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A novel method in the field of genetic engineering of higher plants is presented: microinjection into multicellular structures which have a high competence for plant regeneration through embryogenesis. Microspore-derived embryoids of Brassica napus L. were individually selected and microinjected with NPT II gene constructions. High frequency regeneration of haploid plants through embryogenesis was achieved within 8 weeks. Transformation efficiencies between 27% and 51% were determined by DNA dot blot analysis of primary regenerants. Stable integration of fulllength microinjected genes into high molecular weight DNA was proven by Southern analysis of genomic DNA isolated from regenerated plants. Transformed plants were tested for expression of the NPT II gene by enzyme assay. The chimeric nature of the primary regenerants was demonstrated after their in vitro segregation through secondary embryogenesis into pure transformants. [ABSTRACT FROM AUTHOR]

Abstract This article is a response to Wang and Luo. See correspondence article http://www.biomedcentral.com/1741-7007/10/30/ [WEBCITE] and the original research article http://www.biomedcentral.com/1741-7007/9/24 [WEBCITE].

Abstract Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

Secondary dormancy is an adaptive trait that increases reproductive success by aligning seed germination with permissive conditions for seedling establishment. Aethionema arabicum is an annual plant and member of the Brassicaceae that grows in environments characterized by hot and dry summers. Aethionema arabicum seeds may germinate in early spring when seedling establishment is permissible. We demonstrate that long-day light regimes induce secondary dormancy in the seeds of Aethionema arabicum (CYP accession), repressing germination in summer when seedling establishment is riskier. Characterization of mutants screened for defective secondary dormancy demonstrated that RGL2 mediates repression of genes involved in gibberellin (GA) signaling. Exposure to high temperature alleviates secondary dormancy, restoring germination potential. These data are consistent with the hypothesis that long-day-induced secondary dormancy and its alleviation by high temperatures may be part of an adaptive response limiting germination to conditions permissive for seedling establishment in spring and autumn., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Efficient repair of DNA lesions is essential for the faithful transmission of genetic information between somatic cells and for genome integrity across generations. Plants have multiple, partially redundant, and overlapping DNA repair pathways, probably due to the less constricted germline and the inevitable exposure to light including higher energy wavelengths. Many proteins involved in DNA repair and their mode of actions are well described. In contrast, a role for DNA damage-associated RNA components, evident from many other organisms, is less well understood. Here, we have challenged young Arabidopsis thaliana plants with two different types of genotoxic stress and performed de novo assembly and transcriptome analysis. We identified three long noncoding RNAs (lncRNAs) that are lowly or not expressed under regular conditions but up-regulated or induced by DNA damage. We generated CRISPR/Cas deletion mutants and found that the absence of the lncRNAs impairs the recovery capacity of the plants from genotoxic stress. The genetic loci are highly conserved among world-wide distributed Arabidopsis accessions and within related species in the Brassicaceae group. Together, these results suggest that the lncRNAs have a conserved function in connection with DNA damage and provide a basis for mechanistic analysis of their role., Competing Interests: Conflicts of interest: The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

The view on the role of light during seed germination stems mainly from studies with Arabidopsis (Arabidopsis thaliana), where light is required to initiate this process. In contrast, white light is a strong inhibitor of germination in other plants, exemplified by accessions of Aethionema arabicum, another member of Brassicaceae. Their seeds respond to light with gene expression changes of key regulators converse to that of Arabidopsis, resulting in opposite hormone regulation and prevention of germination. However, the photoreceptors involved in this process in A. arabicum remain unknown. Here, we screened a mutant collection of A. arabicum and identified koy-1, a mutant that lost light inhibition of germination due to a deletion in the promoter of HEME OXYGENASE 1, the gene for a key enzyme in the biosynthesis of the phytochrome chromophore. koy-1 seeds were unresponsive to red- and far-red light and hyposensitive under white light. Comparison of hormone and gene expression between wild type and koy-1 revealed that very low light fluence stimulates germination, while high irradiance of red and far-red light is inhibitory, indicating a dual role of phytochromes in light-regulated seed germination. The mutation also affects the ratio between the 2 fruit morphs of A. arabicum, suggesting that light reception via phytochromes can fine-tune several parameters of propagation in adaptation to conditions in the habitat., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The "Mendelian Rules" of inheritance are cornerstones of genetics, described in Mendel's seminal publication from 1866. The experimental results and their interpretation have been discussed in numerous ways. This perspective emphasizes the contribution of Mendel's preparations prior to his crossing experiments to the discovery of Mendelian genetics. This thoughtful experimental design, and some fortune, avoided pitfalls that could have resulted in non-Mendelian inheritance., (� American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediated genome elimination., Competing Interests: The authors have declared that no competing interests exist.

