Transcriptome profiling of dark-induced bleached S. pistillata
To unravel a telomere transcriptomic signature of stressed samples, we performed RNA-seq profiling of three control and three bleached branches from the D1 experiment. We identified 862 Differentially Expressed Genes (DEG) (adj. p<0.05), including 602 downregulated and 260 upregulated genes (Figure 3A and Supplementary Table 2). In order to identify the DEGs that could be directly controlled by the dark condition, we compared the transcriptomic profile of bleached samples to a previous transcriptome study performed on S. pistillatacolonies during day and night cycles (Ottaviani et al. 2020) (Figure 3B). We found 31 genes in common and differentially expressed in the same direction between the two sets of DEGs (Figure 3B). We considered that the expression of these genes was controlled by the dark condition rather than the stress triggered by a long period in darkness and leading to a bleached state.
Using the telomere-related interactome reported in the Uniprot database, we identified among the DEGs nine genes that could be involved in telomere metabolism in the stressed samples (Figure 3C). One of the two upregulated genes (Gnmt ) was predicted to be Glycine-N methyltransferase involved in DNA methylation, suggesting that the telomere changes triggered by the continuous darkness stress are associated with epigenetic changes. The second upregulated gene (Trpc5 ) is part of a ionic transporter family. Among the downregulated genes, five are known to be involved in cellular growth and tissue remodeling (Cenpf, Plk1, Profilin-2, Mapk6 andPlat ) suggesting a link between the telomere shortening occurring in stressed colonies and their reduced growth rate. We also found thatPot2 , one of the two protections of telomere shelterin ofS. pistillata , was significantly downregulated in bleached samples (Figure 3C). We confirmed, for a subset of genes (seven genes), their significantly expression changes by quantitative PCR analysis both in the samples used for RNA-seq and in biological duplicates using two reference genes with constant expression in control and stressed condition (Trpc2 and Pot1 ) (Figure 3D, Supplementary Table 2 and 3). We confirmed a trend toward downregulation of Pot2 that is not significant due to a single control branch (S1n4) that was expressing a very low level of Pot2 (Supplementary Table 3).
The most significantly enriched (p <0.01) categories of gene ontology terms (GO terms) among the DEGs were related to metabolism, protein homeostasis, biosynthesis and oxidative stress (GO:0006979, GO:0055114) (Figure 4A). When compared with heat induced bleaching transcriptomic studies done on various coral species (Figure 4B-C), we found three common GO terms in the Biological process that are related to growth, proteolysis and macromolecule catabolic process (Figure 4B-C and Supplementary Table 4) (Pinzón, 2015, Seneca and Palumbi. 2015, Traylor-Knowles et al., 2017, Zhou et al., 2017, Li et al., 2021).

