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.
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.
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.
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.
Cell cycle dependent phosphorylation of conserved N-terminal tail residues of histone H3 has been described in both animal and plant cells. Through cytogenetic approaches using different plant species we show a detailed description of distribution patterns of phosphorylated histone H3 at either threonine 3 or threonine 32 in mitosis and meiosis. In meristematic cells of the large genome species Secale cereale, Vicia faba and Hordeum vulgare we have found that phosphorylation of both threonine residues begins in prophase, and dephosphorylation occurs in late anaphase. However, in the small genome species Arabidopsis thaliana dephosphorylation occurs at anaphase. In the first division of meiosis of species with large genomes phosphorylation of histone H3 at either threonine 3 or threonine 32 is seen first in diakinesis and extends to anaphase I, whereas in the second division these post-translational modifications are visible at metaphase II through anaphase II. While in A. thaliana dephosphorylation takes place at anaphase I and II. In all species analysed phosphorylated H3 at either threonine 3 or threonine 32 are distributed along the entire length of chromosomes during mitotic metaphase and metaphase I. In the second meiotic division threonine 3 phosphorylation is restricted to the pericentromeric domain, while phosphorylation of threonine 32 is widespread along chromosome arms of all species analysed.
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.
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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