## 3 Results
### Cellular Response to Abiotic Stresses and Importance of D-_Erythroascorbate as a Substrate_  
It was examined whether _DaMDHAR_ expression in transgenic yeast affects the cellular response to oxidative stress and high-temperature. As shown in Figure 8, the survival of _DaMDHAR_-expressing DM cells was better than that of WT cells when exposed to 20 and 25 mM hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) for 1 h at 28 \({}^{\circ}\)C, or challenged for 4 and 6 min at 50 \({}^{\circ}\)C. Further, AsA is the most common antioxidant molecule in the cell; thus, it is a good target for investigating the acquired ability of _DaMDHAR_-expressing cells to detoxy ROS produced by abiotic stress [19; 21]. Yeasts do not typically synthesize AsA; instead, they synthesize a five-carbon analog, D-erythroascorbic acid (eAsA), from D-arabinose dehydrogenase (ARA2) and D-arabino-c-lactone oxidase activity. This eAsA shares structural and physiochemical properties with AsA, including roles in regulating antioxidant activity, hyphal growth, and stress tolerance. Unlike, AsA, the function of eAsA in cellular stress response is mostly unknown. Therefore, we used a yeast strain in which the eAsA biosynthesis-related gene ARA2 was deleted (_ara2_A) to  
Figure 7: Expression analysis at the protein level of 2-fold changed genes (left panel) and signal intensity (right panel) (**A**) and NADPH assay (**B**). Signal intensity was calculated using Image J. Evaluation of intracellular NADPH was assayed by spectrophotometric quantification. Data were analyzed using Origin 2020. The data are the means and standard deviations of three independent experiments with at least two biological replicates each. A probability analysis to determine statistical significance was performed using the Student’s \(t\)-test. Compared to the appropriate control strain, \(p<0.05\) was considered significant in this study. Hxt2, hexokinase 2; Ald3, aldehyde dehydrogenase isoform 3; Adh6, alcohol dehydrogenase isoform 6; Gnd2, 6-phosphogluconate dehydrogenase; Ctt1, cytosolic catalase; Gto1, glutathione-S-transferase isoform 1; Trx3, Mt thioredoxin; Gpd1, glycerolaldehyde-3-phosphate dehydrogenase; Hsp12, heat shock protein 12; Hsp30, heat shock protein 30; Act1, actin isoform 1 was used as loading control; WT, wild-type cells with empty vector; DM, _DaMDHAR_-expressing transgenic cells. demonstrate whether eAsA deficiency in \(ara2\Delta\) affected the cellular response to FT stress. A functional examination of \(ara2\Delta\)-mediated eAsA was confirmed by evaluating empty vector (p426 GPD)- and DaMDHAR:p426 GPD-transformed \(ara2\Delta\) cells (named as A2 and DA, respectively, Table S1) via immunoblotting analysis (Figure 8B). The stress-response assay was performed by spotting assay using WT, DM, A2, and DA. Yeast cells that reached the early exponential phase were serially diluted with YPD broth medium. For H\({}_{2}\)O\({}_{2}\) and low-temperature assay, 5 \(\upmu\)L each of the diluted solution was spotted onto the YPD agar medium in the absence and presence of 3 mM H\({}_{2}\)O\({}_{2}\). FT2 stress was performed as described in Materials and Methods. Among the four transgenic yeast cells, stress tolerance was observed in the order of DM, WT, DA, and A2 under FT2, H\({}_{2}\)O\({}_{2}\), heat, and chilling stress. There was no difference in stress response between A2 and DA cells (Figure 8C-E). Therefore, these results indicated _DaMDHAR_ expression facilitates enhanced tolerance to abiotic stress, including H\({}_{2}\)O\({}_{2}\) and low-temperature, implying that eAsA plays a critical role in the _DaMDHAR_-mediated abiotic stress response. Figure 8: Cellular response in _DaMDHAR_-expressing yeast cells to oxidative stress and heat shock. (**A**) Exponential yeast cells (A\({}_{600}\approx 1.5\)) were exposed to 20 and 25 mM H\({}_{2}\)O\({}_{2}\) for 1 h at 28 \({}^{\circ}\)C with shaking (160 rpm) and diluted to 10\({}^{-4}\) with YPD broth.