The study investigates the impact of targeted chromosome engineering on telomere dynamics, chromatin structure, gene expression, and phenotypic stability in Arabidopsis thaliana. Using precise CRISPR/Cas-based engineering, reciprocal translocations of chromosome arms were introduced between non-homologous chromosomes. The subsequent homozygous generations of plants were assessed for phenotype, transcriptomic changes and chromatin modifications near translocation breakpoints, and telomere length maintenance. Phenotypically, translocated lines were indistinguishable from wild-type plants, as confirmed through morphological assessments and principal component analysis. Gene expression profiling detected minimal differential expression, with affected genes dispersed across the genome, indicating negligible transcriptional impact. Similarly, ChIPseq analysis showed no substantial alterations in the enrichment of key histone marks (H3K27me3, H3K4me1, H3K56ac) near junction sites or across the genome. Finally, bulk and arm-specific telomere lengths remained stable across multiple generations, except for minor variations in one translocation line. These findings highlight the remarkable genomic and phenotypic robustness of A. thaliana despite large-scale chromosomal rearrangements. The study offers insights into the cis-acting mechanisms underlying chromosome arm-specific telomere length setting and establishes the feasibility of chromosome engineering for studies of plant genome evolution and crop improvement strategies.

• Klíčová slova
• Arabidopsis thaliana, chromatin structure, chromosome engineering, chromosome translocation, gene expression, phenotype, telomeres,
• MeSH
• Arabidopsis * genetika   MeSH
• chromatin * genetika   metabolismus   MeSH
• chromozomy rostlin * genetika   MeSH
• CRISPR-Cas systémy MeSH
• fenotyp MeSH
• genetické inženýrství MeSH
• regulace genové exprese u rostlin MeSH
• telomery * genetika   MeSH
• translokace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin * MeSH

Simple telomeric repeats composed of six to seven iterating nucleotide units are important sequences typically found at the ends of chromosomes. Here we analyzed their abundance and homogeneity in 42 gymnosperm (29 newly sequenced), 29 angiosperm (one newly sequenced), and eight bryophytes using bioinformatics, conventional cytogenetic and molecular biology approaches to explore their diversity across land plants. We found more than 10 000-fold variation in the amounts of telomeric repeats among the investigated taxa. Repeat abundance was positively correlated with increasing intragenomic sequence heterogeneity and occurrence at non-telomeric positions, but there was no correlation with genome size. The highest abundance/heterogeneity was found in the gymnosperm genus Cycas (Cycadaceae), in which megabase-sized blocks of telomeric repeats (i.e., billions of copies) were identified. Fluorescent in situ hybridization experiments using variant-specific probes revealed canonical Arabidopsis-type telomeric TTTAGGG repeats at chromosome ends, while pericentromeric blocks comprised at least four major telomeric variants with decreasing abundance: TTTAGGG>TTCAGGG >TTTAAGG>TTCAAGG. Such a diversity of repeats was not found in the sister cycad family Zamiaceae or in any other species analyzed. Using immunocytochemistry, we showed that the pericentromeric blocks of telomeric repeats overlapped with histone H3 serine 10 phosphorylation signals. We show that species of Cycas have amplified their telomeric repeats in centromeric and telomeric positions on telocentric chromosomes to extraordinary high levels. The ancestral chromosome number reconstruction suggests their occurrence is unlikely to be the product of ancient Robertsonian chromosome fusions. We speculate as to how the observed chromosome dynamics may be associated with the diversification of cycads.

• Klíčová slova
• Cycadaceae, centromeres, chromosome rearrangements, epigenetics, genome evolution, gymnosperms, telomeres,
• MeSH
• centromera genetika   MeSH
• cykasy * genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• Magnoliopsida * genetika   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite the essential requirement of telomeric DNA for genome stability, the length of telomere tracts between species substantially differs, raising the question of the minimal length of telomeric DNA necessary for proper function. Here, we address this question using a hypomorphic allele of the telomerase catalytic subunit, TERT. We show that although this construct partially restored telomerase activity to a tert mutant, telomeres continued to shorten over several generations, ultimately stabilizing at a bimodal size distribution. Telomeres on two chromosome arms were maintained at a length of 1 kb, while the remaining telomeres were maintained at 400 bp. The longest telomeres identified in this background were also significantly longer in wild-type populations, suggesting cis-acting elements on these arms either promote telomerase processivity or recruitment. Genetically disrupting telomerase processivity in this background resulted in immediate lethality. Thus, telomeres of 400 bp are both necessary and sufficient for Arabidopsis viability. As this length is the estimated minimal length for t-loop formation, our data suggest that telomeres long enough to form a t-loop constitute the minimal functional length.

