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. 2021 Aug 4;219(2):iyab126. doi: 10.1093/genetics/iyab126

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

J Matthew Watson 1, Johanna Trieb 1, Martina Troestl 1, Kyle Renfrew 2,✉, Terezie Mandakova 3, Jaroslav Fulnecek 3, Dorothy E Shippen 2, Karel Riha 3,✉
Editor: J Birchler
PMCID: PMC8633133  PMID: 34849882

Abstract

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.

Keywords: telomeres, telomerase, genome stability, subtelomere, t-loop

Introduction

The ends of most eukaryotic chromosomes are capped by telomeres that serve two primary functions: they distinguish the natural chromosome end from a DNA double-strand break (DSB) and provide a mechanism for overcoming the end-replication problem (Stewart et al. 2012; Fouquerel et al. 2016). Telomeres are composed primarily of repetitive telomeric DNA and telomere-specific DNA-binding proteins (de Lange 2010). The telomeric repeat is a short G-rich sequence, TTAGGG in vertebrates and TTTAGGG in plants (Fulcher et al. 2014). Telomeric DNA cannot be fully replicated by conventional DNA polymerases, an issue known as the end-replication problem. During lagging strand synthesis, removal of the final RNA primer results in the formation of a gap in the DNA that cannot be replaced, leading to the formation of a 3ʹ single-stranded DNA overhang (G-overhang) and a small loss of terminal sequences (Soudet et al. 2014). The end-replication problem can be counteracted by the enzyme telomerase, consisting minimally of the catalytic subunit TERT (telomerase reverse-transcriptase) and an internal RNA template, TER (telomerase RNA; Greider and Blackburn 1989). Telomerase can anneal to the G-overhang and, using its internal template, processively extend the extreme 3ʹ end (Tomita 2018).

Telomeric repeats serve as a platform for sequence-specific telomere binding proteins that can associate either with double- or single-stranded DNA. In mammals, double-strand telomere binding proteins TRF1 and TRF2 anchor a multi-subunit protein complex termed shelterin to telomeres (de Lange 2010). Shelterin is necessary for preventing cells from recognizing telomeres as DSBs (Sfeir and de Lange 2012), but precisely how this is accomplished is unknown. One model predicts that chromosome ends are protected through the formation of t-loops (Tomaska et al. 2020; Ruis and Boulton 2021). T-loops are higher-order structures in which the G-overhang invades duplex telomeric DNA, forming a looped molecule that hides the chromosome 3ʹ terminal extension (Griffith et al. 1999; Doksani et al. 2013). Although first described in mammals, T-loops have also been identified in other taxa including plants (Munoz-Jordan et al. 2001; Tomaska et al. 2002; Cesare et al. 2003; Raices et al. 2008). T-loops likely do not form in organisms that possess relatively short telomeres, such as Saccharomyces cerevisiae or in the vegetative nuclei of ciliates (Tomaska et al. 2004; Luke-Glaser et al. 2012). In these species, chromosome-end protection appears to largely depend on protein complexes associated with single-stranded telomeric DNA (Greetham et al. 2015).

In the absence of telomerase, telomeric DNA gradually shortens, eventually leading to telomere dysfunction. Thus, the ability of telomeres to protect chromosome ends and allow for cellular viability is a function of their length. The molecular details of this relationship are unclear. Telomeres are maintained at different lengths in different organisms, ranging from 300 bp in yeasts to as long as 150 kb in tobacco and the telomere length can also be highly variable within a species (Fajkus et al. 1995; Shakirov and Shippen 2004; Liti et al. 2009; Fulcher et al. 2015). Most organisms keep telomeres at lengths longer than necessary for function to provide a protective buffer, as defects in telomere maintenance are often only apparent after multiple generations (Lundblad and Szostak 1989; Blasco et al. 1997; Nakamura et al. 1997; Riha et al. 2001; Meier et al. 2006). Several attempts have been made to determine the minimal length of telomeric DNA necessary for telomere function (Heacock et al. 2004; Capper et al. 2007; Xu and Blackburn 2007). These approaches have relied on the identification of the smallest telomere within a population undergoing telomere crisis due to ongoing telomere shortening. The minimal functional lengths inferred from these experiments vary from roughly 100 bp in yeast (Forstemann et al. 2000; Teixeira et al. 2004), 42–78 bp in human cell lines (Capper et al. 2007; Xu and Blackburn 2007), and 360 bp in Arabidopsis (Heacock et al. 2004). However, it has not been possible to determine whether the very short telomeres identified in these studies are actually functional, since they form only a small portion of total telomeres and may represent dysfunctional and nucleolytically processed intermediates. Because variations in minimal telomere length may belie fundamental differences in telomere capping, determining the amount of telomeric DNA necessary for telomere function can provide insight into mechanisms governing chromosome-end protection.

