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Discoveries over the recent decade have demonstrated the unexpected diversity of telomere DNA motifs in nature. However, currently available resources, 'Telomerase database' and 'Plant rDNA database', contain just fragments of all relevant literature published over decades of telomere research as they have a different primary focus and limited updates. To fill this gap, we gathered data about telomere DNA sequences from a thorough literature screen as well as by analysing publicly available NGS data, and we created TeloBase (http://cfb.ceitec.muni.cz/telobase/) as a comprehensive database of information about telomere motif diversity. TeloBase is supplemented by internal taxonomy utilizing popular on-line taxonomic resources that enables in-house data filtration and graphical visualisation of telomere DNA evolutionary dynamics in the form of heat tree plots. TeloBase avoids overreliance on administrators for future data updates by having a simple form and community-curation system for application and approval, respectively, of new telomere sequences by users, which should ensure timeliness of the database and topicality. To demonstrate TeloBase utility, we examined telomere motif diversity in species from the fungal genus Aspergillus, and discovered (TTTATTAGGG)n sequence as a putative telomere motif in the plant family Chrysobalanaceae. This was bioinformatically confirmed by analysing template regions of identified telomerase RNAs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To elucidate the molecular nature of evolutionary changes of telomeres in the plant order Asparagales, we aimed to characterize telomerase RNA subunits (TRs) in these plants. The unusually long telomere repeat unit in Allium plants (12 nt) allowed us to identify TRs in transcriptomic data of representative species of the Allium genus. Orthologous TRs were then identified in Asparagales plants harbouring telomere DNA composed of TTAGGG (human type) or TTTAGGG (Arabidopsis-type) repeats. Further, we identified TRs across the land plant phylogeny, including common model plants, crop plants, and plants with unusual telomeres. Several lines of functional testing demonstrate the templating telomerase function of the identified TRs and disprove a functionality of the only previously reported plant telomerase RNA in Arabidopsis thaliana. Importantly, our results change the existing paradigm in plant telomere biology which has been based on the existence of a relatively conserved telomerase reverse transcriptase subunit (TERT) associating with highly divergent TRs even between closely related plant taxa. The finding of a monophyletic origin of genuine TRs across land plants opens the possibility to identify TRs directly in transcriptomic or genomic data and/or predict telomere sequences synthesized according to the respective TR template region., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are important for the maintenance of genomic stability. Telomeres were considered as typical heterochromatic regions, but in light of recent results, this view should be reconsidered. Asymmetrically located cytosines in plant telomeric DNA repeats may be substrates for a DNA methyltransferase enzyme and indeed, it was shown that these repeats are methylated. Here, we analyse the methylation of telomeric cytosines and the length of telomeres in Arabidopsis thaliana methylation mutants (met 1-3 and ddm 1-8), and in their wild-type siblings that were germinated in the presence of hypomethylation drugs. Our results show that cytosine methylation in telomeric repeats depends on the activity of MET1 and DDM1 enzymes. Significantly shortened telomeres occur in later generations of methylation mutants as well as in plants germinated in the presence of hypomethylation drugs, and this phenotype is stably transmitted to the next plant generation. A possible role of compromised in vivo telomerase action in the observed telomere shortening is hypothesized based on telomere analysis of hypomethylated telomerase knockout plants. Results are discussed in connection with previous data in this field obtained using different model systems.

We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.