressed genes. Examination of the methylation levels in backcrossed plants demonstrates that TCM and TCdM can be maintained in the subsequent generation, but that TCdM is more stable than TCM. Surprisingly, although increased CHH methylation in F1 plants did require Mop1, initiation of the changes in the epigenetic state of TCM DMRs did not require a functional copy of this gene, suggesting that initiation of these changes is not dependent on RNA-directed DNA methylation.

ANTHRACNOSE RESISTANCE GENE2 confers fungal resistance in sorghum.

Sorghum is an important food and feed crop globally; its production is hampered by anthracnose disease caused by the fungal pathogen Colletotrichum sublineola (Cs). Here, we report identification and characterization of ANTHRACNOSE RESISTANCE GENE 2 (ARG2) encoding a nucleotide-binding leucine-rich repeat (NLR) protein that confers race-specific resistance to Cs strains. ARG2 is one of a cluster of several NLR genes initially identified in the sorghum differential line SC328C that is resistant to some Cs strains. This cluster shows structural and copy number variations in different sorghum genotypes. Different sorghum lines carrying independent ARG2 alleles provided the genetic validation for the identity of the ARG2 gene. ARG2 expression is induced by Cs, and chitin induces ARG2 expression in resistant but not in susceptible lines. ARG2-mediated resistance is accompanied by higher expression of defense and secondary metabolite genes at early stages of infection, and anthocyanin and zeatin metabolisms are upregulated in resistant plants. Interestingly, ARG2 localizes to the plasma membrane when transiently expressed in Nicotiana benthamiana. Importantly, ARG2 plants produced higher shoot dry matter than near-isogenic lines carrying the susceptible allele suggesting an absence of an ARG2 associated growth trade-off. Furthermore, ARG2-mediated resistance is stable at a wide range of temperatures. Our observations open avenues for resistance breeding and for dissecting mechanisms of resistance.

Conserved noncoding sequences and de novo Mutator insertion alleles are imprinted in maize.

Genomic imprinting is an epigenetic phenomenon in which differential allele expression occurs in a parent-of-origin-dependent manner. Imprinting in plants is tightly linked to transposable elements (TEs), and it has been hypothesized that genomic imprinting may be a consequence of demethylation of TEs. Here, we performed high-throughput sequencing of ribonucleic acids from four maize (Zea mays) endosperms that segregated newly silenced Mutator (Mu) transposons and identified 110 paternally expressed imprinted genes (PEGs) and 139 maternally expressed imprinted genes (MEGs). Additionally, two potentially novel paternally suppressed MEGs are associated with de novo Mu insertions. In addition, we find evidence for parent-of-origin effects on expression of 407 conserved noncoding sequences (CNSs) in maize endosperm. The imprinted CNSs are largely localized within genic regions and near genes, but the imprinting status of the CNSs are largely independent of their associated genes. Both imprinted CNSs and PEGs have been subject to relaxed selection. However, our data suggest that although MEGs were already subject to a higher mutation rate prior to their being imprinted, imprinting may be the cause of the relaxed selection of PEGs. In addition, although DNA methylation is lower in the maternal alleles of both the maternally and paternally expressed CNSs (mat and pat CNSs), the difference between the two alleles in H3K27me3 levels was only observed in pat CNSs. Together, our findings point to the importance of both transposons and CNSs in genomic imprinting in maize.

A Molecular Cloning and Sanger Sequencing-based Protocol for Detecting Site-specific DNA Methylation.

DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns of DNA methylation in genomes, it is possible to learn about the biological role of cytosine methylation, as well as its involvement in gene regulation and transposon silencing. Advances in whole-genome sequencing have led to the widespread adoption of methods that examine genome-wide patterns of DNA methylation. Achieving sufficient sequencing depth in these types of experiments is costly, particularly for pilot studies in organisms with large genome sizes, or incomplete reference genomes. To overcome this issue, assays to determine site-specific DNA methylation can be used. Although often used, these assays are rarely described in detail. Here, we describe a pipeline that applies traditional TA cloning, Sanger sequencing, and online tools to examine DNA methylation. We provide an example of how to use this protocol to examine the pattern of DNA methylation at a specific transposable element in maize.

Broad-spectrum fungal resistance in sorghum is conferred through the complex regulation of an immune receptor gene embedded in a natural antisense transcript.