Here, we report the creation of Arabidopsis strains that maintain their telomeres at the minimal functional threshold by expressing a hypomorphic allele of TERT. While plants with these shortened telomeres exhibit chromosome end-to-end fusions and ongoing genome instability, they are capable of indefinite survival as long as they are not subjected to further telomere erosion. Thus, we propose that the final telomere length reached in this population, roughly 400 bp, represents the minimal length necessary for proper telomere function in Arabidopsis.

Materials and methods

Plants and growth conditions

Plants were grown in soil under long-day conditions with 16 h daylight at 21°C. All mutants and genotyping conditions have been previously described: tert (Fitzgerald et al. 1999), ku70 (Riha et al. 2002), and pot1a (Surovtseva et al. 2007).

Constructs and plant transformation

Full-length TERT cDNA was obtained from reverse-transcription reactions from wild-type RNA using the RevertAid First-Strand cDNA Synthesis Kit and amplified with Phusion Hot Start II polymerase (Fermentas) using primers TERT cDNA 5ʹ BamHI (5′-GGATCCAAGGAGGAGGAAGGTGTAATG-3′) and TERT cDNA 3’ SacI (5′-GAGCTCAAATGTTACAAATCCATTTTG-3′). Full-length cDNA products were subcloned into pCR2.1-TOPO (Invitrogen) and sequenced. The cDNA was then transferred to binary vectors pCBK05 (35S promoter) forming pCBJ05 or pCBM10 (Actin promoter) forming pCBJ06 using BamHI and SacI sites. Transformation of G4 tert mutants was performed by the floral dip method and transformed plants were selected for resistance to the herbicide Basta.

TRAP assay

TRAP assays were performed as previously described (Kannan et al. 2008).

Telomere length analysis

DNA extraction and TRF analysis were performed as previously described (Watson and Shippen 2007). For both PETRA (Heacock et al. 2004) and STELA (Baird et al. 2003), 20 ng of total genomic DNA were used. For PETRA, the PETRA-T primer (5′-CTCTAGACTGTGAGACTTGGACTACCCTAAACCCT-3′) was annealed to G-overhangs and extended in a 20-µl reaction containing 1x Phi29 buffer (Fermentas), 125 µM dNTPs, 0.5 µM PETRA-T, and 4 U Phi29 at 30°C for 1 h. Samples were heat inactivated at 65°C for 20 min, precipitated with ethanol, and resuspended in 20 µl of water. For STELA, the primer Fok2 (5′-CTCTAGACTGTGAGACTTGGACTACAGGATGTAAACCC-3′) was ligated to genomic DNA in 20-µl reactions containing 1x T4 DNA ligase buffer, 0.5 µM Fok2, and 1 µl T4 DNA ligase (Fermentas) overnight at 16°C. Samples were heat inactivated at 65°C for 20 min, precipitated with ethanol, and resuspended in 20 µl of water. Following this initial processing, STELA and PETRA reactions were identical. One microliters of STELA or PETRA products were PCR amplified in 20-µl reactions containing 1x Phusion GC buffer, 200 µM dNTPs, 0.5 µM PETRA-A (5′-CTCTAGACTGTGAGACTTGGACTA C-3'), 0.5 µM of one subtelomere specific primer (1L: 5′-AGGACCATCCCATATCATTGAGAGA-3′, 1R: 5′-CTATTGCCA GAACCTTGATATTCAT-3′, 2R: 5′-CAACATGGCCCATTTAAGATTGAACGGC-3′, 3L: 5′-CATAATTCTCACAG CAGCACCGTAGA-3′, 4R: 5′-TGGGTGATTGTCATGCTACATGGTA-3′, 5L: 5′-AGGTAGAGTGAACCTAACA CTTGG-3ʹ, 5R: 5′-CAGGACGTGTGAAACAGAAACTACA-3′), 0.4 U Phusion Hot Start II (Fermentas). PCR reactions were performed by incubating samples at 98°C for 3 min followed by 22–25 cycles of 98°C 15 s, 60°C 30 s, 72°C 2 min 30 s, followed by a final incubation at 72°C for 10 min. Reactions were then separated on 1.2% agarose gels cast in 12 × 14 cm trays at 100 V for 2 h 30 min. DNA was transferred to nylon membranes by Southern blotting. Hybridization was performed as previously described (Heacock et al. 2004), membranes were exposed to Kodak phosphoscreens, visualized with a Pharos FX Plus (Biorad) and images were analyzed with Imagelab (Biorad).

