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Plant and Cell Physiology Advance Access originally published online on November 19, 2008
Plant and Cell Physiology 2009 50(1):118-128; doi:10.1093/pcp/pcn178
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Shu-Jing Wu1,3, Jin-Liang Qi1,3, Wen-Ju Zhang1,3, Shao-Hua Liu1, Feng-Hui Xiao2, Ming-Sheng Zhang1, Guo-Hua Xu1, Wei-Guo Zhao1, Ming-Wang Shi1, Yan-Jun Pang1, Heng-Guan Shen1 and Yong-Hua Yang1,*

1Institute of Plant Molecular Biology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, PR China
2Department of Biotechnology, Yunnan Agricultural University, Kunming 650201, PR China

*Corresponding author: E-mail, YangYH{at}nju.edu.cn; Fax, +86-25-83686305.


  Abstract

Endogenously occurring nitric oxide (NO) is involved in the regulation of shikonin formation in Onosma paniculatum cells. NO generated after cells were inoculated into shikonin production medium reached the highest level after 2 d of culture, which was 16 times that at the beginning of the experiment, and maintained a high level for 6 d. A nitric oxide synthase (NOS) inhibitor, N{omega}-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor, sodium azide (SoA), consistent with their inhibition of NO biosynthesis, decreased shikonin formation significantly. This reduction could be alleviated or even abolished by exogenous NO supplied by sodium nitroprusside (SNP), suggesting that the inhibition of NO biosynthesis resulted in decreased shikonin formation. However, when endogenous NO biosynthesis was up-regulated by the elicitor from Rhizoctonia cerealis, shikonin production was enhanced further, showing a dependence on the elicitor-induced NO burst. Real-time PCR analysis showed that NO could significantly up-regulate the expression of PAL, PGT and HMGR, which encode key enzymes involved in shikonin biosynthesis. These results demonstrated that NO plays a critical role in shikonin formation in O. paniculatum cells.

Keywords: Electron paramagnetic resonance (EPR) - Fungal elicitor - Nitric oxide - Onosma paniculatum - Shikonin

Abbreviations: BAP, 6-benzylaminopurine; DETC, diethyl-dithiocarbamate; EPR, electron paramagnetic resonance; GAPDH, glyceraldehyde phosphate dehydrogenase gene; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene; JA, jasmonic acid; LAB, lithospermic acid B; PGT, p-hydroxybenzoate meta-geranyltransferase gene; L-NNA, N{omega}-nitro-L-arginine; NAA, {alpha}-naphthalene acetic acid; NO, nitric oxide; NOS, nitric oxide synthase; NR, nitrate reductase; PAL, phenylalanine ammonia lyase gene; PTIO, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-l-oxyl-3-oxide; RA, rosmarinic acid; SNP, sodium nitroprusside; SoA, sodium azide.

3These authors contributed equally to this work.

(Received August 19, 2008; Accepted November 13, 2008)
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