In the arms race between plants and viruses, two frontiers have been utilized for decades to combat viral infections in agriculture. First, many pathogenic viruses are excluded from plant meristems, which allows the regeneration of virus-free plant material by tissue culture. Second, vertical transmission of viruses to the host progeny is often inefficient, thereby reducing the danger of viral transmission through seeds. Numerous reports point to the existence of tightly linked meristematic and transgenerational antiviral barriers that remain poorly understood. In this review, we summarize the current understanding of the molecular mechanisms that exclude viruses from plant stem cells and progeny. We also discuss the evidence connecting viral invasion of meristematic cells and the ability of plants to recover from acute infections. Research spanning decades performed on a variety of virus/host combinations has made clear that, beside morphological barriers, RNA interference (RNAi) plays a crucial role in preventing-or allowing-meristem invasion and vertical transmission. How a virus interacts with plant RNAi pathways in the meristem has profound effects on its symptomatology, persistence, replication rates, and, ultimately, entry into the host progeny., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed (development, regulation, germination, dormancy) and fruit (development, dehiscence mechanisms) characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae. arabicum gene annotation using 294 RNA-seq libraries and 136 307 full-length PacBio Iso-seq transcripts, increasing BUSCO completeness by 11.6% and featuring 5606 additional genes. Analysis of orthologs showed a lower number of genes in Ae. arabicum than in other Brassicaceae, which could be partially explained by loss of homeologs derived from the At-α polyploidization event and by a lower occurrence of tandem duplications after divergence of Aethionema from the other Brassicaceae. Benchmarking of MADS-box genes identified orthologs of FUL and AGL79 not found in previous versions. Analysis of full-length transcripts related to ABA-mediated seed dormancy discovered a conserved isoform of PIF6-β and antisense transcripts in ABI3, ABI4 and DOG1, among other cases found of different alternative splicing between Turkey and Cyprus ecotypes. The presented data allow alternative splicing mining and proposition of numerous hypotheses to research evolution and functional genomics. Annotation data and sequences are available at the Ae. arabicum DB (https://plantcode.online.uni-marburg.de/aetar_db)., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Paramutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are derived from paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation., Competing Interests: The authors have declared that no competing interests exist.

In plants, aerial organs originate continuously from stem cells in the center of the shoot apical meristem. Descendants of stem cells in the subepidermal layer are progenitors of germ cells, giving rise to male and female gametes. In these cells, mutations, including insertions of transposable elements or viruses, must be avoided to preserve genome integrity across generations. To investigate the molecular characteristics of stem cells in Arabidopsis, we isolated their nuclei and analyzed stage-specific gene expression and DNA methylation in plants of different ages. Stem cell expression signatures are largely defined by developmental stage but include a core set of stem cell-specific genes, among which are genes implicated in epigenetic silencing. Transiently increased expression of transposable elements in meristems prior to flower induction correlates with increasing CHG methylation during development and decreased CHH methylation, before stem cells enter the reproductive lineage. These results suggest that epigenetic reprogramming may occur at an early stage in this lineage and could contribute to genome protection in stem cells during germline development., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The discovery of CRISPR/Cas9 has revolutionized molecular biology, and its impact on plant biotechnology and plant breeding cannot be over-estimated. In many plant species, its application for mutagenesis is now a routine procedure--if suitable target sites, sufficient expression of the Cas9 protein, and functioning sgRNAs are combined. sgRNAs differ in their efficiency, depending on parameters that are only poorly understood. Several software tools and experience from growing databases are supporting the design of sgRNAs, but some seemingly perfect sgRNAs turn out to be inefficient or fail entirely, and most data bases stem from work with mammalian cells. Different in vitro assays testing sgRNAs in reconstituted Cas9 complexes are available and useful to reduce the risk of failure, especially in plants when CRISPR/Cas9 application requires modifications within the germ line and laborious transformation protocols. Low sgRNA efficiency and long generation times in plants can also contribute to the workload and costs of screening for the wanted genome edits. Here, we present a protocol in which a simple, initial in vitro test for suitable sgRNAs is modified to accelerate genotyping of Cas9-induced mutations. We demonstrate applicability of our protocol for mutagenesis and mutation screen for specific genes in Arabidopsis, but the principle should be universally suitable to provide a simple, low-cost, and rapid method to identify edited genes also in other plants and other organisms., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

