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Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

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Molecules and Cells

Abstract

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.

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References

  • Adedapo, A., Jimoh, F., Koduru, S., Afolayan, A., and Masika, P. (2008). Antibacterial and antioxidant properties of the methanol extracts of the leaves and stems of Calpurnia aurea. BMC Complement. Altern. Med. 8, 53.

    Article  PubMed  Google Scholar 

  • Ainsworth, E.A., and Gillespie, K.M. (2007). Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nat. Protoc. 2, 875–877.

    Article  PubMed  CAS  Google Scholar 

  • Asada, K. (1987). Production and scavenging of active oxygen in photosynthesis. Photoinhibition.

    Google Scholar 

  • Asada, K. (1997). The role of ascorbate peroxidase and monodehydroascorbate reductase in H2O2 scavenging in plants. Cold Spring Harbor Monograph Arch. 34, 715–735.

    CAS  Google Scholar 

  • Asada, K. (1999). The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Biol. 50, 601–639.

    Article  CAS  Google Scholar 

  • Baier, M., and Dietz, K.J. (1999). The costs and benefits of oxygen for photosynthesizing plant cells. Prog. Bot. 60, 282–314.

    Article  CAS  Google Scholar 

  • Benzie, I.F.F., and Strain, J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76.

    Article  PubMed  CAS  Google Scholar 

  • Borraccino, G., Dipierro, S., and Arrigoni, O. (1986). Purification and properties of ascorbate free-radical reductase from potato tubers. Planta 167, 521–526.

    Article  CAS  Google Scholar 

  • Bradford, M. (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  PubMed  CAS  Google Scholar 

  • Brehe, J., and Burch, H. (1976). Enzymatic assay for glutathione. Anal. Biochem. 74, 189–197.

    Article  PubMed  CAS  Google Scholar 

  • Chen, H., Nelson, R.S., and Sherwood, J.L. (1994). Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16, 664–668.

    PubMed  CAS  Google Scholar 

  • Clough, S., and Bent, A. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.

    Article  PubMed  CAS  Google Scholar 

  • Dalton, D., Langeberg, L., and Robbins, M. (1992). Purification and characterization of monodehydroascorbate reductase from soybean root nodules1. Arch. Biochem. Biophys. 292, 281–286.

    Article  PubMed  CAS  Google Scholar 

  • Dalton, D., Baird, L., Langeberg, L., Taugher, C., Anyan, W., Vance, C., and Sarath, G. (1993). Subcellular localization of oxygen defense enzymes in soybean (Glycine max [L.] Merr.) root nodules. Plant Physiol. 102, 481–489.

    PubMed  CAS  Google Scholar 

  • Dhindsa, R., Plumb-Dhindsa, P., and Thorpe, T. (1981). Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101.

    Article  CAS  Google Scholar 

  • Dipierro, S., and Borraccino, G. (1991). Dehydroascorbate reductase from potato tubers. Phytochemistry 30, 427–429.

    Article  CAS  Google Scholar 

  • Eltayeb, A.E., Kawano, N., Badawi, G.H., Kaminaka, H., Sanekata, T., Morishima, I., Shibahara, T., Inanaga, S., and Tanaka, K. (2006). Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol. Physiol. Plant. 127, 57–65.

    Article  CAS  Google Scholar 

  • Foyer, C.H., and Halliwell, B. (1976). The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25.

    Article  Google Scholar 

  • Foyer, C.H., and Noctor, G. (2000). Oxygen processing in photosynthesis: regulation and signaling. New Phytol. 146, 359–388.

    Article  CAS  Google Scholar 

  • Fridovich, I. (1986). Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247, 1–11.

    Article  PubMed  CAS  Google Scholar 

  • Gechev, T., Willekens, H., Van Montagu, M., Inze, D., Van Camp, W., Toneva, V., and Minkov, I. (2003). Different responses of tobacco antioxidant enzymes to light and chilling stress. J. Plant Physiol. 160, 509–515.

    Article  PubMed  CAS  Google Scholar 

  • Gillespie, K., and Ainsworth, E. (2007). Measurement of reduced, oxidized and total ascorbate content in plants. Nat. Protoc. 2, 871–874.

