Transcriptional Silencing of 35S rDNA in Tragopogon porrifolius Correlates with Cytosine Methylation in Sequence-Specific Manner.

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.

Remarkable variation of ribosomal DNA organization and copy number in gnetophytes, a distinct lineage of gymnosperms.

INTRODUCTION: Gnetophytes, comprising the genera Ephedra, Gnetum and Welwitschia, are an understudied, enigmatic lineage of gymnosperms with a controversial phylogenetic relationship to other seed plants. Here we examined the organization of ribosomal DNA (rDNA) across representative species. METHODS: We applied high-throughput sequencing approaches to isolate and reconstruct rDNA units and to determine their intragenomic homogeneity. In addition, fluorescent in situ hybridization and Southern blot hybridization techniques were used to reveal the chromosome and genomic organization of rDNA. KEY RESULTS: The 5S and 35S rRNA genes were separate (S-type) in Gnetum montanum, Gnetum gnemon and Welwitschia mirabilis and linked (L-type) in Ephedra altissima. There was considerable variability in 5S rDNA abundance, ranging from as few as ~4000 (W. mirabilis) to >100 000 (G. montanum) copies. A similar large variation was also observed in 5S rDNA locus numbers (two to 16 sites per diploid cell). 5S rRNA pseudogenes were interspersed between functional genes forming a single unit in E. altissima and G. montanum. Their copy number was comparable or even higher than that of functional 5S rRNA genes. In E. altissima internal transcribed spacers of 35S rDNA were long and intrinsically repetitive while in G. montanum and W. mirabilis they were short without the subrepeats. CONCLUSIONS: Gnetophytes are distinct from other gymnosperms and angiosperms as they display surprisingly large variability in rDNA organization and rDNA copy and locus numbers between genera, with no relationship between copy numbers and genome sizes apparent. Concerted evolution of 5S rDNA units seems to have led to the amplification of 5S pseudogenes in both G. montanum and E. altissima. Evolutionary patterns of rDNA show both gymnosperm and angiosperm features underlining the diversity of the group.

Gene conversion events and variable degree of homogenization of rDNA loci in cultivars of Brassica napus.

BACKGROUND AND AIMS: Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. METHODS: We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. KEY RESULTS: Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH ('Darmor', 'Yudal' and 'Asparagus kale') harboured the same number (12 per diploid set) of loci. In B. napus 'Darmor', the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus 'Darmor'. In contrast, B. napus 'Yudal' showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus 'Asparagus kale' showed an intermediate pattern to 'Darmor' and 'Yudal'. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar ('Norin 9') showed co-dominance. CONCLUSIONS: The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion.

Identification of AHK2- and AHK3-like cytokinin receptors in Brassica napus reveals two subfamilies of AHK2 orthologues.

Cytokinin (CK) signalling is known to play key roles in the regulation of plant growth and development, crop yields, and tolerance to both abiotic stress and pathogen defences, but the mechanisms involved are poorly characterized in dicotyledonous crops. Here the identification and functional characterization of sensor histidine kinases homologous to Arabidopsis CK receptors AHK2 and AHK3 in winter oilseed rape are presented. Five CHASE-containing His kinases were identified in Brassica napus var. Tapidor (BnCHK1-BnCHK5) by heterologous hybridization of its genomic library with gene-specific probes from Arabidopsis. The identified bacterial artificial chromosome (BAC) clones were fingerprinted and representative clones in five distinct groups were sequenced. Using a bioinformatic approach and cDNA cloning, the precise gene and putative protein domain structures were determined. Based on phylogenetic analysis, four AHK2 (BnCHK1-BnCHK4) homologues and one AHK3 (BnCHK5) homologue were defined. It is further suggested that BnCHK1 and BnCHK3, and BnCHK5 are orthologues of AHK2 and AHK3, originally from the B. rapa A genome, respectively. BnCHK1, BnCHK3, and BnCHK5 displayed high affinity for trans-zeatin (1-3nM) in a live-cell competitive receptor assay, but not with other plant hormones (indole acetic acid, GA3, and abscisic acid), confirming the prediction that they are genuine CK receptors. It is shown that BnCHK1 and BnCHK3, and BnCHK5 display distinct preferences for various CK bases and metabolites, characteristic of their AHK counterparts, AHK2 and AHK3, respectively. Interestingly, the AHK2 homologues could be divided into two subfamilies (BnCHK1/BnCK2 and BnCHK3/BnCHK4) that differ in putative transmembrane domain topology and CK binding specificity, thus implying potential functional divergence.

The Utility of Graph Clustering of 5S Ribosomal DNA Homoeologs in Plant Allopolyploids, Homoploid Hybrids, and Cryptic Introgressants.

