Hydrogen sulfide treatment at the late growth stage of Saccharomyces cerevisiae extends chronological lifespan

Abstract

Previous studies demonstrated that lifelong treatment with a slow H₂S releasing donor extends yeast chronological lifespan (CLS), but it is not clear when the action of H₂S benefits to CLS during yeast growth. Here, we show that short H₂S treatments by using NaHS as a fast H₂S releasing donor at 96 hours after inoculation extended yeast CLS while NaHS treatments earlier than 72 hours after inoculation failed to do so. To reveal the mechanism, we analyzed the transcriptome of yeast cells with or without the early and late NaHS treatments. We found that both treatments had similar effects on pathways related to CLS regulation. Follow-up qPCR and ROS analyses suggest that altered expression of some antioxidant genes by the early NaHS treatments were not stable enough to benefit CLS. Moreover, transcriptome data also indicated that some genes were regulated differently by the early and late H₂S treatment. Specifically, we found that the expression of YPK2, a human SGK2 homolog and also a key regulator of the yeast cell wall synthesis, was significantly altered by the late NaHS treatment but not altered by the early NaHS treatment. Finally, the key role of YPK2 in CLS regulation by H₂S is revealed by CLS data showing that the late NaHS treatment did not enhance the CLS of a ypk2 knockout mutant. This study sheds light on the molecular mechanism of CLS extension induced by H₂S, and for the first time addresses the importance of H₂S treatment timing for lifespan extension.

Keywords: NaHS; Saccharomyces cerevisiae; aging; chronological lifespan; hydrogen sulfide.

Figure 1

CLS extension is dependent upon the timing of NaHS treatment. Viabilities of cells treated without (black plots) or with 100 μM NaHS at the earlier (blue plots) or the later (red plots) phases of growth were plotted. Specifically, NaHS was added once or twice into cell cultures at the 12 hours (A), 24 and 48 hours (B), 84 hours (C), 96 hours (D), or 72 and 96 hours (E) after inoculation. (F) NaHS was added into cell cultures every 24 hours after 72 hours of inoculation. Arrows indicate the time of NaHS treatment. Triplicate cultures were used to achieve mean ± SD of viabilities. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2

CLS in response to various concentrations of NaHS. Cells were treated with different concentration of NaHS at the early and the late phases of growth. (A–G) Viabilities of Cells treated with or without NaHS at the indicated time were plotted. Arrows indicate the time of NaHS treatment. Triplicate cultures were used to achieve mean ± SD of viabilities. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

Transcriptomic alterations by the early and late NaHS treatments. (A) Schematic of the early and late NaHS treatments in relation to their untreated controls. 100 μM of NaHS were used for each dosing. (B, C) The volcano plots represent DEGs of indicated controls and treatments. (D, E) The bubble charts representing KEGG enrichment analysis of indicated DEGs. The rich factor indicates the degree of enrichment represented by the ratio of DEGs in a pathway to the number of total genes annotated to that pathway. (F) Comparison of enriched pathways in (D and E).

Figure 4

Analysis of common and specific DEGs in the early and late NaHS treatments. (A–C) The bubble charts representing KEGG enrichment analysis of common or specific DEGs in the early and late NaHS treatments. (D) Comparison of enriched pathways in (A–C).

Figure 5

The late NaHS treatment is more cyto-protective. (A and B) Heat maps of antioxidant DEGs in the early and late NaHS treatments. (C) Venn diagram representing overlapped antioxidant DEGs. (D and E) Heat maps of HSP DEGs in the early and late NaHS treatments. (F) Venn diagram representing overlapped HSP DEGs. (G) The generation of Reactive oxygen species (ROS) in the early and late NaHS treatments at day 5 (120 hours after inoculation) was imaged by fluorescence microscope (left) and quantified by calculating the ratio of positively stained cells (right). (H–J) qPCR analysis of GPX2 (H) HSP78 (I) and HSP104 (J) at 53 hours or 120 hours after inoculation with or without the indicated NaHS treatment. The expression of these genes were normalized with the expression of actin (ACT).^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001

Figure 6

The late NaHS treatment regulates the cell wall integrity for the extension of CLS. (A) Gene ontology analysis of genes expressed oppositely in response to the early and late NaHS treatments. (B) The qPCR analysis of YPK2, an AGC-type protein kinase regulating cell wall integrity. (C) CLS of the late NaHS treated RCD490, a ypk2 mutant in BY4742 background. (D) CLS of the late NaHS treated BY4741. (E) CLS of the late NaHS treated YMR104C, a ypk2 mutant in BY4741 background. Triplicate cultures were used to achieve mean ± SD of viabilities. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ns nonsignificant.