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Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.)

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Abstract

The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O ·−2 , H2O2 and OH· was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.

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References

  • Aono M, Saji H, Fujiyama K, Sugita M, Kondo N, Tanaka K (1995) Decrease in activity of glutathione reductase enhances paraquat sensitivity in transgenic Nicotiana tabacum. Plant Physiol 107:645–648

    PubMed Central  CAS  PubMed  Google Scholar 

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  CAS  PubMed  Google Scholar 

  • Bakker EP (1992) Aminoglycoside and aminocyclitol antibiotics: hygromycin B is an atypical bactericidal compound that exerts effects on cells of Escherichia coli characteristics for bacteriostatic aminocyclitols. J Gen Microbiol 138:563–569

    Article  CAS  PubMed  Google Scholar 

  • Beauchamp CO, Fridovich I (1971) Superoxide dismutase. Improved assays and an assay applicable to acrylamide gel. Anal Biochem 44:276–287

    Article  CAS  PubMed  Google Scholar 

  • Blochinger K, Diggelmann H (1984) Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eukaryotic cells. Mol Cell Biol 4:2929–2931

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Cabañas MJ, Vázquez D, Modolell J (1978) Dual interference of hygromycin B with ribosomal translocation and with aminoacyl-tRNA recognition. Eur J Biochem 87:21–27

    Article  PubMed  Google Scholar 

  • Caplan A, Dekeyser R, Van Montagu M (1992) Selectable markers for rice transformation. Methods Enzymol 216:426–441

    Article  CAS  PubMed  Google Scholar 

  • Chen YP, Xing LP, Wu GJ, Wang HZ, Wang XE, Cao AZ, Chen PD (2007) Plastidial glutathione reductase from Haynaldia villosa is an enhancer of powdery mildew resistance in wheat (Triticum aestivum). Plant Cell Physiol 48:1702–1712

    Article  CAS  PubMed  Google Scholar 

  • Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218

    Article  CAS  PubMed  Google Scholar 

  • Davies J, Gorini L, Davis BD (1965) Misreading of RNA codewords induced by aminoglycoside antibiotics. Mol Pharmacol 1:93–106

    CAS  PubMed  Google Scholar 

  • Davis BD (1987) Mechanism of bactericidal action of aminoglycosides. Microbiol Rev 51:341–350

    PubMed Central  CAS  PubMed  Google Scholar 

  • Dekeyser R, Claes B, Marichal M, Van Montagu M, Caplan A (1989) Evaluation of selectable markers for rice transformation. Plant Physiol 90:217–223

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ding S, Lu Q, Zhang Y, Yang Z, Wen X, Zhang L, Lu C (2009) Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state. Plant Mol Biol 69:577–592

    Article  CAS  PubMed  Google Scholar 

  • Duan H, Ding X (2007) Effects of hygromycin on growth and development of Arabidopsis seedlings roots. Pak J Bot 39:2167–2173

    Google Scholar 

  • Dwyer DJ, Kohanski MA, Hayete B, Collins JJ (2007) Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol Syst Biol 3:91

    Article  PubMed Central  PubMed  Google Scholar 

  • Dwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD, Takahashi N, Chan CT, Lobritz MA, Braff D, Schwarz EG, Ye JD, Pati M, Vercruysse M, Ralifo PS, Allison KR, Khalil AS, Ting AY, Walker GC, Collins JJ (2014) Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci USA 111:E2100–E2109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Elstner EF, Heupel A (1976) Inhibition of nitrite formation from hydroxylammonium-chloride: a simple assay for superoxide dismutase. Anal Biochem 70:616–620

    Article  CAS  PubMed  Google Scholar 

  • Eustice DC, Wilhelm JM (1984) Mechanisms of action of aminoglycoside antibiotics in eukaryotic protein synthesis. Antimicrob Agents Chemother 26:53–60

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ezraty B, Vergnes A, Banzhaf M, Duverger Y, Huguenot A, Brochado AR, Su SY, Espinosa L, Loiseau L, Py B, Typas A, Barras F (2013) Fe–S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway. Science 340:1583–1587

    Article  CAS  PubMed  Google Scholar 

  • Foster JG, Hess JL (1980) Superoxide dismutase and glutathione reductase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. Plant Physiol 66:482–487

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Foti JJ, Devadoss B, Winkler JA, Collins J, Walker GC (2012) Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics. Science 336:315–319

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez A, Jimenez A, Vasquez D, Davies JE, Schindler D (1978) Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim Biophys Acta 521:459–469

    Article  CAS  PubMed  Google Scholar 

  • Grant SS, Kaufmann BB, Chand NS, Haseley N, Hung DT (2012) Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci USA 109:12147–12152

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179–188

    Article  CAS  PubMed  Google Scholar 

  • Hagio T, Hirabayashi T, Machii H, Tomotsune H (1995) Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker. Plant Cell Rep 14:329–334

    Article  CAS  PubMed  Google Scholar 

  • Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

    Article  CAS  PubMed  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumanshiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of T-DNA. Plant J 6:271–282

    Article  CAS  PubMed  Google Scholar 

  • Hoefnagel MHN, Millar AH, Wiskich JT, Day DA (1995) Cytochrome and alternative respiratory pathways compete for electrons in the presence of pyruvate in soybean mitochondria. Arch Biochem Biophys 318:394–400

    Article  CAS  PubMed  Google Scholar 

  • Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF, Yu SM (2004) Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 13:29–33

    Article  CAS  PubMed  Google Scholar 

  • Kalghatgi S, Spina CS, Costello JC, Liesa M, Morones-Ramirez JR, Slomovic S, Molina A, Shirihai OS, Collins JJ (2013) Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells. Sci Transl Med 5:192ra85

    Article  PubMed Central  PubMed  Google Scholar 

  • Kato M, Shimizu S (1987) Chlorophyll metabolism in higher plant. VII. Chlorophyll degradation in senescing tobacco leaves; phenolic-dependent peroxidative degradation. Can J Bot 65:729–735

    Article  CAS  Google Scholar 

  • Keren I, Wu Y, Inocencio J, Mulcahy LR, Lewis K (2013) Killing by bactericidal antibiotics does not depend on reactive oxygen species. Science 339:1213–1216

    Article  CAS  PubMed  Google Scholar 

  • Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ (2007) A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130:797–810

    Article  CAS  PubMed  Google Scholar 

  • Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ (2008) Mistranslation of membrane proteins and two-component system activation trigger antibiotic mediated cell death. Cell 135:679–690

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kohanski MA, DePristo MA, Collins JJ (2010) Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol Cell 37:311–320

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Le Martret B, Poage M, Shiel K, Nugent GD, Dix PJ (2011) Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance. Plant Biotechnol J 9:661–673

    Article  PubMed  Google Scholar 

  • Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Imlay JA (2013) Cell death from antibiotics without the involvement of reactive oxygen species. Science 339:1210–1213

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Liu Y, Liu X, Qu Y, Wang X, Li L, Zhao X (2012) Inhibitors of reactive oxygen species accumulation delay and/or reduce the lethality of several antistaphylococcal agents. Antimicrob Agents Chemother 56:6048–6050

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mann RL, Bromer WW (1958) The isolation of a second antibiotic from Streptomyces ygroscopicus. J Am Chem Soc 80:2714–2716

    Article  CAS  Google Scholar 

  • Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498

    Article  CAS  PubMed  Google Scholar 

  • Møller IM (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 52:561–591

    Article  PubMed  Google Scholar 

  • Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880

    CAS  Google Scholar 

  • Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279

    Article  CAS  PubMed  Google Scholar 

  • Noctor G, Mhamdi A, Chaouch S, Han Y, Neukermans J, Marquez-Garcia B, Queval G, Foyer CH (2012) Glutathione in plants: an integrated overview. Plant Cell Environ 35:454–484

    Article  CAS  PubMed  Google Scholar 

  • Orozco-Cárdenas ML, Ryan C (1999) Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc Natl Acad Sci USA 96:6553–6557

    Article  PubMed Central  PubMed  Google Scholar 

  • Purvis AC, Shewfelt RL (1993) Does the alternative pathway ameliorate chilling injury in sensitive plant tissues? Physiol Plant 88:712–718

