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In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.

Telomeres are essential structures formed from satellite DNA repeats at the ends of chromosomes in most eukaryotes. Satellite DNA repeat sequences are useful markers for karyotyping, but have a more enigmatic role in the eukaryotic cell. Much work has been done to investigate the structure and arrangement of repetitive DNA elements in classical models with implications for species evolution. Still more is needed until there is a complete picture of the biological function of DNA satellite sequences, particularly when considering non-model organisms. Celebrating Gregor Mendel’s anniversary by going to the roots, this review is designed to inspire and aid new research into telomeres and satellites with a particular focus on non-model organisms and accessible experimental and in silico methods that do not require specialized equipment or expensive materials. We describe how to identify telomere (and satellite) repeats giving many examples of published (and some unpublished) data from these techniques to illustrate the principles behind the experiments. We also present advice on how to perform and analyse such experiments, including details of common pitfalls. Our examples are a selection of recent developments and underexplored areas of research from the past. As a nod to Mendel’s early work, we use many examples from plants and insects, especially as much recent work has expanded beyond the human and yeast models traditional in telomere research. We give a general introduction to the accepted knowledge of telomere and satellite systems and include references to specialized reviews for the interested reader.

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.

ARM was identified previously as an interaction partner of the telomerase protein subunit (TERT) in Arabidopsis thaliana. To investigate the interconnection between ARM and telomerase and to identify ARM cellular functions, we analyzed a set of arm mutant lines and arm/tert double mutants. Telomere length was not affected in arm single mutant plants, in contrast to double mutants. In the second generation of homozygous arm-1/tert double mutants following the heterozygous state during the double mutant construction, telomeres shortened dramatically, even below levels in tert plants displaying severe morphological defects. Intriguingly, homozygous arm-1/tert double mutants with short telomeres grew without obvious phenotypic changes for next two generations. Then, in agreement with the onset of phenotypic changes in tert, morphological defects were timed to the 5th arm-1/tert homozygous generation. RNAseq analyses of arm-1/tert and respective single mutants displayed markedly overlapping sets of differentially expressed genes in arm-1/tert double mutant and arm-1 single mutant lines, indicating a dominant effect of the ARM mutation. RNAseq data further implied ARM involvement in circadian rhythms, responses to drugs and to biotic and abiotic stimuli. In agreement with it, we observed sensitivity of arm-1 single mutant to the heat stress during germination. Altogether, our results suggest ARM involvement in crucial cellular processes without evidencing its role in the telomerase canonical function.

ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens., Cytokinin and auxin are two major hormonal regulators of plant growth. Here the authors identify SYAC1, a gene that is synergistically activated by the two hormones being applied together, and show that it is required for normal growth while negatively impacting pathogen resistance.

Histone chaperones mediate assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. InArabidopsis thaliana,loss-of-functions of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1(CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. Altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage clearly demonstrate the severity of CAF-1 dysfunction. How histone chaperone molecular networks change without a functional CAF-1 remains elusive. Here we present an intriguing observation that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) supressesFAS1loss-of function. The quadruple mutantfas1nap1;1-3shows wild-type growth and decreased sensitivity to genotoxic stress. Chromatin offas1nap1;1-3plants is less accessible to micrococcal nuclease and progressive loss of telomeres and 45S rDNA is supressed. Interestingly, the strong genetic interaction betweenFAS1andNAP1does not occur via direct protein-protein interaction. We propose that NAP1;1-3 play an essential role in nucleosome assembly infas1,thus their disruption abolishesfas1defects. Our data altogether reveal a novel function of NAP1 proteins, unmasked by CAF-1 dysfunction. It emphasizes the importance of a balanced composition of chromatin and shed light on the histone chaperone molecular network.

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.

