1. Introduction
Heavy metals are amongst the most hazardous pollutants to living
organisms due to their high toxicity and persistence in the environment1–4. Multiple studies showed that heavy metals damage
the cell membrane system and negatively affect plant development,
growth, and reproduction 5–8, generate reactive
oxygen species (ROS) 9,10, and may alter the structure
of essential plant biomolecules 11–14. Still, some
species have adapted to living in heavy metal-enriched environments, and
are able to survive and accumulate concentrations of heavy metals that
greatly exceed the tolerance limits of most other plants15–17.
Metal tolerant species often rely on the enhanced expression of genes
related to heavy metal homeostasis – i.e. transport, chelation, and
sequestration of metals – relative to closely related non-tolerant ones18–23. Thus, constitutive overexpression of heavy
metal homeostasis-related genes (e.g. ABC metal transporters and enzymes
involved in oxidative stress relief 20,21) often
underlies differences between tolerant and non-tolerant plants (but see24,25 for the contribution of gene copy number
variation). Yet, the regulatory mechanisms determining the differences
in gene expression are still poorly understood. Gene expression in
eukaryotic genomes is, in part, tightly regulated by epigenetic
mechanisms, the set of chromatin modifications that control chromatin
structure, and thus, the accessibility of the transcriptional machinery
to genes 26,27. In plants, epigenetic mechanisms, such
as DNA methylation, histone modifications, and small non-coding RNAs,
are known to regulate gene expression changes in response to
developmental and environmental cues (e.g. 28–33).
Based on this evidence, epigenetic mechanisms are currently considered
potentially important regulators of plants response to stress.
So far, experimental evidence on the contribution of epigenetic
mechanisms to plant heavy metal tolerance has shown that DNA methylation
could have a direct protective role. For example, metal tolerant plants
like Arabidopsis halleri - in response to Cd - and Noccaea
caerulescens - in response to Ni showed hypermethylation of DNA and no
heavy metal-induced DNA damage 34,35. Non-tolerant
species like the moss Physcomitrium patens , however, showed
overall DNA hypomethylation and significant signs of DNA strand breaks
in response to Mn 36. Studies employing anonymous DNA
methylation markers or immunolabelling techniques reported DNA
hypermethylation in response to exposure to high Al levels in wheat
(Triticum aestivum ) 37 and maize38, to Pb in maize (Zea mays)39, to Cd in Posidonia oceanica40 and Nicotiana benthamiana41, and to Cd, Ni and Cr in Trifolium repensand Cannabis sativa 42. In contrast, DNA
hypomethylation was reported in response to Cd in rapeseed
(Brassica napus ) 43 and the red algaGracilaria dura 44, to Al in triticale (xTriticosecale ) 45,46, to Cu and Zn inPopulus alba 47, and to low Al levels in wheat37. Epigenetic mechanisms like DNA methylation can
thus be modified in response to heavy metal exposure.
More targeted studies have demonstrated that epigenetic changes induced
by heavy metals can lead to changes in the expression of genes involved
in heavy metal tolerance. For example, work with methylation mutants
confirmed that CG and H3K9me2 hypomethylation upstream of the metal
responsive metallochaperone, OsHMP , led to its overexpression and
enhanced Cd tolerance in rice (Oryza sativa )48. Similar DNA and histone hypomethylation led to
increased expression of a Cd tolerance factor (OsCTF ) that
enhanced Cd tolerance in rice 49. Other studies used
transformation of Arabidopsis thaliana with an
S-adenosylmethionine synthetase (SAMS) gene, which encodes an enzyme
that catalyzes the biosynthesis of SAM, the main methyl group donor for
DNA methylation in plants. This transformation conferred Al as well as
Cd, Cu, and Zn tolerance to this species 50. The
overexpression of specific heavy metal detoxification transporters in a
Pb, Cd, and Zn tolerant variety of wheat has been linked to CG
hypomethylation in their promoter region in response to all three metals51. Prolonged Cr exposure during selection of a
Cr-tolerant strain of the green alga Scenedesmus acutus led to
new DNA methylation and expression patterns in protein-coding genes
involved in Cr tolerance. MicroRNAs also seem to contribute to plant
response to Cr stress in rice 52, Cd in rice53,54 and rapeseed 55,56, and Al,
Cd, and Hg in Medicago truncatula 57. Finally,
some studies showed that DNA methylation changes induced during heavy
metal exposure can be transmitted to the offspring in rice58,59 and Arabidopsis 60potentially leading to heritable changes in gene expression patterns.
Collectively, these data suggest that epigenetic regulation could play
an important role in the plant response to heavy metal stress. However,
these studies are based on a phylogenetically restricted set of species,
largely from the Brassicaceae and Poaceae (i.e. crops and
model plants like Arabidopsis ), which may not be representative
of plants as a whole.
Bryophytes, the sister group to the vascular plants, have long been
known for their capacity to tolerate high concentrations of heavy metals
in their tissues 61. Most of what is presently known
about their tolerance mechanisms comes from biochemical and
physiological studies. Mechanisms such as retention of heavy metals in
extracellular wax-like substances 62, sequestration in
the cell wall 63–66, preferential accumulation in
specific parts/organs of the leafy plant – i.e. gametophore67–69, or the activation of the ROS scavenging
machinery and induction of metal-chelating proteins70, have been reported to mitigate heavy metal stress
in bryophytes. However, we lack data concerning the molecular
underpinnings of these anatomical or physiological phenomena. Filling
this gap will enable us to test whether the land plants employ conserved
pathways to tolerate heavy metal stress, or whether each lineage has
evolved novel mechanisms.
In this study, we used a reduced representation bisulfite sequencing
technique (hereafter epiGBS 71), and RNA sequencing
(RNAseq) to quantify the effect of Cd and Cu exposure on DNA methylation
and gene expression in the copper moss Scopelophila cataractae(Mitt.) Broth, a species with high affinity for heavy metal-, especially
Cu-enriched substrates 72. We generated laboratory
cultures, in control or test (either Cd or Cu) conditions, of samples
from four field microsites (maximum distance between microsites
<500 m) within a former copper mine. Using this experimental
set up, we previously found that phenotypic differences for Cd and Cu
tolerance (defined as the ability to maintain vegetative growth in metal
stressed vs. control conditions, sensu 73) in
this species were maintained in the laboratory 68. The
more tolerant plants were found in the center of the mine growing in
microhabitats devoid of any vegetation and exposed to higher metal
concentrations, i.e., harsher environmental conditions, while less
tolerant plants were from the mine edges, located in milder, less
contaminated microhabitats. Here we used epigenetic and transcriptomic
analyses to identify the mechanisms driving intraspecific
differentiation for heavy metal tolerance in S. cataractae .
Specifically, we addressed the following questions: (1) What are
mechanisms driving intraspecific differentiation for heavy metal
tolerance in S. cataractae ? (2) Does heavy metal exposure affect
DNA methylation? (3) What are the main functional changes in S.
cataractae in response to heavy metal exposure? (4) Is there any
association between methylation and expression changes? Our results
provided evidence for non-genetically-based intraspecific phenotypic
differentiation for heavy metal tolerance in this species with the more
tolerant plants investing more in constitutive protection mechanisms and
being more efficient in entering a conservative state when faced with
acute Cu stress.