    Article  CAS  Google Scholar 

  • Rizhsky L, Davletova S, Liang H, Mittler R (2004) The zinc finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J Biol Chem 279:11736–11743

    Article  CAS  PubMed  Google Scholar 

  • Sandalio LM, Rodriguez-Serrano M, Romero-Puertas M, del Rio LA (2008) Imaging of reactive oxygen species and NO in vivo in plant tissues. Methods Enzymol 440:399–409

    Google Scholar 

  • Scandalios JG (2002) The rise of ROS. Trends Biochem Sci 27:483–486

    Article  CAS  PubMed  Google Scholar 

  • Setsukinai KI, Urano Y, Kakinuma K, Majima HJ, Nagano T (2003) Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 278:3170–3175

    Article  CAS  PubMed  Google Scholar 

  • Shu DF, Wang LY, Duan M, Deng YS, Meng QW (2011) Antisense-mediated depletion of tomato chloroplast glutathione reductase enhances susceptibility to chilling stress. Plant Physiol Biochem 49:1228–1237

    Article  CAS  PubMed  Google Scholar 

  • Tsai YC, Hong CY, Liu LF, Kao CH (2004) Relative importance of Na+ and Cl− in NaCl-induced antioxidant systems in roots of rice seedlings. Physiol Plant 122:86–94

    Article  CAS  Google Scholar 

  • Van Den Elzen PJ, Townsend J, Lee KY, Bedbrook JR (1985) A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5:299–302

    Article  PubMed  Google Scholar 

  • Waldron C, Murphy EB, Roberts JL, Gustafson GD, Armour SL, Malcom SK (1985) Resistance to hygromycin B: a new marker for plant transformation. Plant Mol Biol 5:103–108

    Article  CAS  PubMed  Google Scholar 

  • Walters DA, Vetsch CS, Potts DE, Lundquist RC (1992) Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants. Plant Mol Biol 18:189–200

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Zhao X (2009) Contribution of oxidative damage to antimicrobial lethality. Antimicrob Agents Chemother 53:1395–1402

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Weisblum B, Davies J (1968) Antibiotic inhibitors of the bacterial ribosome. Bacteriol Rev 32:493–528

    PubMed Central  CAS  PubMed  Google Scholar 

  • Wu TM, Lin WR, Kao YT, Hsu YT, Yeh CH, Hong CY, Kao CH (2013) Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice. Plant Mol Biol 83:379–390

    Article  CAS  PubMed  Google Scholar 

  • Wu TM, Lin WR, Kao CH, Hong CY (2015) Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice. Plant Mol Biol 87:555–564

    Article  CAS  PubMed  Google Scholar 

  • Zidenga T, Leyva-Guerrero E, Moon H, Siritunga D, Sayre R (2012) Extending cassava root shelf life via reduction of reactive oxygen species production. Plant Physiol 159:1396–1407

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was partly supported by research grant NSC 101-2313-B-002-008-MY3 from the Ministry of Science and Technology (MOST) of the Republic of China to C.Y. Hong. T.M. Wu was supported by a postdoctoral fellowship (NSC 103-2811-B-002-142) from the MOST of the Republic of China. N.N.P. Chandrika was supported by National Taiwan University Postdoctoral Research Fellowship.

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  1. Tsung-Meng Wu

    Present address: Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan

Authors and Affiliations

  1. Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, Taipei, 10617, Taiwan

    Hui-Min Oung, Ke-Chun Lin, Tsung-Meng Wu, Nulu Naga Prafulla Chandrika & Chwan-Yang Hong

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  1. Hui-Min Oung
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  2. Ke-Chun Lin
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Correspondence to Chwan-Yang Hong.

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Hui-Min Oung, Ke-Chun Lin, Tsung-Meng Wu, Nulu Naga Prafulla Chandrika have contributed equally to this work.

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Oung, HM., Lin, KC., Wu, TM. et al. Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.). Plant Mol Biol 89, 577–588 (2015). https://doi.org/10.1007/s11103-015-0380-4

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