Sorghum (Sorghum bicolor), the fifth most widely grown cereal crop globally, provides food security for millions of people. Anthracnose caused by the fungus Colletotrichum sublineola is a major disease of sorghum worldwide. We discovered a major fungal resistance locus in sorghum composed of the nucleotide-binding leucine-rich repeat receptor gene ANTHRACNOSE RESISTANCE GENE1 (ARG1) that is completely nested in an intron of a cis-natural antisense transcript (NAT) gene designated CARRIER OF ARG1 (CARG). Susceptible genotypes express CARG and two alternatively spliced ARG1 transcripts encoding truncated proteins lacking the leucine-rich repeat domains. In resistant genotypes, elevated expression of an intact allele of ARG1, attributed to the loss of CARG transcription and the presence of miniature inverted-repeat transposable element sequences, resulted in broad-spectrum resistance to fungal pathogens with distinct virulence strategies. Increased ARG1 expression in resistant genotypes is also associated with higher histone H3K4 and H3K36 methylation. In susceptible genotypes, lower ARG1 expression is associated with reduced H3K4 and H3K36 methylation and increased expression of NATs of CARG. The repressive chromatin state associated with H3K9me2 is low in CARG-expressing genotypes within the CARG exon and higher in genotypes with low CARG expression. Thus, ARG1 is regulated by multiple mechanisms and confers broad-spectrum, strong resistance to fungal pathogens.

The Epigenome and Beyond: How Does Non-genetic Inheritance Change Our View of Evolution?

Evidence from across the tree of life suggests that epigenetic inheritance is more common than previously thought. If epigenetic inheritance is indeed as common as the data suggest, this finding has potentially important implications for evolutionary theory and our understanding of how evolution and adaptation progress. However, we currently lack an understanding of how common various epigenetic inheritance types are, and how they impact phenotypes. In this perspective, we review the open questions that need to be addressed to fully integrate epigenetic inheritance into evolutionary theory and to develop reliable predictive models for phenotypic evolution. We posit that addressing these challenges will require the collaboration of biologists from different disciplines and a focus on the exploration of data and phenomena without preconceived limits on potential mechanisms or outcomes.

RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.

In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Mediator of Paramutation1 (Mop1), a gene encoding a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

The mop1 mutation affects the recombination landscape in maize.

Meiotic recombination is a fundamental process that generates genetic diversity and ensures the accurate segregation of homologous chromosomes. While a great deal is known about genetic factors that regulate recombination, relatively little is known about epigenetic factors, such as DNA methylation. In maize, we examined the effects on meiotic recombination of a mutation in a component of the RNA-directed DNA methylation pathway, Mop1 (Mediator of paramutation1), as well as a mutation in a component of the trans-acting small interference RNA biogenesis pathway, Lbl1 (Leafbladeless1). MOP1 is of particular interest with respect to recombination because it is responsible for methylation of transposable elements that are immediately adjacent to transcriptionally active genes. In the mop1 mutant, we found that meiotic recombination is uniformly decreased in pericentromeric regions but is generally increased in gene rich chromosomal arms. This observation was further confirmed by cytogenetic analysis showing that although overall crossover numbers are unchanged, they occur more frequently in chromosomal arms in mop1 mutants. Using whole genome bisulfite sequencing, our data show that crossover redistribution is driven by loss of CHH (where H = A, T, or C) methylation within regions near genes. In contrast to what we observed in mop1 mutants, no significant changes were observed in the frequency of meiotic recombination in lbl1 mutants. Our data demonstrate that CHH methylation has a significant impact on the overall recombination landscape in maize despite its low frequency relative to CG and CHG methylation.

Transposable elements employ distinct integration strategies with respect to transcriptional landscapes in eukaryotic genomes.

Transposable elements (TEs) are ubiquitous DNA segments capable of moving from one site to another within host genomes. The extant distributions of TEs in eukaryotic genomes have been shaped by both bona fide TE integration preferences in eukaryotic genomes and by selection following integration. Here, we compare TE target site distribution in host genomes using multiple de novo transposon insertion datasets in both plants and animals and compare them in the context of genome-wide transcriptional landscapes. We showcase two distinct types of transcription-associated TE targeting strategies that suggest a process of convergent evolution among eukaryotic TE families. The integration of two precision-targeting elements are specifically associated with initiation of RNA Polymerase II transcription of highly expressed genes, suggesting the existence of novel mechanisms of precision TE targeting in addition to passive targeting of open chromatin. We also highlight two features that can facilitate TE survival and rapid proliferation: tissue-specific transposition and minimization of negative impacts on nearby gene function due to precision targeting.

