Epigenetic mechanisms affect the curled leaf phenotype in the hypomethylated ddc mutant of Arabidopsis thaliana.

The ddc mutant of Arabidopsis thaliana is characterized by pleiotropic phenotypic alterations including a curl-shaped leaf, previously explained by disturbed auxin metabolism and transport. The present study was aimed at further explore the molecular bases underlying the abnormal phenotype of the ddc leaf. We demonstrated that genes specifically related to leaf fate commitment and morphogenesis were misexpressed on developing ddc leaves, such as upregulation of CURLY LEAF (CLF) and downregulation of ASYMMETRIC LEAVES2 (AS2), KNOTTED-like gene from A. thaliana (KNAT6), TEOSINTE-LIKE1 CYCLOIDEA and PROLIFERATING CELL FACTOR 2 (TCP2) and others. The CLF gene, encoding a component of Polycomb repressive complex 2 (PRC2) which adds trimethylation marks at Lys27 of histone H3, was overexpressed in the ddc mutant and concomitantly was correlated with DNA methylation-dependent repression of its negative regulator UCL1. KNAT6, encoding a class 1 KNOX homeotic gene, had increased H3K27me3 trimethylation levels, suggesting it is a target gene of the CLF containing PRC2 complex in the ddc mutant. We postulate that different epigenetic mechanisms modulate expression of genes related to auxin pathways as well as gene targets of Polycomb repressive action, during leaf morphogenesis.

Influence of selected factors on the Firmicutes, Bacteroidetes phyla and the Lactobacillaceae family in the digestive tract of sheep.

In this study, we used 10 healthy sheep, which gave birth to healthy twins. Stool samples were collected from mothers and their offspring 3 times during the study (0, 28 and 56 day postpartum). Milk samples were taken from the mothers at the same time. RT PCR analysis of faeces and milk was performed in order to assess the level of bacteria from the Firmicutes and Bacteroidetes phyla including the family Lactobacillaceae (phylum Firmicutes). The composition of mother's milk was also analyzed and their BCS. The data were compiled statistically. The obtained results showed that the level of the studied groups of bacteria may change due to the change of diet. Additionally, there were significant differences between lambs and mothers in the levels of the studied groups of bacteria. Analysis also shown that in the digestive system of mothers was a smaller disproportion in the level of the studied bacterial phyla than in lambs. The results also indicated the occurrence of differences in the bacterial composition at the individual level, both in ewes and their offspring. Additionally, in the conducted experiment, there were differences in the level of Firmicutes and Bacteroidetes groups depending on the sex.

New Bird Sexing Strategy Developed in the Order Psittaciformes Involves Multiple Markers to Avoid Sex Misidentification: Debunked Myth of the Universal DNA Marker.

Sexing of birds is indispensable for scientific, breeding and conservation programs but is difficult in many species and is particularly problematic in the case of nestlings showing no sexual dimorphism. Most useful and efficient methods of sex determination are based on unique features of the Z and W sex chromosomes detected via PCR to distinguish males (ZZ) and females (ZW). During the last twenty-five years researchers searched for the universal marker capable of sexing a maximally wide spectrum of species in a single PCR assay. We screened the phylogenetically representative set of 135 Psittaciformes species including 59 species sexed for the first time. Two known (P2P8, CHD1iA) PCR markers and four additional W/Z polymorphisms (CHD1iE, CHD1i16, CHD1i9 and NIPBLi16) located within the Chromo Helicase DNA binding CHD1 or the Nipped-B homolog NIPBL genes were applied. We present the electrophoretic patterns obtained for the PCR products of the analyzed markers including most typical and atypical patterns allowing sex determination, as well as those obtained when the given marker failed in sexing. Technical aspects of molecular sex determination are discussed: the optimization of amplification conditions, direct PCR and potential misinterpretations. A truly universal marker has not been found, and therefore, we propose a sexing strategy based on multiple CHD1i16, NIPBLi16, CHD1i9 and CHD1iE markers. This new strategy confirms the sex of a given bird with at least two markers detecting independent Z/W polymorphisms, reduces the number of necessary PCR reactions and minimizes the risk of sex misidentification.

Effect of Intense Exercise on the Level of Bacteroidetes and Firmicutes Phyla in the Digestive System of Thoroughbred Racehorses.

Exercise significantly affects the body of both animals and humans, including the composition of the digestive microbiome. This study aimed to determine the changes in the composition of the most numerous bacterial phyla (Firmicutes and Bacteroidetes, as well as the level of the Lactobacillaceae family) in the digestive system of horses under the influence of physical effort. The study included a group of 17 Thoroughbred racehorses at the age of 3 years, fed the same forage, from whom feces samples were collected individually before and 48 h after physical effort. The obtained samples were subjected to DNA isolation and RT-PCR analysis. The results showed a significant increase in the level of both phyla after exercise compared to the state before physical effort; there were no such differences in the level of facultative aerobes, i.e., the Lactobacillaceae family (although a decreasing tendency was found after exercise). In addition, the analysis of the level of the studied phyla indicates individual differences in horses' response to the effort.

Evaluation of Changes in the Levels of Firmicutes and Bacteroidetes Phyla of Sheep Feces Depending on the Breed.

Studies carried out so far have indicated the effect of the microbiome on the composition of ruminant products. Recent studies have shown that not only diet, but also genetic factors can affect the microbiological composition of the digestive system. The aim of the study was to determine the differences in the levels of selected bacterial phyla in terms of breed differences. Three sheep breeds, i.e., Olkuska, Romanov, and old-type Polish Merino, differing in their use (meat-wool, meat, prolificacy) and country of breed origin were included in the study. Sheep at the same age and of the same sex were kept for a period of 3 months in the same environmental conditions and fed the same feed in the same proportions. The study included real-time PCR (polymerase chain reaction) analysis of feces collected before the slaughter and measurements of body weight and chilled carcasses. The obtained results showed significant differences between the breeds in the levels of bacterial populations tested. There were also differences in body weight between the breeds during the first weight measurements, however, the final results did not show any differences-after three months of maintenance all of them reached similar body weights, despite differences in fecal microbiological composition. The study suggests that in addition to diet and environmental conditions, the microbiology can also be influenced by breed.

Plant Elongator-Protein Complex of Diverse Activities Regulates Growth, Development, and Immune Responses.

Contrary to the conserved Elongator composition in yeast, animals, and plants, molecular functions and catalytic activities of the complex remain controversial. Elongator was identified as a component of elongating RNA polymerase II holoenzyme in yeast, animals, and plants. Furthermore, it was suggested that Elonagtor facilitates elongation of transcription via histone acetyl transferase activity. Accordingly, phenotypes of Arabidopsis elo mutants, which show development, growth, or immune response defects, correlate with transcriptional downregulation and the decreased histone acetylation in the coding regions of crucial genes. Plant Elongator was also implicated in other processes: transcription and processing of miRNA, regulation of DNA replication by histone acetylation, and acetylation of alpha-tubulin. Moreover, tRNA modification, discovered first in yeast and confirmed in plants, was claimed as the main activity of Elongator, leading to specificity in translation that might also result indirectly in a deficiency in transcription. Heterologous overexpression of individual Arabidopsis Elongator subunits and their respective phenotypes suggest that single Elongator subunits might also have another function next to being a part of the complex. In this review, we shall present the experimental evidence of all molecular mechanisms and catalytic activities performed by Elongator in nucleus and cytoplasm of plant cells, which might explain how Elongator regulates growth, development, and immune responses.

Plant-RRBS: DNA Methylome Profiling Adjusted to Plant Genomes, Utilizing Efficient Endonuclease Combinations, for Multi-Sample Studies.

In plants, methylation at cytosines often leads to changes in gene expression and inactivation of transposable elements. Changes in cytosine methylation (epimutations) might produce epialleles with distinct phenotypes. We present a genome-wide cytosine methylation profiling method based on bisulfite conversion and next-generation sequencing, which is applicable for plant species with available reference genomes. This so-called plant-RRBS profiling method reproducibly covers specific genomic regions and enriches for coverage of cytosine positions that are suitable for comparative analyses in multi-sample studies in basic biology and breeding studies. The plant-RRBS workflow consists of genomic DNA digestion with coverage-efficient restriction endonuclease combinations followed by a performant library generation and next-generation sequencing and a straightforward, publically available methylation data processing pipeline. Plant-RRBS has a twofold higher ratio of cytosine coverage per covered genome as compared to whole-genome bisulfite sequencing, covering tens of millions of cytosine positions, and allows detection of differential cytosine methylation, which was evaluated using rice epilines.

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.

