Loss of telomere function in metazoans results in catastrophic damage to the genome, cell cycle arrest, and apoptosis. Here we show that the mustard weed Arabidopsis thaliana can survive up to 10 generations without telomerase. The last five generations of telomerase-deficient plants endured increasing levels of cytogenetic damage, which was correlated with developmental anomalies in both vegetative and reproductive organs. Mutants ultimately arrested at a terminal vegetative state harboring shoot meristems that were grossly enlarged, disorganized, and in some cases, dedifferentiated into a callusoid mass. Unexpectedly, late-generation mutants had an extended life-span and remained metabolically active. The differences in plant and animal responses to dysfunctional telomeres may reflect the more plastic nature of plant development and genome organization.

End-to-end fusion of critically shortened telomeres in higher eucaryotes is presumed to be mediated by nonhomologous end-joining (NHEJ). Here we describe two PCR-based methods to monitor telomere length and examine the fate of dysfunctional telomeres in Arabidopsis lacking the catalytic subunit of telomerase (TERT) and the DNA repair proteins Ku70 and Mre11. Primer extension telomere repeat amplification relies on the presence of an intact G-overhang, and thus measures functional telomere length. The minimum functional telomere length detected was 300–400 bp. PCR amplification and sequence analysis of chromosome fusion junctions revealed exonucleolytic digestion of dysfunctional ends prior to fusion. In ku70 tert mutants, there was a greater incidence of microhomology at the fusion junction than in tert mutants. In triple ku70 tert mre11 mutants, chromosome fusions were still detected, but microhomology at the junction was no longer favored. These data indicate that both Ku70 and Mre11 contribute to fusion of critically shortened telomeres in higher eucaryotes. Furthermore, Arabidopsis processes critically shortened telomeres as double-strand breaks, using a variety of end-joining pathways

Telomerase is an essential enzyme that maintains telomeres on eukaryotic chromosomes. In mammals, telomerase is required for the lifelong proliferative capacity of normal regenerative and reproductive tissues and for sustained growth in a dedifferentiated state. Although the importance of telomeres was first elucidated in plants 60 years ago, little is known about the role of telomeres and telomerase in plant growth and development. Here we report the cloning and characterization of the Arabidopsis telomerase reverse transcriptase (TERT) gene, AtTERT. AtTERT is predicted to encode a highly basic protein of 131 kDa that harbors the reverse transcriptase and telomerase-specific motifs common to all known TERT proteins. AtTERT mRNA is 10 -20 times more abundant in callus, which has high levels of telomerase activity, versus leaves, which contain no detectable telomerase. Plants homozygous for a transfer DNA insertion into the AtTERT gene lack telomerase activity, confirming the identity and function of this gene. Because telomeres in wild-type Arabidopsis are short, the discovery that telomerase-null plants are viable for at least two generations was unexpected. In the absence of telomerase, telomeres decline by approximately 500 bp per generation, a rate 10 times slower than seen in telomerase-deficient mice. This gradual loss of telomeric DNA may reflect a reduced rate of nucleotide depletion per round of DNA replication, or the requirement for fewer cell divisions per organismal generation. Nevertheless, progressive telomere shortening in the mutants, however slow, ultimately should be lethal.transfer DNA ͉ telomeres ͉ plant ͉ callus T he groundbreaking studies of Barbara McClintock (1, 2) and Hermann Muller (3) demonstrated that genome stability depends on the integrity of the telomere complex at the ends of eukaryotic chromosomes. Although alternative strategies have been reported (4), telomere synthesis by telomerase is the primary mechanism for sustaining chromosome ends in eukaryotes. Telomeres and their maintenance by telomerase comprise a biological clock that influences cellular lifespan in mammals (5). Telomerase expression is confined primarily to the germ line and permanently regenerating tissues of the adult soma. In other cells, telomerase is turned off and telomeres progressively shorten with each division. Once telomeres shorten below a critical length, a DNA damage checkpoint is activated, leading to cellular senescence and death. Telomerase is reactivated in about 85% of all human tumors and telomere function is maintained indefinitely (6).Telomerase is a ribonucleoprotein reverse transcriptase (7). The RNA subunit contains a templating sequence complementary to the G-rich strand of the telomere, whereas the telomerase reverse transcriptase (TERT) harbors the catalytic activity for telomere repeat synthesis. Characterization of TERT subunits from a variety of protozoa, yeasts, and mammals has revealed several distinct reverse transcriptase motifs that comprise the polymerase active site as well ...

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis