A hypomorphic allele of telomerase uncovers the minimal functional length of telomeres in Arabidopsis.

Despite the essential requirement of telomeric DNA for genome stability, the length of telomere tracts between species substantially differs, raising the question of the minimal length of telomeric DNA necessary for proper function. Here, we address this question using a hypomorphic allele of the telomerase catalytic subunit, TERT. We show that although this construct partially restored telomerase activity to a tert mutant, telomeres continued to shorten over several generations, ultimately stabilizing at a bimodal size distribution. Telomeres on two chromosome arms were maintained at a length of 1 kb, while the remaining telomeres were maintained at 400 bp. The longest telomeres identified in this background were also significantly longer in wild-type populations, suggesting cis-acting elements on these arms either promote telomerase processivity or recruitment. Genetically disrupting telomerase processivity in this background resulted in immediate lethality. Thus, telomeres of 400 bp are both necessary and sufficient for Arabidopsis viability. As this length is the estimated minimal length for t-loop formation, our data suggest that telomeres long enough to form a t-loop constitute the minimal functional length.

Germline replications and somatic mutation accumulation are independent of vegetative life span in Arabidopsis.

In plants, gametogenesis occurs late in development, and somatic mutations can therefore be transmitted to the next generation. Longer periods of growth are believed to result in an increase in the number of cell divisions before gametogenesis, with a concomitant increase in mutations arising due to replication errors. However, there is little experimental evidence addressing how many cell divisions occur before gametogenesis. Here, we measured loss of telomeric DNA and accumulation of replication errors in Arabidopsis with short and long life spans to determine the number of replications in lineages leading to gametes. Surprisingly, the number of cell divisions within the gamete lineage is nearly independent of both life span and vegetative growth. One consequence of the relatively stable number of replications per generation is that older plants may not pass along more somatically acquired mutations to their offspring. We confirmed this hypothesis by genomic sequencing of progeny from young and old plants. This independence can be achieved by hierarchical arrangement of cell divisions in plant meristems where vegetative growth is primarily accomplished by expansion of cells in rapidly dividing meristematic zones, which are only rarely refreshed by occasional divisions of more quiescent cells. We support this model by 5-ethynyl-2'-deoxyuridine retention experiments in shoot and root apical meristems. These results suggest that stem-cell organization has independently evolved in plants and animals to minimize mutations by limiting DNA replication.

Cell Proliferation Analysis Using EdU Labeling in Whole Plant and Histological Samples of Arabidopsis.

The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis.

Comparative biology of telomeres: where plants stand.

Telomeres are essential structures at the ends of eukaryotic chromosomes. Work on their structure and function began almost 70 years ago in plants and flies, continued through the Nobel Prize winning work on yeast and ciliates, and goes on today in many model and non-model organisms. The basic molecular mechanisms of telomeres are highly conserved throughout evolution, and our current understanding of how telomeres function is a conglomeration of insights gained from many different species. This review will compare the current knowledge of telomeres in plants with other organisms, with special focus on the functional length of telomeric DNA, the search for TRF homologs, the family of POT1 proteins, and the recent discovery of members of the CST complex.

siRNA-mediated methylation of Arabidopsis telomeres.

Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA-based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat-containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)(n) repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA-dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA-directed mechanism of chromatin maintenance at telomeres in Arabidopsis.

Telomere rapid deletion regulates telomere length in Arabidopsis thaliana.

Telomere length is maintained in species-specific equilibrium primarily through a competition between telomerase-mediated elongation and the loss of terminal DNA through the end-replication problem. Recombinational activities are also capable of both lengthening and shortening telomeres. Here we demonstrate that elongated telomeres in Arabidopsis Ku70 mutants reach a new length set point after three generations. Restoration of wild-type Ku70 in these mutants leads to discrete telomere-shortening events consistent with telomere rapid deletion (TRD). These findings imply that the longer telomere length set point is achieved through competition between overactive telomerase and TRD. Surprisingly, in the absence of telomerase, a subset of elongated telomeres was further lengthened, suggesting that in this background a mechanism of telomerase-independent lengthening of telomeres operates. Unexpectedly, we also found that plants possessing wild-type-length telomeres exhibit TRD when telomerase is inactivated. TRD is stochastic, and all chromosome ends appear to be equally susceptible. The frequency of TRD decreases as telomeres shorten; telomeres less than 2 kb in length are rarely subject to TRD. We conclude that TRD functions as a potent force to regulate telomere length in Arabidopsis.

Telomerase-independent cell survival in Arabidopsis thaliana.

Telomerase is the reverse transcriptase responsible for the maintenance of telomeric repeat sequences in most species that have been studied. Inactivation of telomerase causes telomere shortening and results in the loss of the telomere's protective function, which in mammals leads to cell-cycle arrest and apoptosis. Experiments performed on Arabidopsis thaliana mutants lacking telomerase activity revealed their unusually high tolerance for genome instability. Here we present molecular and cytogenetic analysis of two cell lines (A and B) derived from seeds of late-generation telomerase-deficient A. thaliana. These cultures have survived for about 3 years and are still viable. However, neither culture has adapted mechanisms to maintain terminal telomeric repeats. One culture (B) suffers from severe growth irregularities and a high degree of mortality. Karyological analysis revealed dramatic genomic rearrangements, a large variation in ploidy, and an extremely high percentage of anaphase bridges. The second cell line (A) survived an apparent crisis and phenotypically appears wild-type with respect to growth and morphology. Despite these indications of genome stabilization, a high percentage of anaphase bridges was observed in the A line. We conclude that the restructured chromosome termini provide the A line with partial protection from end-joining repair activities, thus allowing normal growth.

Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70.

The Ku70/80 heterodimer is a critical component of the non-homologous end-joining (NHEJ) pathway and of the telomere cap in yeast and mammals. We report the molecular characterization of the KU70 and KU80 genes in Arabidopsis and describe the consequences of a Ku70 deficiency. Arabidopsis KU70/80 genes are ubiquitously expressed and their products form stable heterodimers in vitro. Plants harboring a T-DNA insertion in KU70 exhibit no growth or developmental defects under standard growth conditions. However, mutant seedlings are hypersensitive to gamma-irradiation-induced double-strand breaks. Unexpectedly, we found that mutants are hypersensitive to methyl methanosulfonate during seed germination, but lose this sensitivity in seedlings, implying that the requirement for NHEJ varies during plant development. Lack of Ku70 results in a dramatic deregulation of telomere length control, with mutant telomeres expanding to more than twice the size of wild type by the second generation. Furthermore, in contrast to the situation in mammals, chromosome fusions are not associated with a Ku deficiency in Arabidopsis. These findings imply that Ku may play a different role in capping plant and animal telomeres.