4. Discussion
In this work, we used epiGBS and RNAseq to study the drivers of intraspecific variation for heavy metal tolerance in the copper mossS. cataractae , and to provide new insights into the molecular mechanisms used by bryophytes to deal with heavy metal toxicity. Our results showed that (i) genetic differentiation did not explain phenotypic differences in Cd and Cu tolerance in S. cataractae ; (ii) heavy metal exposure induced some DNA methylation changes. These, however, were inconsistent within treatment groups leading to an increase in methylation variation in Cd- and/or Cu-treated plants in plants from the most tolerant populations; (iii) single cytosine methylation changes (SMPs) tended to hypomethylation in the most tolerant populations and to hypermethylation in the least tolerant ones; (iv) plants from both the more and the less tolerant populations constitutively expressed multiple genes involved in heavy metal tolerance in the absence of Cu in the laboratory. The most tolerant plants, however, expressed more genes involved in the alleviation of heavy metal stress under control conditions, whereas the less tolerant invested more in growth; and v) upon acute Cu stress, both populations inhibited the expression of these protective genes to nearly equivalent levels. The magnitude of this inhibition was greater in the more tolerant population. Altogether, these results suggest that chronic exposure to different levels of heavy metals in the field is associated with non-genetically-based intraspecific differentiation for heavy metal tolerance in S. cataractae . At the molecular level, this phenotypic differentiation was reflected in a greater constitutive investment in protective mechanisms (e.g. ROS scavenging enzymes, heavy metal transporters, protein chaperones) in the most tolerant population, but maintaining an equivalent response into an energy conservative state when facing an acute Cu stress event in the laboratory.
Scopelophila cataractae is called “copper moss” due to its high affinity for heavy metals, especially Cu. In fact, its worldwide geographical distribution mostly matches that of Cu-enriched substrates72,103,104, and its growth has been consistently favored in the presence of Cu in the laboratory105,106. Yet, previous studies have shown that this metal compromises the species’ growth upon exposure to high concentrations of bioavailable Cu 68,105, and that this acute Cu stress can uncover novel intraspecific variation in Cu tolerance 68. In our previous study, we reported phenotypic differentiation for Cd and Cu tolerance among plants from the same populations studied here: growth of plants from populations that originated from contaminated microhabitats was increased (Sc2) or unaffected (Sc3) in metal-treated compared to control plants, whereas growth of plants from populations that originated from contaminated microhabitats (Sc3, Sc4) was reduced 68. These differences were observed after clonally propagating all plants in control conditions for months, which should have eliminated any potential carryover environmental effects from the field. Taken together, these findings supported a role of natural selection in generating locally adapted genotypes among the four populations. Nonetheless, our current results showed no evidence of genetic differentiation among the populations studied, at least in the portion of the genome interrogated with the epiGBS technique. The lack of genetic differentiation could be explained by the movement of gametophore fragments and asexual propagules down the slope of the field site (⁓500 m long). Thus, our analysis suggested that this phenotypic differentiation could have arisen through mechanisms other than DNA sequence variation.
Epigenetic variation has emerged in the past decades as a potentially important source of phenotypic variation and differentiation in plants107–113. Thus, we assessed whether DNA methylation contributed to the differences in tolerance observed in our common garden experiments. We did not find evidence for epigenetic differentiation among populations when comparing mean methylation levels across all cytosines, and the effect of Cd and Cu exposure on individual cytosine methylation levels was rather limited compared to response to other stresses in other plants (e.g. 114). We propose three possible causes for this limited response. First of all, environmental stress can increase inter-individual methylation variation in plants 115. This type of inconsistency would limit our capacity to find clear directional changes between experimental groups. In this study, we found a significant increase in DNA methylation variability in plants from Sc2 in response to both metal treatments, and a slight increase in variability in plants from Sc1 and Sc3 in response to Cu, and from Sc4 in response to Cd. Although this effect needs to be further validated with a greater number of replicate samples per treatment group, these results support a potential population-specific epigenetic response to heavy metal exposure. Even though it has not been experimentally proven, such stress-induced inter-individual epigenetic variation could contribute to increased population-level phenotypic variation and help plant populations to deal with environmental stress 116.
