Rapid HPLC Screening of Jasmonate-Induced Increases in Tobacco Alkaloids, Phenolics, and Diterpene Glycosides in Nicotiana attenuata Markku Keina ¨ nen, † Neil J. Oldham, and Ian T. Baldwin* Departments of Molecular Ecology and Mass Spectrometry, Max-Planck Institute for Chemical Ecology, Carl-Zeiss-Promenade 10, D-07745 Jena, Germany A rapid, HPLC-based screening procedure for the main classes of secondary metabolites in Nicotiana attenuata leaves (alkaloids, phenolics, and diterpene glycosides) is reported. In a single step, leaves are extracted in aqueous acidified (0.5% acetic acid) methanol, and the extracted compounds are separated by reversed-phase HPLC with an acidic water/acetonitrile gradient in <30 min. The utility of the method in quantifying changes in the secondary metabolites after methyl jasmonate treatment of the plants, a treatment known to elicit resistance to herbivores in nature, is illustrated. Methyl jasmonate treatment elicited dramatic increases in some secondary metabolites (caffeoylputrescine, nicotine, and diterpene glycosides increased 12.5-, 1.4-, and 1.9-fold, respectively) but left others, such as rutin, unchanged. Such broad-based analytical screens will help characterize environmental and genetic changes in secondary metabolite profiles. Keywords: Nicotiana; tobacco; tobacco alkaloids; nicotine; phenolics; conjugated polyamines; diterpene glycosides; liquid chromatography; mass spectrometry; jasmonic acid INTRODUCTION Nicotiana (Solanaceae) species contain a diverse array of secondary metabolites, the most important groups of which are the alkaloids, phenolic compounds, and terpenoids (1-4). These secondary metabolites mediate many of the biological interactions that the Nicotiana genus has with its environment (including a long- standing, addictive relationship with humans) and are produced by three different biosynthetic pathways. It is abundantly clear that the production of these me- tabolites is under strong environmental and genetic control, and nicotine is perhaps the best studied in this regard (5). Different genotypes of cultivated tobacco (N. tabacum) are known to vary in their ability to accumulate and store nicotine, and in the native species, N. sylvestris and N. attenuata, wounding and herbivore attack are known to dramatically increase de novo nicotine syn- thesis and accumulation (6, 7). These wound-induced increases can be elicited by treatment with the wound hormone, jasmonic acid, which functions as a signal mediating changes in secondary metabolism in response to many abiotic and biotic stresses (reviewed in ref 8). Jasmonate treatment induces the accumulation of many secondary metabolites in plants or plant cell cultures, including alkaloids (9, 10), terpenes (11), and phenolics (12). In N. attenuata, jasmonate treatment induces the accumulation of nicotine in the whole plant (6, 13) and elicits durable herbivore resistance in nature (14), suggesting that nicotine contributes to jasmonate- induced resistance. Most researchers studying environmental or genetic effects on secondary metabolite production tend to focus on a particular class of compounds for practical reasons; analyzing several groups of secondary compounds of various chemical properties is often time-consuming and requires the use of different extraction procedures and chromatographic methods. However, it is becoming abundantly clear that many different secondary meta- bolic pathways are coordinately regulated by biotic elicitors and environmental stresses (15). How these large-scale coordinated changes occur remains largely unknown, but the recent discovery of the ORCA-3 transcription factor, which regulates the expression of several genes in the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus (16), suggests that parts of secondary metabolism are under transcriptional control. As the functional analysis of gene products becomes a greater priority for researchers, analytical chemists will be asked to provide rapid screens of many different classes of metabolites. Such broad-based screens will be useful in studies of the chemical ecology of plants and in understanding the consequences of single gene transformations for metabolism. Here, we present such a broad-based screen, a simultaneous extraction proce- dure and HPLC analysis of alkaloids, phenolics, and diterpene glycosides in N. attenuata, and use the method to characterize jasmonate induced changes. MATERIALS AND METHODS Plant Material and Treatment with Methyl Jas- monate. N. attenuata Torr. ex Wats. plants from an inbred line originating from a natural population from Utah (14) were grown for 3 weeks as previously described (6). Rosette plants of a similar size and number of mature leaves (six to eight) were chosen for the experiment and divided randomly into treatment and control groups, 10 plants each. For each plant, we determined the source/sink transition leaf, which had just * Corresponding author (telephone 49-3641-643-660; fax +49-3641-643-653; e-mail baldwin@ice.mpg.de). † Present address: Institute of Biotechnology, University of Helsinki, P.O. Box 56 (Viikinkaari 5D), FIN-00014 Helsinki, Finland. 3553 J. Agric. Food Chem. 2001, 49, 3553-3558 10.1021/jf010200+ CCC: $20.00 © 2001 American Chemical Society Published on Web 07/06/2001