The application of fluorescent tags to generate cell type-specific translational and transcriptional reporter lines is routine in plants, but separation of different cell types for downstream analyses is hampered by the presence of cell walls and tight connections between cells. Enzymatic removal of cell walls induces a wound response, dedifferentiation, or reprogramming of the resulting protoplasts. Their osmotic and mechanical instability and their large size range are challenging for FACS, a flow -sorting procedure based on differential expression of fluorescent tags. In contrast, plant nuclei are relatively robust and easy to isolate. Here, we describe a protocol for fluorescence-activated nuclear sorting (FANS) that allows efficient purification of very few fluorescence-tagged nuclei from a large background of non-labeled tissue. Purified nuclei are suitable for genome, epigenome, transcriptome, or proteome analyses. We describe in detail how to analyze nuclear RNA and DNA methylation from sorted nuclei representing the limited number of stem cells in the shoot apical meristem of Arabidopsis.

Genome stability is constantly threatened by DNA lesions generated by different environmental factors as well as endogenous processes. If not properly and timely repaired, damaged DNA can lead to mutations or chromosomal rearrangements, well-known reasons for genetic diseases or cancer in mammals, or growth abnormalities and/or sterility in plants. To prevent deleterious consequences of DNA damage, a sophisticated system termed DNA damage response (DDR) detects DNA lesions and initiates DNA repair processes. In addition to many well-studied canonical proteins involved in this process, noncoding RNA (ncRNA) molecules have recently been discovered as important regulators of the DDR pathway, extending the broad functional repertoire of ncRNAs to the maintenance of genome stability. These ncRNAs are mainly connected with double-strand breaks (DSBs), the most dangerous type of DNA lesions. The possibility to intentionally generate site-specific DSBs in the genome with endonucleases constitutes a powerful tool to study, in vivo , how DSBs are processed and how ncRNAs participate in this crucial event. In this review, we will summarize studies reporting the different roles of ncRNAs in DSB repair and discuss how genome editing approaches, especially CRISPR/Cas systems, can assist DNA repair studies. We will summarize knowledge concerning the functional significance of ncRNAs in DNA repair and their contribution to genome stability and integrity, with a focus on plants., (Copyright © 2019 Durut and Mittelsten Scheid.)

The timing of seed germination is crucial for seed plants and is coordinated by internal and external cues, reflecting adaptations to different habitats. Physiological and molecular studies with lettuce and Arabidopsis thaliana have documented a strict requirement for light to initiate germination and identified many receptors, signaling cascades, and hormonal control elements. In contrast, seed germination in several other plants is inhibited by light, but the molecular basis of this alternative response is unknown. We describe Aethionema arabicum (Brassicaceae) as a suitable model plant to investigate the mechanism of germination inhibition by light, as this species has accessions with natural variation between light-sensitive and light-neutral responses. Inhibition of germination occurs in red, blue, or far-red light and increases with light intensity and duration. Gibberellins and abscisic acid are involved in the control of germination, as in Arabidopsis, but transcriptome comparisons of light- and dark-exposed A. arabicum seeds revealed that, upon light exposure, the expression of genes for key regulators undergo converse changes, resulting in antipodal hormone regulation. These findings illustrate that similar modular components of a pathway in light-inhibited, light-neutral, and light-requiring germination among the Brassicaceae have been assembled in the course of evolution to produce divergent pathways, likely as adaptive traits., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

