Epigenetic regulation of spurious transcription initiation in Arabidopsis.

In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.

Author Correction: Genome defense against integrated organellar DNA fragments from plastids into plant nuclear genomes through DNA methylation.

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

Divergence of RNA editing among Arabidopsis species.

RNA editing altered the RNA sequence by replacing the C nucleotide to U in the organellar genomes of plants. RNA editing status sometimes differed among distant species. The pattern of conservation and variation of RNA editing status made it possible to evaluate evolutionary mechanisms impacting functional aspects of RNA editing. In this study, divergence of RNA editing in the chloroplast genome among Arabidopsis species was analyzed to determine 9 losses and 1 gain in RNA editing. All changes in A. thaliana lineage resulted from changes to the chloroplast genome sequence, whereas changes in the A. lyrata / halleri lineage were possibly due to exclusive changes in the nuclear editing factors. One loss of RNA editing in A. lyrata was caused by a deficiency in the PPR gene OTP80. The changes in RNA editing occurred approximately every two million years and were not observed at functionally important sites. These results highlight the conserved nature of RNA editing status suggesting the importance of RNA editing during evolution.

Genome defense against integrated organellar DNA fragments from plastids into plant nuclear genomes through DNA methylation.

Nuclear genomes are always faced with the modification of themselves by insertions and integrations of foreign DNAs and intrinsic parasites such as transposable elements. There is also substantial number of integrations from symbiotic organellar genomes to their host nuclear genomes. Such integration might have acted as a beneficial mutation during the evolution of symbiosis, while most of them have more or less deleterious effects on the stability of current genomes. Here we report the pattern of DNA substitution and methylation on organellar DNA fragments integrated from plastid into plant nuclear genomes. The genome analyses of 17 plants show homology-dependent DNA substitution bias. A certain number of these sequences are DNA methylated in the nuclear genome. The intensity of DNA methylation also decays according to the increase of relative evolutionary times after being integrated into nuclear genomes. The methylome data of epigenetic mutants shows that the DNA methylation of organellar DNA fragments in nuclear genomes are mainly dependent on the methylation maintenance machinery, while other mechanisms may also affect on the DNA methylation level. The DNA methylation on organellar DNA fragments may contribute to maintaining the genome stability and evolutionary dynamics of symbiotic organellar and their host's genomes.

DNA Methylation Diversification at the Integrated Organellar DNA-Like Sequence.

Plants have a lot of diversity in epigenetic modifications such as DNA methylation in their natural populations or cultivars. Although many studies observing the epigenetic diversity within and among species have been reported, the mechanisms how these variations are generated are still not clear. In addition to the de novo spontaneous epi-mutation, the intra- and inter-specific crossing can also cause a change of epigenetic modifications in their progenies. Here we report an example of diversification of DNA methylation by crossing and succeeding selfing. We traced the inheritance pattern of epigenetic modification during the crossing experiment between two natural strains Columbia (Col), and Landsberg electa (Ler) in model plant Arabidopsis thaliana to observe the inheritance of DNA methylation in two organellar DNA-like sequence regions in the nuclear genome. Because organellar DNA integration to the nuclear genome is common in flowering plants and these sequences are occasionally methylated, such DNA could be the novel source of plant genome evolution. The amplicon sequencing, using bisulfite-converted DNA and a next-generation auto-sequencer, was able to efficiently track the heredity of DNA methylation in F1 and F2 populations. One region showed hypomethylation in the F1 population and succeeding elevation of DNA methylation with large variance in the F2 population. The methylation level of Col and Ler alleles in F2 heterozygotes showed a significant positive correlation, implying the trans-chromosomal effect on DNA methylation. The results may suggest the possible mechanism causing the natural epigenetic diversity within plant populations.

Genome-Wide Analysis of Parent-of-Origin Allelic Expression in Endosperms of Brassicaceae Species, Brassica rapa.

Uniparental gene expression, observed in both animals and plants, is termed genomic imprinting. Genomic imprinting is a well-known epigenetic phenomenon regulated through epigenetic modifications such as DNA methylation and histone modifications. Recent genome-wide studies of endosperm transcription have revealed the rapid change of imprinted genes between species, suggesting the flexibility of this phenomenon. Although the functional significance and evolutionary trends of imprinted genes are still obscure, it can be clarified by inter-species comparisons. In this study, we analyzed the pattern of genomic imprinting in Brassica rapa, a species related to Arabidopsis thaliana. Compared with the ancient karyotype of A. thaliana and B. rapa, B. rapa has a triplicated genome. Many imprinted genes, beyond the estimated number previously reported in other species, were observed. Several imprinted genes have been conserved among species in Brassicaceae. We also observed rapid molecular evolution of imprinted genes compared to non-imprinted genes in B. rapa. Especially, imprinted gene overlapping between species showed more rapid molecular evolution and preferential expression in endosperms. It may imply that a small number of imprinted genes have retained functional roles among diverged species and have been the target of natural selection.

