전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Research article

References

  1. Kanki T, Furukawa K, Yamashita SI. 2015. Mitophagy in yeast: molecular mechanisms and physiological role. Biochim. Biophys. Acta 1853: 2756-2765.
    Pubmed CrossRef
  2. Tsukada M, Ohsumi Y. 1993. Isolation and characterization of autophagy defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333: 169-174.
    CrossRef
  3. Fracchiolla D, Sawa-Makarska J, Zens B, Ruiter A, Zaffagnini G, Brezovich A, et al. 2016. Mechanism of cargodirected Atg8 conjugation during selective autophagy. Elife 5(pii): e18544.
    Pubmed PMC CrossRef
  4. Weiergräber OH, Schwarten M, Strodel B, Willbold D. 2017. Investigating structure and dynamics of Atg8 family proteins. Methods Enzymol. 587: 115-142.
    Pubmed CrossRef
  5. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal D. 2008. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4: 151-175.
  6. Reggiori F, Klionsky DJ. 2013. Autophagic processes in yeast: mechanism, machinery and regulation. Genetics 194: 341-361.
    Pubmed PMC CrossRef
  7. Wong PM, Puente C, Ganley IG, Jiang X. 2013. The ULK1 complex: sensing nutrient signals for autophagy activation. Autophagy 9: 124-137.
    Pubmed PMC CrossRef
  8. Xiong Y, McCormack M, Li L, Hall Q, Xiang C, Sheen J. 2013. Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature 496: 181-186.
    Pubmed PMC CrossRef
  9. Chew LH, Lu S, Liu X, Li FK, Yu AY, Klionsky DJ, et al. 2015. Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms. Autophagy 11: 891-905.
    Pubmed PMC CrossRef
  10. Perpetuini G, Di Gianvito P, Arfelli G, Schirone M, Corsetti A, Tofalo R, et al. 2016. Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation. Yeast 33: 303-312.
    Pubmed CrossRef
  11. Suzuki H, Osawa T, Fujioka Y, Noda NN. 2017. Structural biology of the core autophagy machinery. Curr. Opin. Struct. Biol. 43: 10-17.
    Pubmed CrossRef
  12. Mendes-Ferreira A, Sampaio-Marques B, Barbosa C, Rodrigues F, Costa V, Mendes-Faia A, et al. 2010. Accumulation of non-superoxide anion reactive oxygen species mediates nitrogen-limited alcoholic fermentation by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 76: 7918-24.
    Pubmed PMC CrossRef
  13. Tesnière C, Brice C, Blondin B. 2015. Responses of Saccharomyces cerevisiae to nitrogen starvation in wine alcoholic fermentation. Appl. Microbiol. Biotechnol. 99: 7025-7034.
    Pubmed CrossRef
  14. Piggott N, Cook MA, Tyers M, Measday V. 2011. Genomewild fitness profiles reveal a requirement for autophagy during yeast fermentation. G3 1: 353-367.
    CrossRef
  15. Horie T, Kawamata T, Matsunami M, Ohsumi Y. 2017. Recycling of iron via autophagy is critical for the transition from glycolytic to respiratory growth. J. Biol. Chem. 292: 8533-8543.
    Pubmed PMC CrossRef
  16. Gibson BR, Lawrence SJ, Boulton CA, Box WG, Graham NS, Linforth S, et al. 2008. The oxidative stress response of a lager brewing yeast strain during industrial propagation and fermentation. FEMS Yeast Res. 8: 574-585.
    Pubmed CrossRef
  17. Landolfo S, Politi H, Angelozzi D, Mannazzu I. 2008. ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium. Biochim. Biophys. Acta 1780: 892-898.
    Pubmed CrossRef
  18. Cheng Y, Du Z, Zhu H, Guo X, He X. 2016. Protective effects of arginine on Saccharomyces cerevisiae against ethanol stress. Sci. Rep. 6: 31311.
    Pubmed PMC CrossRef
  19. Charoenbhakdi S, Dokpiku T, Burphan T, Techo T, Auesukaree C. 2016. Vacuolar H+-ATPase protects Saccharomyces cerevisiae cells against ethanol-induced oxidative and cell wall stresses. Appl. Environ. Microbiol. 82: 3121-3130.
    Pubmed PMC CrossRef
  20. Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 9: 249-279.
    Pubmed CrossRef
  21. Song C, Mitter SK, Qi X, Beli E, Rao HV, Ding J, et al. 2017. Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1and promotes retinal pigmented epithelial cell survival through increased autophagy. PLos One 12: e0171940.
    Pubmed PMC CrossRef
  22. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. 2007. Reactive oxygen species are essential for autophagy and specifically regulate the activity of atg4. EMBO J. 26: 1749-1760.
    Pubmed PMC CrossRef
  23. Xua J, Wua Y, Lu G, Xie S, Ma Z, Chen Z, Shen HM, et al. 2017. Importance of ROS-mediated autophagy in determining apoptotic cell death induced by physapubescin B. Redox. Biol. 12: 198-207.
    Pubmed PMC CrossRef
  24. Chen SY, Chiu LY, Maa MC, Wang JS, Chien CL, Lin WW. 2011. zVAD-induced autophagic cell death requires c-Srcdependent ERK and JNK activation and reactive oxygen species generation. Autophagy 7: 217-28.
    Pubmed PMC CrossRef
  25. Demain AL. 2009. Biosolutions to the energy problem. J. Ind. Microbiol. Biotechnol. 36: 319-332.
    Pubmed CrossRef
  26. Galeote VA, Blondin B, Dequin S, Sablayrolles JM. 2001. Stress effects of ethanol on fermentation kinetics by stationary-phase cells of Saccharomyces cerevisiae. Biotechnol. Lett. 23: 677-681.
    CrossRef
  27. Aguilera F, Peinado RA, Millan C, Ortega JM, Mauricio JC. 2006. Relationship between ethanol tolerance, H+-ATPase activity and the lipid composition of the plasma membrane in different wine yeast strains. Int. J. Food Microbiol. 110: 34-42.
    Pubmed CrossRef
  28. Walker ME, Nguyen TD, Liccioli T, Schmid F, Kalatzis N, Sundstrom JF, et al. 2014. Genome-wild identification of the Fermentome; genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae. BMC Genomics 15: 552.
    Pubmed PMC CrossRef
  29. Almeida B, Sampaio-Marques B, Carvalho J, Silva MT, Leao C, Rodrigues F, et al. 2007. An atypical active cell death process underlies the fungicidal activity of ciclopirox olamine against the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 7: 404-412.
    Pubmed CrossRef
  30. Ludovico P, Rodrigues F, Almeida A, Silva MT, Barrientos A, Corte-Real M. 2002. Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol. Bio. Cell 13: 2598-2606.
    Pubmed PMC CrossRef
  31. Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3: 1101-1108.
    Pubmed CrossRef
  32. Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7: 405-410.
    CrossRef
  33. Barja G. 2004. Free radicals and aging. Trends Neurosci. 27: 595-600.
    Pubmed CrossRef
  34. Yin D, Chen K. 2005. The essential mechanisms of aging: irreparable damage accumulation of biochemical side-reactions. Exp. Gerontol. 40: 455-465.
    Pubmed CrossRef
  35. Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C. 2006. Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bio. Essays. 28: 1091-2101.
    Pubmed CrossRef
  36. Chen HJ, Huang CS, Huang GJ, Chow TJ, Lind YH. 2013. NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas). J. Plant Physiol. 170: 1471-1483.
    Pubmed CrossRef
  37. Rattanawong K, Kerdsomboon K, Auesukaree C. 2015. Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlorin Saccharomy cescerevisiae. Free Radic. Bio. Med. 89: 963-971.
    Pubmed CrossRef
  38. Magrì A, Di Rosa MC, Tomasello MF, Guarino F, Reina S, Messina A, Pinto VD. 2016. Overexpression of human SOD1 in VDAC1-less yeast restores mitochondrial functionality modulating beta-barrel outer membrane protein genes. Biochim. Biophys. Acta 1857: 789-798.
    Pubmed CrossRef
  39. Knuppertz L, Warnsmann V, Hamann A, Grimm C, Osiewacz HD. 2017. Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina. Autophagy 13: 1037-1052.
    Pubmed PMC CrossRef
  40. Murphy MP. 2009. How mitochondria produce reactive oxygen species. Biochem. J. 417: 1-13.
    Pubmed PMC CrossRef
  41. Huang S, Aken OV, Schwarzländer M, Belt K, Millar AH. 2016. The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants. Plant Physiol. 171: 1551-1559.
    Pubmed PMC CrossRef
  42. Li D, Song JZ, Li H, Shan MH, Liang Y, Zhu J, et al. 2015. Storage lipid synthesis is necessary for autophagy induced by nitrogen starvation. FEBS Lett. 89: 269-276.
    Pubmed CrossRef
  43. Cebollero E, Gonzalez R. 2006. Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines. Appl. Environ. Microb. 72: 4121-4127.
    Pubmed PMC CrossRef
  44. Ogawa Y, Nitta A, Uchiyama H, Imamura T, Shimoi H, Ito K. 2000. Tolerance mechanism of the ethanoltolerant mutant of sake yeast. J. Biosci. Bioeng. 90: 313-320.
    Pubmed CrossRef
  45. Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, et al. 2009. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell 21: 2914-2927.
    Pubmed PMC CrossRef
  46. Pérez-Pérez ME, Zaffagnini M, Marchand CH, Crespo JL, Lemaire SD. 2014. The yeast autophagy protease Atg4 is regulated by thioredoxin. Autophagy 10: 1953-1964.
    Pubmed PMC CrossRef
  47. Navarro-Yepes J, Burns M, Anandhan A, Khalimonchuk O, del Razo LM, Quintanilla-Vega B, et al. 2014. Oxidative stress, redox signaling, and autophagy: cell death versus survival. Antioxid. Redox. Signal 21: 66-85.
    Pubmed PMC CrossRef
  48. Zhu Z, Huang Y, Lv L, Tao Y, Shao M, Zhao C, et al. 2017. Acute ethanol exposure-induced autophagy-mediated cardiac injury v ia a ctivation of the ROS-JNK-Bcl-2 pathway. J. Cell Physiol. 233: 924-935.
    Pubmed CrossRef
  49. Kim KY, Park KI, Kim SH, Yu SN, Lee D, Kim YW, et al. 2017. Salinomycin induces reactive oxygen species and apoptosis in aggressive breast cancer cells as mediated with regulation of autophagy. Anticancer Res. 37: 1747-1758.
    Pubmed CrossRef
  50. Shiroma S, Jayakody LN, Horie K, Okamoto K, Kitagakia H. 2014. Enhancement of ethanol fermentation in Saccharomyces cerevisiae sake yeast by disrupting mitophagy function. Appl. Environ. Microbiol. 80: 1002-1012.
    Pubmed PMC CrossRef
  51. Basit F, van Oppen LM, Schöckel L, Bossenbroek HM, van Emst-de Vries SE, Hermeling JC, et al. 2017. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell Death Dis. 8: e2716.
    Pubmed PMC CrossRef
  52. Bhatelia K, Singh K, Prajapati P, Sripada L, Roy M, Singh R. 2017. MITA modulated autophagy flux promotes cell death in breast cancer cells. Cell Signal 35: 73-83.
    Pubmed CrossRef