SMG7 proteins are evolutionary conserved across eukaryotes and primarily known for their function in nonsense mediated RNA decay (NMD). In contrast to other NMD factors, SMG7 proteins underwent independent expansions during evolution indicating their propensity to adopt novel functions. Here we characterized SMG7 and SMG7-like (SMG7L) paralogs in Arabidopsis thaliana . SMG7 retained its role in NMD and additionally appears to have acquired another function in meiosis. We inactivated SMG7 by CRISPR/Cas9 mutagenesis and showed that, in contrast to our previous report, SMG7 is not an essential gene in Arabidopsis. Furthermore, our data indicate that the N-terminal phosphoserine-binding domain is required for both NMD and meiosis. Phenotypic analysis of SMG7 and SMG7L double mutants did not indicate any functional redundancy between the two genes, suggesting neofunctionalization of SMG7L. Finally, protein sequence comparison together with a phenotyping of T-DNA insertion mutants identified several conserved regions specific for SMG7 that may underlie its role in NMD and meiosis. This information provides a framework for deciphering the non-canonical functions of SMG7-family proteins.

The genomes of most plant species are dominated by transposable elements (TEs). Once considered as 'junk DNA', TEs are now known to have a major role in driving genome evolution. Over the last decade, it has become apparent that some stress conditions and other environmental stimuli can drive bursts of activity of certain TE families and consequently new TE insertions. These can give rise to altered gene expression patterns and phenotypes, with new TE insertions sometimes causing flanking genes to become transcriptionally responsive to the same stress conditions that activated the TE in the first place. Such connections between TE-mediated increases in diversity and an accelerated rate of genome evolution provide powerful mechanisms for plants to adapt more rapidly to new environmental conditions. This review will focus on environmentally induced transposition, the mechanisms by which it alters gene expression, and the consequences for plant genome evolution and breeding., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stress defense in plants is elaborated at the level of protection and adaptation. Dynamic changes in sophisticated chromatin substructures and concomitant transcriptional changes play an important role in response to stress, as illustrated by the transient rearrangement of compact heterochromatin structures or the modulation of chromatin composition and modification upon stress exposure. To connect cytological, developmental, and molecular data around stress and chromatin is currently an interesting, multifaceted, and sometimes controversial field of research. This review highlights some of the most recent findings on nuclear reorganization, histone variants, histone chaperones, DNA- and histone modifications, and somatic and meiotic heritability in connection with stress., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

In plants, transposable elements (TEs) are kept inactive by transcriptional gene silencing (TGS). TGS is established and perpetuated by RNA-directed DNA methylation (RdDM) and maintenance methylation pathways, respectively. Here, we describe a novel RdDM function specific for shoot apical meristems that reinforces silencing of TEs during early vegetative growth. In meristems, RdDM counteracts drug-induced interference with TGS maintenance and consequently prevents TE activation. Simultaneous disturbance of both TGS pathways leads to transcriptionally active states of repetitive sequences that are inherited by somatic tissues and partially by the progeny. This apical meristem-specific mechanism is mediated by increased levels of TGS factors and provides a checkpoint for correct epigenetic inheritance during the transition from vegetative to reproductive phase and to the next generation.