Telomerase is essential for the maintenance of telomeres, structures located at the ends of linear eukaryotic chromosomes that are crucial for genomic stability. Telomerase has been frequently explored in mammals because of its activity in many types of cancers, but knowledge in plants is rather sketchy despite plants representing useful models due to peculiarities in their telomeres and telomerase biology. We studied in planta complementation of telomerase in Arabidopsis thaliana mutant plants with disrupted expression of the gene encoding the telomerase protein subunit (AtTERT) and significantly shortened telomeres. We found that the upstream region of AtTERT, previously identified as a putative minimal promoter, was essential for reconstitution of telomerase function, as demonstrated by the full or partial recovery of the telomere phenotype in mutants. In contrast, transformation by the full length AtTERT gene construct resulted in more progressive telomere shortening in mutants and even in wild type plants, despite the high level of AtTERT transcript and telomerase activity detected by in vitro assay. Thus, the telomerase protein subunit putative promoter is essential for in planta telomerase reconstitution and restoration of its catalytical activity. Contributions from other factors, including those tissue-specific, for proper telomerase function are discussed.

Phylogenetic divergence in Asparagales plants is associated with switches in telomere sequences. The last switch occurred with divergence of the genus Allium (Amaryllidaceae) from the other Allioideae (formerly Alliaceae) genera, resulting in uncharacterized telomeres maintained by an unknown mechanism. To characterize the unknown Allium telomeres, we applied a combination of bioinformatic processing of transcriptomic and genomic data with standard approaches in telomere biology such as BAL31 sensitivity tests, terminal restriction fragment analysis, the telomere repeat amplification protocol (TRAP), and fluorescence in situ hybridization (FISH). Using these methods, we characterize the unusual telomeric sequence (CTCGGTTATGGG)n present in Allium species, demonstrate its synthesis by telomerase, and characterize the telomerase reverse transcriptase (TERT) subunit of Allium cepa. Our findings open up the possibility of studying the molecular details of the evolutionary genetic change in Allium telomeres and its possible role in speciation. Experimental studies addressing the implications of this change in terms of the interplay of telomere components may now be designed to shed more light on telomere functions and evolution in general.

Telomeres are nucleoprotein structures that distinguish native chromosomal ends from double-stranded breaks. They are maintained by telomerase that adds short G-rich telomeric repeats at chromosomal ends in most eukaryotes and determines the TnAmGo sequence of canonical telomeres. We employed an experimental approach that was based on detection of repeats added by telomerase to identify the telomere sequence type forming the very ends of chromosomes. Our previous studies that focused on the algal order Chlamydomonadales revealed several changes in telomere motifs that were consistent with the phylogeny and supported the concept of the Arabidopsis-type sequence being the ancestral telomeric motif for green algae. In addition to previously described independent transitions to the Chlamydomonas-type sequence, we report that the ancestral telomeric motif was replaced by the human-type sequence in the majority of algal species grouped within a higher order clade, Caudivolvoxa. The Arabidopsis-type sequence was apparently retained in the Polytominia clade. Regarding the telomere sequence, the Chlorogonia clade within Caudivolvoxa bifurcates into two groups, one with the human-type sequence and the other group with the Arabidopsis-type sequence that is solely formed by the Chlorogonium species. This suggests that reversion to the Arabidopsis-type telomeric motif occurred in the common ancestral Chlorogonium species. The human-type sequence is also synthesized by telomerases of algal strains from Arenicolinia, Dunaliellinia and Stephanosphaerinia, except a distinct subclade within Stephanosphaerinia, where telomerase activity was not detected and a change to an unidentified telomeric motif might arise. We discuss plausible reasons why changes in telomeric motifs were tolerated during evolution of green algae.