Related articles in JMB

More Related Articles

Article

Research article

J. Microbiol. Biotechnol. 2018; 28(12): 1982-1991

Published online December 28, 2018 https://doi.org/10.4014/jmb.1806.06014

Copyright © The Korean Society for Microbiology and Biotechnology.

Ethanol induces autophagy regulated by mitochondrial ROS in Saccharomyces cerevisiae

Hongjuan Jing *, Huanhuan Liu , Lu Zhang , Jie Gao , Haoran Song and Xiaorong Tan

Henan University of Technology, China

Received: June 12, 2018; Accepted: October 22, 2018

Abstract

Ethanol accumulation inhibited the growth of Saccharomyces cerevisiae during wine fermentation. Autophagy and the release of reactive oxygen species (ROSs) were also induced under ethanol stress. However, the relation between autophagy and ethanol stress was still unclear. In the study, expression of autophagy genes ATG1 and ATG8 and production of ROS under ethanol treatment in yeast were measured. The results showed that ethanol stress very significantly induced expression of ATG1 and ATG8 genes and the production of peroxide hydrogen (H2O2) and superoxide anion (O2·-). Moreover, the atg1 and atg8 mutants aggregated more H2O2 and O2·- than the wild type yeast. In addition, inhibitors of the ROS scavenging enzyme induced expression of ATG1 and ATG8 genes through increasing the levels of H2O2 and O2·-. Oppositely, glutathione (GSH) and N-acetylcystine (NAC) decreased the ATG1 and ATG8 expression by reducing H2O2 and O2·- production. Besides that, rapamycin and 3-methyladenine caused an obvious change in autophagy levels and simultaneously altered the release of H2O2 and O2·-. At last, inhibitors of mitochondrial electron transport chain (mtETC) increased the production of H2O2 and O2·- and also promoted expression levels of the ATG1 and ATG8 genes. In conclusion, ethanol stress induced autophagy which was regulated by H2O2 and O2·- derived from mtETC. In turn, the autophagy contributed to the elimination H2O2 and O2·-.

