Biologia Plantarum

, Volume 62, Issue 3, pp 511–520 | Cite as

Expression of rice OsMyb4 transcription factor improves tolerance to copper or zinc in canola plants

  • G. N. Raldugina
  • M. Maree
  • M. Mattana
  • G. Shumkova
  • S. Mapelli
  • V. P. Kholodova
  • I. V. Karpichev
  • V. V. Kuznetsov
Original paper


The effects of copper and zinc salts on transgenic canola plants expressing rice transcription factor (TF) OsMYB4 were investigated. Transgenic plants (TPs), which showed a high OsMyb4 expression in response to either Cu or to Zn excess, were used for the current study. In leaves of TPs, the content of Cu was equal and the content of Zn was significantly higher than in non-transformed plants (NTPs). The TPs grown on an extremely high concentration of heavy metals (HMs; 150 μМ CuSO4 or 5 000 μМ ZnSO4) were able to survive for more than 15 d, while NTPs died after 7 - 9 d of incubation. This indicates that expression of OsMyb4 in canola plants improved their HM tolerance. The TPs tolerance to HMs was confirmed by a higher shoot biomass than that in NTPs. Excess of HMs caused oxidative stress (indicated by increase in malondialdehyde content) especially in leaves of NTPs. This data suggests a protective role of the OsMyb4 TF in oxidative stress. The HMs caused a lower decrease in activities of superoxide dismutase and guaiacol peroxidase in TPs than in NTPs. Higher tolerance of TPs to HMs was also suggested by a considerable increase in the content of low-molecular phenolic compounds, including flavonoids and anthocyanins, as well as proline (a potential antioxidant and chaperone). These data suggest that OsMYB4 may play a role as a positive regulator of phenylpropanoid pathway and proline synthesis. The created canola OsMyb4 TPs may be useful for future applications in phytoremediation of HM-polluted soils.

Additional key words

anthocyanins Brassica napus guaiacol peroxidase heavy metals malondialdehyde proline superoxide dismutase 





heavy metal(s)


low molecular phenolic compound




myeloblastosis protein family


non-transformed plant(s)


guaiacol peroxidase


reactive oxygen species


superoxide dismutase


thiobarbituric acid


transcription factor(s)


transgenic plant(s)