Key messageArabidopsis and human ARM protein interact with telomerase. Deregulated mRNA levels of DNA repair and ribosomal protein genes in an Arabidopsis arm mutant suggest non-telomeric ARM function. The human homolog ARMC6 interacts with hTRF2.AbstractTelomerase maintains telomeres and has proposed non-telomeric functions. We previously identified interaction of the C- terminal domain of Arabidopsis telomerase reverse transcriptase (AtTERT) with an armadillo/β-catenin-like repeat (ARM) containing protein. Here we explore protein– protein interactions of the ARM protein, AtTERT domains, POT1a, TRF-like family and SMH family proteins, and the chromatin remodeling protein CHR19 using bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H) analysis, and co- immunoprecipitation. The ARM protein interacts with both the N- and C-terminal domains of AtTERT in different cellular compartments. ARM interacts with CHR19 and TRF-like I family proteins that also bind AtTERT directly or through interaction with POT1a. The putative human ARM homolog co-precipitates telomerase activity and interacts with hTRF2 protein in vitro. Analysis of Arabidopsis arm mutants shows no obvious changes in telomere length or telomerase activity, suggesting that ARM is not essential for telomere maintenance. The observed interactions with telomerase and Myb-like domain proteins (TRF-like family I) may therefore reflect possible non- telomeric functions. Transcript levels of several DNA repair and ribosomal genes are affected in arm mutants, and ARM, likely in association with other proteins, suppressed expression of XRCC3 and RPSAA promoter constructs in luciferase reporter assays. In conclusion, ARM can participate in non-telomeric functions of telomerase, and can also perform its own telomerase-independent functions.

International audience; An experimental design was set up to understand the influence of five process parameters on the kinetics of struvite precipitation in synthetic swine wastewaters. The responses studied were the kinetics of phosphorus (P) removal, the struvite precipitation rate and the dissolution rate of amorphous calcium phosphates (ACP). The kinetic study showed that the Premoval was complete in less than 1 h and was influenced positively by the added MgO. The precipitation of struvite with MgO was confirmed to follow a first-order kinetic. This study showed that ACP co-precipitated with struvite during the first 30 min. Afterwards, ACP dissolved to maintain the phosphates balance limiting the struvite growth. An initial Mg:Ca > 1.5 induced a complete dissolution of ACP in 1 h. Another experiment was conducted and it validated the results of the statistical model. This experiment also determined that 7–10 h was the best time to recover large crystals. After 10 h, the crystals were broken by stirring.

Telomeres, ubiquitous and essential structures of eukaryotic chromosomes, are known to come in a variety of forms, but knowledge about their actual diversity and evolution across the whole phylogenetic breadth of the eukaryotic life remains fragmentary. To fill this gap, we employed a complex experimental approach to probe telomeric minisatellites in various phylogenetically diverse groups of algae. Our most remarkable results include the following findings: 1) algae of the streptophyte class Klebsormidiophyceae possess the Chlamydomonas-type telomeric repeat (TTTTAGGG) or, in at least one species, a novel TTTTAGG repeat, indicating an evolutionary transition from the Arabidopsis-type repeat (TTTAGGG) ancestral for Chloroplastida; 2) the Arabidopsis-type repeat is also present in telomeres of Xanthophyceae, in contrast to the presence of the human-type repeat (TTAGGG) in other ochrophytes studied, and of the photosynthetic alveolate Chromera velia, consistent with its phylogenetic position close to apicomplexans and dinoflagellates; 3) glaucophytes and haptophytes exhibit the human-type repeat in their telomeres; and 4) ulvophytes and rhodophytes have unusual telomere structures recalcitrant to standard analysis. To obtain additional details on the distribution of different telomere types in eukaryotes, we performed in silico analyses of genomic data from major eukaryotic lineages, utilizing also genome assemblies from our on-going genome projects for representatives of three hitherto unsampled lineages (jakobids, malawimonads, and goniomonads). These analyses confirm the human-type repeat as the most common and possibly ancestral in eukaryotes, but alternative motifs replaced it along the phylogeny of diverse eukaryotic lineages, some of them several times independently.

Many articles have been published on coagulant dosing in membrane bioreactors, though few have been long-term studies examining the treatment of real wastewater. This study summarises the results of a membrane bioreactor pilot-plant (flat sheet membrane, nominal pore size 0.03 μm) that treated real municipal wastewater for two-years. Both influence of phosphorus precipitation by ferric sulphate on membrane permeability (flux decrease) and soluble microbial product concentration (especially on carbohydrates and proteins) were monitored. Flux decrease over work cycles lasting several months without phosphorus precipitation were compared to two periods with precipitation. X-ray elemental diffractometry of the filtration cake showed differences in the main contributors to inorganic fouling, with decreases in Ca and Si during operation with coagulant addition, and an increase in Fe.

Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.

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.

The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5 kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes.