Keywords: Autophagy, ethanol stress, fermentation, hydrogen peroxide, superoxide anion, reactive oxygen specie

Introduction

In order to remove dysfunctional and unneeded constituents and recycle intracellular nutrients, macro-autophagy (hereinafter referred to as autophagy) encapsulates and transfers cytoplasmic material to the vacuole or lysosome for degradation [1]. Autophagy-related genes (ATGs) originally were identified in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) [2]. The most important of these ATGs was the ubiquitin-like protein ATG8 that attached to lipid phosphatidylethanolamine (PE) on the outside of emerging phagophores. The ATG8-PE adduct contributed to expand and seal the vesicle and recruit specific cargo [3, 4]. Therefore, upregulation of ATG8 expression is implicated in the induction of autophagy [5]. Moreover, the initiation of autophagy is regulated by Ser/Thr kinase ATG1 and its accessory regulator ATG13 in fungi [6-10]. It has been proven that the expression of ATG1 is also an indicator of autophagy levels [11].

During the early stage of fermentation, sufficient nitrogen helps the proliferation and growth of yeast. The further consumption of nitrogen then results in sugar fermentation. Therefore, nitrogen limitation has been very common in winemaking. It was also well known that nitrogen starvation could induce autophagy [12, 13]. However, autophagy was induced early in wine fermentation in a nitrogen-replete environment [14]. About the function of autophagy during fermentation, it has been reported that recycling of iron via autophagy is critical for the transition from glycolytic to respiratory growth [15]. Therefore, the function of autophagy and the exact factors inducing it are still unknown during fermentation.

More and more studies showed that reactive oxygen species (ROS) were associated with fermentation [16-19]. ROS were a possible part of the yeast response to a variety of stress factors occurring during most fermentations [20]. ROSs, mainly including the superoxide anion (O2.-) and hydrogen peroxide (H2O2), are generated by cells during normal metabolism [21]. Many studies have shown that ROS play a vital role in regulating autophagy [21-23]. However, which molecule of ROS regulated autophagy was still being debated. For example, it had been proven that H2O2 had the ability to activate autophagy [21]. However, Chen et al. showed that O2-. could induce autophagy [24]. In addition, other ROS except O2-. possibly induced autophagy [12]. Therefore, the exact ROS molecule regulating autophagy still needed to be researched in the future.

Ethanol production is likely to remain one of the most important biotechnological products well into the future, with the continued manufacture of spirits, wine, sake, beer, and so on [25]. But ethanol accumulation in the culture broth is still a significant stress factor during fermentation. Although S. cerevisiae is highly ethanol tolerant high ethanol inhibits cell growth and viability [26-28], and the mechanism of ethanol tolerance is still unclear. The aim of the current study was to obtain new insight into the response of yeast under ethanol stress conditions and to clarify the complicated relationship between ROS and autophagy during fermentation. To achieve these aims, ROS production and the expression of ATG8 or ATG1 genes were evaluated in wild type and mutant yeast strains grown on a medium with ethanol.

Materials and Methods

Strain and Maintenance Medium

The wine yeast strain BY4742 (S. cerevisiae) was supplied by Pro. Zhiwei Huang of East China University. The mutants atg1 and atg8 were purchased from Invitrogen (Carlsbad, CA). The mutants were constructed by hygromycin stripe homologous replacement of the mutation genes of ATG1 or ATG8 and selected by yeast peptone dextrose agar (YPD) medium with 200 μg/ml G418. Wild-type yeasts could not grow on YPD medium containing G418 whereas mutant cells could survive. The mutants were preserved on YPD medium containing G418 in order to prevent back mutation. The wild-type yeast maintained at 4°C on slants of YPD medium contained (g/L): glucose 20, peptone 10, yeast extract 5, and agar 20. Fresh cells grown on YPD slants for 24 h were used in all experiments.

Fermentation Conditions

For all experiments, starter cultures were prepared by growing the yeast cells overnight in 250-ml flasks containing 100 ml of YPD medium. The flasks were incubated at 30°C in an orbital shaker set at 180 rpm. The experimental cultures were inoculated with 5×105 CFU/ml of starter culture. Fermentation was carried out in 500-ml flasks filled to two-thirds of their volume and maintained at 30°C in an orbital shaker at 180 rpm, according to the methodology [12]. With distilled water as a control, 10% ethanol made by adding absolute ethyl alcohol was used as ethanol stress. 100 mM 2-methoxyestradiol (2-ME) was stored in dimethylsulfoxide (DMSO) and final concentration was 100 μM. 1 M 3-amino-1,2,4-triazole (3-AT) was stored in double distilled water and final concentration was 2 mM. 10 mM rapamycin (Rapa) disolved in ethanol and stored in TritonX-100 and final concentration was 5 μM. 3-methyladenine (3-MA) was stored in PBS solution and final concentration was 10 μM. 2.5 mM glutathione (GSH) and 10 mM N-acetyl-cysteine (NAC) were both disolved in double distilled water. 100 mM antimycin A (Anti A) and rotenone (Rote) were both disolved in DMSO and final concentrations for both were 5 mM. Each flask was closed with a rubber stopper. Prior to sampling, the flasks were stirred to ensure homogeneity.

Assessment of Cell Death

Cell death was assessed by propidium iodide (PI) (Sigma, USA) vital staining as described elsewhere [29] with minor adaptations. Briefly, yeast (106 cells/ml) was stained by 20 μM PI for 10 min at 37°C and then observed by fluorescence microscope. The fluorescence intensity was measured by fluorescence 96-well microplate (excitation wavelength 540 nm, emission wavelength 590 nm). The death of cells was with high red fluorescence.

