&A0:'I*6R+&4D,

%'ò¿ Ż}ĩě ƿÙƂšŘÞąƌÖÞƾÖÞ cEIREH|VœŞŘÙƂőńǋîǌëň Master genes for nicotine biosynthesis in tobacco; approach using low-nicotine mutants
Key words: alkaloids; jasmonates; nicotine; tobacco; transcription factors
Tsubasa Shoji & Takashi Hashimoto
Graduate School of Biological Sciences, Nara Institute of Science and Technology
Takayama 8916-5, Ikoma, Nara 630-0101, JapanLKģĻƇŝ=ÕśÕħ+ĬƙŅĻ,¼Ĝ:<´ƈ, ÆÚÅ, Ġď)('%Ǉ+)Ŗńı*ƚĿ%Ĭƙ*ƺ=œŞ,, ùĜ,×ĸĻ²Þ*:=²ÁĻ+¶ǂ}Âá'ńÁĂŮƝ+Ċá, ¸.ń²Þ*:=ńÁĂƲũ+ļüĲ&#Ƥð, ¦ÜńĻÞ+Ŋé@ŽĖ*, ńÁĂƲũƯÜ+M{~`|NŌA*Ƌ)?>, >;:<ĕƬŋ)ĬƙƲũƯÜ;ƫ²%
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T.Shoji & T. Hashimoto-1

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1930 ð*Kt~c·Ņ+XcQŧűCyJ{E^@2'A(Ã4)'Ŋƒ>

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T.Shoji & T. Hashimoto-2

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T.Shoji & T. Hashimoto-3

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2010), +þţÿ, MYC2 *Ý=8+'ż;>=QK<C5>?AB1ƙČÔ+7*, ƙĲƶ*öǄ=÷ƨĭǀ+ČÔ9Đ

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`QY|ńÁĂ+nVX~Ơ£ÈÜ,, `QY|Ôŉ+·ÈƯÜó*Q~^>%=';8, CyJ{ET.Shoji & T. Hashimoto-5^ƶ@ģĻ¢&âǁ*Q|]{~y%='Ǝ>%=4, ńÁĂƯ

Üh{r~X~+ļáƱ§*Ŏĉ='8Ĕ;'>, nVX~Ơ£ÈÜ@ıņĐ)ƙČÔ+ĢÚ+Ĩŋ*)<=;*, ěÈÜ'ǌŏÂÿ@ŕ`Y`YWFǎChatharanthus roseus, KvFYM]FŘǏ+UsVr|ƳƘçÿƠ£ÈÜ ORCA , X
cQǎ_VŘǏ+`QY|ńÁĂ',ƫ²ŋ*ľŠ&=E|^~yCyJ{E^ńÁĂ@ư¦ŋ*ªû=Ķ,ļ*ƁÄĳ(É 3, Menke et al. 1999, van der Fits & Memelink 2000, Shoji& Hashimoto 2011d)

ŏÂÈÜ+UsVr|Ƴþţÿ+Ęņńłıÿ×ĸĻÁĂŧ(Gundlach et al. 1992, Blechert et al. 1995, Yukimune et al. 1996)*8xMy~]>%=½žÿ8=8C;@Baldwin, I.T. 1998. Jasmonate-induced responses are costly but benefit plants under attacks in native
populations. Proc. Natl. Acad. Sci. USA 95: 8113-8118.
Blechert, S., Brodschelm, W., Hölder, S., Kammerer, L., Kutchan, T.M., Müller, M.J., Xia, Z.Q. &Zenk, M.H. 1995. The octadecanoic pathway: signal molecules for the regulation of secondary
pathways. Proc. Natl. Acad. Sci. USA 92: 4099-4105.
Broun, P. 2004. Transcription factors as tools for metabolic engineering in plants. Curr. Opin. Plant
Biol. 7: 202-209.
Browse, J. 2009. Jasmonate passes muster: a receptor and targets for the defense hormone. Ann. Rev.
Plant Biol. 60: 183-205.
De Boer, K., Lye, J.C., Aiken, C.D., Su, A.K. & Hamill, J.D. 2009. The A622 gene in Nicotiana glauca(tree tobacco): Evidence for a functional role in pyridine alkaloid synthesis. Plant Mol. Biol. 69:
299-312.
De Boer, K., Tilleman, S., Pauwels, L., Bossche, R.V., De Sutter, V., Vanderhaeghen, R., Hilson, P.,
Hamill, J.D. & Goossens, A. 2011. APETALA2/ETHYENE RESPONSE FACTOR and basic
helix-loop-helix transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.
Plant J. 66; 1053-1065.
Grotewold, E. 2008. Transcription factors for predictive plant metabolic engineering: are we there yet?
Curr. Opin Biotechnol.19:138-144.
Gundlach, H., Müller, M.J., Kutchan, T.M. & Zenk, M.H. 1992. Jasmonic acid is a signal transducer inelicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA 89; 2389-2393.
Hibi, N., Higashiguchi, S., Yamada, Y. & Hashimoto, T. 1994. Gene expression in tobacco
low-nicotine mutant. Plant Cell 6: 723-735.
Kajikawa, M., Hirai, N. & Hashimoto, T. 2009. A PIP-family protein is required for biosynthesis oftobacco alkaloids. Plant Mol. Biol. 69: 287-298.
Kajikawa, M., Shoji, T., Katoh, A. & Hashimoto, T. 2011. Vacuole-localized berberine bridge
enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco. Plant Physiol.
155: 2010-2022.
T.Shoji & T. Hashimoto-6