    Article  PubMed  CAS  Google Scholar 

  • Grantz, A.A., Brummell, D.A., and Bennett, A.B. (1995). Ascorbate free radical reductase mRNA levels are induced by wounding. Plant Physiol. 108, 411–418.

    Article  PubMed  CAS  Google Scholar 

  • Halliwell, B., and Gutteridge, J.M.C. (1999). Free radicals in biology and medicine. (Oxford University Press New York).

    Google Scholar 

  • Harlow, E., and Lane, D. (1988). Antibodies: a laboratory manual (CSHL Press).

    Google Scholar 

  • Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fujita, Y., Yasuhara, T., Yoshida, T., and Okuda, T. (1989). Effects of the interaction of tannins with co-existing substances. VI: effects of tannins and related polyphenols on superoxide anion radical, and on 1, 1-diphenyl-2-picrylhydrazyl radical. Chem. Pharm. Bull. 37, 2016–2021.

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Hossain, M., and Asada, K. (1984a). Inactivation of ascorbate peroxidase in spinach chloroplasts on dark addition of hydrogen peroxide: its protection by ascorbate. Plant Cell Physiol. 25, 1285–1295.

    CAS  Google Scholar 

  • Hossain, M.A., and Asada, K. (1984b). Purification of dehydroascorbate reductase from spinach and its characterization as a thiol enzyme. Plant Cell Physiol. 25, 85.

    CAS  Google Scholar 

  • Hossain, M., and Asada, K. (1985). Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. J. Biol. Chem. 260, 12920–12926.

    PubMed  CAS  Google Scholar 

  • Hossain, M., Nakano, Y., and Asada, K. (1984). Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol. 25, 385–395.

    CAS  Google Scholar 

  • Inze, D., and Montagu, M.V. (1995). Oxidative stress in plants. Curr. Opin. Biotechnol. 6, 153–158.

    Article  CAS  Google Scholar 

  • Jimenez, A., Hernandez, J., del Rio, L., and Sevilla, F. (1997). Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol. 114, 275.

    PubMed  CAS  Google Scholar 

  • Karimi, M., Inze, D., and Depicker, A. (2002). GATEWAY (TM) vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193–195.

    Article  PubMed  CAS  Google Scholar 

  • Karuppanapandian, T., Sinha, P.B., Haniya, A.M.K., and Mamoharan, K. (2006). Differential antioxidative responses of ascorbate glutathione cycle enzymes and metabolites to chromium stress in green gram (Vigna radiata L. Wilczek) leaves. J. Plant Biol. 49, 440–447.

    Article  CAS  Google Scholar 

  • Kato, Y., Urano, J., Maki, Y., and Ushimaru, T. (1997). Purification and characterization of dehydroascorbate reductase from rice. Plant Cell Physiol. 38, 173–178.

    Article  CAS  Google Scholar 

  • Kendall, E.J., and McKersie, B.D. (1989). Free radical and freezing injury to cell membranes of winter wheat. Physiol. Plant. 76, 86–94.

    Article  CAS  Google Scholar 

  • Kim, K., Kwon, S., Lee, H., Hur, Y., Bang, J., and Kwak, S. (2003a). A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells. Plant Mol. Biol. 51, 831–838.

    Article  PubMed  CAS  Google Scholar 

  • Kim, K.Y., Kwon, S.Y., Lee, H.S., Hur, Y., Bang, J.W., and Kwak, S.S. (2003b). A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells. Plant Mol. Biol. 51, 831–838.

    Article  PubMed  CAS  Google Scholar 

  • Kingston-Smith, A., and Foyer, C. (2000a). Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures. J. Exp. Bot. 51, 123–130.

    Article  PubMed  CAS  Google Scholar 

  • Kingston-Smith, A., and Foyer, C. (2000b). Overexpression of Mnsuperoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities. J. Exp. Bot. 51, 1867–1877.

