Publications by authors named "Ortrun Mittelsten Scheid"

51 Publications

Under siege: virus control in plant meristems and progeny.

Plant Cell 2021 May 20. Epub 2021 May 20.

Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria.

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 and 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.
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http://dx.doi.org/10.1093/plcell/koab140DOI Listing
May 2021

Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure.

PLoS Genet 2021 03 9;17(3):e1009444. Epub 2021 Mar 9.

Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria.

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.
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http://dx.doi.org/10.1371/journal.pgen.1009444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7978347PMC
March 2021

Aethionema arabicum genome annotation using PacBio full-length transcripts provides a valuable resource for seed dormancy and Brassicaceae evolution research.

Plant J 2021 Apr 8;106(1):275-293. Epub 2021 Feb 8.

Plant Cell Biology, Department of Biology, University of Marburg, Marburg, Germany.

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).
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http://dx.doi.org/10.1111/tpj.15161DOI Listing
April 2021

Versatile in vitro assay to recognize Cas9-induced mutations.

Plant Direct 2020 Sep 28;4(9):e00269. Epub 2020 Sep 28.

Gregor Mendel Institute of Molecular Plant Biology Austrian Academy of Sciences Vienna BioCenter (VBC) Vienna Austria.

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.
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http://dx.doi.org/10.1002/pld3.269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522499PMC
September 2020

Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns.

EMBO J 2020 10 20;39(20):e103667. Epub 2020 Aug 20.

Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria.

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.
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http://dx.doi.org/10.15252/embj.2019103667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560203PMC
October 2020

Preparing Chromatin and RNA from Rare Cell Types with Fluorescence-Activated Nuclear Sorting (FANS).

Methods Mol Biol 2020 ;2093:95-105

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria.

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.
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http://dx.doi.org/10.1007/978-1-0716-0179-2_7DOI Listing
December 2020

The Role of Noncoding RNAs in Double-Strand Break Repair.

Front Plant Sci 2019 27;10:1155. Epub 2019 Sep 27.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria.

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, , 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.
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http://dx.doi.org/10.3389/fpls.2019.01155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776598PMC
September 2019

Probing the 3D architecture of the plant nucleus with microscopy approaches: challenges and solutions.

Nucleus 2019 12;10(1):181-212

e Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich , Zürich , Switzerland.

The eukaryotic cell nucleus is a central organelle whose architecture determines genome function at multiple levels. Deciphering nuclear organizing principles influencing cellular responses and identity is a timely challenge. Despite many similarities between plant and animal nuclei, plant nuclei present intriguing specificities. Complementary to molecular and biochemical approaches, 3D microscopy is indispensable for resolving nuclear architecture. However, novel solutions are required for capturing cell-specific, sub-nuclear and dynamic processes. We provide a pointer for utilising high-to-super-resolution microscopy and image processing to probe plant nuclear architecture in 3D at the best possible spatial and temporal resolution and at quantitative and cell-specific levels. High-end imaging and image-processing solutions allow the community now to transcend conventional practices and benefit from continuously improving approaches. These promise to deliver a comprehensive, 3D view of plant nuclear architecture and to capture spatial dynamics of the nuclear compartment in relation to cellular states and responses. 3D and 4D: Three and Four dimensional; AI: Artificial Intelligence; ant: antipodal nuclei (ant); CLSM: Confocal Laser Scanning Microscopy; CTs: Chromosome Territories; DL: Deep Learning; DLIm: Dynamic Live Imaging; ecn: egg nucleus; FACS: Fluorescence-Activated Cell Sorting; FISH: Fluorescent In Situ Hybridization; FP: Fluorescent Proteins (GFP, RFP, CFP, YFP, mCherry); FRAP: Fluorescence Recovery After Photobleaching; GPU: Graphics Processing Unit; KEEs: KNOT Engaged Elements; INTACT: Isolation of Nuclei TAgged in specific Cell Types; LADs: Lamin-Associated Domains; ML: Machine Learning; NA: Numerical Aperture; NADs: Nucleolar Associated Domains; PALM: Photo-Activated Localization Microscopy; Pixel: Picture element; pn: polar nuclei; PSF: Point Spread Function; RHF: Relative Heterochromatin Fraction; SIM: Structured Illumination Microscopy; SLIm: Static Live Imaging; SMC: Spore Mother Cell; SNR: Signal to Noise Ratio; SRM: Super-Resolution Microscopy; STED: STimulated Emission Depletion; STORM: STochastic Optical Reconstruction Microscopy; syn: synergid nuclei; TADs: Topologically Associating Domains; Voxel: Volumetric pixel.
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http://dx.doi.org/10.1080/19491034.2019.1644592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682351PMC
December 2019

Illuminating (White and) Purple Patches.

Plant Cell 2019 06 29;31(6):1208-1209. Epub 2019 Apr 29.

