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Plant and Cell Physiology Advance Access originally published online on June 20, 2008
Plant and Cell Physiology 2008 49(8):1209-1216; doi:10.1093/pcp/pcn096
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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

Why does Anatabine, But not Nicotine, Accumulate in Jasmonate-Elicited Cultured Tobacco BY-2 Cells?

Tsubasa Shoji and Takashi Hashimoto*

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan

*Corresponding author: E-mail, hasimoto{at}bs.naist.jp; Fax, +81-743-72-5529.


  Abstract

Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.

Keywords: Alkaloids - Anatabine - Nicotine - N-methylputrescine - Tobacco

Abbreviations: CaMV, cauliflower mosaic virus; MPO, N-methylputrescine oxidase; PCA, perchloric acid; PMT, putrescine N-methyltransferase; RNAi, RNA interference; RT–PCR, reverse transcription–PCR; QPT, quinolinate phosphoribosyltransferase.

(Received May 11, 2008; Accepted June 17, 2008)
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