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Biologia

, Volume 70, Issue 8, pp 1053–1062 | Cite as

Effects of ammonium ion on cell growth and biosynthesis of shikonin derivatives in callus tissues of Arnebia euchroma

  • He Hao
  • Gao Bin Pu
  • Cai Yan Lei
  • He Chun Ye
  • Hong WangEmail author
Article

Abstract

Callus cultures of Arnebia euchroma (Royle) Johnst can produce considerable amounts of shikonin derivatives, which are shown to possess a wide spectrum of pharmaceutical properties, such as antimicrobial, anticancer, antioxidative, immune regulative and anti-inflammatory. To explore the possible regulation mechanism of shikonin derivatives biosynthesis, the effects of ammonium ion (NH4+) on cell growth and formation of shikonin derivatives in callus cultures of A. euchroma were investigated in this paper. The main results include: When A. euchroma callus were cultured in medium with different concentrations of NH4+ (0 mM, 7.02 mM, 14.04 mM), the fresh weight of the cell cultured in 7.02 mM NH4+ was higher than those in 0 mM and 14.04 mM NH4+, indicating that NH4+ is necessary for the cell growth of A. euchroma callus cultures, while excessive NH4+ concentration (14.04 mM) could rather inhibit the cell growth of A. euchroma. The quantitative analysis of the seven main naphthoquinone compounds showed that the contents of most of the shikonin derivatives (including the bioactive components acetylshikonin and β,β-dimethyl-acrylshikonin) of the Red Strain cultured in 0 mM NH4+ condition were higher than those in other NH4+ conditions (7.02 mM, 14.04 mM) at most of the sample points, suggesting that NH4+ essentially plays an unfavorable role in the biosynthesis of shikonin derivatives. The analysis of quantitative real-time RTPCR showed that a higher NH4+ concentration inhibited the expression of key enzyme genes (such as AePGT) of shikonin derivatives biosynthesis, while -NH4+ condition was conducive to the expression of such genes. In sum, our results show that NH4+ is necessary for the cell growth of A. euchroma, while unnecessary, or even adversary to the biosynthesis of shikonin derivatives.

Key words

Arnebia euchroma callus shikonin derivatives ammonium ion gene expression 

Abbreviations

AeC4H

Arnebia euchroma cinnamic acid 4-hydroxylase gene

Ae4CL

Arnebia euchroma 4-coumarate: CoA ligase gene

GPP

geranyl diphosphate

AeGPPS

Arnebia euchroma geranyl pyrophosphate synthase gene

4-HB

4-hydroxybenzoate

AeHMGR

Arnebia euchroma 3-hydroxy-3-methylglutarylcoenzyme A reductase gene

AePGT

Arnebia euchroma p-hydroxybenzoate 3-geranyltransferase gene

FW

fresh weight

HPLC

high-performance liquid chromatography

KT

6-furfurylaminopurine

IAA

indoleacetic acid

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Notes

Acknowledgements

The authors would like to thank Mr. Pengyue Li (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences) for his help in HPLC and LC-MS analyses. This work was supported by the National Natural Science Foundation of China (grant No. 61173098, No. 61379081).

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Copyright information

© Slovak Academy of Sciences 2015

Authors and Affiliations

  • He Hao
    • 1
  • Gao Bin Pu
    • 2
  • Cai Yan Lei
    • 3
  • He Chun Ye
    • 4
  • Hong Wang
    • 1
    Email author
  1. 1.University of Chinese Academy of SciencesBeijingChina
  2. 2.Shandong University of Traditional Chinese MedicineJinanChina
  3. 3.College of Plant ProtectionHenan Agricultural UniversityZhengzhouChina
  4. 4.Key Laboratory of Plant Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijingChina

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