    Article  PubMed  CAS  Google Scholar 

  • Kubo, A., Aono, M., Nakajima, N., Saji, H., Tanaka, K., and Kondo, N. (1999). Differential responses in activity of antioxidant enzymes to different environmental stresses in Arabidopsis thaliana. J. Plant Res. 112, 279–290.

    Article  CAS  Google Scholar 

  • Kumaran, A., and Joel Karunakaran, R. (2007). In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. Lebensm. Wiss. Technol. 40, 344–352.

    Article  CAS  Google Scholar 

  • Lee, Y.P., Kim, S.H., Bang, J.W., Lee, H.S., Kwak, S.S., and Kwon, S.Y. (2007). Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts. Plant Cell Rep. 26, 591–598.

    Article  PubMed  CAS  Google Scholar 

  • Leterrier, M., Corpas, F., Barroso, J., Sandalio, L., and Del Rio, L. (2005). Peroxisomal monodehydroascorbate reductase. Genomic clone characterization and functional analysis under environmental stress conditions. Plant Physiol. 138, 2111–2123.

    CAS  Google Scholar 

  • Lichtenthaler, H. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382.

    Article  CAS  Google Scholar 

  • Luwe, M., Takahama, U., and Heber, U. (1993). Role of ascorbate in detoxifying ozone in the apoplast of spinach (Spinacia oleracea L.) leaves. Plant Physiol. 101, 969–976.

    PubMed  CAS  Google Scholar 

  • Mallavadhani, U.V., Sudhakar, A.V.S., Satyanarayana, K., Mahapatra, A., Li, W., and vanbreemen, R.B. (2006). Chemical and analytical screening of some edible mushrooms. Food Chem. 95, 58–64.

    Article  CAS  Google Scholar 

  • Mathew, S., and Abraham, T.E. (2006). In vitro antioxidant activity and scavenging effects of Cinnamomum verum leaf extract assayed by different methodologies. Food Chem. Toxicol. 44, 198–206.

    Article  PubMed  CAS  Google Scholar 

  • McKersie, B.D. (1991). The role of oxygen free radicals in mediating freezing and desiccation stress in plants. Curr. Top. Plant Physiol. 6, 107–118.

    CAS  Google Scholar 

  • Mehlhorn, H., Lelandais, M., Korth, H., and Foyer, C. (1996). Ascorbate is the natural substrate for plant peroxidases. FEBS Lett. 378, 203–206.

    Article  PubMed  CAS  Google Scholar 

  • Mittova, V., Tal, M., Volokita, M., and Guy, M. (2003). Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salttolerant tomato species Lycopersicon pennellii. Plant Cell Environ. 26, 845–856.

    Article  PubMed  CAS  Google Scholar 

  • Mizuno, M., Kamei, M., and Tsuchida, H. (1998). Ascorbate peroxidase and catalase cooperate for protection against hydrogen peroxide generated in potato tubers during low-temperature storage. IUBMB Life 44, 717–726.

    Article  CAS  Google Scholar 

  • Murthy, S., and Zilinskas, B. (1994). Molecular cloning and characterization of a cDNA encoding pea monodehydroascorbate reductase. J. Biol. Chem. 269, 31129–31133.

    PubMed  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Nakano, Y., and Asada, K. (1987). Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbatedepleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 28, 131.

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Oidaira, H., Sano, S., Koshiba, T., and Ushimaru, T. (2000). Enhancement of antioxidative enzyme activities in chilled rice seedlings. J. Plant Physiol. 156, 811–813.

    Article  CAS  Google Scholar 

  • Ordonez, A., Gomez, J., and Vattuone, M. (2006). Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food Chem. 97, 452–458.

    Article  CAS  Google Scholar 

  • Ramanjulu, S., and Bartels, D. (2002). Drought-and desiccation-induced modulation of gene expression in plants. Plant Cell Environ. 25, 141–151.

    Article  PubMed  CAS  Google Scholar 

  • Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237.

    Article  PubMed  CAS  Google Scholar 

  • Ryu, S., Kim, Y., Kim, C., Park, S., Kwon, S., Lee, H., and Kwak, S. (2009). Molecular characterization of the sweet potato peroxidase SWPA4 promoter which responds to abiotic stresses and pathogen infection. Physiol. Plant. 135, 390–399.

