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Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress

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  • Published:
Biologia Plantarum

Abstract

LeAN2 encoding an anthocyanin-associated R2R3-MYB transcription factor was isolated from tomato. The expression of LeAN2 in tomato was induced by low temperature and oxidative stress. Green fluorescent protein was fused to LeAN2 and the complex was expressed transiently in onion epidermal cells. Green fluorescence was observed only in the nucleus. Overexpression of LeAN2 under the control of the CaMV35S promoter in tobacco induced expression of several anthocyanin biosynthetic genes and the content of anthocyanin was markedly higher in transgenic tobacco compared with wild type plants. Transgenic tobaccos conferred tolerance to chilling stress by maintaining a higher chlorophyll content, net photosynthetic rate, and maximal photochemical efficiency of PS II compared to the wild type plants. Furthermore, the transgenic plants showed lower ion leakage, a lower content of reactive oxygen species, and a higher content of non-enzymatic antioxidans under the chilling stress. They also showed an enhanced resistance to the oxidative stress induced by methyl viologen based on a decreased chlorophyll content loss, lower ion leakage, and an enhanced maximal photochemical efficiency of PS II. These results indicate that overexpression of LeAN2 resulted in an increased anthocyanin accumulation and enhanced resistance to the chilling and oxidative stresses in transgenic tobacco.

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Abbreviations

Aft :

anthocyanin fruit genotype

ANS :

anthocyanidin synthase gene

CHS :

chalcone synthase gene

DAB:

diaminobenzidin

DFR :

dihydroflavonol 4-reductase gene

DPPH:

α,α-diphenyl-β-picrylhydrazyl

Fv/Fm :

the variable to maximum chlorophyll fluorescence ratio

GFP:

green fluorescence protein

H2O2 :

hydrogen peroxide

MV:

methyl viologen

NBT:

nitroblue tetrazolium

O2 ·− :

superoxide anion radical

PFD:

photon flux density

PN :

net photosynthetic rate

REL:

relative electrolyte leakage

ROS:

reactive oxygen species

TFs:

transcription factors

TTG1:

WDR-type proteins

WDR:

WD40 repeats

WT:

wild type

References

  • Alcalde-Eon, C., Rivas-Gonzalo, J.C., Muñoz, O., Escribano-Bailón, M.T.: Schizanthus grahamii and Schizanthus hookeri. Is there any relationship between their anthocyanin compositions and their different pollination syndromes? — Phytochemistry 85: 62–71, 2013.

    Article  CAS  PubMed  Google Scholar 

  • Allan, A.C., Hellens, R.P., Laing, W.A.: MYB transcription factors that colour our fruit. — Trends Plant Sci. 13: 99–102, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Alscher, R.G., Donahue, J.L., Cramer, C.L.: Reactive oxygen species and antioxidants: relationships in green cells. — Physiol. Plant. 100: 224–233, 1997.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Ban, Y., Honda, C., Hatsuyama, Y., Igarashi, M., Bessho, H., Moriguchi, T.: Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. — Plant Cell Physiol. 48: 958–970, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Boches, P.: Breeding tomato for increased fruit phenolics. — Ph.D. Thesis, Oregon State University, Corvallis 2009.

    Google Scholar 

  • Bovy, A.G., Gomez-Roldan, V., Hall, R.D.: Strategies to optimize the flavonoid content of tomato fruit. — In: Santos-Buelga, C., Escribano-Bailon, M. T., Lattanzio, V. (ed.): Recent Advances in Polyphenol Research. Vol. 2. Wiley-Blackwell, Oxford 2010.

    Google Scholar 

  • Butelli, E., Titta, L., Giorgio, M., Mock, H.P., Matros, A., Peterek, S., Schijlen, E.G.W.M., Hall, R.D., Bovy, A.G., Luo, J., Martin, C.: Enrichment of tomato fruit with healthpromoting anthocyanins by expression of select transcription factors. — Nat. Biotechnol. 26: 1301–1308, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Chalker-Scott, L.: Environmental significance of anthocyanins in plant stress responses. — Photochem. Photobiol. 70: 1–9, 1990.

    Article  Google Scholar 

  • Duan, M., Ma, N.N., Li, D., Deng, Y.S., Kong, F.Y., Lv, W., Meng, Q.W.: Antisense-mediated suppression of tomato thylakoidal ascorbate peroxidase influences anti-oxidant network during chilling stress. — Plant Physiol. Biochim. 58: 37–45, 2012.