Assessment of Intracellular H2O2 and O2.-

H2O2 was monitored with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma) essentially as described elsewhere [30]. DCFH-DA didn’t fluoresce but could transmembrane freely. The dye could be hydrolyzed by esterase to 2, 7-dichlorofluorescin (DCFH) which was arrested in an actively respiring cell. DCFH was oxidized by ROS to a fluorescent compound DCF in cytoplasm. To detect O2.-, dihydroethidium (DHE) (Sigma) was used as a probe. Briefly, yeast (106 cells/ml) was stained by 4 μM DHE for 10 min at 30°C in dark. The fluorescences of DCF and DHE were detected by fluorescence microscope and Fluorescence-activated cell sorter (FACS) analysis.

FACS analysis was carried out with an FACSCalibur Flow Cytometer (Becton, Dickinson and Company, USA) equipped with an argon ion laser emitting a 488-nm beam at 15 mW. The green and red fluorescence were collected through a 488-nm blocking filter. Green and red fluorescence were used for detection by a 550-nm/long-pass dichroic mirror with a 525-nm/band-pass filter and a 590-nm/long-pass with a 620-nm/band-pass filter, respectively. An acquisition protocol was defined to measure green fluorescence (FL1 log) and red fluorescence (FL2 log) on a 4-decade logarithmic scale. Data (20,000 cells per sample) were analyzed with the cell quest pro included in the System II acquisition software for the Flow Jo software.

qRT-PCR

Wild-type yeast was treated with a different reagent from ethanol. RNA was obtained from the sample which was periodically collected. RNA was extracted using Trizol (Invitrogen, Carlsbad, USA) standard procedures. Heat shock treatment (15 min at 42°C followed by 3 min at 95°C) was for cellular disruption. Total RNA (250 ng) was reverse transcribed using a SuperScript III Platinum Two-Step Real-Time Quantitative RT-PCR Kit with SYBR green from Invitrogen. One microliter of the reverse-transcribed RNA was used as a template to amplify the genes, using primers to the ATG8 gene (sense, 5-TTGCTGACAGGTTCA AGAATAGG-3; antisense, 5-ATCAACGCCGCAGTAGGTG-3), ATG1 gene (sense, 5-TACTGTGCTCTTGGGGACCTA-3; antisense, 5-CGGACGCTAACTGCTGTAAATA-3) and the ACT1 gene (sense, 5-GGATTCTGAGGTTGCTGCTTT-3; antisense, 5-TGACCCATA CCGACCATGATAC-3). The expression of the ATG8 and ATG1 genes was assessed by qRT-PCR in a StepOnePlus system (ABI). Results were normalized to the reference gene ACT1. The data were analyzed by applying the Livak method or the 2-ΔΔCT method [31]. The method was as follows: ΔΔCT =(CT(target gene) − CT(reference gene)) test − (CT(target gene) − CT(reference gene)) calibrator, where CT is the threshold cycle.

Statistical Analysis

Data were reported as mean values of at least three independent assays and presented as means ± standard deviations (SD). Statistical analyses were carried out using a Student’s t-test. P values of less than 0.05 or 0.01 were considered statistically significant, or very significant which were shown as “*” and “**” at the tops of the columns in the figures.

Results

Ethanol Stress Induced Autophagy

Ethanol was the main metabolite of S. cerevisiae during the fermentation process [25]. In order to confirm the effect of ethanol stress on autophagy, the expression levels of the ATG1 and ATG8 genes were measured by qRT-PCR. Based on the preliminary experiment, the peak expression value of ATG8 and ATG1 was at 4 h. Therefore, expression of the ATG8 and ATG1 genes was detected at 4 h. The results showed that the expression levels of the ATG1 and ATG8 genes were very significantly increased during ethanol stress (p < 0.01) (Fig. 1). In detail, the expression levels of ATG1 and ATG8 were increased to 153.5% and 252.6% by ethanol stress, respectively (Fig. 1). Therefore, we confirmed that ethanol stress indeed induced autophagy.

Figure 1. Ethanol-induced expression of the ATG1 and ATG8 genes. Normalized fold expression levels of the ATG1 and ATG8 genes were evaluated by qRT-PCR in S. cerevisiae treated with 10% ethanol for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test and was shown as the statistical significance between ethanol with control. Con: control, Eth: treated with ethanol.

Autophagy Contributed to Survival of S. cerevisiae from Ethanol Stress

The above results revealed that ethanol stress markedly induced autophagy. In order to clarify the role of autophagy during ethanol stress, the death rates of the wild type, atg1 and atg8 strains were assessed by PI staining. The results showed that there was no difference between the wild type and the mutant strains during their growth on YPD medium (Fig. 2). However, ethanol stress increased the cell death rate of all strains (Fig. 2). Compared with wild-type yeast, the cell death of atg1 and atg8 mutants was dramatically enhanced by ethanol stress. Therefore, this indicated that autophagy protected yeast cells from ethanol stress.

Figure 2. Cell death of mutants was higher than that of wild type under ethanol stress. Wild type, atg1 and atg8 treated with 10% ethanol for 2 h were stained with PI. Then all strains were analyzed by fluorescence microscope (A) and fluorescence microplate reader (B). Values indicated mean ± standard deviation from (n = 6). Statistical significance (**, p < 0.01) was determined by a Student’s t-test and was shown as the statistical significance between ethanol with control. Con: control, Eth: treated with ethanol.

Ethanol Induced Production of H2O2 and O2.- in S. cerevisiae

To evaluate whether ROS accumulation was accompanied by ethanol stress, ROS production was measured by fluorescence staining and flow cytometry. Production of H2O2 and O2`- reached highest at 2 h according to the preliminary experiment. Therefore, production of H2O2 and O2`- was detected at 2 h. The production of H2O2 and O2.- was induced in both cases by ethanol stress for 2 h (Fig. 3A). H2O2 production in atg1, atg8 mutants was higher than that in wild type yeast under ethanol treatment for 24 h. In addition, the atg1 mutant had the highest H2O2 concentration (Fig. 3B). Similarly, the O2.- content in mutant cells was also higher than that in wild type yeast cells (Fig. 3C). However, the difference in O2.- production between wild type and mutants was not greatly different than that observed in H2O2. Therefore, these results demonstrated that ethanol stress contributed to the accumulation of ROS and autophagy had the ability to eliminate ROS.

Figure 3. Ethanol induced more H2O2 and O2.- in mutants than in wild type. Production of H2O2 and O2`- in wild type treated with 10% ethanol for 2 h were stained by DCFH or DHE (A). Production of H2O2 (B) and O2.- (C) in wild type, atg1 and atg8 treated with 10% ethanol for 24 h were stained by DCFH (B) or DHE (A). All strains were analyzed by flow cytometry. Black bars represent 10 μm.

