Role of DNA methylation in the chromium tolerance of Scenedesmus acutus (Chlorophyceae) and its impact on the sulfate pathway regulation

Highlights

• Cytosine methylation occurred mainly in the symmetric CG sites in Scenedesmus acutus

• DNA is differently methylated in the chromium tolerant strain of Scenedesmus acutus

• The cytosine methylation status impacts on sulfate uptake/assimilation pathway

• Epigenomic marks the resistance to chromium stress in Scenedesmus acutus

Abstract

DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many researches on DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in algae. Even more the complex mechanisms by which DNA methylation modulates stress in algae are yet largely unresolved, mainly with respect to heavy metal stress, for which in plants, metal- and species- specific responses were instead evidenced. In this work, we performed a comparative Whole-Genome Bisulfite Sequencing (WGBS) on two strains of the green alga Scenedesmus acutus with different Cr(VI) sensitivity. The pattern of distribution of 5-mC showed significant differences between the two strains concerning both differentially methylated local contexts (CG, CHG and CHH) and Differentially Methylated Regions (DMRs) as well. We also demonstrated that DNA methylation plays an important role in modulating some genes for sulfate uptake/assimilation confirming the involvement of the sulfate pathway in the Cr-tolerance. Our results suggest that DNA methylation may be of particular importance in defining signal specificity associated with Cr-tolerance and in establishing new epigenetic marks which contribute to the adaptation to metal stress and also to transmit the epigenomic traits to the progeny.

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.