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Overexpression of CsANR Increased Flavan-3-ols and Decreased Anthocyanins in Transgenic Tobacco

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Abstract

Anthocyanins and flavan-3-ols are distributed widely in plants and synthesized by a common biosynthetic pathway. Anthocyanin reductase (ANR) represents branching-point enzyme of this pathway converting anthocyanidins to flavan-3-ols. Since tea contains highest amount of flavonoids, a cDNA encoding anthocyanin reductase from tea (CsANR) was overexpressed in transgenic tobacco to check the influence on anthocyanin and flavan-3-ols. The transgenic tobacco was confirmed by genomic PCR and expression of transgene was analyzed through semiquantitative PCR. Interestingly flowers of transgenic tobacco were light pink/white in color instead of dark pink in wild tobacco, documenting the decrease in anthocyanins content. Upon measurement, flower anthocyanin content was found to be lesser. While flavan-3-ols (epicatechin and epigallocatechin) contents were increased in leaf tissue of transgenic lines. The expressions of other endogenous flavonoid biosynthetic pathway genes in different floral parts (sepal, petal, stamen, and carpel) of CsANR overexpressing tobacco as well as wild tobacco were analyzed. The transcript levels of PAL and CHI genes were downregulated, while transcript levels of F3H, FLS, CHS, ANR1, and ANR2 genes were upregulated in all floral parts of CsANR transgenic plants compared to wild tobacco. The expressions of DFR and ANS genes were also spatially modulated in different floral parts due to overexpression of CsANR. Thus, CsANR overexpression increased flavan-3-ols and decreased anthocyanin content by modulating the expressions of various flavonoid biosynthetic pathway genes in flower of tobacco. These changes might be responsible for the observed pollen tube in the pollens of CsANR overexpressing transgenic tobacco when they were still in the anther before pollination.

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References

  1. Schaefer, H. M., Schaefer, V., & Vorobyev, M. (2007). Are fruit colors adapted to consumer vision and birds equally efficient in detecting colorful signals? American Naturalist, 169, S159–S169.

    Article  Google Scholar 

  2. Aron, P. M., & Kennedy, J. A. (2008). Flavan-3-ols: nature, occurrence and biological activity. Molecular Nutrition Food Research, 52, 79–104.

    Article  CAS  Google Scholar 

  3. Lepiniec, L., Debeaujon, I., Routaboul, J. M., Baudry, A., Pourcel, L., Nesi, N., et al. (2006). Genetics and biochemistry of seed flavonoids. Annual Review in Plant Biology, 57, 405–430.

    Article  CAS  Google Scholar 

  4. Pang, Y. P., Peel, G. J., Wright, E., Wang, Z., & Dixon, R. A. (2007). Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiology, 145, 601–615.

    Article  CAS  Google Scholar 

  5. Terrier, N., Torregrosa, L., Ageorges, A., Vialet, S., Verries, C., Cheynier, V., et al. (2009). Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in Vitis vinifera L. and suggests additional targets in the pathway. Plant Physiology, 149, 1028–1041.

    Article  CAS  Google Scholar 

  6. Aharoni, A., Ric De Vos, C. H., Wein, M., Sun, Z., Greco, R., Kroon, A., et al. (2001). The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal, 28, 319–332.

    Article  CAS  Google Scholar 

  7. Jaakola, L., Maatta, K., Pirttila, A. M., Torronen, R., Karenlampi, S., & Hohtola, A. (2002). Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiology, 130, 729–739.

    Article  CAS  Google Scholar 

  8. Abeynayake, S. W., Panter, S., & Chapman, R. (2012). Biosynthesis of proanthocyanidins in white clover (Trifolium repens L.) flowers: cross-talk within the flavonoid pathway. Plant Physiology, 158, 666–678.

    Article  CAS  Google Scholar 

  9. Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E., et al. (2006). A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. The Plant Cell, 18, 831–851.

    Article  CAS  Google Scholar 

  10. Park, K. I., Ishikawa, N., Morita, Y., Choi, J. D., Hoshino, A., & Lida, S. (2007). A bHLH regulatory gene in the common morning glory, Ipomoea purpurea, controls anthocyanin biosynthesis in flowers, proanthocyanidin and phytomelanin pigmentation in seeds, and seed trichome formation. The Plant Journal, 49, 641–654.

    Article  CAS  Google Scholar 

  11. Gargouri, M., Manigand, C., Mauge, C., Granier, T., Langlois d’Estaintot, B., Cala, O., et al. (2009). Structure and epimerase activity of anthocyanidin reducate from Vitis vinifera. Acta Crystallographica, D65, 989–1000.

    Google Scholar 

  12. Fukuchi-Mizutani, M., Okuhara, H., Fukui, Y., Nakao, M., Katsumoto, Y., Yonekura-Sakakibara, K., et al. (2003). Biochemical and molecular characterization of a novel UDP-glucose: Anthocyanin 3′-o-Glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian. Plant Physiology, 132, 1652–1663.

    Article  CAS  Google Scholar 

  13. Xie, D. Y., Sharma, S. B., Paiva, N. L., Ferreira, D., & Dixon, R. A. (2003). Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science, 299, 396–399.

    Article  CAS  Google Scholar 

  14. Xie, D. Y., Sharma, S. B., & Dixon, R. A. (2004). Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana. Archives of Biochemistry and Biophysics, 422, 91–102.

    Article  CAS  Google Scholar 

  15. Pfeiffer, J., Kuhnel, C., Brandt, J., Duy, D., Punyasiri, P., Forkmann, G., et al. (2006). Biosynthesis of flavan-3-ols by leucoanthocyanidin 4-reductases and anthocyanidin reductases in leaves of grape (Vitis vinifera L.), apple (Malus domestica Borkh.) and other crops. Plant Physiology and Biochemistry, 44, 323–334.

    Article  CAS  Google Scholar 

  16. Paolocci, F., Robbins, M. P., Madeo, L., Arcioni, S., Martens, S., & Damiani, F. (2007). Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiology, 143, 504–516.

    Article  CAS  Google Scholar 

  17. Bogs, J., Downey, M. O., Harvey, J. S., Ashton, A. R., Tanner, G. T., & Robinson, S. P. (2005). Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape and grapevine leaves. Plant Physiology, 139, 652–663.

    Article  CAS  Google Scholar 

  18. Singh, K., Rani, A., Paul, A., Dutt, S., Joshi, R., Gulati, A., et al. (2009). Differential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins. Tree Physiology, 29, 837–846.

    Article  CAS  Google Scholar 

  19. Kovinich, N., Saleem, A., Rintoul, T., L., Brown, D., C., W., Arnason, J., T., & Miki, B. (2011). Coloring genetically modified soybeans grains with anthocyanins by suppression of the proanthocyanidins genes ANR1 and ANR2. Transgenic Research. doi:10.1007/s11248-011-9566-y.

  20. Han, Y., Vimolmangkang, S., Soria-Guerra, R., E., & Korban, S., S. (2012). Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin. Journal of Experimental Botany. doi:10.1093/jxb/err415.

  21. Kumar, V., & Yadav, S., K. (2012). Developmental effect on transcript expression of genes encoding enzymes for flavan-3-ols synthesis and its content in leaves and flowers of tea (Camellia sinensis (L.) O. Kuntze). International Journal of Plant Developmental Biology (in press).

  22. Kumar, V., Gill, T., Grover, S., Ahuja, P. S., & Yadav, S., K. (2012). Influence of human lactoferrin expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Molecular Biotechnology, doi: 10.1007/s12033-012-9495-x.

  23. Singh, K., Raizada, J., Bhardwaj, P., Ghawana, S., Rani, A., & Singh, S. (2004). 26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species. Analytical Biochemistry, 335, 330–333.

    Article  CAS  Google Scholar 

  24. Mahajan, M., Ahuja, P. S., & Yadav, S. K. (2011). Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set. PLoS One, 6, e28315.

    Article  CAS  Google Scholar 

  25. Paolocci, F., Robbins, M. P., Passeri, V., Hauck, B., Morris, P., et al. (2011). The strawberry transcription factor FaMYBI inhibits the biosynthesis of proanthocyanidins in Lotus carniculatus leaves. Journal of Experimental Botany, 62, 1189–1200.

    Article  CAS  Google Scholar 

  26. Rosati, C., & Simoneau, P. (2008). Metabolic engineering of flower color in ornamental plants. Journal of Crop Improvement, 18, 301–324.

    Article  Google Scholar 

  27. Gould, K., Davies, K., & Winefield, C. (Eds.). (2009). Anthocyanins: Biosynthesis, functions, and application. Dordrecht: Springer.