Autophagy Induced by Ethanol Stress Depended on ROS

The above results show that ethanol not only induced the expression of ATG1 and ATG8 but also enhanced the production of H2O2 and O2.-. In addition, accumulating evidence shows that moderate ROS as signal molecules regulated autophagy [12, 21, 23]. Therefore, in order to clarify the relationship between autophagy and ROS, reductants were used to change ROS levels. Obviously, both GSH and NAC reductants decreased very significantly the production of H2O2 and O2.- under ethanol stress (Figs. 4A and 4B). The production of H2O2 was reduced to 55.4% and 44.0% by GSH and NAC (Fig. 4B). GSH and NAC decreased the production levels of O2.- to 43.5% and 29.2% (Fig. 4B). Simultaneously, GSH and NAC markedly decreased the gene expression of ATG1 and ATG8 (Fig. 4C). For example, expression of ATG1 and ATG8 was decreased to 54.7% and 38.8% by GSH. NAC lowered ATG1 and ATG8 expression to 55.6% and 19.3%. Moreover, in wild type, GSH and NAC also decreased the production of H2O2 and O2.- in atg1 and atg8 mutants (Fig. S1).

Figure 4. GSH and NAC decreased production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae under ethanol stress. (A) Production of H2O2 and O2.- in wild type treated with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 μm. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). GSH or NAC decreased expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.

In spite of moderate ROS acting as a signal, excess ROS oxidized nearby biological macromolecules including DNA, proteins and lipids [32-36]. In order to protect themselves from oxidative damage, cells used enzymes to eliminate ROS, such as catalase (CAT) in cytoplasm and manganese superoxide dimutase (Mn-SOD) in mitochondria [37-39]. Therefore, 2-ME and 3-AT were used as the inhibitors of Mn-SOD and CAT, to increase of O2.- and H2O2 levels in cells. The results showed that 2-ME increased the production of H2O2 and O2.- (Figs. 5A and 5B). 3-AT only induced the levels of O2.- to 159.0% but had no effect on the levels of H2O2 (Fig. 5B). 2-ME and 3-AT both activated the expression of ATG1 and ATG8 genes under ethanol stress (Fig. 5C). In detail, the expression of ATG1 and ATG8 was elevated to 216% and 183.9% by 2-ME. 3-AT increased the expression levels of ATG1 and ATG8 to 185.6% and 152.1%, respectively. The production of H2O2 and O2.- in atg1 and atg8 mutants was also induced by 2-ME and 3-AT (Fig. S2).

Figure 5. 2-ME and 3-AT increased production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae under ethanol stress. (A) Production of H2O2 and O2.- in wild type treated with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 um. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). 2-ME and 3-AT increased expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.

To further verify that ROS regulated autophagy in yeast under ethanol stress, the inhibitor of autophagy (3-MA) and the inducer of autophagy (Rapa) were added in the medium under ethanol stress. The results showed that Rapa very significantly promoted the production of H2O2 and O2.- in wild-type yeast (Figs. 6A and 6B) and evoked the expression of ATG1 and ATG8 (Fig. 6C). On the contrary, 3-MA dramatically decreased only the content of H2O2 (Figs. 6A and 6B) and reduced the expression of ATG1 and ATG8 (Fig. 6C). Therefore, autophagy was dependent on levels of H2O2 under ethanol stress. In mutants, Rapa and 3-MA both promoted the production of H2O2 and O2.-under ethanol stress (Fig. S3). Rapa and 3-MA did not change the levels of autophagy in the mutants. Accumulation of more H2O2 and O2.- by Rapa and 3-MA may be attributed to their alternative function.

Figure 6. Rapa and 3-MA regulated production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae. (A) Production of H2O2 and O2.- in wild-type yeast with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 μm. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). Rapa and 3- MA regulated expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.

ROS Induced by Ethanol Stress Mainly Originated from Mitochondria

Since we found that ROS were implicated in autophagy under ethanol stress, the source of ROS was also explored in this study. The respiratory complexes I, II, and III of the mitochondrial electron transport chain (mtETC) were reported to be the major production sites of O2.- [40, 41]. Anti A and Rote, as the inhibitors of complex III and complex I of the mtETC, were used to increase ROS in mitochondria. The results showed that Anti A and Rote both undoubtly increased production of H2O2 and O2.-(Figs. 7A and 7B) and promoted the expression of ATG1 and ATG8 genes under ethanol stress (Fig. 7C). In detail, production of H2O2 was increased to 212.8% and 257.4% by Rote and Anti A, respectively. Rote and Anti A raised production of O2.- to 225.0% and 299.2%, respectively. Expression of ATG1 and ATG8 was increased to 496.4%and 431.9% by Anti A. Rote raised ATG1 and ATG8 expression to 348.5% and 299.3%. These findings indicated that ROS derived from the mtETC-regulated autophagy under ethanol stress. Therefore, ROS accumulation in yeast attributable to H2O2 and O2.-derived from the mtETC. In the mutants, Anti A and Rote also increased production of H2O2 and O2.- (Fig. S4). The results further proved that production of H2O2 and O2.- was mainly caused by the mtETC under ethanol stress.

Figure 7. Rote and Anti A increased production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae. (A) Production of H2O2 and O2.- in wild-type yeast with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 μm. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). Rote and Anti A increased expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.

Discussion

The budding yeast had to escape from nitrogen deficient conditions during most alcoholic fermentations. And it was well known that autophagy could be induced by deficient nitrogen during fermentation [12, 13, 42]. However, Piggott et al. had demonstrated that autophagy was induced at the early stage of wine fermentation in a nitrogen-replete environment [14]. Therefore, it was suggested that autophagy might be triggered by other factors that arose during the early stage of fermentation. Besides that, it had been proven that autophagy was induced in second-fermentation yeasts during sparkling wine production [43]. Cost-effective ethanol production depended on rapid and high-yielding conversion of carbohydrate to ethanol. Therefore, ethanol accumulation in the culture broth appeared at the early stage of fermentation. It was well known that the yeast had the property of ethanol tolerance. However, the mechanism of the ethanol tolerance was not clear. In the current study, the results showed that autophagy was induced by ethanol stress (Fig. 1). And compared with atg1 and atg 8, wild type contributed to cell survival from ethanol stress (Fig. 2). Therefore, yeast was likely surviving ethanol injury by means of increasing the levels of autophagy to clear away the damaged organelles.