Retrotransposons are major components of plant and animal genomes. They amplify by reverse transcription and reintegration into the host genome but their activity is usually epigenetically silenced. In plants, genomic copies of retrotransposons are typically associated with repressive chromatin modifications installed and maintained by RNA-directed DNA methylation. To escape this tight control, retrotransposons employ various strategies to avoid epigenetic silencing. Here we describe the mechanism developed by ONSEN, an LTR-copia type retrotransposon in Arabidopsis thaliana. ONSEN has acquired a heat-responsive element recognized by plant-derived heat stress defense factors, resulting in transcription and production of full length extrachromosomal DNA under elevated temperatures. Further, the ONSEN promoter is free of CG and CHG sites, and the reduction of DNA methylation at the CHH sites is not sufficient to activate the element. Since dividing cells have a more pronounced heat response, the extrachromosomal ONSEN DNA, capable of reintegrating into the genome, accumulates preferentially in the meristematic tissue of the shoot. The recruitment of a major plant heat shock transcription factor in periods of heat stress exploits the plant's heat stress response to achieve the transposon's activation, making it impossible for the host to respond appropriately to stress without losing control over the invader.

All processes requiring interaction with DNA are attuned to occur within the context of the complex chromatin structure. As it does for programmed transcription and replication, this also holds true for unscheduled events, such as repair of DNA damage. Lesions such as double-strand breaks occur randomly; their repair requires that enzyme complexes access DNA at potentially any genomic site. This is achieved by chromatin remodeling factors that can locally slide, evict, or change nucleosomes. Here, we show that the Swi2/Snf2-related (SWR1 complex), known to deposit histone H2A.Z, is also important for DNA repair in Arabidopsis thaliana. Mutations in genes for Arabidopsis SWR1 complex subunits photoperiod-independent Early Flowering1, actin-related protein6, and SWR1 complex6 cause hypersensitivity to various DNA damaging agents. Even without additional genotoxic stress, these mutants show symptoms of DNA damage accumulation. The reduced DNA repair capacity is connected with impaired somatic homologous recombination, in contrast with the hyper-recombinogenic phenotype of yeast SWR1 mutants. This suggests functional diversification between lower and higher eukaryotes. Finally, reduced fertility and irregular gametogenesis in the Arabidopsis SWR1 mutants indicate an additional role for the chromatin-remodeling complex during meiosis. These results provide evidence for the importance of Arabidopsis SWR1 in somatic DNA repair and during meiosis.

Stressful conditions for plants can originate from numerous physical, chemical and biological factors, and plants have developed a plethora of survival strategies including developmental and morphological adaptations, specific signaling and defense pathways as well as innate and acquired immunity. While it has become clear in recent years that many stress responses involve epigenetic components, we are far from understanding the mechanisms and molecular interactions. Extending our knowledge is fundamental, not least for plant breeding and conservation biology. This review will highlight recent insights into epigenetic stress responses at the level of signaling, chromatin modification, and potentially heritable consequences., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Deep sequencing after bisulfite conversion (BS-Seq) is the method of choice to generate whole genome maps of cytosine methylation at single base-pair resolution. Its application to genomic DNA of Arabidopsis flower bud tissue resulted in the first complete methylome, determining a methylation rate of 6.7% in this tissue. BS-Seq reads were mapped onto an in silico converted reference genome, applying the so-called 3-letter genome method. Here, we present BiSS (Bisufite Sequencing Scorer), a new method applying Smith-Waterman alignment to map bisulfite-converted reads to a reference genome. In addition, we introduce a comprehensive adaptive error estimate that accounts for sequencing errors, erroneous bisulfite conversion and also wrongly mapped reads. The re-analysis of the Arabidopsis methylome data with BiSS mapped substantially more reads to the genome. As a result, it determines the methylation status of an extra 10% of cytosines and estimates the methylation rate to be 7.7%. We validated the results by individual traditional bisulfite sequencing for selected genomic regions. In addition to predicting the methylation status of each cytosine, BiSS also provides an estimate of the methylation degree at each genomic site. Thus, BiSS explores BS-Seq data more extensively and provides more information for downstream analysis.

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3' regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles., Competing Interests: The authors have declared that no competing interests exist.

