Role of alternative telomere lengthening unmasked in telomerase knock-out mutant plants

Telomeres in many eukaryotes are maintained by telomerase in whose absence telomere shortening occurs. However, telomerase-deficient Arabidopsis thaliana mutants (Attert(-/-)) show extremely low rates of telomere shortening per plant generation (250-500 bp), which does not correspond to the expected outcome of replicative telomere shortening resulting from ca. 1,000 meristem cell divisions per seed-to-seed generation. To investigate the influence of the number of cell divisions per seed-to-seed generation, Attert(-/-) mutant plants were propagated from seeds coming either from the lower-most or the upper-most siliques (L- and U-plants) and the length of their telomeres were followed over several generations. The rate of telomere shortening was faster in U-plants, than in L-plants, as would be expected from their higher number of cell divisions per generation. However, this trend was observed only in telomeres whose initial length is relatively high and the differences decreased with progressive general telomere shortening over generations. But in generation 4, the L-plants frequently show a net telomere elongation, while the U-plants fail to do so. We propose that this is due to the activation of alternative telomere lengthening (ALT), a process which is activated in early embryonic development in both U- and L-plants, but is overridden in U-plants due to their higher number of cell divisions per generation. These data demonstrate what so far has only been speculated, that in the absence of telomerase, the number of cell divisions within one generation influences the control of telomere lengths. These results also reveal a fast and efficient activation of ALT mechanism(s) in response to the loss of telomerase activity and imply that ALT is probably involved also in normal plant development

Synergism of the Two Myb Domains of Tay1 Protein Results in High Affinity Binding to Telomeres

Double-stranded regions of the telomeres are recognized by proteins containing Myb-like domains conferring specificity toward telomeric repeats. Although biochemical and structural studies revealed basic molecular principles involved in DNA binding, relatively little is known about evolutionary pathways leading to various types of Myb domain-containing proteins in divergent species of eukaryotes. Recently we identified a novel type of telomere-binding protein YlTay1p from the yeast Yarrowia lipolytica containing two Myb domains (Myb1, Myb2) very similar to the Myb domain of mammalian TRF1 and TRF2. In this study we prepared mutant versions of YlTay1p lacking Myb1, Myb2, or both Myb domains and found that YlTay1p carrying either Myb domain exhibits preferential affinity to both Y. lipolytica (GGGTTAGTCA)n and human (TTAGGG)n telomeric sequences. Quantitative measurements of the protein binding to telomeric DNA revealed that the presence of both Myb domains is required for a high affinity of YlTay1p to either telomeric repeat. Additionally, we performed detailed thermodynamic analysis of the YlTay1p interaction with its cognate telomeric DNA, which is to our knowledge the first energetic description of a full-length telomeric-protein binding to DNA. Interestingly, when compared with human TRF1 and TRF2 proteins, YlTay1p exhibited higher affinity not only for Y. lipolytica telomeres but also for human telomeric sequences. The duplication of the Myb domain region in YlTay1p thus produces a synergistic effect on its affinity toward the cognate telomeric sequence, alleviating the need for homodimerization observed in TRF-like proteins possessing a single Myb domain

Telomere maintenance in liquid crystalline chromosomes of dinoflagellates

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

High-throughput non-destructive phenotyping of traits that contribute to salinity tolerance in Arabidopsis thaliana

Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence imaging, which captures the growth, morphology, color and photosynthetic performance of Arabidopsis thaliana plants in response to salt stress. We optimized our salt treatment by controlling the soil-water content prior to introducing salt stress. We investigated these traits over time in two accessions in soil at 150, 100 or 50 mM NaCl to find that the plants subjected to 100 mM NaCl showed the most prominent responses in the absence of symptoms of severe stress. In these plants, salt stress induced significant changes in rosette area and morphology, but less prominent changes in rosette coloring and photosystem II efficiency. Clustering of chlorophyll fluorescence traits with plant growth of nine accessions maintained at 100 mM NaCl revealed that in the early stage of salt stress, salinity tolerance correlated with non-photochemical quenching processes and during the later stage, plant performance correlated with quantum yield. This integrative approach allows the simultaneous analysis of several phenotypic traits. In combination with various genetic resources, the phenotyping protocol described here is expected to increase our understanding of plant performance and stress responses, ultimately identifying genes that improve plant performance in salt stress conditions

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability

Abstract The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.</jats:p