Dorothy E. Shippen

Key message: Quantitative mass spectrometry of Arabidopsis telomerase uncovered AtLa1, a homolog of ciliate and yeast proteins that promotes telomerase maturation. AtLa1 is essential for telomerase function in vivo, and in vitro it engages the same region of AtTR bound by AtNAP57, homologous to a telomerase accessory from mammals. Striking divergence is evident in the biogenesis pathways and protein complements of telomerase from mammals and single-cell organisms. However, little is known about plant telomerase. Along with catalytic subunit TERT and templating RNA TR, we previously showed Arabidopsis thaliana telomerase is also associated with AtNAP57, a dyskerin homolog essential for mammalian telomerase biogenesis. Here we employ quantitative mass spectrometry (qMS) to uncover additional Arabidopsis telomerase constituents. We report AtLa1 as a new telomerase-associated protein. RNA-IP assays confirmed AtLa1 association with AtTR, while transient RNAi-mediated knockdown of AtLa1 strongly diminished telomerase activity, supporting a functional role for AtLa1 in telomere maintenance in vivo. In vitro binding studies revealed AtLa1 contacts AtTR via the UUU-3′OH and a plant-specific P1a-P1b-P4 three-way junction (TWJ). Notably, the TWJ is also required for AtNAP57 association with AtTR. However, this protein was not detected in our qMS experiment using overexpressed AtTERT and AtTR, perhaps because the purification scheme enriched for RNP assembly intermediates. La-related proteins serve as RNA chaperones and are associated with a wide variety of telomerase complexes. Therefore, we postulate that AtNAP57 and AtLa1 compete for AtTR or bind sequentially during telomerase biogenesis. Further exploration of Arabidopsis telomerase may offer novel insights into telomerase evolution and mechanisms of biogenesis.

Telomeres safeguard the genome, acting as sentinels of oxidative stress and preventing chromosome ends from eliciting a DNA damage response. PROTECTION OF TELOMERES 1 (POT1) is a highly conserved telomere protein, essential for chromosome integrity and telomeric DNA replication. Arabidopsis thaliana encodes two divergent POT1 paralogs: AtPOT1a stimulates telomerase activity, but AtPOT1b function is unknown. Here we show that AtPOT1b modulates reactive oxygen species (ROS) homeostasis. Oxidative stress induces AtPOT1b expression and telomeric accumulation, while AtPOT1b inactivation elevates ROS, increases telomeric and genome-wide oxidation, and causes stochastic telomere length changes. To address how AtPOT1b controls ROS, we report its localization in nuclei and peroxisomes, and association with catalases and peroxidases that enhance ROS scavenging. Impairing AtPOT1b-CAT2 interaction increases ROS accumulation and telomeric oxidation. Moss or human POT1 rescues ROS overaccumulation in Arabidopsis pot1b mutants, but not telomere deficiency in pot1a pot1b mutants, supporting a conserved role for POT1 in modulating ROS homeostasis and genome stability, distinct from canonical telomeric functions.

