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Vascular plants appeared ~410 million years ago then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes (1). We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first non-seed vascular plant genome reported. By comparing gene content in evolutionary diverse taxa, we found that the transition from a gametophyte- to sporophyte-dominated life cycle required far fewer new genes than the transition from a non-seed vascular to a flowering plant, while secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in post-transcriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the tasiRNA pathway and extensive RNA editing of organellar genes.

Summary Orthologs of the yeast telomere protein Stn1 are present in plants, but other components of the Cdc13/Stn1/Ten1 (CST) complex have only been found in fungi. Here we report the identification of Conserved Telomere maintenance Component 1 (CTC1) in plants and vertebrates. CTC1 encodes an ∼140 kDa telomere-associated protein predicted to contain multiple OB-fold domains. Arabidopsis mutants null for CTC1 display a severe telomere deprotection phenotype accompanied by a rapid onset of developmental defects and sterility. Telomeric and subtelomeric tracts are dramatically eroded, and chromosome ends exhibit increased G-overhangs, recombination, and end-to-end fusions. AtCTC1 both physically and genetically interacts with AtSTN1. Depletion of human CTC1 by RNAi triggers a DNA damage response, chromatin bridges, increased G-overhangs and sporadic telomere loss. These data indicate that CTC1 participates in telomere maintenance in diverse species and that a CST-like complex is required for telomere integrity in multicellular organisms.

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 ...

Telomerase activity is developmentally regulated in mammals. Here we examine telomerase activity in plants, whose development differs in fundamental ways from that of animals. Using a modified version of the telomere repeat amplification protocol (TRAP) assay, we detected an activity in extracts from carrots, caulif lower, soybean, Arabidopsis, and rice with all the characteristics expected for a telomerase synthesizing the plant telomere repeat sequence TTTAGGG. The activity was dependent on RNA and protein components, required dGTP, dATP, and dTTP, but not dCTP, and generated products with a seven nucleotide periodicity. Telomerase activity was abundant in caulif lower meristematic tissue and undifferentiated cells from Arabidopsis, soybean, and carrot suspension cultures, but was low or not detectable in a sampling of differentiated tissues from mature plants. Telomerase from caulif lower meristematic tissues exhibited relaxed DNA sequence requirements, which might ref lect the capacity to form telomeres on broken chromosomes in vivo. The dramatic differences in telomerase expression and their correlation with cellular proliferation capacity mirror changes in human telomerase levels during differentiation and immortalization. Hence, telomerase activation appears to be a conserved mechanism involved in conferring long-term proliferation capacity.Telomeres are nucleoprotein structures at the ends of chromosomes that are essential for maintaining the integrity of the genome. The primary mechanism for generating and sustaining telomere DNA in eukaryotes is telomerase (1). A ribonucleoprotein with a reverse transcriptase activity, telomerase synthesizes the G-rich strand of telomere DNA using an internal RNA subunit as a template (2). This DNA addition compensates for the loss of terminal sequences that occurs during replication by the conventional cellular machinery (3, 4).Widespread among eukaryotes, telomerase has been detected in protozoa, yeast, amphibians, and mammals (5-11). Telomerase activity is developmentally regulated in humans (12, 13). In the differentiated human soma, telomeres shorten in a phenomenon attributed to the lack of detectable telomerase activity in these cells (14,15). Once telomeres shorten below a critical threshold, they appear to lose the capacity to effectively cap chromosomes and activate a damaged DNA response pathway that causes cell-cycle arrest (12). Hence, telomeres have been proposed to represent a biological clock that determines life span (16). A subset of normal human tissues express telomerase, including the regenerating tissues of the blood and epidermis (17-19) and the germ line (20). In addition, telomerase activity can be found in most human primary malignancies (13,21).The confinement of telomerase expression to the permanently regenerating tissues of the soma is not a feature shared by all organisms. In mice, many somatic tissues have detectable telomerase activity (11, 22) and telomeres do not shorten (23). However, more recent studies indicate that telomer...

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