Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing

It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele

The effect of polyploidy and hybridization on the evolution of floral colour in Nicotiana (Solanaceae)

Background and Aims: We investigate whether changes in floral colour accompany polyploid and homoploid hybridisation, important processes in angiosperm evolution. Potentially, changes in floral colour can facilitate speciation through pollinator shifts. Methods: We examined spectral reflectance of corolla tissue from 60 Nicotiana (Solanaceae) accessions (41 taxa) based on spectral shape (corresponding to pigmentation) as well as bee and hummingbird colour perception to assess patterns of floral colour evolution. We compared polyploid and homoploid hybrid spectra to those of their progenitors to evaluate whether hybridisation has resulted in floral colour shifts. Key Results: Floral colour categories in Nicotiana seem to have arisen multiple times independently during the evolution of the genus. Polyploid and homoploid hybrids can display a floral colour: 1) intermediate between progenitors, 2) like one or other progenitor, or 3) a transgressive or divergent colour not present in either progenitor. Conclusions: Floral colour evolution in Nicotiana is weakly constrained by phylogeny, but colour shifts occur and are sometimes associated with allopolyploid or homoploid speciation. Transgressive floral colour in N. tabacum has arisen by inheritance of anthocyanin pigmentation from its paternal progenitor while having a plastid phenotype like its maternal progenitor. Potentially, floral colour evolution has been driven by, or resulted in, pollinator shifts

Consequences of Lamin B1 and Lamin B Receptor Downregulation in Senescence

Anchoring of heterochromatin to the nuclear envelope appears to be an important process ensuring the spatial organization of the chromatin structure and genome function in eukaryotic nuclei. Proteins of the inner nuclear membrane (INM) mediating these interactions are able to recognize lamina-associated heterochromatin domains (termed LAD) and simultaneously bind either lamin A/C or lamin B1. One of these proteins is the lamin B receptor (LBR) that binds lamin B1 and tethers heterochromatin to the INM in embryonic and undifferentiated cells. It is replaced by lamin A/C with specific lamin A/C binding proteins at the beginning of cell differentiation and in differentiated cells. Our functional experiments in cancer cell lines show that heterochromatin in cancer cells is tethered to the INM by LBR, which is downregulated together with lamin B1 at the onset of cell transition to senescence. The downregulation of these proteins in senescent cells leads to the detachment of centromeric repetitive sequences from INM, their relocation to the nucleoplasm, and distension. In cells, the expression of LBR and LB1 is highly coordinated as evidenced by the reduction of both proteins in LBR shRNA lines. The loss of the constitutive heterochromatin structure containing LADs results in changes in chromatin architecture and genome function and can be the reason for the permanent loss of cell proliferation in senescence

Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture

In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided

Expression of 5 S rRNA genes linked to 35 S rDNA in plants, their epigenetic modification and regulatory element divergence

<p>Abstract</p> <p>Background</p> <p>In plants, the 5 S rRNA genes usually occur as separate tandems (S-type arrangement) or, less commonly, linked to 35 S rDNA units (L-type). The activity of linked genes remains unknown so far. We studied the homogeneity and expression of 5 S genes in several species from family Asteraceae known to contain linked 35 S-5 S units. Additionally, their methylation status was determined using bisulfite sequencing. Fluorescence in situ hybridization was applied to reveal the sub-nuclear positions of rDNA arrays.</p> <p>Results</p> <p>We found that homogenization of L-type units went to completion in most (4/6) but not all species. Two species contained major L-type and minor S-type units (termed L_s-type). The linked genes dominate 5 S rDNA expression while the separate tandems do not seem to be expressed. Members of tribe Anthemideae evolved functional variants of the polymerase III promoter in which a residing C-box element differs from the canonical angiosperm motif by as much as 30%. On this basis, a more relaxed consensus sequence of a plant C-box: (5’-RGSWTGGGTG-3’) is proposed. The 5 S paralogs display heavy DNA methylation similarly as to their unlinked counterparts. FISH revealed the close association of 35 S-5 S arrays with nucleolar periphery indicating that transcription of 5 S genes may occur in this territory.</p> <p>Conclusions</p> <p>We show that the unusual linked arrangement of 5 S genes, occurring in several plant species, is fully compatible with their expression and functionality. This extraordinary 5 S gene dynamics is manifested at different levels, such as variation in intrachromosomal positions, unit structure, epigenetic modification and considerable divergence of regulatory motifs.</p

Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes

Editorial: Molecular organization, evolution, and function of ribosomal DNA

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