INTRODUCTION: Ribosomal DNA (rDNA) loci have been widely used for identification of allopolyploids and hybrids, although few of these studies employed high-throughput sequencing data. Here we use graph clustering implemented in the RepeatExplorer (RE) pipeline to analyze homoeologous 5S rDNA arrays at the genomic level searching for hybridogenic origin of species. Data were obtained from more than 80 plant species, including several well-defined allopolyploids and homoploid hybrids of different evolutionary ages and from widely dispersed taxonomic groups. RESULTS: (i) Diploids show simple circular-shaped graphs of their 5S rDNA clusters. In contrast, most allopolyploids and other interspecific hybrids exhibit more complex graphs composed of two or more interconnected loops representing intergenic spacers (IGS). (ii) There was a relationship between graph complexity and locus numbers. (iii) The sequences and lengths of the 5S rDNA units reconstituted in silico from k-mers were congruent with those experimentally determined. (iv) Three-genomic comparative cluster analysis of reads from allopolyploids and progenitor diploids allowed identification of homoeologous 5S rRNA gene families even in relatively ancient (c. 1 Myr) Gossypium and Brachypodium allopolyploids which already exhibit uniparental partial loss of rDNA repeats. (v) Finally, species harboring introgressed genomes exhibit exceptionally complex graph structures. CONCLUSION: We found that the cluster graph shapes and graph parameters (k-mer coverage scores and connected component index) well-reflect the organization and intragenomic homogeneity of 5S rDNA repeats. We propose that the analysis of 5S rDNA cluster graphs computed by the RE pipeline together with the cytogenetic analysis might be a reliable approach for the determination of the hybrid or allopolyploid plant species parentage and may also be useful for detecting historical introgression events.

Intragenomic heterogeneity of intergenic ribosomal DNA spacers in Cucurbita moschata is determined by DNA minisatellites with variable potential to form non-canonical DNA conformations.

The intergenic spacer (IGS) of rDNA is frequently built of long blocks of tandem repeats. To estimate the intragenomic variability of such knotty regions, we employed PacBio sequencing of the Cucurbita moschata genome, in which thousands of rDNA copies are distributed across a number of loci. The rRNA coding regions are highly conserved, indicating intensive interlocus homogenization and/or high selection pressure. However, the IGS exhibits high intragenomic structural diversity. Two repeated blocks, R1 (300-1250 bp) and R2 (290-643 bp), account for most of the IGS variation. They exhibit minisatellite-like features built of multiple periodically spaced short GC-rich sequence motifs with the potential to adopt non-canonical DNA conformations, G-quadruplex-folded and left-handed Z-DNA. The mutual arrangement of these motifs can be used to classify IGS variants into five structural families. Subtle polymorphisms exist within each family due to a variable number of repeats, suggesting the coexistence of an enormous number of IGS variants. The substantial length and structural heterogeneity of IGS minisatellites suggests that the tempo of their divergence exceeds the tempo of the homogenization of rDNA arrays. As frequently occurring among plants, we hypothesize that their instability may influence transcription regulation and/or destabilize rDNA units, possibly spreading them across the genome.

Effects of conditional IPT-dependent cytokinin overproduction on root architecture of Arabidopsis seedlings.

Cytokinin (CK) has been known to inhibit primary root elongation and suggested to act as an auxin antagonist in the regulation of lateral root (LR) formation. While the role of auxin in root development has been thoroughly studied, the detailed and overall description of CK effects on root system morphology, particularly that of developing lateral root primordia (LRPs), and hence its role in organogenesis is still in progress. Here we examine the effects of conditional endogenous CK overproduction on root architecture and consider its temporal aspect during the early development of Arabidopsis thaliana. We employed the pOp/LhGR system to induce ectopic ipt overexpression with a glucocorticoid dexamethasone at designated developmental points. The transient CaMV 35S>GR>ipt transactivation greatly enhanced levels of biologically active CKs of zeatin (Z)-type and identified a distinct developmental interval during which primary root elongation is susceptible to increases in endogenous CK production. Long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem, disturbed a characteristic graded distribution pattern of auxin response in LRPs and impaired their development. Our findings indicate the impact of perturbed endogenous CK on the regulation of asymmetric auxin distribution during LRP development and imply that there is cross-talk between auxin and CK during organogenesis in A. thaliana.

Cytokinin and auxin interactions in plant development: metabolism, signalling, transport and gene expression.

Auxin and cytokinins have been identified as key regulators of plant development. Recently, these phytohormones have been shown to interact during important developmental processes, including positioning, identity acquisition and maintenance of meristem organizing centres, regulation of balance between cell division and differentiation, and postembryonic de novo organogenesis. Here, we discuss recent advances in our understanding of the underlying molecular mechanisms at the levels of regulating metabolism, signalling, gene expression and protein stability.

Spatiotemporal aspect of cytokinin-auxin interaction in hormonal regulation of the root meristem.

Hormonal regulation of root development is a long known phenomenon. In the past decades, the molecular mechanisms of individual hormonal pathways and their impact on root development have been studied. Recent genetic and molecular studies suggest importance of interactions of the individual hormonal pathways and their components. In our paper we show impact of endogenous cytokinin on the root architecture and its interaction with auxin in Arabidopsis thaliana. In this addendum we discuss our results in the light of significant recent papers that deal with cytokinin-auxin interactions and we point out spatiotemporal specificity of these interactions in the root development.