Analysis of TERT mRNA

Total RNA was isolated from inflorescence of four-week-old plants using RNA Blue (Top-Bio). Remaining DNA in RNA samples was hydrolyzed using TURBO DNA-free Kit (Invitrogen). One μg of RNA was reverse transcribed using Superscript IV and combination of oligo(dT) and randomhexamers according to manufacturer’s recommendation. Quantitative RT-PCR was done using KAPA SYBR FAST universal kit (Kapa Biosystems). TERT cDNA was amplified with the primers Tert-RT-PCR-F 5′-CTGTATTGCACGAACGACCA-3′ and Tert-RT-PCR-R 5′-TCGTCGCCTTGAAACAAACC-3′. The SAND transcript used for normalization was amplified with the primers SAND-qF1 5′-CAAGGCAGGAAATCACCAGGTTG-3′ and the reverse primer SAND-qR1 5′-CTGTACAGCTGATGCAGACCAG-3′. qPCR was performed in three biological and each in three technical replicates. TERT expression was normalized to SAND and relative expression was calculated using the delta–delta Ct method (Livak and Schmittgen 2001).

Chromosome preparation, probe labeling, BAC FISH, and image processing

Whole inflorescences of Arabidopsis thaliana were fixed in freshly prepared ethanol: acetic acid fixative (3:1) overnight, transferred into 70% ethanol and stored at −20°C until use. Selected inflorescences were rinsed in distilled water and citrate buffer (10 mM sodium citrate, pH 4.8), and digested by a 0.3% mix of pectolytic enzymes (cellulase, cytohelicase, pectolyase; all from Sigma) in citrate buffer for c. 3 h. Chromosome preparations were prepared from pistils and pretreated with RNase (100 µg/ml, AppliChem) and pepsin (0.1 mg/ml, Sigma-Aldrich) as previously described (Mandakova et al. 2014). Arabidopsis thaliana BAC clones T25K16 (AC007323), F6F3 (AC023628), F22L4 (AC061957), T1N6 (AC009273), and F22N6 (B98711) were used for in situ identification of chromosome arm 1L, F19K16 (AC011717), F18B13 (AC009322), F5I6 (AC018848), T21F11 (AC018849), and F23A5 (AC011713) for 1R, F13A10 (AC006418), T3A4 (AC005819), F19D11 (AC005310), F14M4 (AC004411), and T8I13 (AC002337) for 2R, and F19H22 (AL035679), T22F8 (AL050351), F23K16 (AL078620), T23P19 (B98327), and T5J17 (AL035708) for 4R. DNA probes were labeled with biotin-dUTP and digoxigenin-dUTP by nick translation as previously described (Mandakova et al. 2010). Differentially labeled BACs corresponding to 1L + 4R and 1R + 2R chromosome arms were pooled, precipitated, and resuspended in 20 µl of hybridization mixture (50% formamide and 10% dextran sulfate in 2× SSC) per slide. Probes and chromosomes were denatured together on a hot plate at 80°C for 2 min and incubated in a moist chamber at 37°C overnight. Posthybridization washing was performed in 20% formamide in 2× SSC at 42°C. Fluorescent detection was as follows: biotin-dUTP was detected by avidin–Texas Red (Vector Laboratories) and amplified by goat anti-avidin–biotin (Vector Laboratories) and avidin–Texas Red; digoxigenin-dUTP was detected by mouse antidigoxigenin (Jackson ImmunoResearch) and goat antimouse Alexa Fluor 488 (Molecular Probes). Chromosomes were counterstained with DAPI (4ʹ,6-diamidino-2-phenylindole; 2 μg/ml) in Vectashield (Vector Laboratories). Fluorescence signals were analyzed with an Olympus BX-61 epifluorescence microscope and CoolCube CCD camera (MetaSystems). Images were acquired separately for the two fluorochromes using appropriate excitation and emission filters (AHF Analysentechnik). The two monochromatic images were pseudo-colored and merged using Adobe Photoshop CS2 software (Adobe Systems).