Characterization of a heat-activated retrotransposon in Vigna angularis.

In plants, several transposable elements are conserved across species. We found a homolog of ONSEN, which is a heat-activated retrotransposon originally isolated from Arabidopsis thaliana, in Vigna. The ONSEN-like elements (VaONS) were detected in all the analyzed Japanese accessions of Vigna angularis (adzuki bean) by Southern blot analysis. However, VaONS sequences were observed to be polymorphic in the different accessions. Interestingly, extrachromosomal DNA (ecDNA) was detected in some accessions of adzuki bean, indicating the conserved heat-activation of VaONS. Furthermore, we successfully induced retrotransposition of VaONS in adzuki plant regenerated through callus. Findings of our study should provide a new tool for molecular breeding of adzuki bean.

Exploring the History of Chloroplast Capture in Arabis Using Whole Chloroplast Genome Sequencing.

Chloroplast capture occurs when the chloroplast of one plant species is introgressed into another plant species. The phylogenies of nuclear and chloroplast markers from East Asian Arabis species are incongruent, which indicates hybrid origin and shows chloroplast capture. In the present study, the complete chloroplast genomes of A. hirsuta, A. nipponica, and A. flagellosa were sequenced in order to analyze their divergence and their relationships. The chloroplast genomes of A. nipponica and A. flagellosa were similar, which indicates chloroplast replacement. If hybridization causing chloroplast capture occurred once, divergence between recipient species would be lower than between donor species. However, the chloroplast genomes of species with possible hybrid origins, A. nipponica and A. stelleri, differ at similar levels to possible maternal donor species A. flagellosa, which suggests that multiple hybridization events have occurred in their respective histories. The mitochondrial genomes exhibited similar patterns, while A. nipponica and A. flagellosa were more similar to each other than to A. hirsuta. This suggests that the two organellar genomes were co-transferred during the hybridization history of the East Asian Arabis species.

The complete chloroplast genome of Sisymbrium irio.

The complete chloroplast genome of Sisymbrium irio was determined. The length of the complete chloroplast genome is 154,001 bp. The whole chloroplast genome consists of 83,891 bp long single copy (LSC) and 17,630 bp small single copy (SSC) regions, separated by a pair of 26,240 bp inverted repeat (IR) regions. The S. irio chloroplast genome encodes 112 annotated known unique genes including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic position of S. irio is sister to Brassiceae and Thlaspideae.

Evolution of sequence-specific anti-silencing systems in Arabidopsis.

The arms race between parasitic sequences and their hosts is a major driving force for evolution of gene control systems. Since transposable elements (TEs) are potentially deleterious, eukaryotes silence them by epigenetic mechanisms such as DNA methylation. Little is known about how TEs counteract silencing to propagate during evolution. Here, we report behavior of sequence-specific anti-silencing proteins used by Arabidopsis TEs and evolution of those proteins and their target sequences. We show that VANC, a TE-encoded anti-silencing protein, induces extensive DNA methylation loss throughout TEs. Related VANC proteins have evolved to hypomethylate TEs of completely different spectra. Targets for VANC proteins often form tandem repeats, which vary considerably between related TEs. We propose that evolution of VANC proteins and their targets allow propagation of TEs while causing minimal host damage. Our findings provide insight into the evolutionary dynamics of these apparently "selfish" sequences. They also provide potential tools to edit epigenomes in a sequence-specific manner.

Analysis of nuclear mitochondrial DNAs and factors affecting patterns of integration in plant species.

Sequences homologous to organellar DNA that have been integrated into nuclear genomes are referred to as nuclear mitochondrial DNAs (NUMTs) and nuclear plastid DNAs (NUPTs). NUMTs in nine plant species were analyzed to reveal the integration patterns and possible factors involved. The cumulative lengths of NUMTs in two-thirds of species analyzed were greater than those of NUPTs observed in a previous study. The age distribution of NUMTs was similar to that of NUPTs, suggesting similar mechanisms for integration and degradation of both NUPTs and NUMTs. Nuclear genome size and the cumulative length of NUMTs showed a significant positive correlation for older but not younger NUMTs. The same correlation was also found between nuclear genome size and older NUPTs in 17 species. These results suggested that genome size is a key factor to determine the cumulative length of relatively older NUPTs/NUMTs. Although the factor(s) determining the cumulative length of younger NUPTs/NUMTs is unclear, these sequences may be more deleterious, which could explain the different manner of determining the cumulative length of younger NUPTs/NUMTs in nuclear genomes. In addition, a relationship between the cumulative length of integrated NUMTs and complexity of mitochondrial genomes (i.e., the number of repeats) was found. The results indicate that the structural complexity of both NUMTs and their original mitochondrial sequences affects integration and degradation processes.