Summary: Analysis of yeast and mammalian telomerase ribonucleoprotein (RNP) complexes reveals striking divergence in their biogenesis and protein complements. However, little is known about telomerase in plants. In addition to the catalytic subunit TERT and the templating RNA TR, we previously reported Arabidopsis thaliana contains two telomerase accessory factors, AtNAP57, a dyskerin homolog that in mammals is essential for telomerase activity, and the telomeric DNA binding protein AtPOT1a. Both proteins stimulate Arabidopsis telomerase repeat addition processivity. Here we employ quantitative mass spectrometry (MS) to further examine telomerase in Arabidopsis. Unexpectedly, dyskerin and AtPOT1a were not detected in our purified complexes, but AtLa1, an RNA-binding factor that recognizes the UUU-3′OH of RNA Pol III transcripts, was highly enriched. RNA-IP assays confirmed AtLa1 association with AtTR in vivo. RNAi-mediated knockdown of AtLa1 strongly diminished telomerase activity, indicating AtLa1 is required for its function in vivo. In vitro binding studies revealed that AtLa1 contacts AtTR via the UUU-3′OH and a plant-specific P1a-P1b-P4 three-way junction (TWJ). Since the TWJ is also required for AtNAP57 binding, the data suggesting that AtNAP57 and AtLa1 compete for AtTR binding or sequentially associate during RNP during biogenesis. In contrast to AtNAP57, AtLa1 did not stimulate telomerase activity when TERT and TR were assembled in vitro, consistent with function during a different step in telomerase assembly. We conclude Arabidopsis telomerase employs multiple accessory factors utilized by both mammalian and single-celled relatives. Further exploration of Arabidopsis telomerase may offer novel insight into telomerase evolution and mechanisms of biogenesis. Significance of Research: Quantitative mass spectrometry of Arabidopsis telomerase uncovered AtLa1, a homolog of ciliate and yeast proteins that promotes telomerase maturation. AtLa1 is essential for telomerase function in vivo, and in vitro it engages the same region of AtTR bound by AtNAP57, homologous to a telomerase accessory from mammals.

Summary: Plants are critical for sustaining human life and planetary health. However, their potential to enable humans to survive and thrive beyond Earth remains unrealized. This Viewpoint presents a collective vision outlining priorities associated with plant science to support a new frontier of human existence. These priorities are drawn from the International Space Life Sciences Working Group (ISLSWG) Plants for Space Exploration and Earth Applications workshop, held at the European Low Gravity Research Association (ELGRA) conference in September 2024. First, we highlight transformative advances gained from using the ‘laboratory of space’ in understanding how plants respond to gravity and other stressors. Second, we introduce a new crop Bioregenerative Life Support System (BLSS) readiness level (BRL) framework – extending the existing Crop Readiness Level (CRL) – to assist in overcoming challenges to establish resilient, sustainable crop production. Materializing the vision of plants as enablers of space exploration will require innovative approaches, including predictive modeling, synthetic biology, robust Earth‐based analogue systems, and reliable space‐based instruments to monitor biological processes. Success relies upon a unified international community to promote sharing of resources, facilities, expertise, and data to accelerate progress. Ultimately, this work will both advance human space exploration and provide solutions to enhance sustainable plant production on Earth.

Colonization of the Moon and other planets is an aspiration of NASA and may yield important benefits for our civilization. The feasibility of such endeavors depends on both innovative engineering concepts and the successful adaptation of life forms that exist on Earth to inhospitable environments. In this study, we investigate the potential of Raman spectroscopy (RS) in a non-invasive and non-destructive assessment of changes in the biochemistry of plants exposed to zero gravity on the International Space Station and during growth on lunar regolith simulants on Earth. We report that RS can sense changes in plant carotenoids, pectin, cellulose, and phenolics, which in turn, could be used to gauge the degree of plant stress in new environments. Our findings also demonstrate that RS can monitor the efficiency of soil supplements that can be used to mitigate nutrient-free regolith media. We conclude that RS can serve as a highly efficient approach for monitoring plant health in exotic environments.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

NASA envisions a future where humans establish a thriving colony on the Moon by 2050. Plants will be essential for this endeavor, but little is known about their adaptation to extraterrestrial bodies. The capacity to grow plants in lunar regolith would represent a major step towards this goal by minimizing the reliance on resources transported from Earth. Recent studies reveal that Arabidopsis thaliana can germinate and grow on genuine lunar regolith as well as on lunar regolith simulant. However, plants arrest in vegetative development and activate a variety of stress response pathways, most notably the oxidative stress response. Telomeres are hotspots for oxidative damage in the genome and a marker of fitness in many organisms. Here we examine A. thaliana growth on a lunar regolith simulant and the impact of this resource on plant physiology and on telomere dynamics, telomerase enzyme activity and genome oxidation. We report that plants successfully set seed and generate a viable second plant generation if the lunar regolith simulant is pre-washed with an antioxidant cocktail. However, plants sustain a higher degree of genome oxidation and decreased biomass relative to conventional Earth soil cultivation. Moreover, telomerase activity substantially declines and telomeres shorten in plants grown in lunar regolith simulant, implying that genome integrity may not be sustainable over the long-term. Overcoming these challenges will be an important goal in ensuring success on the lunar frontier.