Small RNA-Mediated De Novo Silencing of Ac/Ds Transposons Is Initiated by Alternative Transposition in Maize.

Although transposable elements (TEs) comprise a major fraction of many higher eukaryotic genomes, most TEs are silenced by host defense mechanisms. The means by which otherwise active TEs are recognized and silenced remains poorly understood. Here we analyzed two independent cases of spontaneous silencing of the active maize Ac/Ds transposon system. This silencing is initiated by alternative transposition, a type of aberrant transposition event that engages the termini of two nearby separate TEs. Alternative transposition during DNA replication can generate Composite Insertions that contain inverted duplications of the transposon sequences. We show that the inverted duplications of two Composite Insertions are transcribed to produce double-stranded RNAs that trigger the production of two distinct classes of small interfering RNAs: a 24-nt class complementary to the TE terminal inverted repeats and noncoding subterminal regions, and a 21- to 22-nt class corresponding to the TE transcribed regions. Plants containing these small interfering RNA-generating Composite Insertions exhibit decreased levels of Ac transcript and heritable repression of Ac/Ds transposition. Further, we demonstrate that Composite Insertions can heritably silence otherwise active elements in trans This study documents the first case of transposon silencing induced by alternative transposition and may represent a general initiating mechanism for silencing of DNA transposons.

Silencing of Mutator Elements in Maize Involves Distinct Populations of Small RNAs and Distinct Patterns of DNA Methylation.

Transposable elements (TEs) are a ubiquitous feature of plant genomes. Because of the threat they post to genome integrity, most TEs are epigenetically silenced. However, even closely related plant species often have dramatically different populations of TEs, suggesting periodic rounds of activity and silencing. Here, we show that the process of de novo methylation of an active element in maize involves two distinct pathways, one of which is directly implicated in causing epigenetic silencing and one of which is the result of that silencing. Epigenetic changes involve changes in gene expression that can be heritably transmitted to daughter cells in the absence of changes in DNA sequence. Epigenetics has been implicated in phenomena as diverse as development, stress response, and carcinogenesis. A significant challenge facing those interested in investigating epigenetic phenomena is determining causal relationships between DNA methylation, specific classes of small RNAs, and associated changes in gene expression. Because they are the primary targets of epigenetic silencing in plants and, when active, are often targeted for de novo silencing, TEs represent a valuable source of information about these relationships. We use a naturally occurring system in which a single TE can be heritably silenced by a single derivative of that TE. By using this system it is possible to unravel causal relationships between different size classes of small RNAs, patterns of DNA methylation, and heritable silencing. Here, we show that the long terminal inverted repeats within Zea mays MuDR transposons are targeted by distinct classes of small RNAs during epigenetic silencing that are dependent on distinct silencing pathways, only one of which is associated with transcriptional silencing of the transposon. Further, these small RNAs target distinct regions of the terminal inverted repeats, resulting in different patterns of cytosine methylation with different functional consequences with respect to epigenetic silencing and the heritability of that silencing.

Cost-Effective Profiling of Mutator Transposon Insertions in Maize by Next-Generation Sequencing.

Transposable elements can be highly mutagenic because when they transpose they can insert into genes and disrupt their function, a propensity which has been exploited in many organisms to generate tagged mutant alleles. The Mutator (Mu) family transposon is a family of DNA-type transposons in maize with a particularly high duplication frequency, which results in large numbers of new mutations in lineages that carry active Mu elements. Here we describe a rapid and cost-effective Miseq-based Mu transposon profiling pipeline. This method can also be used for identifying flanking sequences of other types of long insertions such as T-DNAs.

The long and short of doubling down: polyploidy, epigenetics, and the temporal dynamics of genome fractionation.

We consider the rapidly advancing discipline of plant evolutionary genomics, with a focus on the evolution of polyploid genomes. In many lineages, polyploidy is followed by 'biased fractionation', the unequal loss of genes from ancestral progenitor genomes. Mechanistically, it has been proposed that biased fractionation results from changes in the epigenetic landscape near genes, likely mediated by transposable elements. These epigenetic changes result in unequal gene expression between duplicates, establishing differential fitness that leads to biased gene loss with respect to ancestral genomes. We propose a unifying conceptual framework and a set of testable hypotheses based on this model, relating genome size, the proximity of transposable elements to genes, epigenetic reprogramming, chromatin accessibility, and gene expression.

