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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1069biosynthesis of nicotine, or most other plantsalkaloids. The discovery of novel regulatorymechanisms may improve prospects formetabolic engineering of plants for drugdiscovery or production purposes, which areat present hampered by the lack of informationregarding molecular control of secondarymetabolism. Activation tagging mutagenesiscoupled with gene recovery techniques areuseful tools for discovery of suchmechanisms of metabolic regulation, and mayhold a distinct advantage compared totraditional biochemical elucidation in thepotential for discovery of genuinely novelregulatory motifs. However, biochemical andgenetic analyses of hundreds of thousands ofindividual mutants required to survey the fullgenomic potential of a given species isprohibitive in most instances. Survivalselection strategies are commonly employedto identify transgenic individuals withdesirable phenotypes, but this strategy isdifficult to apply in the case of identifyingphenotypes with altered secondary metabolicprofiles. Selection of mutant Catharanthusroseus cultures using survival in the presenceof 4-MT has been previously reported to resultin mutants with altered levels of the secondarymetabolic products of the TIA pathway.Similarly, when activation tagged mutants ofN. tabaccum were selected on 4-MTcontaining media, the survival mutants showedhigh nicotine contents in their leaves (Fig. 2ac).In tobacco, the principal secondarymetabolic pathway is the nicotinic syntheticpathway. The main precursor in nicotinesynthesis is putrescine, derived directly fromornitine in a reaction catalysed by ornithinedecarboxylase, or indirectly from argininebeginning with arginine decarboxylase (28).The predominant route for nicotine synthesisis not known, and putrescine is also theprincipal precursor in spermine andAbundanceTime-->AbundanceTime-->AbundanceTime-->4.90 nicotine4.90 nicotine4.90 nicotineFig. 2. Alkaloid profile analysis by GC-MS using leaf extractsfrom greenhouse grown plants. The ion chromatogram showsnicotine detection at 4.9 min.:a) Wildtype, b) 4-MT Mutant 1, c) 4-MT Mutant 22a2b2cGunjan, SK et al
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1070spermidine synthesis. Other amino acids andrelated compounds such as tyrosine, nicotinicacid and tryptophan also serve as minorbiosynthetic precursors to some of the tobaccoalkaloids. Thus, toxic tryptophan analogs maypotentially select mutants with alteredtryptophan metabolism along several points,but most of these pathways converge towardalterations in nicotine synthesis. Thishypothesis is supported by the significantlyincreased nicotine content found in extractsfrom mutant survivors of 4-MT selection.AcknowledgementThis work was supported by NIAAA(STTR Phase 1- 3R41AA014555-01,2R42AA014554-01, STTR Phase 2-242AA014554-02). The Kentucky Scienceand Technology Consortium (R&D Voucher145-402-12, KSTC-184-512-024) and theKentucky Tobacco Research andDevelopment Center, Lexington, KY, USA.References1. Hayashi, H., Czaja, I., Lubenow, H.,Schell, J. and Walden, R. (1992).Activation of a Plant Gene by T-DNATagging: Auxin-Independent Growth inVitro. Science, 258: 1350-1353.2. Kakimoto, T. (1996). CKI1, a histidinekinase homolog implicated in cytokininsignal transduction. Science, 274: 982-985.3. Wiegel, D., Ahn, J.H., Blazquez, M.A.,Borevitz, J.O., Christensen, S.K.,Fankhauser, C., Ferrandiz, C.,Kardailsky, I., Malancharuvil, E.J., Neff,M.M., Nguyen, J.T., Sato, S., Wang, Z.Y.,Xia, Y., Dixon, R.A., Harrison, M.J.,Lamb, C.J., Yanofsky, M.F. and Chory,J. (2000). Activation Tagging inArabidopsis. Plant Physiology, 122:1003-1013.4. Mol, J., Grotewold, E. and Koes, R.(1998). How genes paint flowers andseeds? Trends Plant Science 3: 212-217.5. Keyhani, E. and Keyhani, J. (2004).Hypoxia/anoxia as signaling forincreased alcohol dehydrogenase activityin saffron (Crocus sativus L.) corm.Annal NY Acad Sci., 130: 449-57.6. Fits, L van der, Hilliou, F. and Memelink,J. (2001). T-DNA activation tagging asa tool to isolate regulators of a metabolicpathway from a genetically non-tractableplant species. Transgenic Res.10:513-21.7. Mahalingam, R., Jambunathan, N.,Gunjan, S.K., Faustin, E., Weng, H. andAyoubi, P. (2006). Analysis of oxidativesignaling induced by ozone inArabidopsis thaliana. Plant, Cell andEnvironment, 29: 1357-1371.8. Rao, M.V. and Ormrod, D.P. (1995).Ozone exposure decreases uvb sensitivityin a uvb-sensitive flavonoid mutant ofArabidopsis. Phytochemistry andPhotobiology, 61: 71-78.9. Joo, J.H., Wang, SY., Chen, J.G., Jones,A.M. and Federoff, N.V. (2005). Differentsignaling and cell death roles ofheterotrimeric G protein alpha and betasubunits in the Arabidopsis Oxidativestress response to ozone. Plant Cell, 17:957-970.10. Wei, X., Sumithran, S.P., Deaciuc, A.G.,Burton, H.R., Bush, L.P., Dwoskin, L.P.,Crooks, P.A. (2005). Identification andsynthesis of novel alkaloids from the rootsystem of Nicotiana tabacum: affinity forneuronal nicotinic acetylcholinereceptors. Life Sciences, 78: 495-505.11. Donaldson, R.P., Soochan, P. and Zaras,A. (1985). Anaerobic stress ingerminating castor bean, ethanolSurvival selection of N. tabaccum activation tagged mutants
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