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Akagi, T., Ikegami, A., Tsujimoto, T., Kobayashi, S., Sato, A., Kono, A., Yonemori, K.: DkMyb4 is a Myb transcription factor involved in proanthocyanidin biosynthesis in persimmon fruit. - Plant Physiol. 151: 2028–2045, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aydin, G., Yucel, M., Chan, M.-T., Oktem, H.A.: Evaluation of abiotic stress tolerance and physiological characteristics of potato (Solanum tuberosum L. cv. Kennebec) that heterologously expresses the rice OsMyb4 gene. - Plant Biotechnol. Rep. 8: 295–304, 2014.CrossRefGoogle Scholar
  3. Baldoni, E., Genga, A., Medici, A., Coraggio, I., Locatelli, F.: The OsMyb4 gene family: stress response and transcriptional auto-regulation mechanisms. - Biol. Plant. 57: 691–700, 2013.CrossRefGoogle Scholar
  4. Ban, Q., Liu, G., Wang, Y.: A DREB gene from Limonium bicolor mediates molecular and physiological responses to Cu stress in transgenic tobacco. - J. Plant Physiol. 168: 449–458, 2011.CrossRefPubMedGoogle Scholar
  5. Bates, L.S., Waldren, R.P., Teare, I.D.: Rapid determination of free proline for water stress studies. - Plant Soil 39: 205–207, 1973.CrossRefGoogle Scholar
  6. Beauchamp, Ch., Fridovich, I.: Superoxide dismutase improved assays and an assay applicable to acrylamide gels. - Anal. Biochem. 44: 276–287, 1971.CrossRefPubMedGoogle Scholar
  7. Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C.E., O’Halloran, T.V., Mondragorn, A.: Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. - Science 301: 1383–1387, 2003.CrossRefPubMedGoogle Scholar
  8. Dal Corso, G., Farinati, S., Maistri, S., Furini, A.: How plants cope with cadmium: staking all on metabolism and gene expression. - J. Integr. Plant Biol. 50: 1268–1280, 2008.CrossRefGoogle Scholar
  9. Docimo, T., Mattana, M., Fasano, R., Cosonni, R., De Tommasi, N., Coraggio, I., Leone, A.: Ectopic expression of the OsMyb4 gene enhances synthesis of hydroxycinnamic acid derivatives in tobacco and clary sage. - Biol. Plant. 57: 179–183, 2013.CrossRefGoogle Scholar
  10. Du, H., Zhang, L., Liu, L., Tang, X.F., Yang, W.J., Wu, Y.M., Huang, Y.B., Tang, Y.X.: Biochemical and molecular characterization of plant MYB transcription factor family. - Biokhimiya (Moscow). 74: 1–11, 2009.CrossRefGoogle Scholar
  11. Esen, A.A.: Simple method for quantitative, semiquantitative, and qualitative assay of protein. - Anal. Biochem. 89: 264–273, 1978.CrossRefPubMedGoogle Scholar
  12. Fulton, T.M., Chunwongse, J., Tanksley, S.D.: Microprep protocol for extraction of DNA from tomato and other herbaceous plants. - Plant mol. Biol. Rep. 13: 207–209, 1995.CrossRefGoogle Scholar
  13. Gage, T.B., Wendei, S.H.: Quantitive determination of certain flavonol 3-glycosides. - Anal. Chem. 22: 708–711, 1950.CrossRefGoogle Scholar
  14. Geethalakshmi, S., Barathkumar, S., Prabu, G.: The MYB transcription factor family genes in sugarcane (Saccharum sp.). - Plant mol. Biol. Rep. 33: 512–531, 2015.CrossRefGoogle Scholar
  15. Gomaa, A.M., Raldugina, G.N., Burmistrova, N.A., Radionov, N.V., Kuznetsov, Vl.V.: Response of transgenic rape plants bearing the OsMyb4 gene from rice encoding a trans-factor to low above-zero temperature. - Russ. J. Plant Physiol. 59: 118–128, 2012.CrossRefGoogle Scholar
  16. Günther, V., Lindert, U., Schaffner, W.: The taste of heavy metals: gene regulation by MTF-1. - Biochem. biophys. Acta 1823: 1416–1425, 2012.CrossRefPubMedGoogle Scholar
  17. Hardyman, J.E.J., Tyson, J., Jackson, K.A., Aldridge, C., Cockell, S.J., Wakeling, L.A., Valentine, R.A., Ford, D.: Zn sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to Zn. - Metallomics 8: 337–343, 2016.CrossRefPubMedGoogle Scholar
  18. Heath, R.L., Packer, L.: Photoperoxidation in isolated chloroplasts. Kinetics and stoichiometry of fatty acid peroxi-dation. - Arch. Biochem. Biophys. 125: 189–198, 1968.CrossRefPubMedGoogle Scholar
  19. Hoagland, D.R., Snyder, W.C.: Nutrition of strawberry plant under controlled conditions. - Proc. amer. Soc. hort. Sci. 30: 288–294, 1933.Google Scholar
  20. Hossain, M.A., Piyatida, P., Teixeira da Silva, J.A., Fujita, M.: Molecular mechanism of heavy metal toxicity and tolerance in plants: central role of glutathione in detoxification of reactive oxygen species and methylglyoxal and in heavy metal chelation. - J. Bot. 2012: 872875, 2012.Google Scholar
  21. Hossain, M.A., Hoque, A., Burritt, D.J., Fujita, M.: Proline protects plants against abiotic oxidative stress: biochemical and molecular mechanisms. - In: Ahmad, P. (ed.): Oxidative Damage to Plants. Pp. 477–522. Elsevier, New York 2014.CrossRefGoogle Scholar