Epigenetic factors determine responses to internal and external stimuli in eukaryotic organisms. Whether and how environmental conditions feed back to the epigenetic landscape is more a matter of suggestion than of substantiation. Plants are suitable organisms with which to address this question due to their sessile lifestyle and diversification of epigenetic regulators. We show that several repetitive elements of Arabidopsis thaliana that are under epigenetic regulation by transcriptional gene silencing at ambient temperatures and upon short term heat exposure become activated by prolonged heat stress. Activation can occur without loss of DNA methylation and with only minor changes to histone modifications but is accompanied by loss of nucleosomes and by heterochromatin decondensation. Whereas decondensation persists, nucleosome loading and transcriptional silencing are restored upon recovery from heat stress but are delayed in mutants with impaired chromatin assembly functions. The results provide evidence that environmental conditions can override epigenetic regulation, at least transiently, which might open a window for more permanent epigenetic changes.

Polyploidization, a widespread phenomenon among plants, is considered a major speciation mechanism. Polyploid plants have a high degree of immediate post-zygotic reproductive isolation from their progenitors, as backcrossing to either parent will produce mainly nonviable progeny. This reproductive barrier is called triploid block and it is caused by malfunction of the endosperm. Nevertheless, the main route to polyploid formation is via unreduced gametes and unstable triploid progeny, suggesting that there are ways to overcome the triploid block. Until recently, the mechanistic basis for unreduced gamete formation and the triploid block were completely unknown. Recent developments have revealed genetic pathways leading to unreduced gamete formation as well as the underlying genetic basis for the triploid block in Arabidopsis. These novel findings will provide the basis for a genetic understanding of polyploid formation and subsequent speciation in plants., (Copyright 2009 Elsevier Ltd. All rights reserved.)

It is commonly observed that onset or release of transcriptional gene silencing (TGS) correlates with alteration of repressive epigenetic marks. The TGS regulator MOM1 in Arabidopsis is exceptional since it regulates transcription in intermediate heterochromatin with only minor changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and RNA polymerase-V (Pol-V). Pol-V transcribes heterochromatic loci, which seems to be required for maintenance of their silencing; however, it is still not clear how Pol-V is targeted to heterochromatin. We now provide evidence that Pol-V is required for MOM1-mediated suppression of transcription at a subset of its chromosomal targets. Thus, Pol-V genetically interacts with MOM1 in the control of gene silencing. Interestingly, functional cooperation of MOM1 and Pol-V not only broadens the range of the controlled loci in comparison to each individual factor, but also determines the degree of TGS.

Epigenetic changes of gene expression can potentially be reversed by developmental programs, genetic manipulation, or pharmacological interference. However, a case of transcriptional gene silencing, originally observed in tetraploid Arabidopsis thaliana plants, created an epiallele resistant to many mutations or inhibitor treatments that activate many other suppressed genes. This raised the question about the molecular basis of this extreme stability. A combination of forward and reverse genetics and drug application provides evidence for an epigenetic double lock that is only alleviated upon the simultaneous removal of both DNA methylation and histone methylation. Therefore, the cooperation of multiple chromatin modifications can generate unanticipated stability of epigenetic states and contributes to heritable diversity of gene expression patterns.

Methylation of cytosines is a very important epigenetic modification of genomic DNA in many different eukaryotes, and it is frequently involved in transcriptional regulation of genes. In plants, DNA methylation is regulated by a complex interplay between several methylating and demethylating enzymes. Analysis of the resulting cytosine methylation patterns with the highest resolution is achieved after sodium bisulfite treatment, deaminating nonmethylated cytosines to uracil. Subsequent PCR and sequence analysis of individual amplicons displays the degree, position, and sequence context of methylation of every cytosine residue in individual genomic sequences. We describe the application of bisulfite sequencing for the analysis of DNA methylation at defined individual sequences of plant genomic DNA.

Amplifying and sequencing DNA after bisulfite treatment of genomic DNA reveals the methylation state of cytosine residues at the highest resolution possible. However, a thorough analysis is required for statistical evaluation of methylation at all sites in each genomic region. Several software tools were developed to assist in quantitative evaluation of bisulfite sequencing data from complex methylation patterns occurring in plants. This chapter describes the application of Cytosine Methylation Analysis Tool for Everyone (CyMATE). From aligned sequences, CyMATE quantifies and illustrates general and pattern-specific methylation at CG, CHG, and CHH (H = A, C, or T) sites, both per sequence and per position. CyMATE is also able to perform a quality control of sequences and to detect redundancy among individual clones. The software is able to reveal methylation patterns on complementary strands by handling data from hairpin bisulfite sequencing. The tool is freely available for non-commercial use at http://www.cymate.org .

Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed "triploid block." Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin-specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues., Competing Interests: European patent EP09008196 “Polyploid plants” was deposited by ETH on June 23rd, 2009.

This work examines the role of the Arabidopsis thaliana RING E3 ligase, HISTONE MONOUBIQUITINATION1 (HUB1) in disease resistance. Loss-of-function alleles of HUB1 show increased susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola, whereas HUB1 overexpression conferred resistance to B. cinerea. By contrast, responses to the bacterial pathogen Pseudomonas syringae are unaltered in hub1 plants. hub1 mutants have thinner cell walls but increased callose around an infection site. HUB1 acts independently of jasmonate, but ethylene (ET) responses and salicylate modulate the resistance of hub1 mutants to necrotrophic fungi. The ET response factor ETHYLENE INSENSITIVE2 is epistatic to HUB1 for A. brassicicola resistance but additive to HUB1 for B. cinerea resistance. HUB1 interacts with MED21, a subunit of the Arabidopsis Mediator, a conserved complex that regulates RNA polymerase II. RNA interference lines with reduced MED21 expression are highly susceptible to A. brassicicola and B. cinerea, whereas T-DNA insertion alleles are embryonic lethal, suggesting an essential role for MED21. However, HUB1-mediated histone H2B modification is independent of histone H3 and DNA methylation. In sum, histone H2B monoubiquitination is an important chromatin modification with regulatory roles in plant defense against necrotrophic fungi most likely through modulation of gene expression.

Covalent modification by methylation of cytosine residues represents an important epigenetic hallmark. While sequence analysis after bisulphite conversion allows correlative analyses with single-base resolution, functional analysis by interference with DNA methylation is less precise, due to the complexity of methylation enzymes and their targets. A cytidine analogue, 5-azacytidine, is frequently used as an inhibitor of DNA methyltransferases, but its rapid degradation in aqueous solution is problematic for culture periods of longer than a few hours. Application of zebularine, a more stable cytidine analogue with a similar mode of action that is successfully used as a methylation inhibitor in Neurospora and mammalian tumour cell lines, can significantly reduce DNA methylation in plants in a dose-dependent and transient manner independent of sequence context. Demethylation is connected with transcriptional reactivation and partial decondensation of heterochromatin. Zebularine represents a promising new and versatile tool for investigating the role of DNA methylation in plants with regard to transcriptional control, maintenance and formation of (hetero-) chromatin.

Adverse conditions can trigger DNA damage as well as DNA repair responses in plants. A variety of stress factors are known to stimulate homologous recombination, the most accurate repair pathway, by increasing the concentration of necessary enzymatic components and the frequency of events. This effect has been reported to last into subsequent generations not exposed to the stress. To establish a basis for a genetic analysis of this transgenerational stress memory, a broad range of treatments was tested for quantitative effects on homologous recombination in the progeny. Several Arabidopsis lines, transgenic for well-established recombination traps, were exposed to 10 different physical and chemical stress treatments, and scored for the number of somatic homologous recombination (SHR) events in the treated generation as well as in the two subsequent generations that were not treated. These numbers were related to the expression level of genes involved in homologous recombination and repair. SHR was enhanced after the majority of treatments, confirming previous data and adding new effective stress types, especially interference with chromatin. Compounds that directly modify DNA stimulated SHR to values exceeding previously described induction rates, concomitant with an induction of genes involved in SHR. In spite of the significant stimulation in the stressed generations, the two subsequent non-treated generations only showed a low and stochastic increase in SHR that did not correlate with the degree of stimulation in the parental plants. Transcripts coding for SHR enzymes generally returned to pre-treatment levels in the progeny. Thus, transgenerational effects on SHR frequency are not a general response to abiotic stress in Arabidopsis and may require special conditions.