Results

Following the discovery and analysis of telomerase mutants in Arabidopsis, we attempted to study the fate of shortened telomeres upon reintroduction of telomerase activity. To this end, we transformed fourth-generation tert mutants (G4) with constructs expressing the TERT cDNA under the control of either the cauliflower mosaic virus 35S or actin (ACT2) promoter (Supplementary Figure S1). The 35S:TERT and ACT2:TERT constructs reconstituted the expression of the TERT mRNA to the levels 3–4 and eight folds higher than in wild type, respectively (Figure 1A). Nevertheless, while both constructs restored telomerase activity, TRAP assays revealed that enzyme activity was significantly reduced compared to wild-type plants (Figure 1B). Plants bearing the ACT2:TERT construct showed slightly higher levels of telomerase than the 35S:TERT lines (Figure 1B).

Figure 1.

Figure 1

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Phenotypic analysis of the 35S:TERT allele. (A) Expression of TERT mRNA in flowers of 35S:TERT and ACT2:TERT plants determined by qRT-PCR relative to wild type. Error bars represent standard deviations from three biological replicas. (B) TRAP assay of wild-type seedlings and seedlings transformed with either 35S:TERT or ACT2:TERT constructs. Both constructs show reduced telomerase activity relative to wild type. (C) TRF analysis of successive generations of 35S:TERT or ACT2:TERT plants. Wild-type and tert mutants are shown as controls; the asterisk marks interstitial telomeric DNA. Rescue constructs were transformed into G4 tert mutants. The “T” designation indicates the number of generations post-transformation (Supplementary Figure S1). (D) Phenotypic analysis of successive generations of plants containing the 35S:TERT construct. Two independent lines were analyzed (line 2 and 7) with G6/T2, G7/T3, etc. The total number of plants analyzed in each population is indicated below the bars. (E) Percentage of anaphases containing chromatin bridges over successive generations of plants containing the 35S:TERT construct. Total number of anaphases examined is indicated. Data represent pools from both lines.

We followed two independent transformant lines for both constructs through multiple generations (Supplementary Figure S1) and measured telomere length by terminal restriction fragment length analysis (TRF) in G7/T3 and G8/T4. Telomeres in the two lines expressing TERT driven by the actin promoter showed a gradual lengthening of telomeres (Figure 1C), consistent with restoration of telomerase function in telomere synthesis. In contrast, both lines in which TERT expression was driven by the 35S promoter exhibited continual shortening of telomeres. The telomeric signal was under 1.5 kb in G7/T3 in 35S:TERT plants and telomeres further shortened in G8/T4 (Figure 1C). Telomeres were also substantially shorter than telomeres of parental G5 tert plants. Additionally, telomeres in the 35S:TERT lines displayed a discrete TRF banding pattern, a feature typical for plants with inactive telomerase. These data indicate that despite detectable enzyme activity, telomerase in the 35S:TERT lines is incapable of counteracting the end-replication problem and telomeres continue to shorten.