Gregor Mendel Institute of Molecular Plant BiologyAustrian Academy of Sciences, Vienna BioCenter (VBC)Vienna, Austria

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http://dx.doi.org/10.1105/tpc.19.00308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588294PMC
June 2019

Aethionema arabicum: a novel model plant to study the light control of seed germination.

J Exp Bot 2019 06;70(12):3313-3328

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna BioCenter (VBC), Dr. Bohr-Gasse, Vienna, Austria.

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.
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http://dx.doi.org/10.1093/jxb/erz146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598081PMC
June 2019

Functional Characterization of SMG7 Paralogs in .

Front Plant Sci 2018 6;9:1602. Epub 2018 Nov 6.

Central European Institute of Technology, Masaryk University, Brno, Czechia.

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 . 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.
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http://dx.doi.org/10.3389/fpls.2018.01602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232500PMC
November 2018

Transposons: a blessing curse.

Curr Opin Plant Biol 2018 04 13;42:23-29. Epub 2018 Feb 13.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter (VBC), 1030 Vienna, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pbi.2018.01.003DOI Listing
April 2018

Epigenetic contribution to diversification.

Proc Natl Acad Sci U S A 2017 04 24;114(14):3558-3560. Epub 2017 Mar 24.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter Campus (VBC), 1030 Vienna, Austria

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http://dx.doi.org/10.1073/pnas.1702748114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389299PMC
April 2017

Developmental Control and Plasticity of Fruit and Seed Dimorphism in Aethionema arabicum.

Plant Physiol 2016 11 4;172(3):1691-1707. Epub 2016 Oct 4.

School of Biological Sciences, Plant Molecular Science and Centre for Systems and Synthetic Biology, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (K.G., C.S., T.S., G.L.-M.).

Understanding how plants cope with changing habitats is a timely and important topic in plant research. Phenotypic plasticity describes the capability of a genotype to produce different phenotypes when exposed to different environmental conditions. In contrast, the constant production of a set of distinct phenotypes by one genotype mediates bet hedging, a strategy that reduces the temporal variance in fitness at the expense of a lowered arithmetic mean fitness. Both phenomena are thought to represent important adaptation strategies to unstable environments. However, little is known about the underlying mechanisms of these phenomena, partly due to the lack of suitable model systems. We used phylogenetic and comparative analyses of fruit and seed anatomy, biomechanics, physiology, and environmental responses to study fruit and seed heteromorphism, a typical morphological basis of a bet-hedging strategy of plants, in the annual Brassicaceae species Aethionema arabicum Our results indicate that heteromorphism evolved twice within the Aethionemeae, including once for the monophyletic annual Aethionema clade. The dimorphism of Ae. arabicum is associated with several anatomic, biomechanical, gene expression, and physiological differences between the fruit and seed morphs. However, fruit ratios and numbers change in response to different environmental conditions. Therefore, the life-history strategy of Ae. arabicum appears to be a blend of bet hedging and plasticity. Together with the available genomic resources, our results pave the way to use this species in future studies intended to unravel the molecular control of heteromorphism and plasticity.
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http://dx.doi.org/10.1104/pp.16.00838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100781PMC
November 2016

Editorial.

Semin Cell Dev Biol 2015 Aug;44

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http://dx.doi.org/10.1016/j.semcdb.2015.10.035DOI Listing
August 2015

DNA Damage Repair in the Context of Plant Chromatin.

Plant Physiol 2015 Aug 18;168(4):1206-18. Epub 2015 Jun 18.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter, 1030 Vienna, Austria

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.
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http://dx.doi.org/10.1104/pp.15.00538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528755PMC
August 2015

Stress-induced structural changes in plant chromatin.

Curr Opin Plant Biol 2015 Oct 29;27:8-16. Epub 2015 May 29.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pbi.2015.05.011DOI Listing
October 2015

Epigenetic regulation in plants.

Cold Spring Harb Perspect Biol 2014 Dec 1;6(12):a019315. Epub 2014 Dec 1.

Gregor Mendel-Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Vienna, Austria.

The study of epigenetics in plants has a long and rich history, from initial descriptions of non-Mendelian gene behaviors to seminal discoveries of chromatin-modifying proteins and RNAs that mediate gene silencing in most eukaryotes, including humans. Genetic screens in the model plant Arabidopsis have been particularly rewarding, identifying more than 130 epigenetic regulators thus far. The diversity of epigenetic pathways in plants is remarkable, presumably contributing to the phenotypic plasticity of plant postembryonic development and the ability to survive and reproduce in unpredictable environments.
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http://dx.doi.org/10.1101/cshperspect.a019315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292151PMC
December 2014

Measuring Homologous Recombination Frequency in Seedlings.

Bio Protoc 2014 Apr;4(7)

Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria.

Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to increase in response to various chemical and physical agents that cause DNA damage and/or alter genome stability (reviewed in March-Díaz and Reyes, 2009). We monitor the frequency of SHR in transgenic seedlings containing recombination substrates with two truncated but overlapping parts of the β-glucuronidase (GUS) reporter gene (Orel , 2003; Schuermann , 2005). Upon an SHR event, a functional version of the transgene can be restored (Figure 1A). A histochemical assay applicable to whole plantlets allows the visualization of cells in which the reporter is restored, as the encoded enzyme converts a colorless substrate into a blue compound. This type of reporter has been extensively used to identify gene products required for regulating SHR levels in plants. We analyze plants stimulated for SHR by treatments with DNA damaging agents (bleocin, mitomycin C and UV-C) and compare them to non-treated plants.
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http://dx.doi.org/10.21769/BioProtoc.1094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662141PMC
April 2014

DNA Damage Sensitivity Assays with Seedlings.

Bio Protoc 2014 Apr;4(7)

Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria.

We describe fast and reproducible sensitivity assays to quantify the response of seedlings of different genotypes to a wide range of DNA damaging agents. We apply γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The "true leaf assay" and the "UV resistance assay" are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, wild type, determining the most effective type of induced damage and the potential repair pathway affected.
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http://dx.doi.org/10.21769/BioProtoc.1093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660615PMC
April 2014

Meristem-specific expression of epigenetic regulators safeguards transposon silencing in Arabidopsis.

EMBO Rep 2014 Apr 20;15(4):446-52. Epub 2014 Feb 20.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.

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.
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http://dx.doi.org/10.1002/embr.201337915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989676PMC
April 2014

How a retrotransposon exploits the plant's heat stress response for its activation.

PLoS Genet 2014 Jan 30;10(1):e1004115. Epub 2014 Jan 30.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.

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.
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http://dx.doi.org/10.1371/journal.pgen.1004115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907296PMC
January 2014

The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis.

Plant Cell 2013 Jun 18;25(6):1990-2001. Epub 2013 Jun 18.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Viena, Austria.

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.
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http://dx.doi.org/10.1105/tpc.112.104067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723608PMC
June 2013

Epigenetic responses to stress: triple defense?

Curr Opin Plant Biol 2012 Nov 7;15(5):568-73. Epub 2012 Sep 7.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.pbi.2012.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3508409PMC
November 2012

Advanced methylome analysis after bisulfite deep sequencing: an example in Arabidopsis.

PLoS One 2012 20;7(7):e41528. Epub 2012 Jul 20.

Max F. Perutz Laboratories, Center for Integrative Bioinformatics Vienna, University of Vienna, Vienna, Austria.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401099PMC
February 2013

Stress-induced chromatin changes: a critical view on their heritability.

Plant Cell Physiol 2012 May 28;53(5):801-8. Epub 2012 Mar 28.

Max Planck Institute for Plant Breeding Research, Cologne, Germany.

The investigation of stress responses has been a focus of plant research, breeding and biotechnology for a long time. Insight into stress perception, signaling and genetic determinants of resistance has recently been complemented by growing evidence for substantial stress-induced changes at the chromatin level. These affect specific sequences or occur genome-wide and are often correlated with transcriptional regulation. The majority of these changes only occur during stress exposure, and both expression and chromatin states typically revert to the pre-stress state shortly thereafter. Other changes result in the maintenance of new chromatin states and modified gene expression for a longer time after stress exposure, preparing an individual for developmental decisions or more effective defence. Beyond this, there are claims for stress-induced heritable chromatin modifications that are transmitted to progeny, thereby improving their characteristics. These effects resemble the concept of Lamarckian inheritance of acquired characters and represent a challenge to the uniqueness of DNA sequence-based inheritance. However, with the growing insight into epigenetic regulation and transmission of chromatin states, it is worth investigating these phenomena carefully. While genetic changes (mainly transposon mobility) in response to stress-induced interference with chromatin are well documented and heritable, in our view there is no unambiguous evidence for transmission of exclusively chromatin-controlled stress effects to progeny. We propose a set of criteria that should be applied to substantiate the data for stress-induced, chromatin-encoded new traits. Well-controlled stress treatments, thorough phenotyping and application of refined genome-wide epigenetic analysis tools should be helpful in moving from interesting observations towards robust evidence.
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http://dx.doi.org/10.1093/pcp/pcs044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345370PMC
May 2012

Genetic rearrangements can modify chromatin features at epialleles.

PLoS Genet 2011 Oct 20;7(10):e1002331. Epub 2011 Oct 20.

Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna, Austria.

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.
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http://dx.doi.org/10.1371/journal.pgen.1002331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197671PMC
October 2011

Polyploidization increases meiotic recombination frequency in Arabidopsis.

BMC Biol 2011 Apr 21;9:24. Epub 2011 Apr 21.

Gregor Mendel Institute of Molecular Plant Biology, 1030 Vienna, Austria.

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.
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http://dx.doi.org/10.1186/1741-7007-9-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110136PMC
April 2011

Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis.

Plant Cell 2010 Sep 28;22(9):3118-29. Epub 2010 Sep 28.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Viena, Austria.

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.
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http://dx.doi.org/10.1105/tpc.110.078493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965555PMC
September 2010
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