No end in sight, mysteries of the telomeric variation in plants

The telomere paradox: conserved function but rapid molecular evolution

One of the most fascinating phenomena in evolutionary biology is the rapid evolution of genes with conserved functions across the tree of life. Because the cellular and organismal development processes are highly conserved across eukaryotes, a naive evolutionary expectation is that the genes involved in those processes would also be under high selective constraint and evolve extremely slowly. However, we now know that evolutionarily young genes can rapidly acquire crucial viability functions and even evolutionarily old genes can have unexpected levels of rapid evolution within specific lineages (Talbert et al. 2004). These studies have led to novel insights of function and evolution in molecular systems that are universally important for almost all organisms.

The telomere is one such conserved molecular complex but the adaptive evolutionary history of telomeres has received little attention. Telomeres are nucleoprotein complexes that cap chromosome ends for protection and proper elongation (Fulcher et al. 2014). Because of this vital function, one might expect the components of the telomere would be highly conserved and constrained from evolutionary change. On the surface this seems to be the case, for instance, 27 out of the 29 protein subunits of the mammalian telomerase, a ribonucleoprotein enzyme that ensures the proper synthesis and maintenance of chromosome ends (Osterhage and Friedman 2009), have a homolog in plants (Procházková Schrumpfová et al. 2019), indicating a common ancestral origin for the telomerase enzyme and deep evolutionary conservation. However, telomere protein sequences are highly diverged between species and some of these evolutionary changes were fixed by adaptive evolution (Saint-Leandre and Levine 2020). In addition, the noncoding RNA sequence that is crucial for the de novo synthesis of the telomeric repeats is extremely diverse in size, sequence, and structure (Podlevsky and Chen 2016). What explains the rapid rate of sequence evolution but functional conservation for the molecular components of the telomere?

In this perspective we introduce the fascinating variation observed in plant telomeres. Rapidly developing genomic technology and the availability of abundant genomic data have revealed that plant telomeres are surprisingly variable compared to telomeres of other eukaryotic clades. We argue that understanding the biological basis of plant telomere variation is an important direction for future plant telomere research that will provide new insights on eukaryotic telomere function and evolution.

Plant telomere sequences are highly diverse

All vertebrates have an identical telomere repeat sequence (TTAGGG)_n (Meyne et al. 1989), indicating extreme purifying selection has prohibited evolutionary changes. But in plants, telomere sequences deviate from this ultra-constrained evolutionary history (Podlevsky and Chen 2016; Shakirov et al. 2022). Arabidopsis thaliana was the first plant species to have its telomere sequence decoded (TTTAGGG)_n and subsequent studies revealed many plant species to also harbor the same Arabidopsis-type telomere sequence. But there are multiple lineages deviating from the Arabidopsis-type sequence and these are categorized into two sequence-types (Peska and Garcia 2020): (1) extension/contraction of thymine or guanine nucleotides (e.g. (TTTTTTAGGG)_n in Cestrum elegans), and (2) insertion/substitution of non-(thymine/guanine) nucleotides (e.g. (TTCAGG)_n and (TTTCAGG)_n in the Genlisea group). Extreme deviations, for instance within the Allium group (CTCGGTTATGGG)_n, display minimal resemblance to the Arabidopsis-type sequence.

It is unclear what evolutionary pressure has driven the diversification of plant telomere sequences. One hypothesis could be linked to molecular activity of the telomerase. Telomerase is comprised of two main components, the telomere reverse transcriptase (TERT) that synthesizes the telomere and the telomerase RNA (TR) that acts as a template (Shakirov et al. 2022) (Figure 1). Alignment between the telomere DNA and TR is crucial for proper extension of the telomere and the plant species that have diverged from the Arabidopsis-type telomere DNA sequence have also evolved the same changes in the TR template sequence (Fajkus et al. 2021). Why the TR sequence has evolved those changes is not known. Hints, however, can be gained from mammalian studies, where mutations in TR can influence the enzymatic activity of TERT (Chen and Greider 2003). Whether the evolution of plant telomere sequences is linked to telomerase activity will need further study.

Figure 1.

Schematic representation of the plant telomerase reaction cycle (Podlevsky and Chen 2012; Peska and Garcia 2020). The telomerase catalytic unit consists of the Telomerase Reverse Transcriptase (TERT) and the Telomerase RNA subunit (TR) represented by grey and purple color. Telomere repeat sequences are synthesized at chromosomal ends and added on to the existing telomeric DNA sequence (blue). The Arabidopsis-type telomere sequence consists of seven nucleotides (red) and is the most commonly observed telomere repeat sequence across the plant kingdom. The proper binding between the TR and the telomeric DNA sequence facilitates TERT to synthesize new telomere repeat sequences, using the intrinsic RNA template sequence of the TR (orange). The telomerase has a unique property of translocating the template after reaching the end of the synthesis and reopening the template RNA sequence for further telomere DNA synthesis. This process of nucleotide addition and template translocation results in longer telomere repeats at chromosome ends. Recently, TERT has been associated with natural telomere length variation in A. thaliana (Choi et al. 2021), suggesting the activity of the telomerase may underlie the possible mechanism of telomere length homeostasis in plants.