Spaceflight-induced changes in astronaut telomeres have garnered significant attention in recent years. While plants represent an essential component of future long-duration space travel, the impacts of spaceflight on plant telomeres and telomerase have not been examined. Here we report on the telomere dynamics of Arabidopsis thaliana grown aboard the International Space Station. We observe no changes in telomere length in space-flown Arabidopsis seedlings, despite a dramatic increase in telomerase activity (up to 150-fold in roots), as well as elevated genome oxidation. Ground-based follow up studies provide further evidence that telomerase is induced by different environmental stressors, but its activity is uncoupled from telomere length. Supporting this conclusion, genetically engineered super-telomerase lines with enhanced telomerase activity maintain wildtype telomere length. Finally, genome oxidation is inversely correlated with telomerase activity levels. We propose a redox protective capacity for Arabidopsis telomerase that may promote survivability in harsh environments.

Telomere maintenance is a fundamental cellular process conserved across all eukaryotic lineages. Although plants and animals diverged over 1.5 billion years ago, lessons learned from plants continue to push the boundaries of science, revealing detailed molecular mechanisms in telomere biology with broad implications for human health, aging biology, and stress responses. Recent studies of plant telomeres have unveiled unexpected divergence in telomere sequence and architecture, and the proteins that engage telomeric DNA and telomerase. The discovery of telomerase RNA components in the plant kingdom and some algae groups revealed new insight into the divergent evolution and the universal core of telomerase across major eukaryotic kingdoms. In addition, resources cataloging the abundant natural variation in Arabidopsis thaliana, maize (Zea mays), and other plants are providing unparalleled opportunities to understand the genetic networks that govern telomere length polymorphism and, as a result, are uncovering unanticipated crosstalk between telomeres, environmental factors, organismal fitness, and plant physiology. Here we recap current advances in plant telomere biology and put this field in perspective relative to telomere and telomerase research in other eukaryotic lineages.

Chemical modifications in DNA impact gene regulation and chromatin structure. DNA oxidation, for example, alters gene expression, DNA synthesis and cell cycle progression. Modification of telomeric DNA by oxidation is emerging as a marker of genotoxic damage and is associated with reduced genome integrity and changes in telomere length and telomerase activity. 8-oxoguanine (8-oxoG) is the most studied and common outcome of oxidative damage in DNA. The G-rich nature of telomeric DNA is proposed to make it a hotspot for oxidation, but because telomeres make up only a tiny fraction of the genome, it has been difficult to directly test this hypothesis by studying dynamic DNA modifications specific to this region in vivo. Here, we present a new, robust method to differentially enrich telomeric DNA in solution, coupled with downstream methods for determination of chemical modification. Specifically, we measure 8-oxoG in Arabidopsis thaliana telomeres under normal and oxidative stress conditions. We show that telomere length is unchanged in response to oxidative stress in three different wild-type accessions. Furthermore, we report that while telomeric DNA comprises only 0.02–0.07% of the total genome, telomeres contribute between 0.2 and 15% of the total 8-oxoG. That is, plant telomeres accumulate 8-oxoG at levels approximately 100-fold higher than the rest of the genome under standard growth conditions. Moreover, they are the primary targets of further damage upon oxidative stress. Interestingly, the accumulation of 8-oxoG in the chromosome body seems to be inversely proportional to telomere length. These findings support the hypothesis that telomeres are hotspots of 8-oxoG and may function as sentinels of oxidative stress in plants.