The histone variant H2A.Z has been implicated in numerous chromatin-mediated processes, including transcriptional activation, euchromatin maintenance, and heterochromatin formation. In yeast and humans, H2A.Z is deposited into chromatin by a conserved protein complex known as SWR1 and SRCAP, respectively. Here, we show that mutations in the Arabidopsis thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN6 (ARP6) and PHOTO-PERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), produce similar developmental phenotypes and result in the misregulation of a common set of genes. Using H2A.Z-specific antibodies, we demonstrate that ARP6 and PIE1 are required for the deposition of H2A.Z at multiple loci, including the FLOWERING LOCUS C (FLC) gene, a central repressor of the transition to flowering. Loss of H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and premature flowering, indicating that this histone variant is required for high-level expression of FLC. In addition to defining a novel mechanism for the regulation of FLC expression, these results support the existence of a SWR1-like complex in Arabidopsis and show that H2A.Z can potentiate transcriptional activation in plants. The finding that H2A.Z remains associated with chromatin throughout mitosis suggests that it may serve an epigenetic memory function by marking active genes and poising silenced genes for reactivation.

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-offunction phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long-and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.

SummaryArabidopsis has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by ACT2 and ACT8, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene-specific RNA gel blot hybridization and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays showed that ACT2 and/or ACT8 mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most Arabidopsis organs. The 5' flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a 13-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The ACT2/GUS construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the ACT8/GUS construct was weaker. It was observed only in a subset of the organs and tissues expressing ACT2/GUS and was not significantly expressed in the flower. ACT2, ACT8, and ACT8/GUS mRNAs were present at moderate to high levels in pollen, and yet neither ACT2/GUS nor ACT8/GUS enzyme expression could be detected in pollen.This suggested a mechanism of translational control affecting ACT2 and ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.

Parenting in the burying beetle Nicrophorus vespilloides is complex and, unusually, the sex and number of parents that can be present is flexible. Such flexibility is expected to involve specialized behaviour by the two sexes under biparental conditions. Here, we show that offspring fare equally well regardless of the sex or number of parents present. Comparing transcriptomes, we find a largely overlapping set of differentially expressed genes in both uniparental and biparental females and in uniparental males including vitellogenin, associated with reproduction, and takeout, influencing sex-specific mating and feeding behaviour. Gene expression in biparental males is similar to that in non-caring states. Thus, being ‘biparental’ in N. vespilloides describes the family social organization rather than the number of directly parenting individuals. There was no specialization; instead, in biparental families, direct male parental care appears to be limited with female behaviour unchanged. This should lead to strong sexual conflict.

SummaryThe Actin Depolymerizing Factor (ADF) gene family of Arabidopsis thaliana encodes 11 functional protein isovariants in four ancient subclasses. We report the characterization of the tissue-specific and developmental expression of all Arabidopsis ADF genes and the subcellular localization of several protein isovariants. The four subclasses exhibited distinct expression patterns as examined by qRT-PCR and histochemical assays of a GUS reporter gene under the control of individual ADF regulatory sequences. Subclass I ADFs were expressed strongly and constitutively in all vegetative and reproductive tissues except pollen. Subclass II ADFs were expressed specifically in mature pollen and pollen tubes or root epidermal trichoblast cells and root hairs, and these patterns evolved from an ancient dual expression pattern comprised of both polar tip growth cell types, still observed in the monocot Oryza sativa. Subclass III ADFs were expressed weakly in vegetative tissues, but were strongest in fast growing and/or differentiating cells including callus, emerging leaves, and meristem regions. The single subclass IV ADF was constitutively expressed at moderate levels in all tissues, including pollen. Immunocytochemical analysis with subclass-specific monoclonal antibodies demonstrated that subclass I isovariants localize to both the cytoplasm and the nucleus of leaf cells, while subclass II isovariants predominantly localize to the cytoplasm at the tip region of elongating root hairs and pollen tubes. The distinct expression patterns of the ADF subclasses support a model of ADF s co-evolving with the ancient and divergent actin isovariants.

