Amplification of monomer sequences into long contiguous arrays is the main feature distinguishing satellite DNA from other tandem repeats, yet it is also the main obstacle in its investigation because these arrays are in principle difficult to assemble. Here we explore an alternative, assembly-free approach that utilizes ultra-long Oxford Nanopore reads to infer the length distribution of satellite repeat arrays, their association with other repeats and the prevailing sequence periodicities. Using the satellite DNA-rich legume plant Lathyrus sativus as a model, we demonstrated this approach by analyzing 11 major satellite repeats using a set of nanopore reads ranging from 30 to over 200 kb in length and representing 0.73× genome coverage. We found surprising differences between the analyzed repeats because only two of them were predominantly organized in long arrays typical for satellite DNA. The remaining nine satellites were found to be derived from short tandem arrays located within LTR-retrotransposons that occasionally expanded in length. While the corresponding LTR-retrotransposons were dispersed across the genome, this array expansion occurred mainly in the primary constrictions of the L. sativus chromosomes, which suggests that these genome regions are favourable for satellite DNA accumulation.
- MeSH
- centromera MeSH
- chromozomy rostlin MeSH
- DNA rostlinná genetika MeSH
- frekvence genu * MeSH
- genom rostlinný MeSH
- heterochromatin MeSH
- Lathyrus genetika MeSH
- molekulární evoluce MeSH
- nanopóry * MeSH
- retroelementy * MeSH
- satelitní DNA * MeSH
- tandemové repetitivní sekvence * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co-expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N-terminal tail and the histone fold domain of non-nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.
Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.
- MeSH
- chromozomy rostlin genetika MeSH
- délka genomu genetika MeSH
- DNA rostlinná chemie genetika MeSH
- fazol genetika MeSH
- genom rostlinný genetika MeSH
- mapování chromozomů MeSH
- retroelementy genetika MeSH
- rostlinné geny genetika MeSH
- sekvenční analýza DNA metody MeSH
- syntenie MeSH
- vigna genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.
- MeSH
- biologický transport MeSH
- buněčné linie MeSH
- fylogeneze MeSH
- homeostáza MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák genetika fyziologie MeSH
- teoretické modely MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Repetitive sequences are ubiquitous components of all eukaryotic genomes. They contribute to genome evolution and the regulation of gene transcription. However, the uncontrolled activity of repetitive sequences can negatively affect genome functions and stability. Therefore, repetitive DNAs are embedded in a highly repressive heterochromatic environment in plant cell nuclei. Here, we analyzed the sequence, composition and the epigenetic makeup of peculiar non-pericentromeric heterochromatic segments in the genome of the Australian crucifer Ballantinia antipoda. By the combination of high throughput sequencing, graph-based clustering and cytogenetics, we found that the heterochromatic segments consist of a mixture of unique sequences and an A-T-rich 174 bp satellite repeat (BaSAT1). BaSAT1 occupies about 10% of the B. antipoda nuclear genome in >250 000 copies. Unlike many other highly repetitive sequences, BaSAT1 repeats are hypomethylated; this contrasts with the normal patterns of DNA methylation in the B. antipoda genome. Detailed analysis of several copies revealed that these non-methylated BaSAT1 repeats were also devoid of heterochromatic histone H3K9me2 methylation. However, the factors decisive for the methylation status of BaSAT1 repeats remain currently unknown. In summary, we show that even highly repetitive sequences can exist as hypomethylated in the plant nuclear genome.
- MeSH
- Arabidopsis genetika MeSH
- cévnaté rostliny chemie genetika metabolismus MeSH
- epigeneze genetická MeSH
- fylogeneze MeSH
- genom rostlinný MeSH
- heterochromatin genetika metabolismus MeSH
- histony chemie metabolismus MeSH
- metylace DNA genetika MeSH
- satelitní DNA chemie genetika metabolismus MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.
- MeSH
- Arabidopsis genetika růst a vývoj fyziologie MeSH
- cytokininy metabolismus MeSH
- genová ontologie MeSH
- listy rostlin genetika růst a vývoj fyziologie MeSH
- proliferace buněk MeSH
- proteom * MeSH
- regulátory růstu rostlin metabolismus MeSH
- signální transdukce * MeSH
- transkriptom * MeSH
- zvětšování buněk MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double-stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51-1-2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51-1-2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One-Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell-cycle checkpoint allows the cell-cycle progression manifested as a phenotype resistant to bleomycin.
- MeSH
- DNA-helikasy genetika metabolismus MeSH
- genetické lokusy MeSH
- mechy enzymologie genetika MeSH
- mutace MeSH
- nestabilita genomu * MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální 18S genetika MeSH
- RNA ribozomální 5S genetika MeSH
- RNA ribozomální genetika MeSH
- rostlinné proteiny genetika metabolismus MeSH
- telomery genetika MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telomerase maturation and recruitment to telomeres is regulated by several telomerase- and telomere-associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome-wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T-DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- DNA-helikasy metabolismus MeSH
- fylogeneze MeSH
- jaderné proteiny MeSH
- katalytická doména MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteiny vázající RNA metabolismus MeSH
- proteiny vázající telomery metabolismus MeSH
- telomerasa metabolismus MeSH
- telomery metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromosomal inversions occur in natural populations of many species, and may underlie reproductive isolation and local adaptation. Traditional methods of inversion discovery are labor-intensive and lack sensitivity. Here, we report the use of three-dimensional contact probabilities between genomic loci as assayed by chromosome-conformation capture sequencing (Hi-C) to detect multi-megabase polymorphic inversions in four barley genotypes. Inversions are validated by fluorescence in situ hybridization and Bionano optical mapping. We propose Hi-C as a generally applicable method for inversion discovery in natural populations.
The small genus Ricotia (nine species, Brassicaceae) is confined to the eastern Mediterranean. By comparative chromosome painting and a dated multi-gene chloroplast phylogeny, we reconstructed the origin and subsequent evolution of Ricotia. The ancestral Ricotia genome originated through hybridization between two older genomes with n = 7 and n = 8 chromosomes, respectively, on the Turkish mainland during the Early Miocene (c. 17.8 million years ago, Ma). Since then, the allotetraploid (n = 15) genome has been altered by two independent descending dysploidies (DD) to n = 14 in Ricotia aucheri and the Tenuifolia clade (2 spp.). By the Late Miocene (c. 10 Ma), the latter clade started to evolve in the most diverse Ricotia core clade (6 spp.), the process preceded by a DD event to n = 13. It is noteworthy that this dysploidy was mediated by a unique chromosomal rearrangement, merging together the same two chromosomes as were merged during the origin of a fusion chromosome within the paternal n = 7 genome c. 20 Ma. This shows that within a time period of c. 8 Myr genome evolution can repeat itself and that structurally very similar chromosomes may originate repeatedly from the same ancestral chromosomes by different pathways (end-to-end translocation versus nested chromosome insertion).
- MeSH
- Brassicaceae klasifikace genetika MeSH
- chromozomální aberace MeSH
- chromozomy rostlin * MeSH
- fylogeneze MeSH
- genom rostlinný MeSH
- hybridizace genetická * MeSH
- karyotypizace MeSH
- malování chromozomů MeSH
- molekulární evoluce * MeSH
- ploidie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Turecko MeSH