On the other hand, the limited epigenetic response found here could also be due, in part, to fundamental differences between the epigenomic landscape of bryophytes and angiosperms, combined with the technical characteristics of the epiGBS technique. Overall, bryophyte genomes are much less methylated than angiosperm genomes (117,118, but see 119 for differences among stages of the bryophyte life cycle), and cytosine methylation tends to be segregated away from genic regions 120. For example, in contrast to flowering plants, gene body methylation (gbM) is absent in most genes of the model moss P. patens 117 and the model liverwort Marchantia polymorpha 118 (but see119). The epiGBS technique interrogates a biased subset of the genome, as the restriction enzyme that we used, PstI, preferentially targets coding regions 71. Together with the low gbM found for other bryophytes, this would explain the very low DNA methylation levels found here, and the limited response to heavy metal exposure within the interrogated genomic regions. Further, our epiGBS fragments did not overlap with our DETs so we had no information about the methylation status of those specific transcripts (as reported in 121,122. Finally, it is also possible that epigenetic mechanisms other than DNA methylation, or even point mutations located in genomic regions other than that interrogated here, could determine the phenotypic differences observed here. For example, the population-specific expression of proteins involved in chromatin reorganization (only differentially expressed in the more tolerant population), like the histone modifying enzyme histone deacetylase 6 and its associated WD-40 repeat-containing protein MSI1, the histone chaperone peptidyl-prolyl cis-trans isomerase FKBP53, and the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5, points towards a potential role of histone modifications in heavy metal stress tolerance in S. cataractae .
Some authors pointed out that changes in DNA methylation in response to heavy metal stress could be due to a side effect of metal-induced ROS production 42. However, some of the patterns observed here indicate otherwise. First, exposure to Cd induced more methylation changes despite the lack of a significant increase in oxidative damage in any of the populations studied (Fig. 3H in 68). Second, the effect of Cd and Cu on DNA methylation was population-specific, with more methylation variability induced in one of the most tolerant moss populations (Sc2). Third, more cytosines were hypo- than hypermethylated in plants from the most tolerant populations (Sc2, Sc3), while we observed the opposite trend in the less tolerant ones (Sc1, Sc4). Finally, when compared within each population, both metals induced numerous common DMPs. Altogether, these findings suggest that the DNA methylation changes observed here were not random, as expected if generated through metal-induced ROS production, but likely dependent on the previous stress experience of the plants.
The set of DETs found in this study provided us with valuable information about the adaptive mechanisms to deal with chronic Cu exposure developed by S. cataractae in natural conditions. For example, 16.2% and 23.6% of all DETs in Sc3 and Sc4 respectively, were involved in cellular protein homeostasis including protein and RNA biosynthesis/degradation, and protein repair. Heavy metals can disrupt protein folding and cause aggregation 10,123–125. Aggregation and accumulation of unfolded proteins triggers the unfolded protein response (UPR) pathway, which enhances protein folding and degradation, and reduces protein production under stress conditions123. Multiple components of the UPR pathway were constitutively expressed in S. cataractae , including components of the calnexin/calreticulin complex as well as protein chaperones involved in protein folding, the Ubiquitin-26S proteasome proteolytic system, and multiple translation and elongation initiation factors involved in protein synthesis.
Similarly, a considerable number of constitutively expressed transcripts were involved in direct or indirect ROS detoxification (e.g. glutathione S-transferases, L-ascorbate peroxidases, superoxide dismutases), cellular signaling, trafficking and metal transport (e.g. calmodulins, clathrins, ras-related proteins, V-type proton ATPases, zinc finger proteins, ABC transporters), fatty acid oxidation enzymes related to the jasmonic acid biosynthetic pathway (e.g. peroxisomal acyl-CoA oxidase, 3-ketoacyl-CoA thiolase), and purine metabolism (adenosine kinases, inosine-5’-monophosphate dehydrogenase). Polyamine metabolism, including enzymes like ornithine decarboxylase, S-adenosylmethionine synthase, and spermidine synthase, was also associated with the adaptive response to Cu in S. cataractae . These organic compounds have been shown to increase heavy metal tolerance in plants through ROS detoxification126–128 and decreased metal accumulation126,129. The functioning of all these stress alleviation mechanisms is energy and carbon consuming, which explains the increased constitutive expression of enzymes involved in energy production like the glycolysis, penthose-phosphate, and tricarboxylic acid pathways 130.
In this study, we found evidence of potentially non-genetically-based phenotypic differentiation for heavy metal tolerance in bryophytes. Our results indicated that chronic exposure to high concentrations of metals in natural conditions led to constitutive overexpression of genes that contribute to heavy metal stress tolerance in several ways (e.g. regulation of cellular protein homeostasis, ROS detoxification, metal transport). Importantly, plants that had grown under higher stress conditions in the field were more tolerant to acute Cu stress events like that used in our experiments, possibly due to their greater capacity to decrease production of energetically expensive products as a strategy to reduce energy consumption in a situation in which the ability of these mechanisms to provide protection against the stress would be overwhelmed. Based on the higher epigenetic response of the most tolerant populations (greater changes in DNA methylation and constitutive expression of chromatin remodeling proteins) we propose that their increased capacity to regulate the expression of heavy metal stress-related genes could have an epigenetic basis that we could not detect with our approach. This work thus serves as a proof of concept for future studies which should focus on the targeted evaluation of the regulatory mechanisms presented here, and evaluate their relative importance in heavy metal stress tolerance in bryophytes.