Fig3

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.