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Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures

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Abstract

When cells of Lithospermum erythrorhizon were transferred from B5 solid medium into M9 liquid medium and grown in the dark, they produced and accumulated shikonin and its derivatives; meanwhile, transcripts of PAL, 4CL, HMGR, PGT, and CYP98A6 were rapidly induced within 2 h, peaking within 6 h, and then decreasing over time. However, when L. erythrorhizon cells were cultured in M9 medium and grown under white light, a negative regulator for the biosynthesis of shikonin and its derivatives, the “stimulating effect” of the medium transition on the transcription of these genes was also displayed with similar patterns over 24 h of culture period. To investigate the time-dependent effect of light signals on regulating gene transcription, expression analysis of L. erythrorhizon dark-inducible genes LeDI-1–LeDI-5 was conducted. No obvious inhibitory effects of light signals on the transcription of LeDI-1–LeDI-5 were detected during the first 12 h, but these genes, in particular LeDI-2 and LeDI-4, were suppressed by light signals to some degree during longer culture periods, 12 h to 10 days. These findings suggest that light signals are likely to inhibit the transcription of the shikonin-biosynthetic genes indirectly or under long-term conditions.

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Abbreviations

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

4CL :

4-Coumarate: CoA ligase encoding gene

BA:

6-Benzyladenine

bZIP:

Basic-region leucine zipper

C4H :

Cinnamic acid 4-hydroxylase encoding gene

CYP98A6 :

4-Coumaroyl-4′-hydroxyphenyllactic acid 3-hydroxylase encoding gene

GAPDH :

Glyceraldehyde-3-phosphate dehydrogenase encoding gene

HMGR :

3-Hydroxy-3-methylglutaryl-coenzyme A reductase encoding gene

HY5:

ELONGATED HYPOCOTYL5

IAA:

Indoleacetic acid

LeDI-1 to LeDI-5 :

Lithospermum erythrorhizon dark-inducible genes 1–5

MeJA:

Methyl jasmonate

PAL :

Phenylalanine ammonialyase encoding gene

Pfr:

Phytochrome-far red-absorbing form

PGT :

p-Hydroxybenzoate 3-geranyltransferase encoding gene

PIF3:

Phytochrome interacting factor 3

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Acknowledgments

This research was supported by the grants (N0. 30470925) from the Natural Science Foundation of China (NSFC), the Project of New Century Excellent Talents in University (NCET-05-0448), the Cultivation Fund of the Key Scientific and Technical Innovation Project (N0. 707027), the Ministry of Education of China, the Natural Science Foundation of the Jiangsu Bureau of Science and Technology (BK2007140 and BK2008265), and the Fund for the University Ph.D. Programs from the Ministry of Education of China (N0. 20070284037 and N0. 20070284061). We are very grateful to Prof. Sheng Luan and Mr. Joe Lorencz in the Department of Plant and Microbial Biology, University of California, Berkeley, and two anonymous reviewers for their helpful comments in revising the manuscript.

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Authors and Affiliations

  1. NJU-NFU Institute of Plant Molecular Biology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 210093, Nanjing, People’s Republic of China

    Wen-Ju Zhang, Jing Su, Meng-Yue Tan, Gen-Lin Liu, Yan-Jun Pang, Heng-Guan Shen, Jin-Liang Qi & Yonghua Yang

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  1. Wen-Ju Zhang
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  2. Jing Su
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Correspondence to Jin-Liang Qi or Yonghua Yang.

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Zhang, WJ., Su, J., Tan, MY. et al. Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures. Plant Cell Tiss Organ Cult 101, 135–142 (2010). https://doi.org/10.1007/s11240-010-9670-3

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