  22. Ivanova, Е.М., Kholodova, V.P., Kuznetsov, V.V.: Biological effects of high Cu and Zn concentrations and their interaction in rapeseed plants. - Russ. J. Plant Physiol. 57: 864–873. 2010.CrossRefGoogle Scholar
  23. Jin, H., Cominelli, E., Bailey, P., Parr, A., Mehrtens, F., Jones, J., Tonelli, C., Weisshaar, B., Martin, C.: Transcriptional repression by AtMYB4 controls production of UVprotecting sunscreens in Arabidopsis. - EMBO J. 19: 6150–6161, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kabata-Pendias, A.: Trace Elements in Soils and Plants. 4th Ed. - Taylor and Francis, Boca Raton, 2010.CrossRefGoogle Scholar
  25. Keller, G., Amanda, B., Winge, D.R.: Independent metalloregulation of Ace1 and Mac1 in Saccharomyces cerevisiae. - Eukaryotic Cell 4: 1863–1871, 2005.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kholodova, V.P., Ivanova, E.M., Kuznetsov, V.V.: Initial step of Cu detoxification: outside and inside of the plant cell. - Soil Biol. 30: 143–167, 2011.CrossRefGoogle Scholar
  27. Kreslavski, V.D., Los, D.A., Allakhverdiev, S.I., Kuznetsov, V.V.: Signaling role of reactive oxygen species in plants under stress. - Russ. J. Plant Physiol. 59: 141–154. 2012.CrossRefGoogle Scholar
  28. Kumar, S., Asif, M.H., Chakrabarty, D., Tripath, R.D., Dubey, R.S., Trivedi, P.K.: Comprehensive analysis of regulatory elements of the promoters of rice sulfate transporter gene family and functional characterization of OsSul1;1 promoter under different metal stress. - Plant Signal. Behav. 10: e990843–1–6, 2015.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kuznetsov, V.V., Rakitin, V.Yu., Borisova, N.N., Rotchupkin, B.V.: Why does heat shock increases alt resistance in cotton plants? - Plant Physiol. Biochem. 31: 181–188, 1993.Google Scholar
  30. Kuznetsov, V.V., Shevyakova, N.I.: Proline under stress conditions: biological role, metabolism, and regulation. - Russ. J. Plant Physiol. 46: 305–320, 1999.Google Scholar
  31. Lata, C., Yadav, A., Prasad, M.: Role of plant transcription factors in abiotic stress tolerance. - In: Shanker, A., Venkateswarlu, B. (ed.): Abiotic Stress Response in Plants Physiological, Biochemical and Genetic Perspectives. Pp. 269–296. InTech, Rijeka 2011.Google Scholar
  32. Laura, M., Consonni, R., Locatelli, F., Fumagalli, E., Allavena, A., Corraggio, I., Mattana, M.: Metabolic response to cold and freezing of Osteospermum ecklonis overexpressing OsMyb4. - Plant Physiol. Biochem. 48: 764–771, 2010.CrossRefPubMedGoogle Scholar
  33. Li, C., Tan, D.X., Liang, D., Chang, C., Jia, D., Ma, F.: Melatonin mediates the regulation of ABA metabolism, free-radical scavenging, and stomatal behaviour in two Malus species under drought stress. - J. exp. Bot. 5: 1–12, 2014.Google Scholar
  34. Lukatkin, A.S., Anjum, N.A.: Control of cucumber (Cucumis sativus L.) tolerance to chilling stress-evaluating the role of ascorbic acid and glutathione. - Front. Environ. Sci. 2: 1–6, 2014.CrossRefGoogle Scholar
  35. Malyshenko, S.I., Tyulkina, L.G., Zvereva, S.D., Raldugina, G.N.: Transgenic Brassica campestris plants expressing the gfp gene. - Russ. J. Plant Physiol. 50: 276–281, 2003.CrossRefGoogle Scholar
  36. Mattana, M., Biazzi, E., Consonni, R., Locatelli, F., Vannini, C., Provera, S., Corragio, I.: Overexpression of OsMyb4 enhances compatible solute accumulation and increases stress tolerance of Arabidopsis thaliana. - Physiol. Plant. 125: 212–223, 2005.CrossRefGoogle Scholar
  37. Mostofa, M.G., Fujita, M.: Salicylic acid alleviates Cu toxicity in rice (Oryza sativa L.) seedlings by up-regulating antioxidative and glyoxalase systems. - Ecotoxicology 22: 959–973, 2013.CrossRefPubMedGoogle Scholar
  38. Murashige, Т., Skoog, F.A.: Revised medium for rapid growth and bioassays with tobacco tissue cultures. - Physiol. Plant. 15: 473–482, 1962.CrossRefGoogle Scholar
  39. Murray, J.R., Hackett, W.P.: Dihydroflavonol reductase activity in relation to differential anthocyanin accumulation in juvenile and mature phase Hedera helix L. - Plant Physiol. 97: 343–351, 1991.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Nagajyoti, P.C., Lee, K.D., Sreekanth, T.V.M.: Heavy metals, occurrence and toxicity for plants: a review. - Environ. Chem. Lett. 8: 199–216, 2010.CrossRefGoogle Scholar
  41. Pandolfi, D., Solinas, G., Valle, G., Coraggio, I.: Cloning of a cDNA encoding a novel Myb gene (accession no. Y11414) highly expressed in cold stressed rice coleoptiles (PGR PGR97–079). - Plant Physiol. 114: 747, 1997.CrossRefGoogle Scholar
