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.