Hypomethylated drm1 drm2 cmt3 mutant phenotype of Arabidopsis thaliana is related to auxin pathway impairment.

DNA methylation carried out by different methyltransferase classes is a relevant epigenetic modification of DNA which plays a relevant role in the development of eukaryotic organisms. Accordingly, in Arabidopsis thaliana loss of DNA methylation due to combined mutations in genes encoding for DNA methyltransferases causes several developmental abnormalities. The present study describes novel growth disorders in the drm1 drm2 cmt3 triple mutant of Arabidopsis thaliana, defective both in maintenance and de novo DNA methylation, and highlights the correlation between DNA methylation and the auxin hormone pathway. By using an auxin responsive reporter gene, we discovered that auxin accumulation and distribution were affected in the mutant compared to the wild type, from embryo to adult plant stage. In addition, we demonstrated that the defective methylation status also affected the expression of genes that regulate auxin hormone pathways from synthesis to transport and signalling and a direct relationship between differentially expressed auxin-related genes and altered auxin accumulation and distribution in embryo, leaf and root was observed. Finally, we provided evidence of the direct and organ-specific modulation of auxin-related genes through the DNA methylation process.

Elongator promotes germination and early post-germination growth.

The Elongator complex interacts with RNA polymerase II and via histone acetylation and DNA demethylation facilitates epigenetically the transcription of genes involved in diverse processes in plants, including growth, development, and immune response. Recently, we have shown that the Elongator complex promotes hypocotyl elongation and photomorphogenesis in Arabidopsis thaliana by regulating the photomorphogenesis and growth-related gene network that converges on genes implicated in cell wall biogenesis and hormone signaling. Here, we report that germination in the elo mutant was delayed by 6 h in the dark when compared to the wild type in a time lapse and germination assay. A number of germination-correlated genes were down-regulated in the elo transcriptome, suggesting a transcriptional regulation by Elongator. We also show that the hypocotyl elongation defect observed in the elo mutants in darkness originates very early in the post-germination development and is independent from the germination delay.

The Elongator complex regulates hypocotyl growth in darkness and during photomorphogenesis.

The Elongator complex (hereafter Elongator) promotes RNA polymerase II-mediated transcript elongation through epigenetic activities such as histone acetylation. Elongator regulates growth, development, immune response and sensitivity to drought and abscisic acid. We demonstrate that elo mutants exhibit defective hypocotyl elongation but have a normal apical hook in darkness and are hyposensitive to light during photomorphogenesis. These elo phenotypes are supported by transcriptome changes, including downregulation of circadian clock components, positive regulators of skoto- or photomorphogenesis, hormonal pathways and cell wall biogenesis-related factors. The downregulated genes LHY, HFR1 and HYH are selectively targeted by Elongator for histone H3K14 acetylation in darkness. The role of Elongator in early seedling development in darkness and light is supported by hypocotyl phenotypes of mutants defective in components of the gene network regulated by Elongator, and by double mutants between elo and mutants in light or darkness signaling components. A model is proposed in which Elongator represses the plant immune response and promotes hypocotyl elongation and photomorphogenesis via transcriptional control of positive photomorphogenesis regulators and a growth-regulatory network that converges on genes involved in cell wall biogenesis and hormone signaling.This article has an associated First Person interview with the first author of the paper.

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions.

BACKGROUND: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. METHODS: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. RESULTS: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. CONCLUSIONS: Plant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

Plant Elongator-mediated transcriptional control in a chromatin and epigenetic context.

Elongator (Elp) genes were identified in plants by the leaf growth-altering elo mutations in the yeast (Saccharomyces cerevisiae) gene homologs. Protein purification of the Elongator complex from Arabidopsis thaliana cell cultures confirmed its conserved structure and composition. The Elongator function in plant growth, development, and immune response is well-documented in the elp/elo mutants and correlated with the histone acetyl transferase activity of the ELP3/ELO3 subunit at the coding part of key regulatory genes of developmental and immune response pathways. Here we will focus on additional roles in transcription, such as the cytosine demethylation activity of ELP3/ELO3 at gene promoter regions and primary microRNA transcription and processing through the ELP2 subunit interaction with components of the small interference RNA machinery. Furthermore, specific interactions and upstream regulators support a role for Elongator in transcription and might reveal mechanistic insights into the specificity of the histone acetyl transferase and cytosine demethylation activities for target genes.

