Telomeres and their associated factors in Arabidopsis thaliana

Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary for genome integrity. Telomeres are maintained by intermittent action of telomerase. I explored the kinetics of telomere length homeostasis in the model plant Arabidopsis thaliana by crossing wild type plants to different generations of telomerase deficient plants, and then analyzing telomere length in the resulting progeny. Unexpectedly, I found plants lacking telomerase for seven generations can lengthen telomeres when telomerase is reintroduced, but one generation is not sufficient to reestablish the telomere set point. Est1 is a non-catalytic component of the Saccharomyces cerevisiae telomerase holoenzyme. To investigate the role of Est1 in higher eukaryotes, I identified two putative Est1 homologues in Arabidopsis, AtEST1a and AtEST1b. Plants deficient in AtEST1a displayed no vegetative or reproductive defects. However, plants deficient for AtEST1b were sterile and had severe vegetative and reproductive irregularities. Surprisingly, no defects in telomere maintenance were observed in any single or double mutant line. This suggests that the Est1- like proteins in plants have evolved new functions outside of telomere length maintenance and end protection.One consequence of telomere dysfunction is end-to-end chromosome fusion. In mammals, telomere fusion is mediated through NHEJ and requires DNA Ligase IV (Lig4). Lig4 is an essential component of the NHEJ pathway along with the Ku70/Ku80 heterodimer and DNA-PKcs. To address the mechanism of chromosome fusion in Arabidopsis, we investigated the role of Lig4 in mutant combinations lacking TERT, the catalytic subunit of telomerase, and Ku70. Surprisingly, telomere end-to-end fusions were observed in ku70 tert lig4 triple mutants, suggesting that neither Lig4 nor Ku70 are required for the fusion of critically shortened telomeres in Arabidopsis. To investigate the origin of genome instability, terminal restriction fragment analysis was performed on triple mutants. Strikingly, telomeres diminished five to six-fold faster than in a tert single mutant. Moreover, in the triple mutants, telomere tracts were extremely heterogeneous, suggesting that the telomeres were exposed to catastophic nucleolytic attack. These data provide the first evidence that Lig4 contributes to telomere maintenance and chromosome end protection.