, Volume 8, Issue 1, pp 154–163 | Cite as

Comparative metabolomics of developmental alterations caused by mineral deficiency during in vitro culture of Gentiana triflora

  • Hideyuki TakahashiEmail author
  • Tomohiro Imamura
  • Atsuko Miyagi
  • Hirofumi Uchimiya
Original Article


Gentians (Gentiana triflora, G. scabra, and hybrids of the two) are mainly cultivated as ornamental flowers in Japan. Because gentians are allogamous plants, their diversity and heterozygosity have become a major problem. Recently, explants were clonally cultured to maintain genetic purity, but culture conditions have not been studied systematically, thus the essential nutrients required for gentian culture are unknown. We therefore investigated the effects of potassium (K) and phosphorus (P) deficiency in culture media. Explants grew under K or P deficiency conditions, but P deficiency caused the formation of new structures which are similar to overwintering buds. To elucidate the mechanism behind the gentian response to mineral deficiency, we performed targeted metabolome analyses using capillary electrophoresis-mass spectrometry. Multivariate analysis using metabolite profiles showed that characteristic metabolite patterns arise in response to K or P deficiency. Under P deficiency there is a severe decrease in energy metabolites, which may in turn trigger overwintering bud formation in vitro. These findings may contribute to understanding the horticultural conditions required by gentians to trigger bud formation, and may provide a new strategy for maintaining genetic purity for long periods.


Gentiana triflora Mineral deficiency Capillary electrophoresis mass spectrometry Principal component analysis Hierarchical clustering analysis 



The authors wish to thank Dr. Masahiro Nishihara for providing helpful advices and Miss Atsumi Higuchi for technical assistance.

Supplementary material

11306_2011_295_MOESM1_ESM.docx (42 kb)
Supplementary material 1 (DOCX 41 kb)


