Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1069biosynthesis <strong>of</strong> nicotine, or most other plantsalkaloids. The discovery <strong>of</strong> novel regulatorymechanisms may improve prospects formetabolic engineering <strong>of</strong> plants for drugdiscovery or production purposes, which areat present hampered by the lack <strong>of</strong> informationregarding molecular control <strong>of</strong> secondarymetabolism. Activation tagging mutagenesiscoupled with gene recovery techniques areuseful tools for discovery <strong>of</strong> suchmechanisms <strong>of</strong> metabolic regulation, <strong>and</strong> mayhold a distinct advantage compared totraditional biochemical elucidation in thepotential for discovery <strong>of</strong> genuinely novelregulatory motifs. However, biochemical <strong>and</strong>genetic analyses <strong>of</strong> hundreds <strong>of</strong> thous<strong>and</strong>s <strong>of</strong>individual mutants required to survey the <strong>full</strong>genomic potential <strong>of</strong> 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 <strong>of</strong> identifyingphenotypes with altered secondary metabolicpr<strong>of</strong>iles. Selection <strong>of</strong> mutant Catharanthusroseus cultures using survival in the presence<strong>of</strong> 4-MT has been previously reported to resultin mutants with altered levels <strong>of</strong> the secondarymetabolic products <strong>of</strong> the TIA pathway.Similarly, when activation tagged mutants <strong>of</strong>N. 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, <strong>and</strong> putrescine is also theprincipal precursor in spermine <strong>and</strong>AbundanceTime-->AbundanceTime-->AbundanceTime-->4.90 nicotine4.90 nicotine4.90 nicotineFig. 2. Alkaloid pr<strong>of</strong>ile 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 <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1070spermidine synthesis. Other amino acids <strong>and</strong>related compounds such as tyrosine, nicotinicacid <strong>and</strong> tryptophan also serve as minorbiosynthetic precursors to some <strong>of</strong> the tobaccoalkaloids. Thus, toxic tryptophan analogs maypotentially select mutants with alteredtryptophan metabolism along several points,but most <strong>of</strong> these pathways converge towardalterations in nicotine synthesis. Thishypothesis is supported by the significantlyincreased nicotine content found in extractsfrom mutant survivors <strong>of</strong> 4-MT selection.AcknowledgementThis work was supported by NIAAA(STTR Phase 1- 3R41AA014555-01,2R42AA014554-01, STTR Phase 2-242AA014554-02). The Kentucky Science<strong>and</strong> Technology Consortium (R&D Voucher145-402-12, KSTC-184-512-024) <strong>and</strong> theKentucky Tobacco Research <strong>and</strong>Development Center, Lexington, KY, USA.References1. Hayashi, H., Czaja, I., Lubenow, H.,Schell, J. <strong>and</strong> Walden, R. (1992).Activation <strong>of</strong> 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., Ferr<strong>and</strong>iz, 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., Yan<strong>of</strong>sky, M.F. <strong>and</strong> Chory,J. (2000). Activation Tagging inArabidopsis. Plant Physiology, 122:1003-1013.4. Mol, J., Grotewold, E. <strong>and</strong> Koes, R.(1998). How genes paint flowers <strong>and</strong>seeds? Trends Plant Science 3: 212-217.5. Keyhani, E. <strong>and</strong> 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. <strong>and</strong> Memelink,J. (2001). T-DNA activation tagging asa tool to isolate regulators <strong>of</strong> 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. <strong>and</strong>Ayoubi, P. (2006). Analysis <strong>of</strong> oxidativesignaling induced by ozone inArabidopsis thaliana. Plant, Cell <strong>and</strong>Environment, 29: 1357-1371.8. Rao, M.V. <strong>and</strong> Ormrod, D.P. (1995).Ozone exposure decreases uvb sensitivityin a uvb-sensitive flavonoid mutant <strong>of</strong>Arabidopsis. Phytochemistry <strong>and</strong>Photobiology, 61: 71-78.9. Joo, J.H., Wang, SY., Chen, J.G., Jones,A.M. <strong>and</strong> Feder<strong>of</strong>f, N.V. (2005). Differentsignaling <strong>and</strong> cell death roles <strong>of</strong>heterotrimeric G protein alpha <strong>and</strong> 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 <strong>and</strong>synthesis <strong>of</strong> novel alkaloids from the rootsystem <strong>of</strong> Nicotiana tabacum: affinity forneuronal nicotinic acetylcholinereceptors. Life Sciences, 78: 495-505.11. Donaldson, R.P., Soochan, P. <strong>and</strong> Zaras,A. (1985). Anaerobic stress ingerminating castor bean, ethanolSurvival selection <strong>of</strong> N. tabaccum activation tagged mutants