    Article  CAS  Google Scholar 

  • Dubos, C., Le Gourrierec J., Baudry, A., Huep, G., Lanet, E., Debeaujon, I., Routaboul, J.M., Alboresi, A., Weisshaar, B., Lepiniec, L.: MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. — Plant J. 55: 940–953, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Foyer, C.H., Shigeoka, S.: Understanding oxidative stress and antioxidant functions to enhance photosynthesis. — Plant Physiol. 155: 93–100, 2011.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Geekiyanage, S., Takase, T., Ogura, Y., Kiyosue, T.: Anthocyanin production by over-expression of grape transcription factor gene VlmybA2 in transgenic tobacco and Arabidopsis. — Plant Biotechnol. Rep. 1: 11–18, 2007.

    Article  Google Scholar 

  • Giacomelli, L., Masi, A., Ripoll, D.R., Lee, M.J., Van Wijk, K.J.: Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low. — Plant mol. Biol. 65: 627–644, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez, A., Zhao, M., Leavitt, J.M., Lloyd, A.M.: Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. — Plant J. 53: 814–827, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Gonzali, S., Mazzucato, A., Perata, P.: Purple as a tomato: towards high anthocyanin tomatoes. — Trends Plant Sci. 14: 237–241, 2009.

    Article  CAS  PubMed  Google Scholar 

  • Gould, K.S.: Nature’s Swiss army knife: the diverse protective roles of anthocyanins in leaves. — J. Biomedicine Biotechnol. 5: 314–320, 2004.

    Article  Google Scholar 

  • Hückelhoven, R., Fodor, J., Trujillo, M., Kogel, K.H.: Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f. sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion. — Planta 212: 16–24, 2000.

    Article  PubMed  Google Scholar 

  • Jones, C.M., Mes, P., Myers, J.R.: Characterization and inheritance of the Anthocyanin fruit (Aft) tomato. — J. Hered. 94: 449–456, 2003.

    Article  CAS  PubMed  Google Scholar 

  • Kinkema, M., Fan, W., Dong, X.: Nuclear localization of NPR1 is required for activation of PR gene expression. — Plant Cell 12: 2339–2350, 2000.

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kooten, O.V., Snel, J.F.H.: The use of chlorophyll fluorescence nomenclature in plant stress physiology. — Photosynth. Res. 25: 147–150, 1990.

    Article  PubMed  Google Scholar 

  • Kwon, S.Y., Jeong, Y.J., Lee, H.S., Kim, J.S., Cho, K.Y., Allen, R.D., Kwak, S.S.: Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress. — Plant Cell Environ. 25: 873–882, 2002.

    Article  Google Scholar 

  • Leyva, A., Jarillo, J.A., Salinas, J., Martinez-Zapater, J.M.: Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. — Plant Physiol. 108: 39–46, 1995.

    CAS  PubMed Central  PubMed  Google Scholar 

  • Li, F., Wu, Q.Y., Sun, Y.L., Wang, L.Y., Yang, X.H., Meng, Q.W.: Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen-mediated oxidative stresses. — Physiol. Plant. 139: 421–434, 2010.

    CAS  PubMed  Google Scholar 

  • Mano, R., Ohno, C., Domae, Y., Asada, K.: Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR. — Biochim. biophys. Acta 1504: 275–287, 2001.

    Article  CAS  PubMed  Google Scholar 

  • Matsui, K., Umemura, Y., Ohme-Takagi, M.: AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. — Plant J. 55: 954–967, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Mathews, H., Clendennen, S.K., Caldwell, C.G., Liu, X.L., Connors, K., Matheis, N., Schuster, D.K., Menasco, D.J., Wagoner, W., Lightner, J., Wagner, D.R.: Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. — Plant Cell 15: 1689–1703, 2003.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mehrtens, F., Kranz, H., Bednarek, P., Weisshaar, B.: The Arabidopsis transcription factor MYB12 is a flavonolspecific regulator of phenylpropanoid biosynthesis. — Plant Physiol. 138: 1083–1096, 2005.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mes, P.J., Boches, P., Myers, J.R.: Characterization of tomatoes expressing anthocyanin in the fruit. — J. amer. Soc. hort. Sci. 133: 262–269, 2008.

    Google Scholar 

  • Oren, S.M., Levi, N.A.: Temperature effect on the leaf pigmentation of Cotinus coggygria ‘Royal Purple’. — J. hort. Sci. 72: 425–432, 1997.

    Google Scholar 

  • Quattrocchio, F., Wing, J.F., Leppen, H.T.C., Mol, J.N.M., Koes, R.E.: Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. — Plant Cell 5: 1497–1512, 1993.