The rapidly evolving telomere binding proteins

Since telomere sequences are highly variable in plants, a natural question that arises is whether the proteins that bind to the telomere are also rapidly evolving. Very few studies in plants, however, have investigated the evolution of telomere binding proteins. Protection of Telomeres 1 (POT1) is part of a nucleoprotein complex that binds and protects chromosome ends (de Lange 2018). POT1 is a highly conserved telomere protein found in almost all eukaryotes and interestingly in plants the POT1 gene has duplicated multiple times (Beilstein et al. 2015). In A. thaliana, the POT1 paralogs are highly diverged (~50% amino acid similarity) from each other (Shakirov et al. 2005), and the divergence was partly driven by positive selection to enhance the binding of the POT1 paralog with other chromosome end protecting proteins. (Beilstein et al. 2015). Importantly, investigating the evolution of the POT1 gene has led to a deeper understanding of telomere gene functions and the interaction among the molecular components of the telomere.

Evolutionary analysis of telomere binding proteins in animals have also discovered rapid evolution in several telomere genes (Saint-Leandre and Levine 2020). What evolutionary driver could explain the rapid evolution of telomere binding proteins in plants and animals? One hypothesis, the telomere conflict model (Saint-Leandre and Levine 2020), postulates the rapid evolution arising from an evolutionary arms race with subtelomeric transposable elements. Another model postulates adaptation to biotic and abiotic stress as the evolutionary driver of telomere protein adaptive evolution (Shakirov et al. 2022). Many telomere proteins have pleiotropic roles, in particular responding to oxidative stresses (Barnes et al. 2019), but the evolutionary link between the telomerase and stress response has not been tested. It’s also possible the telomere sequence and telomere binding protein are subjected to the same evolutionary driver in plants and relates to the molecular activity of the telomerase. Clearly, there is a need for exploring multiple models of telomere binding protein evolution in plants, which we predict will uncover novel functional discoveries of telomerase.

Why do telomere lengths vary in plants?

Misregulation of telomere length jeopardizes genomic stability and results in highly deleterious consequences for any organism. These deleterious fitness consequences might suggest telomere length would be under strong evolutionary and molecular constraints. However, telomere length varies significantly among species (e.g. A. thaliana telomere length is ~3 kbp while in tobacco 150 kbp) and even between individuals within the same species (e.g. A. thaliana ecotypes can differ up to 10 fold) (Shakirov et al. 2022). Quantitative genetics and functional validation studies have shown that plant telomere length variation is a heritable complex trait and controlled by natural genetic polymorphisms (Abdulkina et al. 2019).

The highly variable telomere lengths in plants are intriguing, but also perplexing. What is the functional consequence of variable telomere lengths and is natural selection involved in shaping the length variation? A popular model to explain telomere length variation hypothesizes telomere length to be an evolutionary outcome of life-history tradeoffs (Young 2018). In mammals, the association between lifespan and telomere length suggests telomere length has a role in aging and is subject to natural selection. But aging in plants is fundamentally different from mammals (Watson and Riha 2011), hence various telomere length evolution models that were developed based on animal systems are not completely applicable for plants. In addition, there are several life-history traits that fundamentally differ between plant and animals, for instance the annual versus perennial life-history variation within the same species (Friedman 2020) or the sessile nature of plants place the environment as a strong factor that controls many plant traits. Hence, how plant telomere lengths can vary across space and time, can vary at the population-level, and can vary at the tissue-specific level within and between individuals are all largely unanswered questions that could lead to a unique understanding of plant telomere biology. It is clear there is a need for considering alternative models to explain telomere length variation for plants. Recent studies have discovered links between telomere length and plant reproductive fitness and physiological traits (Choi et al. 2021; Campitelli et al. 2022). Importantly, these studies have shown variation in plant life-history strategies can potentially influence the evolution of the telomere through natural selection. What mechanism is involved in the link between the telomere length and life-history trait is unknown, and elucidating the molecular link will uncover novel insights of plant adaptation and life-history trait evolution.

Future outlook

In this perspective we present more questions than answers regarding plant telomeric variation, but it also highlights the exciting future research that could be conducted to understand plant telomere evolution and function. The plant telomere biology field has a deep history of molecular and biochemical research (Shakirov et al. 2022). Combining this rich functional genetic knowledge of plant telomeres with evolutionary genetic approaches would be a powerful tool to understand the biology underlying the diversity of telomeric variation in plants. Importantly, understanding plant telomeres could have far reaching relevance especially for understanding the evolution of all eukaryotic telomeres.