BackgroundThe function of cytosine (DNA) methylation in insects remains inconclusive due to a lack of mutant and/or genetic studies.ResultsHere, we provide evidence for the functional role of the maintenance DNA methyltransferase 1 (Dnmt1) in an insect using experimental manipulation. Through RNA interference (RNAi), we successfully posttranscriptionally knocked down Dnmt1 in ovarian tissue of the hemipteran Oncopeltus fasciatus (the large milkweed bug). Individuals depleted for Dnmt1, and subsequently DNA methylation, failed to reproduce. Eggs were inviable and declined in number, and nuclei structure of follicular epithelium was aberrant. Erasure of DNA methylation from gene or transposon element bodies did not reveal a direct causal link to steady-state mRNA levels in somatic cells. These results reveal an important function of Dnmt1 seemingly not contingent on directly controlling gene expression.ConclusionsThis study provides direct experimental evidence for a functional role of Dnmt1 in egg production and embryo viability and uncovers a trivial role, if any, for DNA methylation in control of gene expression in O. fasciatus.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0246-5) contains supplementary material, which is available to authorized users.

Endogenous plant arsenate reductase (ACR) activity converts arsenate to arsenite in roots, immobilizing arsenic below ground. By blocking this activity, we hoped to construct plants that would mobilize more arsenate aboveground. We have identified a single gene in the Arabidopsis thaliana genome, ACR2, with moderate sequence homology to yeast arsenate reductase. Expression of ACR2 cDNA in Escherichia coli complemented the arsenate-resistant and arsenate-sensitive phenotypes of various bacterial ars operon mutants. RNA interference reduced ACR2 protein expression in Arabidopsis to as low as 2% of wild-type levels. The various knockdown plant lines were more sensitive to high concentrations of arsenate, but not arsenite, than wild type. The knockdown lines accumulated 10-to 16-fold more arsenic in shoots (350 -500 ppm) and retained less arsenic in roots than wild type, when grown on arsenate medium with <8 ppm arsenic. Reducing expression of ACR2 homologs in tree, shrub, and grass species should play a vital role in the phytoremediation of environmental arsenic contamination.Escherichia coli ArsC ͉ drinking water ͉ CDC25 ͉ toxicant ͉ arsenic pollution E nvironmental arsenic pollution is widely recognized as a global health problem (1) (www.epa.gov͞ogwdw͞ars͞arsenic.html). High levels of arsenic in soil and drinking water have been reported around the world, but the situation is worst in India and Bangladesh, where Ͼ400 million people are at risk of arsenic poisoning (2). The World Health Organization predicts that long-term exposure to arsenic could reach epidemic proportions, estimating that 1 in 10 people in the most contaminated areas may ultimately die from diseases related to arsenic poisoning (3). The high financial cost associated with repairing the environmental damage by using physical remediation methods such as excavation and reburial make these technologies unacceptable for cleaning up the vast areas of the planet that need arsenic remediation. As a result, the overwhelming majority of arsenic-contaminated sites are not being cleaned up.Phytoremediation is the use of plants to clean up environmental pollutants and is considered an important alternative to physical methods for cleaning up arsenic (4). Our objective is to develop a genetics-based arsenic phytoremediation strategy that can be used in any plant species. Plants that hyperaccumulate arsenic to high levels aboveground would be harvested and the arsenic further concentrated by incineration. In previous studies, we engineered model plants expressing a bacterial arsenate reductase (ArsC; EC 1.20.4.1) aboveground and constitutively expressing ␥-glutamylcysteine synthetase (5). By reducing arsenate to arsenite in leaves and trapping arsenite in thiol-peptide complexes, these plants accumulate 3-fold more arsenic aboveground than wild type and are also highly tolerant to toxic levels of arsenic. The research described herein extends these observations and attacks a particular problem limiting the engineered phytoremediation of arsenic: its transpor...

The relative significance of gene regulation and protein isovariant differences remains unexplored for most gene families, particularly those participating in multicellular development. Arabidopsis thaliana encodes three vegetative actins, ACT2, ACT7, and ACT8, in two ancient and highly divergent subclasses. Mutations in any of these differentially expressed actins revealed only mild phenotypes. However, double mutants were extremely dwarfed, with altered cell and organ morphology and an aberrant F-actin cytoskeleton (e.g., act2-1 act7-4 and act8-2 act7-4) or totally root-hairless (e.g., act2-1 act8-2). Our studies suggest that the three vegetative actin genes and protein isovariants play distinct subclass-specific roles during plant morphogenesis. For example, during root development, ACT7 was involved in root growth, epidermal cell specification, cell division, and root architecture, and ACT2 and ACT8 were essential for root hair tip growth. Also, genetic complementation revealed that the ACT2 and ACT8 isovariants, but not ACT7, fully rescued the root hair growth defects of single and double mutants. Moreover, we synthesized fully normal plants overexpressing the ACT8 isovariant from multiple actin regulatory sequences as the only vegetative actin in the act2-1 act7-4 background. In summary, it is evident that differences in vegetative actin gene regulation and the diversity in actin isovariant sequences are essential for normal plant development.