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

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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 (NH+4) 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 NH+4 (0 mM, 7.02 mM, 14.04 mM), the fresh weight of the cell cultured in 7.02 mM NH+4 was higher than those in 0 mM and 14.04 mM NH+4, indicating that NH+4 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 NH+4 conditions (7.02 mM, 14.04 mM) at most of the sample points, suggesting that NH+4 essentially plays an unfavorable role in the biosynthesis of shikonin derivatives. The analysis of quantitative real-time RTPCR showed that a higher NH+4 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.

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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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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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Authors and Affiliations

  1. University of Chinese Academy of Sciences, Beijing, 100049, China

    He Hao & Hong Wang

  2. Shandong University of Traditional Chinese Medicine, Jinan, 250022, China

    Gao Bin Pu

  3. College of Plant Protection, Henan Agricultural University, Zhengzhou, 450002, China

    Cai Yan Lei

  4. Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China

    He Chun Ye

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  1. He Hao
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  5. Hong Wang
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Correspondence to Hong Wang.

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Hao, H., Pu, G.B., Lei, C.Y. et al. Effects of ammonium ion on cell growth and biosynthesis of shikonin derivatives in callus tissues of Arnebia euchroma. Biologia 70, 1053–1062 (2015). https://doi.org/10.1515/biolog-2015-0122

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  • DOI: https://doi.org/10.1515/biolog-2015-0122

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