The ends of linear eukaryotic chromosomes are protected by specific chromatin structures called telomeres that are composed of tandemly repeated telomeric DNA and proteins. In vertebrates, six specific proteins associate with telomeres having affinity to either single-stranded or double-stranded telomeric DNA and they are collectively called shelterin . These complex structures are essential for chromosome stability, as they differentiate chromosome ends from DNA double-strand breaks (DSBs) . They protect chromosome termini from nucleolytic attack and undesirable recombination. Telomeres also counterbalance incomplete replication of terminal DNA by conventional DNA polymerase ; cells have evolved specific telomerase reverse transcriptase (TERT), which can synthesise telomeric repeats using its own RNA template thus ensuring proper telomere length. In general, eukaryotic telomeres are composed of tandem G/C rich repeats that end in a single strand 3' overhang which can fold back and invade the duplex repeats to form the so-called T-loop . In the absence of telomerase, telomeres become non-functional, shorten with successive cell divisions, and chromosome termini can fuse as a consequence of de-protection. Their fusion is a result of the non-homologous-end-joining (NHEJ) which is the prevailing mechanism of DSB healing in plants. Although numerous attempts have been made to assess shelterin counterparts in plants, to date these proteins have not been identified (see  for review). Together with proteins invariantly occurring at the telomeres and providing a telomere "capping" function, many additional protein complexes are regularly found in eukaryotic telomeres. Paradoxically, many of these proteins are involved in DNA repair or recombination and this seems inappropriate at the telomere ends which should be hidden from recombination events and end-to-end fusions.
Over the past decade, an increasing body of data has accumulated on the concerted network of DNA repair factors and various protein kinases following the disruption of DNA integrity, including noxious extrinsic factors, DNA replication errors, checkpoint signalling and, meiotic and somatic recombination. One system that plays an essential role in DNA repair, recombination, DNA replication as well as in the cell cycle checkpoint activation and telomere maintenance, is the MRN complex (for recent reviews see [6, 7]). This consists of three subunits (MRE11-RAD50-NBS1). A single NBS1 molecule is associated with two dimers of MRE11 and RAD50. The MRE11 and RAD50 proteins form a heterotetramer which contains two DNA-binding and processing domains that can bridge free DNA ends . In Saccharomyces cerevisiae, this complex comprises subunits MRE11, RAD50, and XRS2. Whereas both proteins, MRE11 and RAD50, share a high homology across various eukaryotes, the XRS2 protein has a lower degree of homology with NBS1, which is specific for mammals and plants , although a functional homologue of NBS1 has been found in Schizosaccharomyses pombe.
In humans, mutation in the NBS1 gene leads to the chromosomal instability disorder, Nijmegen breakage syndrome 1. Besides other clinical hallmarks, this syndrome is associated with enhanced sensitivity to ionizing radiation and chromosomal instability which leads to early developing cancer even in NBS1
+/− heterozygotes . Murine NBS1
+/− heterozygotes are phenotypically normal although complete removal of the NBS1 is embryonically lethal in mice . Accumulating evidence demonstrates that NBS1 interacts with telomeres and contributes to their stability, at least in human and mouse cells (reviewed in ). Direct interaction of NBS1with telomere repeat-binding factor 1, TRF1, has been shown for immortalized telomerase negative cells  implying that this interaction might be involved in the alternative lengthening of telomeres. Moreover, it has been shown by indirect immuno-fluorescence that NBS1 co-localise with a shelterin constituent, telomere repeat-binding factor 2 (TRF2), during the S phase in cultured HeLa cells , possibly by modulating t-loop formation. Similarly, in mouse embryonal fibroblasts, active recruitment of NBS1 to dysfunctional telomeres has been observed .
It is known that MRE11 and RAD50 together with protein kinases ATM and ATR, are also essential for proper telomere maintenance in plants (Reviewed in [5, 16, 17]). Inactivation of Arabidopsis RAD50 or MRE11 leads to hypersensitivity to ionizing radiation or radiomimetics and reduced plant health and even sterility. In knockout RAD50 or MRE11 mutant Arabidopsis plants genome instabilities are induced, including chromosome end-to-end fusions [18–21]. Absence of RAD50 led to rapid shortening of telomeres and loss of telomere repeats accompanied by chromosome-end fusions, while in double mutant plants (rad50/tert) a synergistic effects of RAD50 and telomerase on the frequency of bridges have been found , demonstrating the dual role of the RAD50 protein in plants. A homolog of the third MRN constituent, NBS1, has been isolated in the higher plants, Arabidopsis thaliana and Oryza sativa. The NBS1 proteins from both plant species were shown to be smaller in size than animal NBS1, but both contained typical domains such as the FHA (forkhead-associated), BRCT (BRCA1 C Terminus) domain, the MRE11-binding domain, and the ATM-interacting domain. Functional analysis using yeast two-hybrid assay showed that the OsNBS1 protein interacted not only with plant MRE11 but also with animal MRE11. OsNBS1 mRNA expression was found to be higher in the shoot apex and young flower and AtNBS1 expression increased when plants were exposed to X-rays . Cytogenetic analyses showed numerous anomalies including the fragmentation of meiotic chromosomes in Atnbs1 knockout mutants , although on simultaneous inactivation of plant ATM.
In this study, we examined the role of NBS1 in telomere maintenance in the plant model species Arabidopsis thaliana. Using plants deficient in both genes, NBS1 and TERT, we found that plants exhibited severe genomic instability even in early generations. These phenotypes worsened with increasing generations on self-pollination and plants developed serious developmental defects leading to sterility in the 6th generation. By comparing the length of telomeres in double and single mutants we observed accelerated and more frequent telomere shortening in the former.