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein- or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ~2 × 10(5) cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

Telomerase is essential for proper functioning of telomeres in eukaryotes. We cloned and characterised genes for the protein subunit of telomerase (TERT) in the allotetraploid Nicotiana tabacum (tobacco) and its diploid progenitor species Nicotiana sylvestris and Nicotiana tomentosiformis with the aim of determining if allopolyploidy (hybridisation and genome duplication) influences TERT activity and divergence. Two of the three sequence variants present in the tobacco genome (NtTERT-C/s and NtTERT-D) revealed similarity to two sequence variants found in N. sylvestris and another variant (NtTERT-C/t) was similar to TERT of N. tomentosiformis. Variants of N. sylvestris origin showed less similarity to each other (80.5 % in the genomic region; 90.1 % in the coding sequence) than that between the NtTERT-C/s and NtTERT-C/t variants (93.6 and 97.2 %, respectively). The NtTERT-D variant was truncated at the 5' end, and indels indicated that it was a pseudogene. All tobacco variants were transcribed and alternatively spliced sequences were detected. Analysis of gene arrangements uncovered a novel exon in the N-terminal domain of TERT variants, a feature that is likely to be commonly found in Solanaceae species. In addition, species-specific duplications were observed within exon 5. The putative function, copy number and evolutionary origin of these NtTERT sequence variants are discussed.

Telomerase, an enzyme responsible for the maintenance of linear chromosome ends, is precisely regulated during plant development. In animals, involvement of the epigenetic state of the telomerase reverse transcriptase (TERT) gene in the complex regulation of telomerase activity has been reported. To reveal whether epigenetic mechanisms participate in the regulation of plant telomerase, the relationship between telomerase activity in tissues of Arabidopsis thaliana and DNA methylation and histone modifications in the A. thaliana TERT (AtTERT) upstream region was studied. As expected, a gradual decrease of telomerase activity during leaf maturation was observed. A different pattern with a more progressive loss of telomerase activity and AtTERT transcription during leaf development was revealed in MET1 gene-knockout mutants. Analysis of DNA methylation in the AtTERT upstream region showed low levels of methylated cytosines without notable differences between telomerase-positive and telomerase-negative wild-type tissues. Surprisingly, a high level of CG methylation was found in the AtTERT coding region, although this type of methylation is a characteristic attribute of constitutively expressed genes. Analysis of chromatin modifications in the AtTERT upstream region and in exon 5 showed increased loading of the H3K27me3 mark in the telomerase-negative mature leaf compared to telomerase-positive seedlings, whereas H3K4me3, H3K9Ac, and H3K9me2 were approximately at the same level. Consistently, the chromatin structure of the AtTERT gene was maintained. These results are discussed in the context of the general involvement of epigenetic mechanisms in the regulation of gene expression and with respect to similar studies performed in animal models.

Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

The organisation of dinoflagellate chromosomes is exceptional among eukaryotes. Their genomes are the largest in the Eukarya domain, chromosomes lack histones and may exist in liquid crystalline state. Therefore, the study of the structural and functional properties of dinoflagellate chromosomes is of high interest. In this work, we have analysed the telomeres and telomerase in two Dinoflagellata species, Karenia papilionacea and Crypthecodinium cohnii. Active telomerase, synthesising exclusively Arabidopsis-type telomere sequences, was detected in cell extracts. The terminal position of TTTAGGG repeats was determined by in situ hybridisation and BAL31 digestion methods and provides evidence for the linear characteristic of dinoflagellate chromosomes. The length of telomeric tracts, 25-80 kb, is the largest among unicellular eukaryotic organisms to date. Both the presence of long arrays of perfect telomeric repeats at the ends of dinoflagellate chromosomes and the existence of active telomerase as the primary tool for their high-fidelity maintenance demonstrate the general importance of these structures throughout eukaryotes. We conclude that whilst chromosomes of dinoflagellates are unique in many aspects of their structure and composition, their telomere maintenance follows the most common scenario.

In the published online version, the affiliations were mixed up. Corrected affiliation section is shown below. Also, the update has also been reflected in the author group section above.

