Transcriptomic Insights into the Effect of Melatonin in Saccharomyces cerevisiae in the Presence and Absence of Oxidative Stress

Abstract

Melatonin is a ubiquitous indolamine that plays important roles in various aspects of biological processes in mammals. In Saccharomyces cerevisiae, melatonin has been reported to exhibit antioxidant properties and to modulate the expression of some genes involved in endogenous defense systems. The aim of this study was to elucidate the role of supplemented melatonin at the transcriptional level in S. cerevisiae in the presence and absence of oxidative stress. This was achieved by exposing yeast cells pretreated with different melatonin concentrations to hydrogen peroxide and assessing the entry of melatonin into the cell and the yeast response at the transcriptional level (by microarray and qPCR analyses) and the physiological level (by analyzing changes in the lipid composition and mitochondrial activity). We found that exogenous melatonin crossed cellular membranes at nanomolar concentrations and modulated the expression of many genes, mainly downregulating the expression of mitochondrial genes in the absence of oxidative stress, triggering a hypoxia-like response, and upregulating them under stress, mainly the cytochrome complex and electron transport chain. Other categories that were enriched by the effect of melatonin were related to transport, antioxidant activity, signaling, and carbohydrate and lipid metabolism. The overall results suggest that melatonin is able to reprogram the cellular machinery to achieve tolerance to oxidative stress.

Keywords: antioxidant; bioactive compound; hydrogen peroxide; hypoxia; melatonin; mitochondria; oxidative stress; yeast.

Figure 1

Distribution of differentially expressed genes in yeast cells grown with and without melatonin (5 μM or 25 μM), in stressed (2 mM of H₂O₂) and in unstressed conditions (Control, Mel, MelH, 25MelH and H). (A) The number of differentially expressed genes between different conditions (fold change −1 ≤ FC ≥ 1; p-value < 0.05). (B,C) Venn diagram showing the number of common genes found among the Mel vs. Control, MelH vs. Control and H vs. Control comparisons (up- (B) and down (C) regulated genes). (D) Venn diagram showing the number of common genes found among the up- and downregulated genes identified in the Mel vs. Control and MelH vs. H comparisons. (E) List of genes in some groups indicated in (B–D). The list of genes and the GO enrichment analysis of all the groups is shown in Table S1.

Figure 2

Categories enriched by differentially expressed genes that were up- (values to the left) and down- (values to the right) regulated (1 ≤ fold change ≥1; p-value < 0.05) among nonstressed (blue) and stressed (orange) (2 mM H₂O₂) cells in the absence or presence of melatonin (5 μM). Percentages of enrichment are calculated as the ratio of the number of up- or downregulated genes in relation to the total number of genes involved in each molecular function or pathway in S. cerevisiae. The numbers correspond to the p-values. When two p-values are presented, the left one corresponds to Mel vs. Control and the right to MelH vs. H. (A) Biological process function enrichment from Gene Ontology (GO) analysis, (B) cellular components enriched according to GO analysis, (C) pathway enrichment analyzed with the DAVID tool. Abbreviations: ETC, electron transport chain; ER, endoplasmic reticulum; TCA, citrate cycle, IMtM, inner mitochondrial membrane.

Figure 3

Intracellular melatonin quantification in cells untreated and treated with 5 μM of exogenous melatonin, before (Control and Mel) and after (H and MelH) being exposed to oxidative stress with 2 mM of H₂O₂. Error bars represent standard deviation, letters show statistical differences with p-value < 0.05.

Figure 4

Lipid composition of nonstressed and stressed yeast cells with 2 mM of H₂O₂, grown with and without 5 μM of melatonin (Control, Mel, H, MelH). (A) Sterols. (B) Phospholipids. (C) UFA/SFA ratio (from Vázquez et al. [15]) and unsaturation index defined as follows: [(% C16:1 + % C18:1) + 2 (% C18:2) + 3 (% C18:3)]/100. (D) Medium-chain-length of FA (mCL, from Vázquez et al. [15]). Letters indicate significant differences between lipid concentrations (p-value < 0.05).

Figure 5

Expression of six selected genes over a time course. (A) Kinetics of yeast growth according to OD_600nm values. The cells were incubated with or without melatonin (5 µM) for 6 h, and two conditions (Mel, MelH) were subjected to 2 mM H₂O₂ treatment (0 h). (B) Expression of each gene of interest at different time points in the nonstressed and stressed conditions (Control, H), expressed as log₁₀(ratio). The ratio represents the 2^−ΔΔCt value corrected to the 2^−ΔΔCt value of the Control at 0 h. (C) Heat map of the effect of melatonin on the expression of the six genes during the time course analysis in nonstressed and stressed cells. The color corresponds to the log₁₀ of the ratio. The ratio represents the 2^−ΔΔCt value for the condition with melatonin corrected to the 2^−ΔΔCt value for the condition without melatonin. Genes are presented in rows, and the time points under both conditions are presented in columns. Exp, exponential phase; stat, stationary phase. High-intensity colors refer to values >0.35 (green) or < −0.35 (red) * significance level of < 0.05. Error bars represent the standard deviation, and the statistical analysis of B and C is presented in Table S3.

Figure 6

Effect of H₂O₂ and melatonin on the number of mitochondria per cell. The values are expressed as the mean fluorescence intensity (MFI) of the different conditions compared to the control. The cells were incubated with different melatonin concentrations (0 µM, 5 µM and 50 µM) and then subjected to treatment with different H₂O₂ concentrations (0 mM, 2 mM and 5 mM). Error bars represent the standard deviation, and * indicates statistically significant differences between media (p < 0.05).

Figure 7

Genetic interactions given by Phenetics using mitochondrial genes with −1 < FC < 1, p-value < 0.05. Set of genes used in each comparation: (A) Mel vs. Control. (B) MelH vs. H. Nodes represent molecular entities, and the intensity of its color represents the level of differential expression, green being under-expression and red over-expression. The edges indicate the interaction between the genes (blue indicates protein-DNA and orange protein-protein), and the arrows the direction of the interaction.