Ethanol Stress Induced ROS in S. cerevisiae

CTT1, encoding a kind of cytosolic CAT, was found to be highly expressed only in ethanol-tolerant sake yeast mutants or after exposure to ethanol [44]. Therefore, eliminating of ROS by CTT1 helped sake yeast survive ethanol stress. Otherwise, ROS were normally induced during fermentation [18, 19]. However, the exact relationship between ROS and ethanol stress was still unclear. In this study, ROS production in wild-type yeast (Figs. 4-7) and mutant cells (Figs. S1, S2, and S4) was induced by ethanol stress. However, the role of ROS was still unknown under ethanol stress.

Compared to wild-type yeast, the atg1 and atg8 mutants had higher production of H2O2 and O2.- under ethanol stress (Fig. 3). These results were in agreement with previous reports. For instance, it had been shown that the mutants atg2 and atg5 accumulated high levels of H2O2 [45]. Thus, autophagy was in favor of eliminating ROS under ethanol stress. In addition, atg1 and atg8 mutants had a higher cell death rate than-wild type yeast (Fig. 2). These findings suggest that wild-type yeast markedly decreased cell death under ethanol by eliminating ROS by autophagy.

ROS Derived from mtETC Regulated Autophagy under Ethanol Stress

Mounting evidence suggested that ROS might play a role in the control of autophagy [21-23, 46, 47]. And we had proven that ethanol activated production of H2O2 and O2.-(Fig. 3A) and autophagy (Fig. 1). In addition, GSH and NAC decreased autophagy by decreasing the release of H2O2 and O2.- under ethanol stress (Fig. 4). In agreement, NAC had been reported to reduce ethanol-induced autophagy [48] and also to decrease salinomycin-induced autophagy [49]. On the contrary, in our study 2-ME induced autophagy by means of increasing production of H2O2 and O2.-whereas 3-AT increased autophagy probably just through production of O2.- (Fig. 5). Therefore, our results revealed that H2O2 and O2.- regulated autophagy levels in the yeasts under ethanol stress.

Moreover, Rapa promoted the production of H2O2 and O2.- and increased the expression of the ATG1 and ATG8 genes (Fig. 6). However, 3-MA decreased the levels of H2O2 and lowered the expression of the ATG1 and ATG8 genes (Fig. 6). The difference was that both Rapa and 3-MA enhanced production of H2O2 and O2.- in atg1 and atg8 mutants under ethanol stress (Fig. S3). Rapa and 3-MA did not affect the level of autophagy in mutants. Alternative functions of Rapa or 3-MA maybe associated with the increased accumulation of H2O2 and O2.-.

Although ROS have been previously reported to be implicated in autophagy, specifically which ROS were playing this crucial regulatory role was still disputed. Chen et al. considered that O2.- was the main kind of ROS inducing autophagy [24]. However, Mendes-Ferreira et al reported that all kinds of ROS except O2.- had vital roles in regulating autophagy [12]. There were also some reports showing that autophagy was induced by H2O2 [21, 46]. In our study, H2O2 and O2.- both participated in regulating autophagy in yeast under ethanol stress.

Mitochondrion was the main souce of ROS [40, 41]. In the current study, Anti A and Rote also increased the prodution of H2O2 and O2-. in atg1 and atg8 mutants under ethanol (Fig. S4). The results proved that H2O2 and O2- were mainly derived from the mtETC under ethanol. Anti A and Rote enhanced the production of H2O2 and O2-. and activated the expression of the ATG1 and ATG8 genes in wild type (Fig. 7). Therefore, ROS derived from mitochondria had vital roles in regulating autophagy under ethanol stress. Many studies have shown that ROS from mitochondria regulated autophagy or mitophagy [39, 50-52]. For example, MITA expression modulated autophagy flux through enhancing mitochondrial ROS by increasing complex-I activity [51]. In addition, mitochondrial complex I inhibition triggered a mitophagy-dependent ROS increase [52].

Overall, ethanol stress induced autophagy and increased the production of H2O2 and O2.- which were originated from the mtETC, although other sources of ROS are not excluded. Under ethanol stress, the high levels of H2O2 and O2.- markedly induced autophagy. Subsequently, the autophagy contributed to the elimination of H2O2 and O2.-. Ultimately, the autophagy assisted yeast in surviving ethanol stress during fermentation.

Supplemental Materials

Acknowledgments

This work was supported by projects of the National Natural Science Foundation of China (31201409; 31471296), Natural Science Project of Henan Science and Technology Department (162300410175), National Engineering Laboratory for Wheat & Corn Further Processing, Henan University of Technology (NL2016011), and Fundamental Research Funds for the Henan Provincial Colleges and Universities in Henan University of Technology (2016QNJH20).

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Ethanol-induced expression of the ATG1 and ATG8 genes. Normalized fold expression levels of the ATG1 and ATG8 genes were evaluated by qRT-PCR in S. cerevisiae treated with 10% ethanol for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test and was shown as the statistical significance between ethanol with control. Con: control, Eth: treated with ethanol.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

Fig 2.

Figure 2.Cell death of mutants was higher than that of wild type under ethanol stress. Wild type, atg1 and atg8 treated with 10% ethanol for 2 h were stained with PI. Then all strains were analyzed by fluorescence microscope (A) and fluorescence microplate reader (B). Values indicated mean ± standard deviation from (n = 6). Statistical significance (**, p < 0.01) was determined by a Student’s t-test and was shown as the statistical significance between ethanol with control. Con: control, Eth: treated with ethanol.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

Fig 3.

Figure 3.Ethanol induced more H2O2 and O2.- in mutants than in wild type. Production of H2O2 and O2`- in wild type treated with 10% ethanol for 2 h were stained by DCFH or DHE (A). Production of H2O2 (B) and O2.- (C) in wild type, atg1 and atg8 treated with 10% ethanol for 24 h were stained by DCFH (B) or DHE (A). All strains were analyzed by flow cytometry. Black bars represent 10 μm.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

Fig 4.

Figure 4.GSH and NAC decreased production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae under ethanol stress. (A) Production of H2O2 and O2.- in wild type treated with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 μm. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). GSH or NAC decreased expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

Fig 5.

Figure 5.2-ME and 3-AT increased production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae under ethanol stress. (A) Production of H2O2 and O2.- in wild type treated with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 um. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). 2-ME and 3-AT increased expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

Fig 6.