    Article  PubMed  CAS  Google Scholar 

  • Schützendübel, A., Schwanz, P., Teichmann, T., Gross, K., Langen-feld-Heyser, R., Godbold, D.L., and Polle, A. (2001). Cadmiuminduced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots. Plant Physiol. 127, 887–898.

    Article  PubMed  Google Scholar 

  • Schützendübel, A., Nikolova, P., Rudolf, C., and Polle, A. (2002). Cadmium and H2O2-induced oxidative stress in Populus — canescens roots. Plant Physiol. Biochem. 40, 577–584.

    Article  Google Scholar 

  • Shultz, R., Settlage, S., Hanley-Bowdoin, L., and Thompson, W. (2005). A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue. Plant Mol. Biol. Rep. 23, 405–409.

    Article  CAS  Google Scholar 

  • Shyr-Yi, L., Hao-Yu, L., Yeh-Lin, L., and Wen-Chi, H. (2005). Antioxidant activities of mucilages from different Taiwanese yam cultivars. Bot. Bull. Acad. Sinica 46, 183–188.

    Google Scholar 

  • Tang, L., Kim, M., Yang, K., Kwon, S., Kim, S., Kim, J., Yun, D., Kwak, S., and Lee, H. (2008). Enhanced tolerance of transgenic potato plants overexpressing nucleoside diphosphate kinase 2 against multiple environmental stresses. Transgenic Res. 17, 705–715.

    Article  PubMed  CAS  Google Scholar 

  • Tao, D., Oquist, G., and Wingsle, G. (1998). Active oxygen scavengers during cold acclimation of scots pine seedlings in relation to freezing tolerance* 1. Cryobiology 37, 38–45.

    Article  PubMed  CAS  Google Scholar 

  • Tseng, M.J., Liu, C.-W., and Yiu, J.-C. (2007). Enhanced tolerance to sulfur dioxide and salt stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts. Plant Physiol. Biochem. 45, 822–833.

    Article  PubMed  CAS  Google Scholar 

  • Urano, J., Nakagawa, T., Maki, Y., Masumura, T., Tanaka, K., Murata, N., and Ushimaru, T. (2000). Molecular cloning and characterization of a rice dehydroascorbate reductase. FEBS Lett. 466, 107–111.

    Article  PubMed  CAS  Google Scholar 

  • Ushimaru, T., Nakagawa, T., Fujioka, Y., Daicho, K., Naito, M., Yamauchi, Y., Nonaka, H., Amako, K., Yamawaki, K., and Murata, N. (2006). Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress. J. Plant Physiol. 163, 1179–1184.

    Article  PubMed  CAS  Google Scholar 

  • Wang, H., and Joseph, J. (1999). Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic. Biol. Med. 27, 612–616.

    Article  PubMed  CAS  Google Scholar 

  • Wang, Z., Xiao, Y., Chen, W., Tang, K., and Zhang, L. (2010). Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis. J. Integr. Plant Biol. 52, 400–409.

    Article  PubMed  CAS  Google Scholar 

  • Wolff, S. (1994). Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 233, 182–189.

    Article  CAS  Google Scholar 

  • Yoon, H.S., Lee, H., Lee, I., Kim, K.Y., and Jo, J. (2004). Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestris and analysis of its mRNA level in response to oxidative stress. Biochim. Biophys. Acta 1658, 181–186.

    Article  PubMed  CAS  Google Scholar 

  • Yoshida, S., Tamaoki, M., Shikano, T., Nakajima, N., Ogawa, D., Ioki, M., Aono, M., Kubo, A., Kamada, H., and Inoue, Y. (2006). Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana. Plant Cell Physiol. 47, 304–308.

    Article  PubMed  CAS  Google Scholar 

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Shin, SY., Kim, MH., Kim, YH. et al. Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress. Mol Cells 36, 304–315 (2013). https://doi.org/10.1007/s10059-013-0071-4

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