To follow the fate of lines expressing the hypomorphic TERT allele, we continued to propagate the two 35S:TERT lines, named 2 and 7, for multiple generations. Unexpectedly, despite the progressive loss of telomeric DNA observed in the initial TRFs (Figure 1), we were able to self-propagate both line 2 and line 7 through generation G18/T14, when the experiment was discontinued. This is 10 generations longer than tert null mutants, which arrest growth in a terminal generation of G8–G10 (Riha et al. 2001). Notably, out-segregation of the 35S:TERT construct in G7/T3 resulted in the termination of the population in the next generation (Supplementary Figure S1), confirming that the 35S:TERT construct was responsible for the survival of complemented tert mutants. Despite the ability of the 35S:TERT lines to survive for multiple generations, they began to display phenotypes characteristic of genomic instability within two generations after transformation (G7/T3, Figure 1). These phenotypes including wrinkled leaves, fascinated stems, altered phyllotaxy, and reduced seed set. Plants with these phenotypes have previously been categorized based on increasing severity into Type I, Type II, and Terminal phenotypes (Riha et al. 2001), with Type I plants showing mild defects in leaf morphology and phyllotaxy, Type II plants exhibiting severe developmental defects and reduced fertility, and dwarfed terminal plants that do not produce any seeds. We scored populations of plants from both 35S:TERT lines for the frequency of these phenotypic categories over multiple generations (Figure 1D). The severity of the phenotypes increased with each successive generation until G12/T8, indicative of increasing telomere dysfunction. In accordance with the increasing severity of the phenotypes, we observed a gradual increase in the frequency of anaphase bridges through G12/T8 (Figure 1E), suggesting that the worsening phenotypes are due to increased genomic instability caused by telomere erosion. Notably, the fraction of plants within the respective phenotypic categories became stabilized in subsequent generations after G12/T8, with approximately one-third of plants exhibiting wild type or Type I phenotypes (Figure 1C and Supplementary Figure S2). This observation suggests that surviving populations exhibit ongoing genome instability and partial telomere dysfunction.

To determine whether the 35S:TERT construct acts through the canonical telomere maintenance pathway, we examined its genetic interaction with other factors known to be involved in regulating telomerase activity at telomeres in Arabidopsis. We first asked whether the 35S:TERT allele could extend telomeres in the absence of KU70, a negative regulator of telomerase whose inactivation results in telomerase-dependent telomere lengthening (Riha et al. 2002; Riha and Shippen 2003). We generated ku70 tert 35:TERT plants by crossing (Supplementary Figure S3). Loss of KU70 in the 35S:TERT background led to an immediate and obvious lengthening of telomeres, demonstrating that despite its reduced activity (Figure 1A), the 35S:TERT telomerase is capable of extending telomeres. Next, we asked whether survival of the 35S:TERT lines depends on POT1A, an OB-fold containing protein that directly interacts with the telomerase RNP and is required for telomerase repeat addition processivity (Surovtseva et al. 2007; Renfrew et al. 2014). Plants deficient for POT1A exhibit an ever-shorter telomere phenotype, and this phenotype is epistatic with TERT (Surovtseva et al. 2007). We crossed third-generation tert pot1a double mutants to T2 35S:TERT plants from line 2 (Supplementary Figure S4) and propagated the progeny for an additional three generations as POT1A+/−, allowing the longer telomeres originating from the tert pot1a parent to shorten. We then out segregated pot1a mutants in F5 and F6 and scored the population for growth phenotypes (Supplementary Figures S4 and Figure 2C). Population of POT1A−/− mutants in both generations showed more severe phenotypes than either the POT1A+/+ or POT1A+/− plants, and all pot1a−/− plants in the F6 population displayed terminal phenotype. These results demonstrate that the survival of 35S:TERT lines requires POT1A.

Figure 2.

Figure 2

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Genetic analysis of the 35S:TERT allele. (A) Phenotypic analysis of successive generations of 35S:TERT(D680N) transformants. In the absence of telomerase catalytic activity, two independent transformant lines (5 and 17) show rapid loss of viability. (B) TRF analysis of 35S:TERT containing siblings segregating for ku70 (see Supplementary Figure S3 for details of the cross). Loss of KU70 leads to immediate lengthening of telomeres. Asterisks mark interstitial telomeric DNA. (C) Phenotypic analysis of 35S:TERT plants segregating for POT1A. See Supplementary Figure S4 for details for the cross. Loss of POT1A increases the severity of the phenotypes in 35S:TERT background.