Figure 6.Rapa and 3-MA regulated production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae. (A) Production of H2O2 and O2.- in wild-type yeast with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 μm. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). Rapa and 3- MA regulated expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

Fig 7.

Figure 7.Rote and Anti A increased production of H2O2 and O2.- and expression of ATG1 and ATG8 genes in S. cerevisiae. (A) Production of H2O2 and O2.- in wild-type yeast with 10% ethanol for 2 h was stained by DCFH or DHE, respectively. Black bars represent 10 μm. Relative fluorescence of DCFH and DHE is shown in (B). Values indicated mean ± standard deviation (n = 6). Rote and Anti A increased expression of ATG1 and ATG8 (C). Normalized fold expression levels of ATG1 and ATG8 were evaluated by qRT-PCR in S. cerevisiae under ethanol stress for 4 h. ACT1, encoded actin, was used as internal reference. Values indicated mean ± standard deviation (n = 3). Statistical significance (**, p < 0.01) was determined by a Student’s t-test.
Journal of Microbiology and Biotechnology 2018; 28: 1982-1991https://doi.org/10.4014/jmb.1806.06014

References

  1. Kanki T, Furukawa K, Yamashita SI. 2015. Mitophagy in yeast: molecular mechanisms and physiological role. Biochim. Biophys. Acta 1853: 2756-2765.
    Pubmed CrossRef
  2. Tsukada M, Ohsumi Y. 1993. Isolation and characterization of autophagy defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333: 169-174.
    CrossRef
  3. Fracchiolla D, Sawa-Makarska J, Zens B, Ruiter A, Zaffagnini G, Brezovich A, et al. 2016. Mechanism of cargodirected Atg8 conjugation during selective autophagy. Elife 5(pii): e18544.
    Pubmed KoreaMed CrossRef
  4. Weiergräber OH, Schwarten M, Strodel B, Willbold D. 2017. Investigating structure and dynamics of Atg8 family proteins. Methods Enzymol. 587: 115-142.
    Pubmed CrossRef
  5. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal D. 2008. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4: 151-175.
  6. Reggiori F, Klionsky DJ. 2013. Autophagic processes in yeast: mechanism, machinery and regulation. Genetics 194: 341-361.
    Pubmed KoreaMed CrossRef
  7. Wong PM, Puente C, Ganley IG, Jiang X. 2013. The ULK1 complex: sensing nutrient signals for autophagy activation. Autophagy 9: 124-137.
    Pubmed KoreaMed CrossRef
  8. Xiong Y, McCormack M, Li L, Hall Q, Xiang C, Sheen J. 2013. Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature 496: 181-186.
    Pubmed KoreaMed CrossRef
  9. Chew LH, Lu S, Liu X, Li FK, Yu AY, Klionsky DJ, et al. 2015. Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms. Autophagy 11: 891-905.
    Pubmed KoreaMed CrossRef
  10. Perpetuini G, Di Gianvito P, Arfelli G, Schirone M, Corsetti A, Tofalo R, et al. 2016. Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation. Yeast 33: 303-312.
    Pubmed CrossRef
  11. Suzuki H, Osawa T, Fujioka Y, Noda NN. 2017. Structural biology of the core autophagy machinery. Curr. Opin. Struct. Biol. 43: 10-17.
    Pubmed CrossRef
  12. Mendes-Ferreira A, Sampaio-Marques B, Barbosa C, Rodrigues F, Costa V, Mendes-Faia A, et al. 2010. Accumulation of non-superoxide anion reactive oxygen species mediates nitrogen-limited alcoholic fermentation by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 76: 7918-24.
    Pubmed KoreaMed CrossRef
  13. Tesnière C, Brice C, Blondin B. 2015. Responses of Saccharomyces cerevisiae to nitrogen starvation in wine alcoholic fermentation. Appl. Microbiol. Biotechnol. 99: 7025-7034.
    Pubmed CrossRef
  14. Piggott N, Cook MA, Tyers M, Measday V. 2011. Genomewild fitness profiles reveal a requirement for autophagy during yeast fermentation. G3 1: 353-367.
    CrossRef
  15. Horie T, Kawamata T, Matsunami M, Ohsumi Y. 2017. Recycling of iron via autophagy is critical for the transition from glycolytic to respiratory growth. J. Biol. Chem. 292: 8533-8543.
    Pubmed KoreaMed CrossRef
  16. Gibson BR, Lawrence SJ, Boulton CA, Box WG, Graham NS, Linforth S, et al. 2008. The oxidative stress response of a lager brewing yeast strain during industrial propagation and fermentation. FEMS Yeast Res. 8: 574-585.
    Pubmed CrossRef
  17. Landolfo S, Politi H, Angelozzi D, Mannazzu I. 2008. ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium. Biochim. Biophys. Acta 1780: 892-898.
    Pubmed CrossRef
  18. Cheng Y, Du Z, Zhu H, Guo X, He X. 2016. Protective effects of arginine on Saccharomyces cerevisiae against ethanol stress. Sci. Rep. 6: 31311.
    Pubmed KoreaMed CrossRef
  19. Charoenbhakdi S, Dokpiku T, Burphan T, Techo T, Auesukaree C. 2016. Vacuolar H+-ATPase protects Saccharomyces cerevisiae cells against ethanol-induced oxidative and cell wall stresses. Appl. Environ. Microbiol. 82: 3121-3130.
    Pubmed KoreaMed CrossRef
  20. Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 9: 249-279.
    Pubmed CrossRef
  21. Song C, Mitter SK, Qi X, Beli E, Rao HV, Ding J, et al. 2017. Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1and promotes retinal pigmented epithelial cell survival through increased autophagy. PLos One 12: e0171940.
    Pubmed KoreaMed CrossRef
  22. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. 2007. Reactive oxygen species are essential for autophagy and specifically regulate the activity of atg4. EMBO J. 26: 1749-1760.
    Pubmed KoreaMed CrossRef
  23. Xua J, Wua Y, Lu G, Xie S, Ma Z, Chen Z, Shen HM, et al. 2017. Importance of ROS-mediated autophagy in determining apoptotic cell death induced by physapubescin B. Redox. Biol. 12: 198-207.
    Pubmed KoreaMed CrossRef