Single-stranded DNA-binding proteins form protective caps at the chromosome ends and their binding is important in the regulation of telomerase access to telomeres. This group of proteins is represented by POT1 proteins described in yeast, humans and other model organisms. Here we review recent findings obtained in Arabidopsis POT1-like paralogs, namely the observed diversity in their interaction features and corresponding functions.

While most Solanaceae genera (e.g.Solanum, Nicotiana) possess Arabidopsis-type telomeres of (TTTAGGG)n maintained by telomerase, the genera Cestrum, Vestia and Sessea (Cestrum group) lack these telomeres. Here we show that in the Cestrum-group the activity of telomerase has been lost. Nevertheless, proteins binding the single-stranded G-rich strand of the Arabidopsis-type and related human-type (TTAGGG)n telomeric sequences are present in nuclear extracts of both Nicotiana and Cestrum species. These proteins may have a role in telomere function or other cellular activities. In addition to characterizing DNA binding specificity and molecular weights of these proteins, we searched in both N. tabacum (tobacco) and C. parqui for the presence of POT1-like proteins, involved in telomere capping and telomerase regulation. Analysis of POT1-like proteins available on public databases and cloned by us from C. parqui, revealed the N-terminal OB folds typical for this protein family and a novel, plant-specific conserved C-terminal OB-fold domain (CTOB). We propose that CTOB is involved in protein-protein interactions.

A conserved feature of telomeres is the 3'-overhang of their G-rich strand. These G-overhangs function as substrates for telomerase-mediated strand extension, and are critical for end-protection of telomeres. These functions and their regulations are mediated by specific G-overhang binding proteins. In species of the plant order Asparagales, telomere motifs have diverged from a type typical of the plant Arabidopsis thaliana (TTTAGGG)(n) to a type typical of human (TTAGGG)(n). Presumably, this change in motif had an impact on the structure of the telomere and/or the binding of telomeric proteins, including the G-overhang binding proteins. Therefore, we analyse here nucleoprotein complexes formed by protein extracts from plants possessing human-type telomeres (Muscari armeniacum and Scilla peruviana). Proteins were characterized that bind to the G-rich strand of both telomere motifs, or to the ancestral Arabidopsis-type motif alone, but none bound to double-stranded or C-rich complementary strand telomere motifs. We demonstrate the size, sequence-specificity and thermostability of these DNA-binding proteins. We also analysed the formation of complexes from renatured protein fractions after SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide-gel-electrophoresis). We discuss the evolutionary consequences of protein binding flexibility, to act on both ancestral and present telomeric sequences. Of particular interest is that the ancestral repeat, which is thought not to form the telomere, binds the proteins most strongly. These data are discussed in line with other known plant telomere-binding proteins and with the complex nature of the telomere in Asparagales carrying a human-type motif.

The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next-generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)n telomeric motif in C. elegans represents a switch in motif from the 'typical' angiosperm telomere (TTTAGGG)n . That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.

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.

International audience; A sustainable way to recover phosphorus (P) in swine wastewater involves a preliminary step of P dissolution followed by the separation of particulate organic matter (OM). The next two steps are firstly the precipitation of struvite crystals done by adding a crystallization reagent (magnesia) and secondly the filtration of the crystals. To develop the process successfully at an industrial scale, the control of the mechanisms of precipitation is the key point in order to obtain high value-added products, that is, big struvite crystals easy to harvest and handle. Experiments with process parameters optimized previously in a synthetic swine wastewater were performed on real swine wastewater to assess the role of the OM on struvite crystallization. After 24'h, with a pH increase to 6.8 only, 90% of the initial P was precipitated and 60% was precipitated as struvite. 80% of the solid recovered was in the fraction'>'100'µm. The other forms recovered were brushite, amorphous calcium phosphate, NaCl, KCl and OM. The influence of OM on struvite precipitation in acidified swine wastewater was negative on the reaction kinetics but positive on the size of the struvite crystals. The presence of colloidal particles increased the size of the struvite crystals but slowed down the kinetics due to the viscosity induced by the repulsive force of the colloids. The maximum size of single struvite crystals (200'µm) was observed with the presence of particulate OM. © 2015 IRSTEA. Published by Taylor & Francis.