Taken together, these genetic experiments suggest that the long-term survival of the 35S:TERT lines depends on the ability of telomerase to add telomeric repeats to the telomeres. We hypothesized that the 35S:TERT construct leads to the formation of a hypomorphic telomerase enzyme that is unable to rescue the shortening of telomeres in tert mutants, but supports continuous growth in a significant fraction of the population by maintaining telomeres once they reach the minimal length necessary for telomere protection. To test this prediction, we analyzed telomeres in 35S:TERT lines by PETRA, a PCR-based method that permits precise analysis of telomere length at individual chromosome arms (Heacock et al. 2004). We measured the length of telomeres at seven chromosome arms in four to five sibling plants from each line, every two generations, between G6/T2 and G14/T10 (Figure 3A). The plants selected for analysis were randomly sampled from the pool of plants displaying Type I and Type II phenotypes. At the beginning of the experiment in G6/T2, telomeres ranged in size from a low of 285 bp to a high of 1797 bp with an average length of 951 ± 350 bp. In line 2, telomeres that began with a length above 1 kb shortened at an average rate of 243 ± 102 bp per generation, and this rate continued through G10/T6 in the longest telomeres (1R, 2R, and 5L). We observed a similar trend in line 7, where telomeres longer than 1 kb (1L, 2R, and 5L) shortened with an average rate of 198 ± 80 bp per generation. A similar rate of telomere shortening has been reported for tert mutants (Riha et al. 2001; Watson and Shippen 2007), suggesting that the hypomorphic telomerase is not extending these telomeres. In contrast to the longest telomeres, the shortest telomeres in both lines remained essentially unchanged throughout the eight generations of the experiment (Figure 3A, 1L and 5R in line 2, 5R in line 7), indicating that they are sustained at these lengths by the hypomorphic telomerase. Additionally, while the longest telomeres shortened quite rapidly between G6/T2 and G10/T6, the rate of shortening became negligible from G10/T6 onwards, and telomere length was stable for the remaining four generations of the experiment.

Figure 3.

Figure 3

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Analysis of individual telomeres in 35S:TERT plants. (A) Measurement of individual telomere lengths over successive generations in lines 2 and 7 by PETRA. Measurements are the average of four plants, with the exception of G6/T2 in line where only one plant was available, and G12/T8 and G14/T10 where five plants were measured. (B) Comparison of telomere lengths in G14/T10 plants. Data are the same as in (A) but plotted as a bar graph for clarity. Error bars represent the standard deviation for all data points in (A). (C) Representative figures of FISH analysis. Anaphase bridges showing telomere fusions of 2R and 4R are shown.

While the distribution of starting telomere lengths in the two lines varied from 300 to 1800 bp, the final distribution was strongly bimodal, with most telomeres clustered around 400 bp (Figure 3B, 1L, 1R, 3L, 5L, 5R; 421 ± 121 bp), and two telomeres clustered around 900 bp (Figure 3B, 2R, 4R; 910 ± 115 bp). Interestingly, despite varying starting lengths between the two lines, each individual telomere shortened to a similar final length in both lines. As apparent from Figure 3A, the length of each telomere at G14/T10, was already reached in most cases by G10/T6, and for the shortest telomeres even from the beginning of the experiment in G6/T2. Although we terminated the experiment at generation G18/T14, the relative stability of telomeres from G10/T6 onwards implies that the hypomorphic telomerase was capable of maintaining telomeres at this length indefinitely. Our results further suggest that the hypomorphic telomerase is capable of maintaining telomeres only when they reach a chromosome-arm-specific length. Since these late-generation 35S:TERT lines exhibit ongoing chromosome end-to-end fusions (Figure 1E), it is likely that the ability of telomerase to extend particular telomeres directly correlates with their capping status; fully functional telomeres are not extendable, but telomeres that become partially deprotected are substrates for the hypomorphic telomerase. Along with the observation that further disruption of the telomerase pathway leads to lethality, we conclude that the 35S:TERT lines maintain telomeres close to their minimal functional length.

The bimodal size distribution of telomere lengths in G14/T10 was intriguing, as it suggested that the length at which chromosome ends become accessible to the hypomorphic telomerase is telomere-specific. Therefore, we asked whether the two longer telomeres, 2R and 4R, were more stably capped than the others. To test this, we performed pairwise comparison of the frequency of anaphase bridges involving short and long telomeres by FISH in G14/T10 35S:TERT lines (Figure 3C). As expected based on the phenotypic defects as well as the large numbers of anaphase bridges detected in late-generation 35:TERT plants, telomere to telomere fusions were readily detectable. Despite the large difference in telomere length, 2R and 4R were equally as likely to be involved in chromosome fusions as the shorter arms 1R and 1L (Table 1). This indicates that 2R and 4R telomeres become deprotected at longer lengths relative to the other chromosome arms.

Table 1.

Dual-label FISH analysis of anaphase bridges in Type II G14/T10 mutants with probes recognizing either chromosome arms 4R and 5L, or 2R and 1R

Anaphases with bridges Bridges with signal Percentage Fisher’s exact test
4R 55 8 14.50 0.3879
1L 55 6 10.9
2R 52 20 38.5 0.34534
1R 52 23 44.2
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No significant difference was observed between telomeres 4R (long) and 1L (short), and between 2R (long) and 1R (long).

