Role of DNA methylation in the chromium tolerance of Scenedesmus acutus (Chlorophyceae) and its impact on the sulfate pathway regulation
Graphical abstract
Introduction
DNA methylation, generally referred as an addition of a methyl group onto the C5 position of cytosine to form 5-methylcytosine (5-mC), is a conserved epigenetic mechanisms that contributes to modulate gene expression without altering the DNA sequence. DNA methylation, together with other epigenetic marks, such as the histone code (post-translational modifications in histone proteins) and non-coding RNAs (ncRNAs), contributes to establish the “epigenome” that, unlike the stable genome, is dynamically altered during developmental processes and in response to different environmental factors and/or stresses [1,2]. In plants, DNA methylation occurs in symmetric CG and CHG contexts but also in asymmetric CHH contexts (H = A, C, or T) [3]. Albeit DNA methylation has been studied in many plant and animal species and also in unicellular eukaryotes [[4], [5], [6], [7], [8], [9]], it is still poorly understood in microalgae [[10], [11], [12]]. Flowering plants have generally high methylated genomes and extensive variation has been found between species, reflecting their evolution, both in the level and in the patterns of DNA methylation with the greatest variations observed in non-CG context [13]. In contrast, in Chlamydomonas reinhardtii a very low DNA methylation such as 5.4%, 2.6%, and 2.5% respectively in the CG, CHG and CHH contexts, has been detected [14].
DNA methylation is crucial for various biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation [15,16]. Dynamic DNA methylation is also fundamental in the response to both external and internal stimuli thus playing a relevant role in plant diversity and development [[17], [18], [19], [20], [21]].
Methylation profile can change in response to environmental stressor and it has been widely investigated in plants, but few data exist in algae [22]. It is known that abiotic stress, including heavy metals, drought and cold, can trigger DNA methylation changes at a genome-wide scale in plants [23]. In fact, stresses can induce changes in gene expression through hypomethylation or hypermethylation of DNA [24], especially in genome regions showing an adaptive significance during stress responses and which can direct genome evolution [25]. GC methylation has different effects on gene expression: in promoters and transposable elements (TEs), is correlated to gene and transposon silencing, while in gene body often interfere with gene regulation and is presumably associated to the suppression of spurious transcription from cryptic promoters [14,20,26]. Moreover, in presence of a persistent stress an epigenetic mechanism could establishes a DNA methylation-dependent stress memory, mainly due to GC-rich sequences methylation [[27], [28], [29]]; this mechanism ensures the faithfully transfer of the “stress memory” to the offspring [30]. However, epigenetic factors involved in the stress response and their implications in algae remain poorly understood [31,32].
In this context, the aim of the present work is to increase the knowledge on the epigenetic mechanisms and responses to heavy metals in microalgae. Our attention has been focused on stress induced by chromium (VI), which has been recognized as one of the most toxic and widespread metal in the environment [[33], [34], [35], [36]]. To achieve this aim we used two strains of the unicellular green alga Scenedesmus acutus with different chromium sensitivity: the wild type and a Cr(VI) tolerant strain, previously selected by treating the cells with a sub-lethal concentration of chromium (1 mg Cr(VI) L–1) for a long period (3 months) [37,38]. In this strain the Cr-tolerance was heritable, since the chromium tolerant strain (Cr-t) was able to grow in the presence of Cr(VI) even after prolonged culturing in Cr(VI)-free medium [37,38]. Previous analysis by 5-mC immunocytochemical localization revealed differences between the two strains in control conditions and changes in the 5-mC methylation patterns/level induced by Cr(VI) exposure [39]. These evidences suggested that epigenetic mechanisms could be at the basis of the Cr-tolerance acquisition in S. acutus. The Cr-tolerance could have been established during the selection in Cr (VI) supplemented medium fixed in the algal population, inherited and maintained through the progeny even in absence of the metal. To verify this hypothesis and assess the involvement of some epigenetic information in the transgenerational transfer of the Cr (VI) tolerance, we generated a whole genome bisulfite sequencing (WGBS) in order to analyse methylome of S. acutus Cr-tolerant strain (Cr-t) vs sensible strain (wt). In addition, since previous works suggest a relationship between chromium tolerance and sulfate reductive assimilation pathway in S. acutus [[40], [41], [42]], the methylation of genes involved in S uptake and assimilation was analysed. To this aim, we characterized 30 sulfate pathway related genes and evaluated the expression of the genes that resulted differentially methylated in Cr-t vs wt strain. The role of their differential methylation and expression, as well as their complex regulation network in the sulfate assimilation pathway, are discussed.