Genome-wide Estimation of Evolutionary Distance and Phylogenetic Analysis of Homologous Genes.

Homologous genes, including paralogs and orthologs, are genes that share sequence homologies within or between different species. Homologous genes originate from a common origin through speciation, genetic duplication or horizontal gene transfer. Estimation of the sequence divergence of homologous genes help us to understand divergence time, which makes it possible to understand the evolutionary patterns of speciation, gene duplication and gene transfer events. This protocol will provide a detailed bioinformatics pipeline on how to identify the homologous genes, compare their sequence divergence and phylogenetic relationships, focusing on homologous genes that show syntenic relationships using soybean (Glycine max) and common bean (Phaseolus vulgaris) as example species.

Patterns and Consequences of Subgenome Differentiation Provide Insights into the Nature of Paleopolyploidy in Plants.

Polyploidy is an important feature of plant genomes, but the nature of many polyploidization events remains to be elucidated. Here, we demonstrate that the evolutionary fates of the subgenomes in maize (Zea mays) and soybean (Glycine max) have followed different trajectories. One subgenome has been subject to relaxed selection, lower levels of gene expression, higher rates of transposable element accumulation, more small interfering RNAs and DNA methylation around genes, and higher rates of gene loss in maize, whereas none of these features were observed in soybean. Nevertheless, individual gene pairs exhibit differentiation with respect to these features in both species. In addition, we observed a higher number of chromosomal rearrangements and higher frequency of retention of duplicated genes in soybean than in maize. Furthermore, soybean "singletons" were found to be more frequently tandemly duplicated than "duplicates" in soybean, which may, to some extent, counteract the genome imbalance caused by gene loss. We propose that unlike in maize, in which two subgenomes were distinct prior to the allotetraploidization event and thus experienced global differences in selective constraints, in soybean, the two subgenomes were far less distinct prior to polyploidization, such that individual gene pairs, rather than subgenomes, experienced stochastic differences over longer periods of time, resulting in retention of the majority of duplicates.

Natural antisense transcripts are significantly involved in regulation of drought stress in maize.

Natural antisense transcripts (NATs) are a prominent and complex class of regulatory RNAs. Using strand-specific RNA sequencing, we identified 1769 sense and antisense transcript pairs (NAT pairs) in two maize inbreds with different sensitivity to drought, as well as in two derivative recombination inbred lines (RILs). A significantly higher proportion of NATs relative to non-NATs are specifically expressed under water stress (WS). Surprisingly, expression of sense and antisense transcripts produced by NAT pairs is significantly correlated, particularly under WS. We found an unexpected large proportion of NATs with protein coding potential, as estimated by ribosome release scores. Small RNAs significantly accumulate within NAT pairs, with 21 nt smRNA particularly enriched in overlapping regions of these pairs of genes. The abundance of these smRNAs is significantly altered in the leafbladeless1 mutant, suggesting that these genes may be regulated by the tasiRNA pathway. Further, NATs are significantly hypomethylated and include fewer transposable element sequences relative to non-NAT genes. NAT gene regions also exhibit higher levels of H3K36me3, H3K9ac, and H3K4me3, but lower levels of H3K27me3, indicating that NAT gene pairs generally exhibit an open chromatin configuration. Finally, NAT pairs in 368 diverse maize inbreds and 19 segregating populations were specifically enriched for polymorphisms associated with drought tolerance. Taken together, the data highlight the potential impact of that small RNAs and histone modifications have in regulation of NAT expression, and the significance of NATs in response to WS.

Creating Order from Chaos: Epigenome Dynamics in Plants with Complex Genomes.

Flowering plants have strikingly distinct genomes, although they contain a similar suite of expressed genes. The diversity of genome structures and organization is largely due to variation in transposable elements (TEs) and whole-genome duplication (WGD) events. We review evidence that chromatin modifications and epigenetic regulation are intimately associated with TEs and likely play a role in mediating the effects of WGDs. We hypothesize that the current structure of a genome is the result of various TE bursts and WGDs and it is likely that the silencing mechanisms and the chromatin structure of a genome have been shaped by these events. This suggests that the specific mechanisms targeting chromatin modifications and epigenomic patterns may vary among different species. Many crop species have likely evolved chromatin-based mechanisms to tolerate silenced TEs near actively expressed genes. These interactions of heterochromatin and euchromatin are likely to have important roles in modulating gene expression and variability within species.