Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA

Abstract

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.

Figure 1

Identification of the AtTERT gene. (A) Schematic representation of the AtTERT gene. Open boxes represent exons, black lines, introns, and gray boxes, conserved TERT motifs. The position of PCR primers (arrows) and a T-DNA insertion are indicated. (B) Alignment of AtTERT with TERT proteins from human (hTERT) (9), mouse (mTERT) (18), Schizosaccharomyces pombe (SpTERT) (9), Saccharomyces cerevisiae (ScTERT) (8), Euplotes aediculatus (EaTERT) (8), Oxytricha trifallax (OtTERT) (12), and Tetrahymena thermophila (TtTERT) (11, 12).

Figure 2

AtTERT mRNA levels correlate with telomerase activity. (A) Telomerase regulation in Arabidopsis. TRAP results from different organs. Elongation ladders correspond to the addition of TTTAGGG repeats. Silque (seed pod); inflor. (inflorescence) bolt. (B) RT-PCR products from callus (C) and leaf (L) mRNA. Primer pairs used to generate AtTERT products (*) are indicated. Bottom band represents the glyceraldehyde-3-phosphate dehydrogenase quantitation control.

Figure 3

Plants with a homozygous disruption in the AtTERT gene lack telomerase. TRAP assays were performed on the floral buds from plants that are wild type (+/+), heterozygous (+/−), or homozygous (−/−) for the T-DNA disruption in AtTERT.

Figure 4

Progressive telomere shortening in telomerase-deficient plants. (A) TRF analysis. Shown are results with Tru9I digestion of DNA from rosette leaves (lane 1) or floral buds and siliques (lane 2) of wild-type plants and rosette leaves of heterozygous plants (lane 5). Results with rosette leaf DNA from first-generation (G₁) and second-generation (G₂) plants homozygous AtTERT disruption are shown in lanes 3 and 4. (B) Quantitative analysis of TRF data from wild-type (wt) and G₂ homozygous mutant plants digested with Tru9I or HaeIII (C) are shown. * indicate interstitial DNA with homology to the telomeric repeat. The rate of telomere shortening was calculated by comparing the size of the smallest telomere fragments in each generation of the mutants with telomeres from wild-type plants.