• Klíčová slova
• genome stability, subtelomere, t-loop, telomerase, telomeres,
• MeSH
• Arabidopsis MeSH
• homeostáza telomer * MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny huseníčku MeSH
• telomerasa MeSH
• TERT protein, Arabidopsis MeSH Prohlížeč

BACKGROUND: Telomeres, nucleoprotein structures comprising short tandem repeats and delimiting the ends of linear eukaryotic chromosomes, play an important role in the maintenance of genome stability. Therefore, the determination of the length of telomeres is of high importance for many studies. Over the last years, new methods for the analysis of the length of telomeres have been developed, including those based on PCR or analysis of NGS data. Despite that, terminal restriction fragment (TRF) method remains the gold standard to this day. However, this method lacks universally accepted and precise tool capable to analyse and statistically evaluate TRF results. RESULTS: To standardize the processing of TRF results, we have developed WALTER, an online toolset allowing rapid, reproducible, and user-friendly analysis including statistical evaluation of the data. Given its web-based nature, it provides an easily accessible way to analyse TRF data without any need to install additional software. CONCLUSIONS: WALTER represents a major upgrade from currently available tools for the image processing of TRF scans. This toolset enables a rapid, highly reproducible, and user-friendly evaluation of almost any TRF scan including in-house statistical evaluation of the data. WALTER platform together with user manual describing the evaluation of TRF scans in detail and presenting tips and troubleshooting, as well as test data to demo the software are available at https://www.ceitec.eu/chromatin-molecular-complexes-jiri-fajkus/rg51/tab?tabId=125#WALTER and the source code at https://github.com/mlyc93/WALTER .

• Klíčová slova
• Online toolset, Telomere length, Terminal restriction fragments,
• MeSH
• software * MeSH
• telomery * genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Knowledge of the fascinating world of DNA repeats is continuously being enriched by newly identified elements and their hypothetical or well-established biological relevance. Genomic approaches can be used for comparative studies of major repeats in any group of genomes, regardless of their size and complexity. Such studies are particularly fruitful in large genomes, and useful mainly in crop plants where they provide a rich source of molecular markers or information on indispensable genomic components (e.g., telomeres, centromeres, or ribosomal RNA genes). Surprisingly, in Allium species, a comprehensive comparative study of repeats is lacking. Here we provide such a study of two economically important species, Allium cepa (onion), and A. sativum (garlic), and their distantly related A. ursinum (wild garlic). We present an overview and classification of major repeats in these species and have paid specific attention to sequence conservation and copy numbers of major representatives in each type of repeat, including retrotransposons, rDNA, or newly identified satellite sequences. Prevailing repeats in all three studied species belonged to Ty3/gypsy elements, however they significantly diverged and we did not detect them in common clusters in comparative analysis. Actually, only a low number of clusters was shared by all three species. Such conserved repeats were for example 5S and 45S rDNA genes and surprisingly a specific and quite rare Ty1/copia lineage. Species-specific long satellites were found mainly in A. cepa and A. sativum. We also show in situ localization of selected repeats that could potentially be applicable as chromosomal markers, e.g., in interspecific breeding.

• Klíčová slova
• Allium, RepeatExplorer, TAREAN, plant genome, rDNA, repeats, retrotransposon, satellite, telomere,
• MeSH
• Allium klasifikace   genetika   MeSH
• chromozomy rostlin MeSH
• genom rostlinný * MeSH
• genomika * metody   MeSH
• hybridizace in situ fluorescenční MeSH
• nukleotidové motivy MeSH
• retroelementy MeSH
• satelitní DNA MeSH
• tandemové repetitivní sekvence MeSH
• telomery MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• retroelementy MeSH
• satelitní DNA MeSH

Parallel research on multiple model organisms shows that while some principles of telomere biology are conserved among all eukaryotic kingdoms, we also find some deviations that reflect different evolutionary paths and life strategies, which may have diversified after the establishment of telomerase as a primary mechanism for telomere maintenance. Much more than animals, plants have to cope with environmental stressors, including genotoxic factors, due to their sessile lifestyle. This is, in principle, made possible by an increased capacity and efficiency of the molecular systems ensuring maintenance of genome stability, as well as a higher tolerance to genome instability. Furthermore, plant ontogenesis differs from that of animals in which tissue differentiation and telomerase silencing occur during early embryonic development, and the "telomere clock" in somatic cells may act as a preventive measure against carcinogenesis. This does not happen in plants, where growth and ontogenesis occur through the serial division of apical meristems consisting of a small group of stem cells that generate a linear series of cells, which differentiate into an array of cell types that make a shoot and root. Flowers, as generative plant organs, initiate from the shoot apical meristem in mature plants which is incompatible with the human-like developmental telomere shortening. In this review, we discuss differences between human and plant telomere biology and the implications for aging, genome stability, and cell and organism survival. In particular, we provide a comprehensive comparative overview of telomere proteins acting in humans and in Arabidopsis thaliana model plant, and discuss distinct epigenetic features of telomeric chromatin in these species.