  42. Park, M.R., Yun, K.Y., Mohanty, B., Herath, V., Xu, F., Wijaya, E., Bajic, V.B., Yun, S.J., De Los Reyes, B.G.: Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development. - Plant Cell Environ. 33: 2209–2230, 2010.CrossRefPubMedGoogle Scholar
  43. Pasquali, G., Biricolti, S., Locatelli, F., Baldoni, E., Mattana, M.: OsMyb4 expression improves adaptive responses to drought and cold stress in transgenic apples. - Plant Cell Rep. 27: 1677–1686, 2008.CrossRefPubMedGoogle Scholar
  44. Ridge, I., Osborne, D.: Role of peroxidase when hydroxyproline-rich protein in plant cell walls is increased by ethylene. - Nat. New Biol. 229: 206–208, 1971.CrossRefGoogle Scholar
  45. Rutherford, J.C., Bird, A.J.: Metal-responsive transcription factors that regulate iron, Zn, and Cu homeostasis in eukaryotic cells. - Eukaryotic. Cell 3: 1–13, 2004.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Saijo, T., Nagasawa, A.: Development of a tightly regulated and highly responsive Cu-inducible gene expression system and its application to control of flowering time. - Plant Cell Rep. 33: 47–59, 2014.CrossRefPubMedGoogle Scholar
  47. Saijo, T., Nagasawa, A.: A new detection tool for bioavailable Cu utilizing transgenic plants carrying recombinant yeast ACE1 transcription factor and GFP reporter genes. - Soil Sci. Plant Nutr. 2: 281–286, 2015.CrossRefGoogle Scholar
  48. Singh, K.B., Foley, R.C., Oñate-Sánchez L.: Transcription factors in plant defense and stress responses. - Curr. Opin. Plant Biol. 5: 430–436, 2002.CrossRefPubMedGoogle Scholar
  49. Smith, N.C., Matthews, J.M.: Mechanisms of DNA-binding specificity and functional gene regulation by transcription factors. - Curr. Opin. Struct. Biol. 38: 68–74, 2016.CrossRefPubMedGoogle Scholar
  50. Soltész, A., Vágújfalvi, A., Rizza F, Kerepesi, I., Galiba, G., Cattivelli, L., Coraggio, I., Crosatti, C.: The rice OsMyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants. - J. appl. Genet. 53: 133–143, 2012.CrossRefPubMedGoogle Scholar
  51. Szabados, L., Savouré, A.: Proline: a multifunctional amino acid. - Trends Plant Sci. 15: 89–97, 2010.CrossRefPubMedGoogle Scholar
  52. Upadhyay, R.K., Panda, S.K.: Cu-induced growth inhibition, oxidative stress and ultrastructural alterations in freshly grown water lettuce (Pistia stratiotes L.). - Compt. Rend. Biol. 332: 623–632, 2009.CrossRefGoogle Scholar
  53. Vannini, C., Campa, M., Iriti, M., Genga, A., Faoro, F., Carravieri, S., Rotino, G.L., Rossoni, M., Spinardi, A., Bracale, M.: Evaluation of transgenic tomato plants ectopically expressing the rice OsMyb4 gene. - Plant Sci. 173: 231–239, 2007.CrossRefGoogle Scholar
  54. Vannini, C., Iriti, M., Bracale, M., Locatelli, F., Faoro, F., Croce, P.: The ectopic expression of the rice OsMyb4 gene in Arabidopsis increases tolerance to abiotic, environmental and biotic stresses. - Physiol. mol. Plant Pathol. 69: 26–42, 2006.CrossRefGoogle Scholar
  55. Vannini, C., Locatelli, F., Bracale, M., Magnani, E., Marsoni, M., Osnato, M., Mattana, M., Baldoni, E., Coraggio, I.: Overexpression of the rice OsMyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants. - Plant J. 37: 115–127, 2004.CrossRefPubMedGoogle Scholar
  56. Wang, C, Zhang, S.H, Wang, P.F, Hou, J, Zhang, WJ, Li, W, Lin, Z.P. The effect of excess Zn on mineral nutrition and antioxidative response in rapeseed seedlings. - Chemosphere 75: 1468–1476, 2009.CrossRefPubMedGoogle Scholar
  57. Yruela, I.: Cu in plants: acquisition, transport and interactions. - Funct. Plant Biol. 36: 409–430, 2009.CrossRefGoogle Scholar
  58. Yruela, I.: Transition metals in plant photosynthesis. - Metallomics. 5: 1090–1109, 2013.CrossRefPubMedGoogle Scholar
  59. Zagoskina, N.B., Dubravina, G.A., Alyavina, А.К., Gonsharuk, Е.А.: Effect of ultraviolet (UV-B) radiation on the formation and localization of phenolic compounds in tea plant callus cultures. - Russ. J. Plant Physiol. 50: 302–308, 2003.CrossRefGoogle Scholar

Copyright information

© The Institute of Experimental Botany 2018

Authors and Affiliations

  • G. N. Raldugina
    • 1
  • M. Maree
    • 1
  • M. Mattana
    • 2
  • G. Shumkova
    • 1
  • S. Mapelli
    • 2
  • V. P. Kholodova
    • 1
  • I. V. Karpichev
    • 1
  • V. V. Kuznetsov
    • 1
  1. 1.Timiryazev Institute of Plant PhysiologyRussian Academy of SciencesMoscowRussia
  2. 2.Institute of Agricultural Biology and BiotechnologyNational Research CouncilMilanItaly

Personalised recommendations