A dehydrin gene isolated from feral olive enhances drought tolerance in Arabidopsis transgenic plants.

Dehydrins belong to a protein family whose expression may be induced or enhanced by developmental process and environmental stresses that lead to cell dehydration. A dehydrin gene named OesDHN was isolated and characterized from oleaster (Olea europaea L. subsp. europaea, var. sylvestris), the wild form of olive. To elucidate the contribution of OesDHN in the development of drought tolerance, its expression levels were investigated in oleaster plants during development and under drought stress condition. The involvement of OesDHN in plant stress response was also evaluated in Arabidopsis transgenic lines, engineered to overexpress this gene, and exposed to a controlled mild osmotic stress. OesDHN expression was found to be modulated during development and induced under mild drought stress in oleaster plants. In addition, the Arabidopsis transgenic plants showed a better tolerance to osmotic stress than wild-type plants. The results demonstrated that OesDHN expression is induced by drought stress and is able to confer osmotic stress tolerance. We suggest a role for OesDHN, as a putative functional marker of plant stress tolerance.

Elongator and SPT4/SPT5 complexes as proxy to study RNA polymerase II transcript elongation control of plant development.

The elongation phase of the RNA polymerase II (RNAPII) transcription process is dynamic and regulated. Elongator and SUPPRESSOR OF Ty4 (SPT4)/SPT5 are transcript elongation factors that contribute to the regulation of mRNA synthesis by RNA polymerase II in the chromatin context. Recently, the Elongator complex consisting of six subunits and the SPT4/SPT5 heterodimer were isolated from Arabidopsis. Mutant plants affected in the expression of Elongator or SPT4/SPT5 share various auxin-signaling phenotypes. In line with that observation, auxin-related genes are prominent among the genes differentially expressed in these mutants. Exemplified by Elongator and SPT4/SPT5, we discuss here the role that transcript elongation factors may play in the control of plant growth and development.

Changes in accumulation of heteroplasmic mitochondrial DNA and frequency of recombination via short repeats during plant lifetime in Phaseolus vulgaris.

Recombination via short repeats in plant mitochondrial genomes results in sublimons--DNA molecules with a copy number much lower compared to the main mitochondrial genome. Coexistence of stoichiometrically different mitotypes, called heteroplasmy, plays an important evolutionary role, since sublimons occasionally replace the main genome resulting in a new plant phenotype. It is not clear, how frequency of recombination and sublimon production is regulated and how it is related to changes in the quantity of the main genome and sublimons. We analyzed the accumulation of two recombining main genome sequences and two resulting sublimons in apical meristems, undifferentiated tissues and leaves of different age of Phaseolus vulgaris. Copy numbers of the main genome sequences varied greatly depending on tissue type and organ age while accumulation of sublimons remained much more stable. Although the overall accumulation of plant mtDNA decreased with the leaf age, the quantity of sublimons increased relative to the main genome indicating a higher frequency of recombination via the short 314 bp repeat. Recombination was symmetrical in young developing leaves while in senescent tissues it shifted towards asymmetric events resulting in overrepresentation of one product. We propose that during plant lifetime replication and recombination frequencies change oppositely sustaining heteroplasmic compositions of the genome, which are favorable for inheritance and maintenance of complex plant mtDNA.

Histone H2B monoubiquitination is required to reach maximal transcript levels of circadian clock genes in Arabidopsis.

Previously, we identified HISTONE MONOUBIQUITINATION1 (HUB1) as an unconventional ubiquitin E3 ligase that is not involved in protein degradation but in the histone H2B modification that is implicated in transcriptional activation in plants. HUB1-mediated regulation of gene expression played a role in periodic and inducible processes such as the cell cycle, dormancy, flowering time and defense responses. Here, we determined the effects of the hub1-1 mutation on expression of a set of diurnally induced circadian clock genes identified from a comparative microarray analysis between the hub1-1 mutant and an HUB1 over-expression line. The hub1-1 mutation reduced the amplitudes of a number of induced clock gene expression peaks, as well as the HUB1-mediated histone H2BUb and H3K4Me3 marks associated with the coding regions, suggesting a role for HUB1 in facilitating transcriptional elongation in plants. Furthermore, double mutants between hub1-1 and elongata (elo) showed an embryo-lethal phenotype, indicating a synergistic genetic interaction. The double mutant embryos arrested at the torpedo stage, implying that together histone ubiquitination and acetylation marks are essential to activate expression of target genes in multiple pathways.