Characterization of a heat-activated retrotransposon in natural accessions of Arabidopsis thaliana.

Natural accessions are used for studying intraspecies genetic variation in the model plant Arabidopsis thaliana in order to address fundamental questions of evolution. Transposable elements are responsible for a wide range of mutations and play significant roles in shaping a genome over evolutionary time. In the present study, we aimed to characterize ONSEN, a heat-activated long terminal repeat (LTR) retrotransposon, in natural A. thaliana accessions. Southern blot analysis demonstrated that ONSEN was present in all the studied accessions, but the copy number was diverse. Olympia-1 contained a single ONSEN copy, located in the centromere of Chromosome 3. A premature stop codon in Olympia-1 ONSEN presumably abolishes integrase activity, which in turn presumably renders the retrotransposon non-functional. Hybridization of Col-0 with Olympia-1 showed that several ONSEN copies in Col-0 were activated by heat stress and maintained their transpositional activity in the progeny.

Gene duplication, silencing and expression alteration govern the molecular evolution of PRC2 genes in plants.

PRC2 genes were analyzed for their number of gene duplications, dN/dS ratios and expression patterns among Brassicaceae and Gramineae species. Although both amino acid sequences and copy number of the PRC2 genes were generally well conserved in both Brassicaceae and Gramineae species, we observed that some rapidly evolving genes experienced duplications and expression pattern changes. After multiple duplication events, all but one or two of the duplicated copies tend to be silenced. Silenced copies were reactivated in the endosperm and showed ectopic expression in developing seeds. The results indicated that rapid evolution of some PRC2 genes is initially caused by a relaxation of selective constraint following the gene duplication events. Several loci could become maternally expressed imprinted genes and acquired functional roles in the endosperm.

Patterns of genomic integration of nuclear chloroplast DNA fragments in plant species.

The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts.

Importance of gene duplication in the evolution of genomic imprinting revealed by molecular evolutionary analysis of the type I MADS-box gene family in Arabidopsis species.

The pattern of molecular evolution of imprinted genes is controversial and the entire picture is still to be unveiled. Recently, a relationship between the formation of imprinted genes and gene duplication was reported in genome-wide survey of imprinted genes in Arabidopsis thaliana. Because gene duplications influence the molecular evolution of the duplicated gene family, it is necessary to investigate both the pattern of molecular evolution and the possible relationship between gene duplication and genomic imprinting for a better understanding of evolutionary aspects of imprinted genes. In this study, we investigated the evolutionary changes of type I MADS-box genes that include imprinted genes by using relative species of Arabidopsis thaliana (two subspecies of A. lyrata and three subspecies of A. halleri). A duplicated gene family enables us to compare DNA sequences between imprinted genes and its homologs. We found an increased number of gene duplications within species in clades containing the imprinted genes, further supporting the hypothesis that local gene duplication is one of the driving forces for the formation of imprinted genes. Moreover, data obtained by phylogenetic analysis suggested "rapid evolution" of not only imprinted genes but also its closely related orthologous genes, which implies the effect of gene duplication on molecular evolution of imprinted genes.

Mobilization of a plant transposon by expression of the transposon-encoded anti-silencing factor.

Transposable elements (TEs) have a major impact on genome evolution, but they are potentially deleterious, and most of them are silenced by epigenetic mechanisms, such as DNA methylation. Here, we report the characterization of a TE encoding an activity to counteract epigenetic silencing by the host. In Arabidopsis thaliana, we identified a mobile copy of the Mutator-like element (MULE) with degenerated terminal inverted repeats (TIRs). This TE, named Hiun (Hi), is silent in wild-type plants, but it transposes when DNA methylation is abolished. When a Hi transgene was introduced into the wild-type background, it induced excision of the endogenous Hi copy, suggesting that Hi is the autonomously mobile copy. In addition, the transgene induced loss of DNA methylation and transcriptional activation of the endogenous Hi. Most importantly, the trans-activation of Hi depends on a Hi-encoded protein different from the conserved transposase. Proteins related to this anti-silencing factor, which we named VANC, are widespread in the non-TIR MULEs and may have contributed to the recent success of these TEs in natural Arabidopsis populations.

Evolution of the ONSEN retrotransposon family activated upon heat stress in Brassicaceae.

A Ty1/Copia-like retrotransposon, ONSEN, is activated by heat stress in Arabidopsis thaliana, and its de novo integrations that were observed preferentially within genes implies its regulation of neighboring genes. Here we show that ONSEN related copies were found in most species of Brassicaceae, forming a cluster with each species in phylogenetic tree. Most copies were localized close to genes in Arabidopsis lyrata and Brassica rapa, suggesting conserved integration specificity of ONSEN family into genic or open chromatin. In addition, we found heat-induced transcriptional activation of ONSEN family in several species of Brassicaceae. These results suggest that ONSEN has conserved transcriptional activation promoted by environmental heat stress in some Brassicaceae species.