Section snippets
In vitro culture of Scenedesmus acutus
Synchronized axenic cultures of the wild-type (wt) and Cr-tolerant (Cr-t) strains [38] of the unicellular green alga S. acutus were maintained in sterile liquid culture medium (US EPA, 1978; pH 7.7 ± 0.1; modified by dissolving in distilled water both micro and macronutrients to obtain a final concentration double of that indicated), in a climate-controlled chamber (23 ± 1 ◦C, 230 μmol m−2 s−1 light intensity irradiance, white-cool fluorescent lamps, 16:8 h photoperiod). The cultures were
Quality control of the Whole Genome Bisulfite Sequencing data
To profile DNA methylation patterns at single-nucleotide resolution in wt vs Cr-t strain, WGBS library constructions and sequencing was performed on three biological replicates per strain. Single-base DNA methylation BS-Seq of these libraries generated up to ∼ 42 million sequencing reads (2 × 150 bp paired end) for each replicate (Table S.2). For each sample, the bisulfite conversion rate (%) determined using the unmethylated λ phage genome, was higher than 98% (Table S.2). After quality
Discussion
In order to give additional information on the involvement of DNA methylation mechanisms in heavy metal tolerance of the freshwater green alga S. acutus, a WGBS was performed in the two strains with different Cr(VI) sensitivity (Cr-t vs wt) of this unicellular microalga. Notwithstanding the studies on stress-responsive epigenomes and transcriptomes are important to understand the evolution of mechanisms related to stress adaptation in photosynthetic organisms, very few data have indeed been
Conclusions
In conclusion, we provide a high-resolution methylation status of individual cytosines throughout the genome of two strains of S. acutus with different sensitivity to Cr (VI). The results bring substantial first information about the S. acutus methylome and show that the two strains have a different 5-mC distribution pattern regard to both methylated local contexts (CG, CHG and CHH) and differentially methylated regions. The significant differences in methylation-mediated gene expression
CRediT authorship contribution statement
Michele Ferrari: Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing. Anna Torelli: Conceptualization, Visualitazion, Writing - review & editing. Matteo Marieschi: Investigation, Resource. Radiana Cozza: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This research was supported by University of Calabria grants (FIL 2017-2019). The authors thank Mrs Simona Lari for the revision of English language of the manuscript.
References (81)
- et al.
Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution
Cell.
(2015) - et al.
Epigenetic control of gene regulation in plants
Biochim. Biophys. Acta - Gene Regul. Mech.
(2011) - et al.
Epigenetics in an ecotoxicological context
Mutat. Res. - Genet. Toxicol. Environ. Mutagen.
(2014) - et al.
Plant stress biology in epigenomic era
Plant Sci.
(2020) - et al.
Epigenetic regulation of stress responses in plants
Curr. Opin. Plant Biol.
(2009) - et al.
Genome instability and epigenetic modification-heritable responses to environmental stress?
Curr. Opin. Plant Biol.
(2011) - et al.
Gene body DNA methylation in plants
Curr. Opin. Plant Biol.
(2017) - et al.
Transgenerational Stability of the Arabidopsis Epigenome Is Coordinated by CG Methylation
Cell.
(2007) - et al.
Chromium(VI) is more toxic than chromium(III) to freshwater algae: A paradigm to revise?
Ecotoxicol. Environ. Saf.
(2010) - et al.
Cyto-histological and morpho-physiological responses of common duckweed (Lemna minor L.) to chromium
Chemosphere.
(2016)
Chromium speciation, bioavailability, uptake, toxicity and detoxification in soil-plant system: A review
Chemosphere.
Hexavalent Chromium Induces Gametogenesis in the Freshwater Alga Scenedesmus acutus
Ecotoxicol. Environ. Saf.
Chromium-induced sexual reproduction gives rise to a cr-tolerant progeny in Scenedesmus acutus
Ecotoxicol. Environ. Saf.
Sulfur starvation and chromium tolerance in Scenedesmus acutus: A possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes
Aquat. Toxicol.
Increase of chromium tolerance in Scenedesmus acutus after sulfur starvation : Chromium uptake and compartmentalization in two strains with different sensitivities to Cr (VI)
Aquat. Toxicol.
The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase
Aquat. Toxicol.
Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis
Cell.
Changes in cysteine and O-acetyl-L-serine levels in the microalga Chlorella sorokiniana in response to the S-nutritional status
J. Plant Physiol.
Establishing, maintaining and modifying DNA methylation patterns in plants and animals
Nat. Rev. Genet.
Regulation and function of DNA methylation in plants and animals
Cell Res.
Epigenetic inheritance in plants
Nature.
Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning
Nature.
Conservation and divergence in eukaryotic DNA methylation
Proc. Natl. Acad. Sci. U. S. A.
Inheritance and Evolution in Primates
Cell.
Trans chromosomal methylation in Arabidopsis hybrids
Proc. Natl. Acad. Sci. U. S. A.
Charting a dynamic DNA methylation landscape of the human genome
Nature.
Single-base methylome profiling of the giant kelp Saccharina japonica reveals significant differences in DNA methylation to microalgae and plants
New Phytol.
A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri
Nucleic Acids Res.
Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum
Nat. Commun.
Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype
Biotechnol. Biofuels.
Widespread natural variation of DNA methylation within angiosperms
Genome Biol.
Conservation and divergence of methylation patterning in plants and animals
Proc. Natl. Acad. Sci.
Variation of DNA methylation patterns associated with gene expression in rice (Oryza sativa) exposed to cadmium
Plant Cell Environ.
Spontaneous epigenetic variation in the Arabidopsis thaliana methylome
Nature.
Transgenerational epigenetic instability is a source of novel methylation variants
Science (80-.)
Dynamics and function of DNA methylation in plants
Nat. Rev. Mol. Cell Biol.
Dynamic DNA methylation in plant growth and development
Int. J. Mol. Sci.
Environmentally responsive genome-wide accumulation of de novo Arabidopsis thaliana mutations and epimutations
Genome Res.
Heat-Induced Release of Epigenetic Silencing Reveals the Concealed Role of an Imprinted Plant Gene
PLoS Genet.
Hyperosmotic stress memory inArabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity
Elife.
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