Fig 1.
Construction of an OsMDHAR-expressing yeast vector and the stress response of OsMDHAR-expressing yeast to hydrogen peroxide.
(A) Schematic diagram of the p426GPD::OsMDHAR construct. The OsMDHAR gene (approximately 1.5 kbp) was subcloned to generate the p426GPD::OsMDHAR construct with OsMDHAR under the control of the constitutive GPD promoter. Semi-quantitative RT-PCR (B), immunoblotting (C), and an enzymatic assay (D) were performed to examine whether OsMDHAR is expressed in this yeast strain. PDA1 and tubulin (Tub) were used as housekeeping controls for RT-PCR and western blotting, respectively. The molecular size of the PCR product and molecular weight of the detected band were approximately 494 bp and 47 kDa, respectively. Stress tolerance to hydrogen peroxide was evaluated by cell survival, growth kinetics, and spotting assays. (E) To monitor cell viability, yeast cells precultured in YPD medium were inoculated into fresh YPD medium and exposed to different concentrations of H2O2 for 16 h at 28°C. Then, the optical density at 600 nm (OD600) was measured. Circles, cells transformed with p426GPD-OsMDHAR (TC cells); squares, wild-type (WT) cells transformed with an empty vector. (F) For the growth kinetics assay, precultured yeast cells were inoculated into YPD medium containing 5 mM H2O2, and the OD600 was measured at 2-h intervals for 36 h. A streaking assay was also performed, in which mid-log phase yeast cells (OD600 ≈ 2.0) were streaked onto YPD agar plates supplemented with 5 mM H2O2. WT (squares) and TC (circles) cells in the absence of 5 mM H2O2; WT (upward triangles) and TC (diamonds) cells in the presence of 5 mM H2O2. (G) Mid-log phase yeast cells were exposed to 20 mM H2O2 for 1 h with shaking, and serially diluted with YPD medium. A 5-μL aliquot of each dilution was spotted onto YPD agar plates.
Fig 2.
Analyses of cell rescue proteins, redox state, and protein oxidation under oxidative conditions.
(A) Expression changes in antioxidant and metabolic enzymes in mid-log phase yeast cells exposed to 20 mM H2O2 for 1 h with shaking. Tubulin (Tub) was used as a loading control. (B) Hydroperoxide levels in TC cells in the absence (red bar) and presence (green bar) of 20 mM H2O2 were assessed using FOX reagent and were calculated relative to that in WT cells grown under normal conditions, which was set to 100%. (C) Mid-log phase yeast were exposed to 20 mM H2O2 for 1 h at 28°C with shaking. Redox state was analyzed by measuring DCFHDA oxidation as an indicator of cytosolic ROS. (D) Sensitivity of mutants (sod1Δ, tsa1Δ, por1Δ, and por2Δ) to oxidative stress. Yeast cells (OD600 ≈ 1.0) were exposed to 10 mM H2O2 for 1 h at 28°C with shaking, serially diluted with YPD medium, spotted onto YPD agar plates, and incubated for 2–3 days. (E) Expression changes in molecular chaperones in mid-log phase yeast cells exposed to 20 mM H2O2 for 1 h with shaking. Tubulin (Tub) was used as a loading control. (F) Protein carbonylation in yeast cells exposed to 20 mM H2O2 for 1 h was calculated relative to that in WT cells under normal conditions, which was set to 100%. Red bar, normal conditions; green bar, H2O2 treatment; WT, yeast cells with an empty vector; TC, OsMDHAR-expressing yeast cells; N, normal conditions; S, H2O2 treatment.
Fig 3.
Stress response related to ascorbate (AsA)-like molecules.
(A) AsA-like content in yeast cells exposed to 20 mM H2O2 for 1 h was analyzed and is shown as nmol per mg protein. The ratio shown is that of the reduced form to oxidized form. (B) ARA2 expression was evaluated by semi-quantitative RT-PCR. PDA1 was used as a control. (C) Oxidative stress response of yeast cells in the absence and presence of ARA2. Mid-log phase cells were serially diluted, and 5 μL of the diluted solutions were spotted onto YPD agar plates containing 4 mM H2O2 (upper panels). Mid-log phase cells were treated with 20 mM H2O2 for 1 h with shaking, diluted with YPD medium, and spotted onto YPD agar plates. The plates were incubated for 2–3 days and photographed. (D) Stress sensitivity of ara2Δ yeast cells, in which the erythroascorbate (EAA) biosynthesis gene was deleted. Yeast cells (A600 ≈ 1.0) were exposed to 10 mM H2O2 for 1 h at 28°C with shaking, serially diluted with YPD medium, spotted onto YPD agar plates, and incubated for 2–3 days. (E) The redox state of ara2Δ yeast cells under oxidative conditions. Yeast cells (OD600 ≈ 1.0) were exposed to 10 mM H2O2 for 1 h after DCFHDA and DHAR 123 treatment for 30 min and washed twice with phosphate-buffered saline (PBS). Probe intensity was observed by fluorescence microscopy. BY, wild-type cells without an empty vector; ara2Δ, cells with a deletion of the EAA biosynthetic gene ARA2; WT, wild-type yeast cells with an empty vector; TC, yeast cells with p426GPD::OsMDHAR; WA, ara2Δ yeast cells with an empty vector; TA, ara2Δ yeast cells with p426GPD::OsMDHAR; N, normal conditions; S, in the presence of H2O2.
Fig 4.
Fermentative capacity and the survival of OsMDHAR-expressing yeast cells during batch fermentation.
(A) Fermentative capacity was analyzed by measuring the alcohol (AC) and residual glucose (RG) concentrations in YG medium after fermentation for 72 h at 30°C. Upward triangles, AC of WT cells; circles, AC of TC cells; squares, RG of WT cells; diamonds, RG of TC cells. (B) Time-dependent OsMDHAR expression during batch fermentation was evaluated by western blotting. Tubulin (Tub) was used as a loading control. (C) Growth kinetics during fermentation was assessed by measuring the OD600 at 2-h intervals for the indicated time. Squares, WT cells; circles, TC cells. (D) Cell viability during fermentation at 30°C was assessed by a spotting assay. Cells were harvested after 24 h (upper panel), 48 h (middle panel), and 72 h (lower panel) of fermentation and serially diluted to 10−9. A 5-μL aliquot of each diluted solution was spotted onto YPD agar plates. After incubation for 3 days, the plates were photographed. (E) Stress response to ethanol. Mid-log phase yeast cells (OD600 ≈ 2.0) were exposed to different concentrations of ethanol (0, 15%, and 20%) for 1 h, serially diluted, and spotted onto YPD agar plates. WT, yeast cells with an empty vector; TC, OsMDHAR-expressing yeast cells.