Although telomere sequences are considered to be highly conserved, there are switch-points in plant telomere evolution that are congruent with species' phylogenies. When Asparagales diverged, the Arabidopsis-type telomeric minisatellite repeat (TTTAGGG)(n) was first replaced by a human-type (TTAGGG)(n) repeat, and both were lost in Allium cepa (Alliaceae). We aimed to discover (1) when this loss occurred during divergence of Alliaceae and, (2) if (TTAGGG)(n) repeats were replaced by other known telomeric minisatellites. Slot-blot hybridization, fluorescent in situ hybridization, BAL31 digestion, asymmetric PCR, and cloning were used to identify and localize candidate telomeric sequences in species of Nothoscordum, Miersia, Ipheion, Tulbaghia, Gethyum, Gilliesia, Leucocoryne, Tristagma, and representatives of the three major Allium clades. Alliaceae genera other than Allium have human (TTAGGG)-type telomeric repeats that form telomeres. In Allium, only Tetrahymena-type (TTGGGG) repeats were ubiquitous in the genome, but they were not localized to telomeres. Likewise, the consensus telomeric repeats in Arabidopsis, human, Bombyx (TTAGG), Chlamydomonas (TTTTAGGG), and Oxytricha (TTTTGGGG) are absent in Allium telomeres. Alliaceae with human-type telomeres share telomere structures with related Asparagales species. We demonstrate that in the Allium ancestor human-type telomeric repeats were lost from telomeres and were not replaced by any investigated alternative minisatellite repeats. However, human and other types of minisatellite telomeric repeats are interspersed in some Allium genomes and their genomic signatures coincide with Allium clades.

The order of monocotyledonous plants Asparagales is attractive for studies of telomere evolution as it includes three phylogenetically distinct groups with telomeres composed of TTTAGGG (Arabidopsis-type), TTAGGG (human-type) and unknown alternative sequences, respectively. To analyze the molecular causes of these switches in telomere sequence (synthesis), genes coding for the catalytic telomerase subunit (TERT) of representative species in the first two groups have been cloned. Multiple alignments of the sequences, together with other TERT sequences in databases, suggested candidate amino acid substitutions grouped in the Asparagales TERT synthesizing the human-type repeat that could have contributed to the changed telomere sequence. Among these, mutations in the C motif are of special interest due to its functional importance in TERT. Furthermore, two different modes of initial elongation of the substrate primer were observed in Asparagales telomerases producing human-like repeats, which could be attributed to interactions between the telomerase RNA subunit (TR) and the substrate.

Subtelomeric regions reveal a relatively high degree of polymorphism due to the increased frequency of recombination events in these chromosome loci. In a search for molecular markers applicable to genotyping of potato varieties, we focused on two possible sources of polymorphism occurring in this region: (i) arrangement of blocks of subtelomeric chromatin; (ii) structure of telomere-subtelomere boundary. Analysis of the internal arrangement of subtelomeric sequences showed several types of cultivar-specific spectra of PCR products aris- ing from the variant orientation of sequence units of the ST3-subtelomeric sequence, or from different lengths of regions linking the individual sequence units. Further, the telomere-subtelomere boundary sequences were amplified using telomeric and ST3-specific primers and the obtained products were cloned. Sequence analysis of the clones resulted in characterisation of a novel telomere-associated sequence (FIN2). Primers derived from this sequence were then used alone or in combination with telomeric or ST3-specific primers to generate cultivar- specific spectra of PCR products. The described combinations of sequence-specific primers may be used for the fast, cheap and reproducible PCR-genotyping of selected potato varieties.