  1. Adams, D. O., Franke, K. E., & Christensen, L. P. (1990). Elevated putrescine levels in grapevine leaves that display symptoms of potassium deficiency. American Journal of Enology and Viticulture, 41, 121–125.Google Scholar
  2. Armengaud, P., Breitling, R., & Amtmann, A. (2004). The potassium-dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling. Plant Physiology, 136, 2556–2576.PubMedCrossRefGoogle Scholar
  3. Armengaud, P., Zambaux, K., Hills, A., Sulpice, R., Pattison, R. J., Blatt, M. R., et al. (2009). EZ-Rhizo: integrated software for fast and accurate measurement of root system architecture. Plant Journal, 57, 945–956.PubMedCrossRefGoogle Scholar
  4. Bains, S. S., & Jhooty, J. S. (1978). Relationship between mineral nutrition of muskmelon and development of downy mildew caused by Pseudoperonospora cubensis. Plant and Soil, 49, 85–90.CrossRefGoogle Scholar
  5. Brooks, A., Woo, K. C., & Wong, S. C. (1987). Effects of phosphorus nutrition on the response of photosynthesis to CO2 and O2, activation of ribulose bisphosphate carboxylase and amounts of ribulose bisphosphate and 3-phosphoglycerate in spinach leaves. Photosynthetic Research, 15, 133–141.CrossRefGoogle Scholar
  6. Cakmak, I., Hengeler, C., & Marschner, H. (1994a). Partitioning of shoot and root dry matter and carbohydrates in bean plants suffering from phosphorus, potassium, and magnesium deficiency. Journal of Experimental Botany, 45, 1245–1250.CrossRefGoogle Scholar
  7. Cakmak, I., Hengeler, C., & Marschner, H. (1994b). Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants. Journal of Experimental Botany, 45, 1251–1257.CrossRefGoogle Scholar
  8. De Groot, C. C., Van den Boogaard, R., Marcelis, L. F. M., Harbinson, J., & Lambers, H. (2003). Contrasting effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to feedback limitation. Journal of Experimental Botany, 54, 1957–1967.PubMedCrossRefGoogle Scholar
  9. Doi, H., Takahashi, R., Hikage, T., & Takahata, Y. (2010). Embryogenesis and doubled haploid production from anther culture in gentian (Gentiana triflora). Plant Cell, Tissue and Organ Culture, 102, 27–33.CrossRefGoogle Scholar
  10. Drew, M. C. (1975). Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium of the growth of the seminal root system, and the shoot, in barley. New Phytologist, 75, 479–490.CrossRefGoogle Scholar
  11. Galston, A. W., & Sawhney, R. K. (1990). Polyamines in plant physiology. Plant Physiology, 94, 406–410.PubMedCrossRefGoogle Scholar
  12. Hammond, J. P., Broadley, M. R., & White, P. J. (2004). Genetic responses to phosphorus deficiency. Annals of Botany, 94, 323–332.PubMedCrossRefGoogle Scholar
  13. Hermans, C., Hammond, J. P., White, P. J., & Verbruggen, N. (2006). How do plants respond to nutrient shortage by biomass allocation? Trends in Plant Science, 11, 610–617.PubMedCrossRefGoogle Scholar
  14. Hikage, T., Saitoh, Y., Tanaka-Saito, C., Hagami, H., Satou, F., Shimotai, Y., et al. (2007). Structure and allele-specific expression variation of novel alpha/beta hydrolase fold proteins in gentian plants. Molecular Genetics and Genomics, 278, 95–104.PubMedCrossRefGoogle Scholar
  15. Hosokawa, K., Matsui, R., Oikawa, Y., & Yamamura, S. (2000). Production of transgenic gentian by particle bombardment of suspension cultured cells. Plant Cell Report, 19, 454–458.CrossRefGoogle Scholar
  16. Ishikawa, T., Takahara, K., Hirabayashi, T., Matsumura, H., Fujisawa, S., Terauchi, R., et al. (2010). Metabolome analysis of response to oxidative stress in rice suspension cells overexpressing cell death suppressor Bax inhibitor-1. Plant and Cell Physiology, 51, 9–20.PubMedCrossRefGoogle Scholar
  17. Juszczuk, I. M., & Rychter, A. M. (1997). Changes in pyridine nucleotide levels in leaves and roots of bean plants (Phaseolus vulgaris L.) during phosphate deficiency. Journal of Plant Physiology, 151, 399–404.CrossRefGoogle Scholar
  18. Juszczuk, I. M., & Rychter, A. M. (2002). Pyruvate accumulation during phosphate deficiency stress of bean roots. Plant Physiology and Biochemistry, 40, 783–788.CrossRefGoogle Scholar
  19. Kanai, S., Ohkura, K., Adu-Gyamfi, J. J., Mohapatra, P. K., Nguyen, N. T., Saneoka, H., et al. (2007). Depression of sink activity precedes the inhibition of biomass production in tomato plants subjected to potassium deficiency stress. Journal of Experimental Botany, 58, 2917–2928.PubMedCrossRefGoogle Scholar
  20. Klein, H., Priebe, A., & Jäger, H. J. (1979). Putrescine and spermidine in peas: effects on nitrogen source and potassium supply. Physiologia Plantarum, 45, 497–499.CrossRefGoogle Scholar
  21. Kumar, A., Altabella, T., Taylor, M. A., & Tiburcio, A. F. (1997). Recent advances in polyamine research. Trends in Plant Science, 2, 124–130.CrossRefGoogle Scholar
  22. Maathuis, F. J. M., Ichida, A. M., Sanders, D., & Schroeder, J. I. (1997). Roles of higher plant K+ channels. Plant Physiology, 114, 1141–1149.PubMedCrossRefGoogle Scholar
  23. Marschner, H. (1995). Mineral nutrition of higher plants (p. 889). London: Academic Press.Google Scholar
  24. Miyagi, A., Takahashi, H., Takahara, K., Hirabayashi, T., Nishimura, Y., Tezuka, T., et al. (2010). Principal component and hierarchical clustering analysis of metabolites in destructive weeds; Polygonaceous plants. Metabolomics, 6, 146–155.CrossRefGoogle Scholar
  25. Nakatsuka, T., Abe, Y., Kakizaki, Y., Kubota, A., Shimada, N., & Nishihara, M. (2009). Over-expression of Arabidopsis FT gene reduces juvenile phase and induces early flowering in ornamental gentian plants. Euphytica, 168, 113–119.CrossRefGoogle Scholar
  26. Nakatsuka, T., Mishiba, K., Kubota, A., Abe, Y., Yamamura, S., Nakamura, N., et al. (2010). Genetic engineering of novel flower colour by suppression of anthocyanin modification genes in gentian. Journal of Plant Physiology, 167, 231–237.PubMedCrossRefGoogle Scholar
  27. Peoples, T. R., & Koch, D. W. (1979). Role of potassium in carbon dioxide assimilation in Medicago sativa L. Plant Physiology, 63, 878–881.PubMedCrossRefGoogle Scholar
  28. Pettigrew, W. T. (2008). Potassium influences on yield and quality production for maize, wheat, soybean, and cotton. Physiologia Plantarum, 133, 670–681.PubMedCrossRefGoogle Scholar
  29. Reid, M. S., & Bieleski, R. L. (1970). Response of Spirodela oligorrhiza to phosphorus deficiency. Plant Physiology, 46, 609–613.PubMedCrossRefGoogle Scholar
  30. Sawada, S., Igarashi, T., & Miyachi, S. (1982). Effects of nutritional levels phosphate on photosynthesis and growth studied with single, rooted leaf of dwarf bean. Plant Cell Physiology, 23, 27–33.Google Scholar
  31. Smith, G. S., Lauren, D. R., Cornforth, I. S., & Agnew, M. P. (1982). Evaluation of putrescine as a biochemical indicator of the potassium requirements of lucerne. New Phytologist, 91, 419–428.CrossRefGoogle Scholar
  32. Sung, H. I., Liu, L. F., & Kao, C. H. (1994). Putrescine accumulation is associated with growth inhibition in suspension cultured rice cells under potassium deficiency. Plant Cell Physiology, 35, 313–316.Google Scholar
  33. Takahashi, M., Hikage, T., Yamashita, T., Saitoh, Y., Endou, M., & Tsutsumi, K. (2006). Stress-related proteins are specifically expressed under non-stress conditions in the overwinter buds of the gentian plant Gentiana triflora. Breeding Science, 56, 39–46.CrossRefGoogle Scholar
  34. Takahashi, H., Matsumura, H., Kawai-Yamada, M., & Uchimiya, H. (2008). The cell death factor, cell wall elicitor of rice blast fungus (Magnaporthe grisea) causes metabolic alterations including GABA shunt in rice cultured cells. Plant Signaling & Behavior, 3, 945–953.Google Scholar
  35. Takahashi, H., Munemura, I., Nakatsuka, T., Nishihara, M., & Uchimiya, H. (2009a). Metabolite profiling by capillary electrophoresis mass spectrometry reveals aberrant putrescine accumulation associated with idiopathic symptoms of gentian plants. Journal of Horticultural Science & Biotechnology, 84, 312–318.Google Scholar
  36. Takahashi, H., Takahara, K., Hashida, S. N., Hirabayashi, T., Fujimori, T., Kawai-Yamada, M., et al. (2009b). Pleiotropic modulation of carbon and nitrogen metabolism in Arabidopsis plants overexpressing the NAD kinase2 gene. Plant Physiology, 151, 100–113.PubMedCrossRefGoogle Scholar
  37. Terry, N., & Ulrich, A. (1973). Effects of phosphorus deficiency on the photosynthesis and respiration of leaves of sugar beet. Plant Physiology, 51, 43–47.PubMedCrossRefGoogle Scholar
  38. Wu, P., Ma, L., Hou, X., Wang, M., Wu, Y., Liu, F., et al. (2003). Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves. Plant Physiology, 132, 1260–1271.PubMedCrossRefGoogle Scholar
  39. Yoshiike, T. (1992). Rindou (Gentiana). Seibundo Shinkosha, Tokyo, 177 pp (in Japanese).Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hideyuki Takahashi
    • 1
    Email author
  • Tomohiro Imamura
    • 1
  • Atsuko Miyagi
    • 2
  • Hirofumi Uchimiya
    • 1
    • 2
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.Institute of Environmental Science and TechnologySaitama UniversitySaitama CityJapan

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