Centromere-targeted de novo integrations of an LTR retrotransposon of Arabidopsis lyrata.

The plant genome evolves with rapid proliferation of LTR-type retrotransposons, which is associated with their clustered accumulation in gene-poor regions, such as centromeres. Despite their major role for plant genome evolution, no mobile LTR element with targeted integration into gene-poor regions has been identified in plants. Here, we report such targeted integrations de novo. We and others have previously shown that an ATCOPIA93 family retrotransposon in Arabidopsis thaliana is mobilized when the DNA methylation machinery is compromised. Although ATCOPIA93 family elements are low copy number in the wild-type A. thaliana genome, high-copy-number related elements are found in the wild-type Arabidopsis lyrata genome, and they show centromere-specific localization. To understand the mechanisms for the clustered accumulation of the A. lyrata elements directly, we introduced one of them, named Tal1 (Transposon of Arabidopsis lyrata 1), into A. thaliana by transformation. The introduced Tal1 was retrotransposed in A. thaliana, and most of the retrotransposed copies were found in centromeric repeats of A. thaliana, suggesting targeted integration. The targeted integration is especially surprising because the centromeric repeat sequences differ considerably between A. lyrata and A. thaliana. Our results revealed unexpectedly dynamic controls for evolution of the transposon-rich heterochromatic regions.

Bursts of retrotransposition reproduced in Arabidopsis.

Retrotransposons, which proliferate by reverse transcription of RNA intermediates, comprise a major portion of plant genomes. Plants often change the genome size and organization during evolution by rapid proliferation and deletion of long terminal repeat (LTR) retrotransposons. Precise transposon sequences throughout the Arabidopsis thaliana genome and the trans-acting mutations affecting epigenetic states make it an ideal model organism with which to study transposon dynamics. Here we report the mobilization of various families of endogenous A. thaliana LTR retrotransposons identified through genetic and genomic approaches with high-resolution genomic tiling arrays and mutants in the chromatin-remodelling gene DDM1 (DECREASE IN DNA METHYLATION 1). Using multiple lines of self-pollinated ddm1 mutant, we detected an increase in copy number, and verified this for various retrotransposons in a gypsy family (ATGP3) and copia families (ATCOPIA13, ATCOPIA21, ATCOPIA93), and also for a DNA transposon of a Mutator family, VANDAL21. A burst of retrotransposition occurred stochastically and independently for each element, suggesting an additional autocatalytic process. Furthermore, comparison of the identified LTR retrotransposons in related Arabidopsis species revealed that a lineage-specific burst of retrotransposition of these elements did indeed occur in natural Arabidopsis populations. The recent burst of retrotransposition in natural population is targeted to centromeric repeats, which is presumably less harmful than insertion into genes. The ddm1-induced retrotransposon proliferations and genome rearrangements mimic the transposon-mediated genome dynamics during evolution and provide experimental systems with which to investigate the controlling molecular factors directly.

Patterns of polymorphism and demographic history in natural populations of Arabidopsis lyrata.

BACKGROUND: Many of the processes affecting genetic diversity act on local populations. However, studies of plant nucleotide diversity have largely ignored local sampling, making it difficult to infer the demographic history of populations and to assess the importance of local adaptation. Arabidopsis lyrata, a self-incompatible, perennial species with a circumpolar distribution, is an excellent model system in which to study the roles of demographic history and local adaptation in patterning genetic variation. PRINCIPAL FINDINGS: We studied nucleotide diversity in six natural populations of Arabidopsis lyrata, using 77 loci sampled from 140 chromosomes. The six populations were highly differentiated, with a median FST of 0.52, and structure analysis revealed no evidence of admixed individuals. Average within-population diversity varied among populations, with the highest diversity found in a German population; this population harbors 3-fold higher levels of silent diversity than worldwide samples of A. thaliana. All A. lyrata populations also yielded positive values of Tajima's D. We estimated a demographic model for these populations, finding evidence of population divergence over the past 19,000 to 47,000 years involving non-equilibrium demographic events that reduced the effective size of most populations. Finally, we used the inferred demographic model to perform an initial test for local adaptation and identified several genes, including the flowering time gene FCA and a disease resistance locus, as candidates for local adaptation events. CONCLUSIONS: Our results underscore the importance of population-specific, non-equilibrium demographic processes in patterning diversity within A. lyrata. Moreover, our extensive dataset provides an important resource for future molecular population genetic studies of local adaptation in A. lyrata.