A group of monocotyledonous plants within the order Asparagales, forming a distinct clade in phylogenetic analyses, was reported previously to lack the ‘typical’ Arabidopsis –type telomere (TTTAGGG) n . This stimulated us to determine what has replaced these sequences. Using slot–blot and fluorescent in situ hybridization (FISH) to species within this clade, our results indicate the following. 1. The typical Arabidopsis –type telomeric sequence has been partly or fully replaced by the human–type telomeric sequence (TTAGGG) n . Species in Allium lack the human–type variant. 2. In most cases the human variant occurs along with a lower abundance of two or more variants of the minisatellite sequences (of seven types evaluated), usually these being the consensus telomeric sequence of Arabidopsis , Bombyx (TTAGG) n and Tetrahymena (TTGGGG n . FISH shows that the variants can occur mixed together at the telomere. 3. Telomerases generate products with a 6 base pair periodicity and when sequenced they reveal predominantly a reiterated human–type motif. These motifs probably form the ‘true telomere’ but the error rate of motif synthesis is higher compared with ‘typical’ plant telomerases. The data indicate that the Asparagales clade is unified by a mutation resulting in a switch from synthesis of Arabidopsis –like telomeres to a low–fidelity synthesis of human–like telomeres.

Změny v genech pro telomerazu měly za nasledek změnu nebo ztratu minisatelitnich telomerových sekvenci.

The manner of packing of the terminal DNA loci into nucleosomes and higher order structures may strongly influence their functional interactions. Besides the structural flexibility of telomeric DNA sequences, conserved features of their chromatin including short nucleosome phasing (157 bp) and nucleosome sliding have been described previously. To gain a complementary knowledge of subtelomeres, we have analysed the chromatin structure of two subtelomeric tandem repeats from the plant Silene latifolia: X43.1 and 15Ssp. X43.1 shows two distinct nucleosome periodicities – 157 and 188 bp. Preferred positions of its two nucleosomes have been mapped at both low and high resolution and the experimental results correspond to computer-predicted positions. 15Ssp is a newly-discovered sequence showing a telomere-associated position by PCR and a subtelomeric location by pulsed-field gel electrophoresis and fluorescence in situ hybridisation. Its 159 bp sequence unit shows a tandem arrangement and the presence of micrococcal nuclease-hypersensitive sites when either naked DNA or chromatin is digested. Use of a chemical nuclease results in a regular nucleosome ladder of 157 bp periodicity. Moreover, 15Ssp mononucleosomes show instability and absence of specific positioning, features typical for telomeric chromatin.

Complex of osmium tetroxide and bipyridine (Os,bipy), KMnO4, and diethyl pyrocarbonate (DEPC) were used to probe curved DNA at single nucleotide resolution. The DNA was constructed from repeated dAn:dTn-blocks with dATATA and dAGAGA interblock sequences. The DNA was probed in the linear and supercoiled form at various salt concentrations. While all purines were available for DEPC attack, the thymines within the blocks were resistant to chemical probing by KMnO4 and Os,bipy. Only the 3'-flanking dTs were available for modification. The thymines within dTC and dTA sequences showed modification indicating that these thymines display an unstacked structure allowing both probes to attack. Under destabilizing conditions, at low ionic strength and superhelical stress, considerable unstacking was observed. We found experimental indications that under these destabilizing conditions unpaired regions might appear, probably within the dATATA sequence.

Telomere minisatellites could be present in both terminal and internal chromosomal regions. We monitored the progress of BAL-31 nuclease digestion on Arabidopsis thaliana genomic DNA prepared by standard isolation techniques to verify its cleavage at terminal and internal genomic regions. A subtelomeric position of candidate sequences was validated using conventional polymerase chain reaction (PCR), combining the C-strand-specific telomeric primer with a subtelomeric reverse primer, and confirmed by quantitative PCR (qPCR) using sequence-specific primer pairs on DNA samples after BAL-31 digestion. qPCR amplification showed a gradual decrease in subtelomeric sequence signals, in contrast to interstitial telomeric sequences from pericentromere and control sequences.