The dramatic difference in telomere length of 2R and 4R suggests that cis-acting elements on these two subtelomeric arms regulate telomere homeostasis differently than on the other chromosome arms we have tested. We therefore examined telomere length in wild-type plants to determine whether these two telomeres are differentially regulated under normal conditions as well. To accomplish this, we measured telomere length by STELA in 32 wild-type plants from four bulk seed stocks. While in PETRA assay are the PCR adaptors anchored to the chromosome end via annealing to a G-overhang and primer extension through telomeres, ligation of the annealed primer to the chromosome end is used in STELA (Heacock et al. 2004; Capper et al. 2007). We chose STELA over PETRA in this analysis due to concern that the primer extension reaction may not proceed through the entire length of wild-type telomeres. Telomere length was measured as the length of the peak signal, and for allelic telomeres, the length of the two alleles was averaged. As shown in Figure 4A, the 2R and 4R telomeres were consistently longer than other telomeres in wild-type plants as judged by two alternate methods. These data argue that cis-acting elements affect the length of telomere homeostasis in the hypomorphic mutant as well as in wild type.

Figure 4.

Figure 4

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Single telomere analysis by STELA in wild-type plants. (A) Telomere length as measured by STELA. Data represent the average of 32 plants. Error bars represent the standard error of the mean. (B) Average telomere rank. As the average telomere length between plants varied, we ranked telomeres within each plant, telomeres were ranked from 1 = shortest to 7 = longest and the ranks were averaged. Error bars represent the standard error of the mean.

Discussion

In this study, we describe a hypomorphic allele of TERT that produces a partially functional telomerase. Although this enzyme is incapable of maintaining telomeres within the wild-type size range, it nevertheless sustains telomeres when they shorten to a length at which they become fusogenic. The enzyme also retains the capability to extend telomeres in plants depleted of Ku70. The Ku complex inhibits telomerase in Arabidopsis, and it also protects telomeres by preventing their exonucleolytic degradation and homologous recombination (Riha and Shippen 2003; Kazda et al. 2012). Together, our data indicate that telomerase derived from the 35S:TERT allele only accesses telomeres that are not fully capped. The molecular mechanism underlying the impaired telomerase performance in the 35S:TERT lines is not known. It may be due to a reduced level of telomerase that is apparent from in vitro activity assay in comparison with wild type and ACT2:TERT plants. Notably, while the 35S:TERT construct lacks introns, the ACT2:TERT construct contains an intron in the 5′ UTR. It is well-established that the presence of introns enhances transgene expression in plants (Laxa 2017), and therefore, the intronless 35S:TERT construct may produce a suboptimal amount of TERT. Alternatively, the 35S promoter may not be efficiently active in tissues where telomerase is most needed, such as rapidly growing embryos (Sunilkumar et al. 2002).

Although the hypomorphic TERT allele could not restore telomeres to wild-type length, it was capable of maintaining telomeres at a shorter length set point. This is evidenced by the fact that 35S:TERT plants can survive for 18 generations, and once the new shorter telomere length set point is established around G10/T6, plants maintain that telomere length for multiple generations. Thus, while the hypomorphic 35S:TERT allele is sufficient to indefinitely prolong plant lifespan, it can only sustain telomeres close to their minimal functional length. Additional support for this conclusion comes from the observation that in late-generation 35S:TERT lines deficient in the POT1A processivity factor lethality is immediate. We conclude that critically short telomeres in these lines cannot be maintained by telomerase and the plants succumb to telomere dysfunction.

The telomeres of late-generation 35S:TERT lines were maintained at a bimodal size distribution with arm-specific lengths corresponding to either 400 (average 421, arms 1L, 1R, 3L, 5L, and 5R) or 900 bp (910, arms 2R and 4R). FISH analysis unexpectedly demonstrated the shorter telomeres were no more prone to fusion than longer telomeres, implying that, despite their longer length, the capping status of the 2R and 4R telomeres is similar to other arms. Interestingly, the 2R and 4R telomeres are also maintained at longer lengths in wild-type plants expressing normal TERT. Hence, cis-regulatory elements on these arms may not only modulate capping status, but also telomere length. A unique feature of the 2R and 4R telomeres is the immediate proximity of abundantly expressed protein-coding genes (Vrbsky et al. 2010), indicating that transcription of canonical transcripts may influence the status of neighboring telomeres. We note that regulation of telomere length by cis-elements has been reported in humans (Britt-Compton et al. 2006).