Heteroplasmy and stoichiometric complexity of plant mitochondrial genomes--though this be madness, yet there's method in't.

Mitochondrial heteroplasmy is defined as the coexistence of divergent mitochondrial genotypes in a cell. The ratio of the alternative genomes may be variable, but in plants, the usually prevalent main genome is accompanied by sublimons--substoichiometric mitochondrial DNA (mtDNA) molecules. Plant mitochondrial heteroplasmy was originally viewed as being associated with pathological mutations or was found in non-natural plant populations. Currently, it is considered to be a common situation in plants. Recent years have changed the previous view on the role of homologous recombination, small-scale mutations, and paternal leakage of mtDNA in the generation of heteroplasmy. Newly developed sensitive techniques have allowed the precise estimation of mtDNA stoichiometry. Mechanisms of maintenance and transmission of heteroplasmic genomes, including DNA recombination and replication, as well as mitochondrial fusion and fission, have been studied. This review describes the high level of plant mitochondrial genome complication--the 'madness' resulting from the heteroplasmic state and explains the method hidden in this madness. Heteroplasmy is described as the evolutionary strategy of uniparentally inherited plant mitochondrial genomes which do not undergo sexual recombination. In order to compensate for this deficiency, alternative types of mtDNA are substoichiometrically accumulated as a reservoir of genetic variability and may undergo accelerated evolution. Occasionally, sublimons are selected and amplified in the process called substoichiometric shifting, to take over the role of the main genome. Alternative mitochondrial genomes may recombine, yielding new mtDNA variants, or segregate during plant growth resulting in plants with mosaic phenotypes. Two opposite roles of mitochondrial heteroplasmy with respect to acceleration or counteracting of mutation accumulation are also discussed. Finally, nuclear control of heteroplasmy and substoichiometric shifting is described.

Counting mtDNA molecules in Phaseolus vulgaris: sublimons are constantly produced by recombination via short repeats and undergo rigorous selection during substoichiometric shifting.

Sublimons are substoichiometric DNA molecules which are generated by recombinations across short repeats, located in main mitochondrial genome of plants. Since short repeats are believed to recombine irreversibly and to be usually inactive, it is unknown how substoichiometric sequences are maintained. Occasionally, sublimons are amplified during substoichiometric shifting (SSS) and take the role of the main genome. Using the Phaseolus vulgaris system, we have addressed the questions concerning accumulation of sublimons, the role of recombination in their maintenance and selective amplification during SSS. We found that sublimons accompanied by parental recombination sequences were maintained by constant recombination across a short 314-bp repeat. The abundance of these sublimons was three orders of magnitude higher than accumulation of those which could not be maintained by recombination because their parental forms were absent from the main genome. As expected for active recombination, two recombination-derived sublimons were equimolar and so were their parental forms. One parental and one substoichiometric form shared the A/C polymorphism indicating their frequent inter-conversion. Only the C variant of the sublimon was amplified during substoichiometric shift implying strong selection of DNA molecules operating during SSS.

Developmentally early and late onset of Rps10 silencing in Arabidopsis thaliana: genetic and environmental regulation.

Transgene dosage, silencing competence of the transgene loci, and photoperiod conditions were found to regulate the onset and efficiency of Rps10 silencing in two independent transgenic lines of Arabidopsis thaliana. The Rps10 gene encodes the S10 protein which is part of the small subunit of mitochondrial ribosomes. Homozygous plants presented developmentally early onset of silencing, a very efficient decrease in the level of Rps10 transcripts, as well as a severe and uniform phenotype called P1. P1 plants either died during the vegetative growth phase or were rescued by reversion resulting from inactivation of silencing. A wide variety of morphological and developmental abnormalities observed within the hemizygous transformants allowed their classification into three categories P2, P3, and P4. The most severe and early was the P2 phenotype found in only one transgenic line and most probably resulting from high competence of the transgene loci. Developmentally late onset of silencing occurred only in the short day photoperiod and was characteristic for the P3 and P4 plants. This phenomenon was attributed to conditions favourable to silencing achieved in the short day photoperiod, e.g. a greatly prolonged vegetative phase accompanied by a gradual increase of the level of Rps10 transcripts. To the best of our knowledge, this is the first report indicating that the onset of silencing depends on the photoperiod conditions in A. thaliana.