• Klíčová slova
• Arabidopsis, aging, chromatin, epigenetics, human, review, telomerase, telomere,
• MeSH
• chromatin metabolismus   MeSH
• epigeneze genetická MeSH
• lidé MeSH
• rostliny metabolismus   MeSH
• stárnutí buněk genetika   MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• chromatin MeSH
• telomerasa MeSH

Standard pathways involved in the regulation of telomere stability do not contribute to gradual telomere elongation observed in the course of A. thaliana calli propagation. Genetic and epigenetic changes accompanying the culturing of plant cells have frequently been reported. Here we aimed to characterize the telomere homeostasis during long term callus propagation. While in Arabidopsis thaliana calli gradual telomere elongation was observed, telomeres were stable in Nicotiana tabacum and N. sylvestris cultures. Telomere elongation during callus propagation is thus not a general feature of plant cells. The long telomere phenotype in Arabidopsis calli was correlated neither with changes in telomerase activity nor with activation of alternative mechanisms of telomere elongation. The dynamics of telomere length changes was maintained in mutant calli with loss of function of important epigenetic modifiers but compromised in the presence of epigenetically active drug zebularine. To examine whether the cell culture-induced disruption of telomere homeostasis is associated with the modulated structure of chromosome ends, epigenetic properties of telomere chromatin were analysed. Albeit distinct changes in epigenetic modifications of telomere histones were observed, these were broadly stochastic. Our results show that contrary to animal cells, the structure and function of plant telomeres is not determined significantly by the epigenetic character of telomere chromatin. Set of differentially transcribed genes was identified in calli, but considering the known telomere- or telomerase-related functions of respective proteins, none of these changes per se was apparently related to the elongated telomere phenotype. Based on our data, we propose that the disruption in telomere homeostasis in Arabidopsis calli arises from the interplay of multiple factors, as a part of reprogramming of plant cells to long-term culture conditions.

• Klíčová slova
• Arabidopsis thaliana, Callus, Chromosome stability, Epigenetics, Regenerated plants, Telomere,
• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• chromatin genetika   MeSH
• cytidin analogy a deriváty   farmakologie   MeSH
• druhová specificita MeSH
• ekotyp MeSH
• epigeneze genetická účinky léků   MeSH
• histony metabolismus   MeSH
• homeostáza telomer * účinky léků   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• regenerace účinky léků   MeSH
• rostlinné geny MeSH
• tabák genetika   MeSH
• techniky tkáňových kultur * MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• cytidin MeSH
• histony MeSH
• messenger RNA MeSH
• proteiny huseníčku MeSH
• pyrimidin-2-one beta-ribofuranoside MeSH Prohlížeč
• telomerasa MeSH

In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance. Nicotiana species have again proved to be appropriate and useful model plants in telomere biology studies.

• Klíčová slova
• Gene sequence variant, Pollen, Polyploids, Telomerase, Telomere, Transcription,
• MeSH
• buněčné jádro genetika   MeSH
• chromatinová imunoprecipitace MeSH
• euchromatin metabolismus   MeSH
• genetická transkripce MeSH
• genetická variace * MeSH
• histony metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• orgánová specificita genetika   MeSH
• polyribozomy metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• pylová láčka růst a vývoj   MeSH
• regulace genové exprese u rostlin * MeSH
• tabák genetika   MeSH
• telomerasa genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• euchromatin MeSH
• histony MeSH
• messenger RNA MeSH
• telomerasa MeSH

Telomeres, as physical ends of linear chromosomes, are targets of a number of specific proteins, including primarily telomerase reverse transcriptase. Access of proteins to the telomere may be affected by a number of diverse factors, e.g., protein interaction partners, local DNA or chromatin structures, subcellular localization/trafficking, or simply protein modification. Knowledge of composition of the functional nucleoprotein complex of plant telomeres is only fragmentary. Moreover, the plant telomeric repeat binding proteins that were characterized recently appear to also be involved in non-telomeric processes, e.g., ribosome biogenesis. This interesting finding was not totally unexpected since non-telomeric functions of yeast or animal telomeric proteins, as well as of telomerase subunits, have been reported for almost a decade. Here we summarize known facts about the architecture of plant telomeres and compare them with the well-described composition of telomeres in other organisms.

• Klíčová slova
• plant, shelterin, telomerase, telomere, telomeric proteins, telomeric repeat binding (TRB),
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

• MeSH
• Arabidopsis genetika   MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• dvouřetězcové zlomy DNA MeSH
• fenotyp MeSH
• fylogeneze MeSH
• homeostáza telomer genetika   MeSH
• homologní rekombinace MeSH
• mechy genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• oprava DNA * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• rostlinné proteiny MeSH
• telomerasa MeSH