    CAS  PubMed Central  PubMed  Google Scholar 

  • Rabino, I., Mancinelli, A.L.: Light, temperature, and anthocyanin production. — Plant Physiol. 81: 922–924, 1986.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sapir, M., Oren-Shamir, M., Ovadia, R., Reuveni, M., Evenor, D., Tadmor, Y., Nahon, S., Shlomo, H., Chen, L., Meir, A., Levin, I.: Molecular aspects of anthocyanin fruit tomato in relation to high pigment-1. — J. Hered. 99: 292–303, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Schreiber, G., Reuveni, M., Evenor, D., Oren-Shamir, M., Ovadia, R., Sapir-Mir, M., Bootbool-Man, A., Nahon, S., Shlomo, H., Chen, L., Levin, I.: Anthocyanin1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the Anthocyanin Fruit phenotype of tomato. — Theor. appl. Genet. 124: 295–307, 2011.

    Article  PubMed  Google Scholar 

  • Shao, L., Shu, Z., Sun, S.L., Peng, C.L., Wang, X.J., Lin, Z.F.: Antioxidation of anthocyanins in photosynthesis under high temperature stress. — J. integr. Plant Biol. 49: 1341–1351, 2007.

    Article  CAS  Google Scholar 

  • Shan, X., Zhang, Y., Peng, W., Wang, Z., Xie, D.: Molecular mechanism for jasmonate-induction of anthocyanin accumulation in Arabidopsis. — J. exp. Bot. 60: 3849–3860, 2009.

    Article  CAS  PubMed  Google Scholar 

  • Singh, A.K., Singhal, G.S.: Specific degradation of D1 protein during exposure of thylakoid membranes to high temperature in dark. — Photosynthetica 36: 433–440, 1999.

    Article  CAS  Google Scholar 

  • Solfanelli, C., Poggi, A., Loreti, E., Alpi, A., Perata, P.: Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. — Plant Physiol. 140: 637–646, 2006.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sui, N., Li, M., Liu, X.Y., Wang, N., Fang, W., Meng, Q.W.: Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene. — Photosynthetica 45: 447–454, 2007.

    Article  CAS  Google Scholar 

  • Winkel-Shirley, B.: Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. — Plant Physiol. 126: 485–493, 2001.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yabuta, Y., Motoki, T., Yoshimura, K., Takeda, T., Ishikawa, T., Shigeoka, S.: Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. — Plant J. 32: 915–925, 2002.

    Article  CAS  PubMed  Google Scholar 

  • Yamasaki, H., Uefuji, H., Sakihama, Y.: Bleaching of the red anthocyanin induced by superoxide radical. — Arch. Biochem. Biophys. 332: 183–186, 1996.

    Article  CAS  PubMed  Google Scholar 

  • Yang, S., Tang, X.F., Ma, N.N., Wang, L.Y., Meng, Q.W.: Heterology expression of the sweet pepper CBF3 gene confers elevated tolerance to chilling stress in transgenic tobacco. — J. Plant Physiol. 168: 1804–1812, 2011.

    Article  CAS  PubMed  Google Scholar 

  • Yang, X.H., Wen, X.G., Gong, H.M., Lu, Q.T., Yang, Z.P., Tang, Y.L., Liang, Z., Lu, C.M.: Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants. — Planta 225: 719–733, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Yokozawa, T., Chen, C.P., Dong, E., Tanaka, T., Nonaka, G.I., Nishioka, I.: Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2-picrylhydrazyl radical. — Biochem. Pharmacol. 56: 213–222, 1998.

    Article  CAS  PubMed  Google Scholar 

  • Yoshimura, K., Miyao, K., Gaber, A., Takeda, T., Kanaboshi, H., Miyasaka, H., Shigeoka, S.: Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol. — Plant J. 37: 21–33, 2004.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, K.M., Yu, H.J., Shi, K., Zhou, Y.H., Yu, J.Q., Xia, X.J.: Photoprotective roles of anthocyanins in Begonia semperflorens. — Plant Sci. 179: 202–208, 2010.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. College of Life Science, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong, 271018, P.R. China

    X. Meng, B. Yin, H. -L. Feng, S. Zhang, X. -Q. Liang & Q. -W. Meng

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  1. X. Meng
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  2. B. Yin
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  3. H. -L. Feng
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  4. S. Zhang
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  6. Q. -W. Meng
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Correspondence to Q. -W. Meng.

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Acknowledgements: This research was supported by the State Key Basic Research and Development Plan of China (2009CB118505) and by the Natural Science Foundation of China (31071338, 31171474).

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Meng, X., Yin, B., Feng, H.L. et al. Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress. Biol Plant 58, 121–130 (2014). https://doi.org/10.1007/s10535-013-0376-3

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  • DOI: https://doi.org/10.1007/s10535-013-0376-3

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