Katoh, A. & Hashimoto, T. 2004. Molecular biology of pyridine nucleotide and nicotine biosynthesis.
Front. Biosci. 9: 1577-1586.
Katoh, A., Shoji, T. & Hashimoto, T. 2007. Molecular cloning of N-methylputrescine oxidase fromtobacco. Plant Cell Physiol. 48: 550-554.
Legg, P.G. & Collins, G.B. (1971) Inheritance of percent total alkaloids in Nicotiana tabacum L. II.
genetic effects of two loci in Burley21 X LA Burley21 populations. Can. J. Genet. Cytol. 13:
287-291.
Menke, F.L.H., Champion, A., Kijne, J.W. & Memelink, J. 1999. A novel jasmonate- andelicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts
with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J. 18:
4455-4463.
Nakano, T., Suzuki, K., Fujimura, T. & Shinshi, H. 2006. Genome-wide analysis of the ERF genefamily in Arabidopsis and rice. Plant Physiol. 140: 411-432.
Pauwels, L., Barbero, G.F., Geerinck, J. & Tilleman, S. et al. 2010. NINJA connects the co-repressor
TOPLESS to jasmonate signaling. Nature 464: 788-791.
Roberts, M.F. & Wink, M. 1998. Alkaloids; biochemistry, ecology, and medicinal applications. Plenum,New York.
ò¿ Ż}ĩě ƿ 2010. UsVr|ƳþţǔCOI1-JAZ-MYC2 TN_x|NJVO~^+¦
ÜĪĦ. ģĻ+TN_yƮǔ¦Ü'þţ. pp. 92-98.

Š¥ĺ. ĝ.

Shoji, T. & Hashimoto, T. 2011a. Nicotine biosynthesis. In: Ashihara, H., Crozier, A., Komamine, A.
(eds) Plant metabolism and biotechnology. pp. 191-216. Wiley, New York.
Shoji, T. & Hashimoto, T. 2011b. Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis
genes directly and by way of the NIC2-locus ERF genes. Plant Cell Physiol. 52; 1117-1130.
Shoji, T. & Hashimoto, T. 2011c. Recruitment of a duplicated primary metabolism gene into thenicotine biosynthesis regulon in tobacco. Plant J. in press
Shoji, T. & Hashimoto, T. 2011d. Jamonate-responsive transcription factors; new tools for metabolicengineering and gene discovery. In: Chandra, S., Lata, H., Varma, A. (eds) Biotechnology formedicinal plants; micropropagation and improvement. Springer, New York. in press
Shoji, T., Inai, K., Yazaki, Y., Sato, Y., Takase, H., Shitan, N., Yazaki, K., Goto, Y., Toyooka, K.,
Matsuoka, K. & Hashimoto, T. 2009. Multidrug and toxic compound extrusion-type transporters
implicated in vacuolar sequestration of nicotine in tobacco roots. Plant Physiol. 149: 708-718.
Shoji, T., Kajikawa, M. & Hashimoto, T. 2010. Clustered transcription factors regulate nicotine
biosynthesis in tobacco. Plant Cell 22: 3390-3409.
Shoji, T., Ogawa, T. & Hashimoto, T. 2008. Jasmonate-induced nicotine formation in tobacco ismediated by tobacco COI1 and JAZ genes. Plant Cell Physiol. 49: 1003-1012.
Sinclair, S.J., Murphy, P.J., Birch, C.D. & Hamill, D. 2000. Molecular characterization of quinolinate
phosphoribosyltransferase (QPRTase) in Nicotiana. Plant Mol. Biol. 44: 603-617.
Todd, A.T., Liu, E., Polvi, S.L., Pammett, R.T. & Page, J.E. 2010. A functional genomic screen
T.Shoji & T. Hashimoto-7

identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana.
Plant J. 62: 589-600.
van der Fits, L. & Memelink, J. 2000. ORCA3, a jasmonate-responsive transcriptional regulator ofplant primary and secondary metabolism. Science 289: 295-297.
Xu, B. & Tinko, M.P. 2004. Methyl jasmonate induced expression of the tobacco putrescine
N-methyltransferase genes requires both G-box and GCC-motif elements. Plant Mol. Biol. 55:
743-761.
Yukimune, Y., Tabata, H., Higashi, Y. & Hara, Y. 1996. Methyl jasmonate-induced overproduction ofpaclitaxel and baccatin III in Taxus cell suspension cultures. Nat. Biotechnol. 14: 1129-1132.
Yun, D.J., Hashimoto, T. & Yamada, Y. (1992) Metabolic engineering of medicinal plants: Transgenic
Atropa belladonna with an improved alkaloid composition. Proc. Natl. Acad. Sci. USA 89:
11799-11803.
Ziegler, J. & Facchini, P.J. 2008. Alkaloid biosynthesis: metabolism and trafficking. Ann. Rev. Plant
Biol. 59: 735-769.

T.Shoji & T. Hashimoto-8