  24. Chen SY, Chiu LY, Maa MC, Wang JS, Chien CL, Lin WW. 2011. zVAD-induced autophagic cell death requires c-Srcdependent ERK and JNK activation and reactive oxygen species generation. Autophagy 7: 217-28.
    Pubmed KoreaMed CrossRef
  25. Demain AL. 2009. Biosolutions to the energy problem. J. Ind. Microbiol. Biotechnol. 36: 319-332.
    Pubmed CrossRef
  26. Galeote VA, Blondin B, Dequin S, Sablayrolles JM. 2001. Stress effects of ethanol on fermentation kinetics by stationary-phase cells of Saccharomyces cerevisiae. Biotechnol. Lett. 23: 677-681.
    CrossRef
  27. Aguilera F, Peinado RA, Millan C, Ortega JM, Mauricio JC. 2006. Relationship between ethanol tolerance, H+-ATPase activity and the lipid composition of the plasma membrane in different wine yeast strains. Int. J. Food Microbiol. 110: 34-42.
    Pubmed CrossRef
  28. Walker ME, Nguyen TD, Liccioli T, Schmid F, Kalatzis N, Sundstrom JF, et al. 2014. Genome-wild identification of the Fermentome; genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae. BMC Genomics 15: 552.
    Pubmed KoreaMed CrossRef
  29. Almeida B, Sampaio-Marques B, Carvalho J, Silva MT, Leao C, Rodrigues F, et al. 2007. An atypical active cell death process underlies the fungicidal activity of ciclopirox olamine against the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 7: 404-412.
    Pubmed CrossRef
  30. Ludovico P, Rodrigues F, Almeida A, Silva MT, Barrientos A, Corte-Real M. 2002. Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol. Bio. Cell 13: 2598-2606.
    Pubmed KoreaMed CrossRef
  31. Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3: 1101-1108.
    Pubmed CrossRef
  32. Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7: 405-410.
    CrossRef
  33. Barja G. 2004. Free radicals and aging. Trends Neurosci. 27: 595-600.
    Pubmed CrossRef
  34. Yin D, Chen K. 2005. The essential mechanisms of aging: irreparable damage accumulation of biochemical side-reactions. Exp. Gerontol. 40: 455-465.
    Pubmed CrossRef
  35. Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C. 2006. Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bio. Essays. 28: 1091-2101.
    Pubmed CrossRef
  36. Chen HJ, Huang CS, Huang GJ, Chow TJ, Lind YH. 2013. NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas). J. Plant Physiol. 170: 1471-1483.
    Pubmed CrossRef
  37. Rattanawong K, Kerdsomboon K, Auesukaree C. 2015. Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlorin Saccharomy cescerevisiae. Free Radic. Bio. Med. 89: 963-971.
    Pubmed CrossRef
  38. Magrì A, Di Rosa MC, Tomasello MF, Guarino F, Reina S, Messina A, Pinto VD. 2016. Overexpression of human SOD1 in VDAC1-less yeast restores mitochondrial functionality modulating beta-barrel outer membrane protein genes. Biochim. Biophys. Acta 1857: 789-798.
    Pubmed CrossRef
  39. Knuppertz L, Warnsmann V, Hamann A, Grimm C, Osiewacz HD. 2017. Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina. Autophagy 13: 1037-1052.
    Pubmed KoreaMed CrossRef
  40. Murphy MP. 2009. How mitochondria produce reactive oxygen species. Biochem. J. 417: 1-13.
    Pubmed KoreaMed CrossRef
  41. Huang S, Aken OV, Schwarzländer M, Belt K, Millar AH. 2016. The roles of mitochondrial reactive oxygen species in cellular signaling and stress response in plants. Plant Physiol. 171: 1551-1559.
    Pubmed KoreaMed CrossRef
  42. Li D, Song JZ, Li H, Shan MH, Liang Y, Zhu J, et al. 2015. Storage lipid synthesis is necessary for autophagy induced by nitrogen starvation. FEBS Lett. 89: 269-276.
    Pubmed CrossRef
  43. Cebollero E, Gonzalez R. 2006. Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines. Appl. Environ. Microb. 72: 4121-4127.
    Pubmed KoreaMed CrossRef
  44. Ogawa Y, Nitta A, Uchiyama H, Imamura T, Shimoi H, Ito K. 2000. Tolerance mechanism of the ethanoltolerant mutant of sake yeast. J. Biosci. Bioeng. 90: 313-320.
    Pubmed CrossRef
  45. Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, et al. 2009. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell 21: 2914-2927.
    Pubmed KoreaMed CrossRef
  46. Pérez-Pérez ME, Zaffagnini M, Marchand CH, Crespo JL, Lemaire SD. 2014. The yeast autophagy protease Atg4 is regulated by thioredoxin. Autophagy 10: 1953-1964.
    Pubmed KoreaMed CrossRef
  47. Navarro-Yepes J, Burns M, Anandhan A, Khalimonchuk O, del Razo LM, Quintanilla-Vega B, et al. 2014. Oxidative stress, redox signaling, and autophagy: cell death versus survival. Antioxid. Redox. Signal 21: 66-85.
    Pubmed KoreaMed CrossRef
  48. Zhu Z, Huang Y, Lv L, Tao Y, Shao M, Zhao C, et al. 2017. Acute ethanol exposure-induced autophagy-mediated cardiac injury v ia a ctivation of the ROS-JNK-Bcl-2 pathway. J. Cell Physiol. 233: 924-935.
    Pubmed CrossRef
  49. Kim KY, Park KI, Kim SH, Yu SN, Lee D, Kim YW, et al. 2017. Salinomycin induces reactive oxygen species and apoptosis in aggressive breast cancer cells as mediated with regulation of autophagy. Anticancer Res. 37: 1747-1758.
    Pubmed CrossRef
  50. Shiroma S, Jayakody LN, Horie K, Okamoto K, Kitagakia H. 2014. Enhancement of ethanol fermentation in Saccharomyces cerevisiae sake yeast by disrupting mitophagy function. Appl. Environ. Microbiol. 80: 1002-1012.
    Pubmed KoreaMed CrossRef
  51. Basit F, van Oppen LM, Schöckel L, Bossenbroek HM, van Emst-de Vries SE, Hermeling JC, et al. 2017. Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells. Cell Death Dis. 8: e2716.
    Pubmed KoreaMed CrossRef
  52. Bhatelia K, Singh K, Prajapati P, Sripada L, Roy M, Singh R. 2017. MITA modulated autophagy flux promotes cell death in breast cancer cells. Cell Signal 35: 73-83.
    Pubmed CrossRef