Despite extensive analysis of chromosome-end protection in yeast and mammals, the relationship between the length of a telomere and its ability to serve as a functional cap on the DNA terminus remains enigmatic. Previous efforts to define the minimal functional length of telomeres have relied on the identification of the shortest telomeres in a population of cells undergoing telomere crisis (Heacock et al. 2004, 2007; Capper et al. 2007; Xu and Blackburn 2007). A drawback to this approach is that cannot assess whether very short telomeres were captured as they were being processed by DNA repair machinery, or were the result of compromised checkpoint pathways. Other experiments have measured the amount of telomeric DNA retained at cloned chromosome fusion junctions, but in these cases, it is impossible to determine telomere length prior to fusion (Heacock et al. 2004, 2007). In contrast, in 35S:TERT lines all telomeres are maintained at a length sufficient to confer long-term survival, but insufficient for buffering any additional loss of telomeric repeats.

For human cells, previous estimates of the minimal functional telomere size are between 42 (Xu and Blackburn 2007) and 72 nt (Capper et al. 2007), lengths which are still sufficient for binding at least one TRF1 or TRF2 molecule. These observations, along with the fact that 72 nt of telomeric DNA bound by TRF2 could inhibit NHEJ in vitro (Bae and Baumann 2007), have led to the proposal that the minimal functional length is defined by the ability to bind TRF2. Similarly, yeast cells continue to grow and divide until telomeres reach less than 100 bp (Forstemann et al. 2000). Our results argue that the minimal functional length in Arabidopsis is significantly longer at approximately 400 bp, consistent with previous analysis of telomere fusion junctions reporting a minimal functional length of 360 bp (Heacock et al. 2004). While several telomere-specific binding components have been described for Arabidopsis (Song et al. 2008; Surovtseva et al. 2009; Schrumpfova et al. 2014), the limited biochemical data available for these factors suggest the binding sites are similar to the yeast and mammalian telomere proteins, and hence much smaller than 400 bp. In contrast, 400 bp is the approximate length required for the circularization of double-stranded DNA. Experiments on naked DNA have suggested that DNA molecules with lengths of roughly 300–400 bp are optimal for circular ligation (Shore et al. 1981). In addition, in vitro experiments with TRF2 revealed that telomeric tracts of 500 bp are sufficient to form t-loops (Griffith et al. 1999; Stansel et al. 2001). Although the length of looped DNA in a t-loop is proportional to total telomere length, the loops in the 500 bp artificial constructs averaged 338 bp, very close to the minimal telomere length detected at multiple chromosome arms in this study. Therefore, we postulate that formation of a secondary structure such as a t-loop is required for proper capping of chromosome ends, and thus defines the minimal functional length for telomeres in Arabidopsis.

Data availability

Strains and plasmids are available upon request. Supplementary files available at figshare: https://doi.org/10.25386/genetics.15042714. Supplementary file contains Supplementary Figures S1–S4 that show crossing schemes used to generate material described in this study and picture of 35S:TERT plants.

Acknowledgments

The authors acknowledge Plant Sciences Facility at Vienna BioCenter Core Facilities (VBCF), and the Plant Sciences Core Facility of CEITEC MU for support with plant cultivation.

Funding

The work was supported from the European Regional Development Fund-Project “REMAP” (No. CZ.02.1.01/0.0/0.0/15_003/0000479), by the Czech Science Foundation (grants 18-20134S and 19-21961S), by EMBO Installation Grant (1304130933), by the Austrian Academy of Sciences and by the National Institutes of Health (GM R01-GM065383). Microscopy was performed in the core facility CELLIM of CEITEC supported by MEYS CR (LM2018129 Czech-BioImaging).

Conflicts of interest

The authors declare that there is no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Strains and plasmids are available upon request. Supplementary files available at figshare: https://doi.org/10.25386/genetics.15042714. Supplementary file contains Supplementary Figures S1–S4 that show crossing schemes used to generate material described in this study and picture of 35S:TERT plants.

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