Discussion

This study reveals that a reef coral experiencing a prolonged darkness leading to symbiosis disruption and bleaching exhibits signs of telomere dysfunctions. Notably a shortening of its mean telomere DNA length and a decreased proportion of long telomeres as well as the downregulation of the Pot2 gene encoding a putative subunit of the telomere protective shelterin complex in coral. This finding is a new example of the intimate link between stress response and telomere dysfunction. In the context of the massive coral bleaching events due to the extreme rise of sea water temperatures, these results suggest that long term symbiosis disruption can affect host telomere state and should be taken into account to evaluate the fitness of coral reef survivors.
The enrichment of GO terms linked to oxidative stress response could reflect an increase in the production of reactive oxygen species (ROS), which are likely candidates to cause the observed telomere DNA shortening (Barnes et al., 2019). An increased production of ROS is also the cause of heat-induced bleaching in the wild suggesting a potential telomere dysfunction due to oxidative stress in heat-induced bleaching as what we observed in dark-induced bleaching. Since oxidative stress can be both the cause and the consequence of dysregulated telomeres (Jacome-Burbano and Gilson, 2020), bleaching could, through an increased oxidative environment, create a positive loop accelerating telomere dysfunction and favoring diseases. If oxidative stress is well documented to be a major cause of symbiosis breakdown due to an increase in photosynthetic activity (Lesser, 1997, Dias et al., 2019), the cause of the oxidative stress observed here after six months of darkness is not known. It could result from the maintenance of an oxidative stress response responsible for the initial symbiont loss and/or from a continuous hypoxic condition that can increase ROS production (Lewis et al., 2012).
Like in rodents and the nematode C. elegans , the coral genome contains two genes coding for human POT1 orthologs. Interestingly, the second coral Pot gene (named here Pot2 ) seems to have arisen after the divergence of Cnidarian organisms since H. vulgaris exhibits only one Pot1 ortholog. In mammals, Pot1 forms a heterodimeric complex with Tpp1 and has a crucial role in regulating the resection of the telomere DNA overhang and in recruiting telomerase at telomeres (Nandakumar et al., 2012). The host telomere DNA length shortening observed in bleached samples could be explained by the downregulation of Pot2 that would fail to recruit telomerase at long telomeres resulting in an overall decrease in telomere length after six months. Interestingly in another invertebrate that exhibits two Pot proteins, C. elegans telomeres harbor both 5’ C- and 3’ G-rich overhangs respectively bound by Pot2 and Pot1 (Raices et al., 2008). Since the 5’ C-strand overhang can be involved in telomere elongation by recombination (Zhang et al., 2019), it is possible that a function of Pot2 in C. elegans is to regulate telomere elongation by recombination. To investigate whether corals exhibit both types of overhangs and how they are bound by the two Pot proteins could give interesting insights into the mechanisms of telomere DNA regulation in coral.
There are several limits to the conclusion that can be drawn from these results in terms of mechanisms of coral bleaching. First, the bleaching state studied here was triggered by continuous darkness, which is not a classical stressor acting in the wild (Sully et al., 2019; Hughes et al., 2018a). Second, the telomere and transcriptomic response could be more due to long term darkness than to the bleaching per se . Third, the bleached corals were kept alive by external feeding, and the shift from a partial to a complete heterotrophic state modified the coral metabolism and could be responsible for the observed telomere shortening.
The impact of long term darkness was visible in 31 DEGs previously identified in a study on S. pistillata looking at the day-night transcriptome (Figure 3B). When compared with temperature induced bleaching GO term enrichment from studies in several coral species (Acropora aculeus , Acropora hyacinthus , Pocillopora damicornis, Orbicella faveolata ), it appears that the common ones were first mostly linked to metabolism and second more numerous in the study using the closely related Pocillopora damicornis (Li et al., 2021). It would be of interest to test whether the combined effect of temperature and holobiont disruption can have a short term effect on telomere length regulation. It is important to point out that even under a constant feeding regime, bleached corals experienced a delay in growth, an impaired function found in enriched GO terms of different bleaching studies. Shallow corals have co-evolved with their symbionts and this relationship became mandatory as corals can’t properly live without them.
Bleaching recovery is dependent on heat-sensitive to heat-tolerant symbiont turnover, showing the importance of the interplay between symbiont physiology and coral metabolism in survival (Claar et al., 2020). Thus, it would be of interest to investigate whether different symbionts association can lead to different telomere length regulation upon stress.
Nevertheless, the association between telomere DNA shortening,Pot2 downregulation and oxidative stress response in an experimentally controlled darkness situation contributes to our understanding of the telomere changes occurring in coral that have experienced a stress and to the predictions of the impacts of climate change to telomere homeostasis and genome maintenance. Our results suggest that after a bleaching event, the telomere shortening that can persist with time, will affect the long-term health of coral reefs with a higher sensitivity to diseases. Moreover, the inheritability of parental stress bleaching experiences to offspring (Puisay et al., 2020) could be conveyed by telomere dysfunction and impact several generations among the surviving individuals.

Acknowledgement

We acknowledge the coral culture facility at the Centre Scientifique de Monaco and specifically Dominique Desgre as well as Nathalie Técher and Natacha Segonds. The work in EG lab is supported by the ANR CoralGene and the Inserm cross-cutting program on aging AGEMED. We acknowledge the Bioinformatic and Genomic facility platform of the IRCAN institute as well as Gianni Liti’s team for lending its CHEF device to run Southern Blot.