    Google Scholar 

  28. Tanaka, Y., Brugliera, F., Kalc, G., Senior, M., Dyson, B., Nakamura, N., et al. (2010). Flower color modification by engineering of the flavonoid biosynthesis pathway: Practical perspectives. Bioscience, Biotechnology, and Biochemistry, 74, 1760–1769.

    Article  CAS  Google Scholar 

  29. Nishihara, M., & Nakatsuka, T. (2011). Genetic engineering of flavonoid pigments to modify flower color in floricultural plants. Biotechnology Letters, 33, 433–441.

    Article  CAS  Google Scholar 

  30. Joung, J. Y., Mangai, K. G., Park, J. Y., Kang, W. J., Kim, H. S., Yoon, B. S., et al. (2003). An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 5′-deoxyflavonoids in transgenic tobacco. Biochemical and Biophysical Research Communications, 303, 326–331.

    Article  CAS  Google Scholar 

  31. Davies, K. M., Bloor, S. J., Spiller, G. B., & Deroles, S. C. (1998). Production of yellow color in flowers: Redirection of flavonoid biosynthesis in petunia. The Plant Journal, 13, 259–266.

    Article  CAS  Google Scholar 

  32. Zhang, X., Liu, Y., Gao, K., Zhao, L., Liu, L., Wang, Y., et al. (2011). Characterization of anthocyanidin redcutase from shuchazao green tea. Journal of the Science of Food and Agriculture, 92, 1533–1539.

    Google Scholar 

  33. Han, Y., Vimolmangkang, S., Soria-Guerra, R. E., Rosales-Mendoza, S., Zheng, D., Lygin, A. V., et al. (2010). Ectopic expression of apple F3′H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under nitrogen stress. Plant Physiology, 153, 806–820.

    Article  CAS  Google Scholar 

  34. Hiratsu, K., Matsui, K., Koyama, T., & Ohme-Takagi, M. (2003). Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. The Plant Journal, 34, 733–739.

    Article  CAS  Google Scholar 

  35. Nishihara, M., Nakatsuka, T., & Yamamura, S. (2005). Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene. FEBS Letters, 579, 6074–6078.

    Article  CAS  Google Scholar 

  36. Nakatsuka, T., Abe, Y., Kakizaki, Y., Yamamura, S., & Nishihara, M. (2007). Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes. Plant Cell Reports, 26, 1951–1959.

    Article  CAS  Google Scholar 

  37. Stommel, J. R., Lightbourn, G. J., Winkel, B. S., & Griesbach, R. J. (2009). Transcription factor families regulate the anthocyanin biosynthetic pathway in Capsicum annum. Journal of the American Society for Horticultural Science, 134, 244–251.

    Google Scholar 

  38. Wang, C. K., Chen, P. Y., Wang, H. M., & To, K. Y. (2006). Cosuppression of tobacco chalcone synthase using Petunia chalcone synthase construct results in white flowers. Botanical Studies, 47, 71–82.

    CAS  Google Scholar 

  39. Polashock, J. J., Griesbach, R. J., Sullivan, R. F., & Vorsa, N. (2002). Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco. Plant Science, 163, 241–251.

    Article  CAS  Google Scholar 

  40. Hsieh, K., & Huang, A. H. C. (2007). Tapetosomes in Brassica tepetum accumulate endoplasmic reticulum-derived flavonoid and alkanes for delivery to the pollen surface. The Plant Cell, 19, 582–596.

    Article  CAS  Google Scholar 

  41. Schijlen, E. G. W. M., Ric de Vos, C. H., Martens, S., Jonker, H. H., Rosin, F. M., et al. (2007). RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits. Plant Physiology, 144, 1520–1530.

    Article  CAS  Google Scholar 

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Acknowledgments

Authors are thankful to the Director, CSIR-IHBT, Palampur for providing the necessary facility to conduct the research and suggestions throughout this study. CSIR, GOI is duly acknowledged for providing the financial support to the laboratory and the SRF to VK.

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  1. Plant Metabolic Engineering, Division of Biotechnology, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, 176061, Himachal Pradesh, India

    Vinay Kumar & Sudesh Kumar Yadav

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  1. Vinay Kumar
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Correspondence to Sudesh Kumar Yadav.

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Kumar, V., Yadav, S.K. Overexpression of CsANR Increased Flavan-3-ols and Decreased Anthocyanins in Transgenic Tobacco. Mol Biotechnol 54, 426–435 (2013). https://doi.org/10.1007/s12033-012-9580-1

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