International audience; A sustainable way to recover phosphorus (P) in swine wastewater involves a preliminary step of P dissolution followed by the separation of particulate organic matter. The next two steps are firstly the precipitation of struvite crystals done by adding a crystallization reagent (magnesia) and secondly the filtration of the crystals. A design of experiments with five process parameters was set up to optimize the size of the struvite crystals in a synthetic swine wastewater. More than 90% of P was recovered as large crystals of struvite in optimal conditions which were: low Mg:Ca ratio (2.25:1), the leading parameter, high N:P ratio (3:1), moderate stirring rate (between 45 and 90. rpm) and low temperature (below 20 °C).These results were obtained despite the presence of a large amount of calcium and using a cheap reactant (MgO). The composition of the precipitates was identified by Raman analysis and solid dissolution. Results showed that amorphous calcium phosphate (ACP) co-precipitated with struvite and that carbonates were incorporated with solid fractions.

Telomerase maintains the ends of eukaryotic chromosomes, and its activity is an important parameter correlating with the proliferative capacity of cells. We have investigated cell cycle-specific changes in telomerase activity using cultures of Desmodesmus quadricauda, a model alga naturally synchronized by light/dark entrainment. A quantitative telomerase assay revealed high activity in algal cultures, with slight changes during the light period. Significantly increased telomerase activity was observed at the end of the dark phase, when cell division was complete. In contrast to other models, a natural separation between nuclear and cellular division typical for the cell cycle in D. quadricauda made this observation possible.

Telomeres, which form the protective ends of eukaryotic chromosomes, are a ubiquitous and conserved structure of eukaryotic genomes but the basic structural unit of most telomeres, a repeated minisatellite motif with the general consensus sequence T(n)A(m)G(o), may vary between eukaryotic groups. Previous studies on several species of green algae revealed that this group exhibits at least two types of telomeric sequences, a presumably ancestral type shared with land plants (Arabidopsis type, TTTAGGG) and conserved in, for example, Ostreococcus and Chlorella species, and a novel type (Chlamydomonas type, TTTTAGGG) identified in Chlamydomonas reinhardtii. We have employed several methodical approaches to survey the diversity of telomeric sequences in a phylogenetically wide array of green algal species, focusing on the order Chlamydomonadales. Our results support the view that the Arabidopsis-type telomeric sequence is ancestral for green algae and has been conserved in most lineages, including Mamiellophyceae, Chlorodendrophyceae, Trebouxiophyceae, Sphaeropleales, and most Chlamydomonadales. However, within the Chlamydomonadales, at least two independent evolutionary changes to the Chlamydomonas type occurred, specifically in a subgroup of the Reinhardtinia clade (including C. reinhardtii and Volvox carteri) and in the Chloromonadinia clade. Furthermore, a complex structure of telomeric repeats, including a mix of the ancestral Arabidopsis-type motifs and derived motifs identical to the human-type telomeric repeats (TTAGGG), was found in the chlamydomonadalean clades Dunaliellinia and Stephanosphaeria. Our results indicate that telomere evolution in green algae, particularly in the order Chlamydomonadales, is far more dynamic and complex than thought before. General implications of our findings for the mode of telomere evolution are discussed.

Analysis of plants bearing a T-DNA insertion is a potent tool of modern molecular biology, providing valuable information about the function and involvement of genes in metabolic pathways. A collection of 12 Arabidopsis thaliana lines with T-DNA insertions in the gene coding for the catalytic subunit of telomerase (AtTERT) and in adjacent regions was screened for telomerase activity [telomere repeat amplification protocol (TRAP) assay], telomere length (terminal restriction fragments), and AtTERT transcription (quantitative reverse transcription-PCR). Lines with the insertion located upstream of the start codon displayed unchanged telomere stability and telomerase activity, defining a putative minimal AtTERT promoter and the presence of a regulatory element linked to increased transcription in the line SALK_048471. Lines bearing a T-DNA insertion inside the protein-coding region showed telomere shortening and lack of telomerase activity. Transcription in most of these lines was unchanged upstream of the T-DNA insertion, while it was notably decreased downstream. The expression profile varied markedly in mutant lines harbouring insertions at the 5' end of AtTERT which showed increased transcription and abolished tissue specificity. Moreover, the line FLAG_385G01 (T-DNA insertion inside intron 1) revealed the presence of a highly abundant downstream transcript with normal splicing but without active telomerase. The role of regulatory elements found along the AtTERT gene is discussed in respect to natural telomerase expression and putative intron-mediated enhancement.