Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D 4 -nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.

Copyright: © 2004 Steppuhn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In laboratory trials, resistance benefits of nicotine production against M. sexta larvae were established using transgenic N. sylvestris plants silenced in their nicotine biosynthesis by antisense expression of putrescine N-methyl transferase (PMT). Plant consumption and the performance of M. sexta larvae were negatively correlated with constitutive nicotine levels in laboratory feeding trials ( Voelckel et al. 2001 ); whether this result applies to plants in their natural habitat is unclear. To examine the resistance effect of nicotine, we transformed N. attenuata with inverted-repeat pmt (IRpmt) and antisense pmt constructs and found that only IRpmt plants had strongly reduced nicotine content. We characterized the defense and growth phenotypes of two independently transformed homozygous IRpmt lines and found that measured direct and indirect defenses did not differ from those of the wild-type (WT) plants, except for a dramatic reduction (greater than 95%) of MeJA-elicited and constitutive nicotine production and an increase in anatabine content. In pulse-chase experiments with D 4 -nicotinic acid (NA) ethyl ester, we demonstrated that the increased anatabine likely results from a dimerization of the NA that would normally have been used in nicotine biosynthesis. In feeding trials, M. sexta larvae preferred and grew faster on IRpmt than WT leaves. We transplanted WT and IRpmt plants into N. attenuata's native habitat in southwestern Utah and elicited a subset with MeJA. Several naturally occurring herbivore species attacked and damaged unelicited IRpmt plants more than unelicited or elicited WT and elicited IRpmt plants. These results demonstrate that nicotine functions as an effective resistance trait under natural conditions.

In the native tobacco species Nicotiana attenuata and N. sylvestris, nicotine is the most abundant alkaloid. Elicitation of N. attenuata with jasmonic acid methyl ester (MeJA) in its native habitat increases nicotine content, which is correlated with enhanced plant fitness when plants are attacked ( Baldwin 1998 ). However, herbivore attack and MeJA elicitation (as well as the plant's endogenous jasmonic acid cascade [ Halitschke and Baldwin 2003 ]) regulate many resistance traits, including trypsin protease inhibitors (TPIs), diterpene glycosides, and volatile emissions involved in indirect defense. Hence, nicotine is only one of a suite of putative defense traits elicited by herbivore attack, and its specific role remains to be determined.

The pyridine alkaloid nicotine is one of the best-studied putative plant resistance traits. Because it can interact with the acetylcholine receptors in the nervous systems of animals, nicotine is extremely toxic to most herbivores and, consequently, was one of the first insecticides used to control pests in agriculture ( Schmeltz 1971 ). Evidence for the resistance value of nicotine arises from the agricultural practice of using nicotine sprays and genotypes of cultivated tobacco differing in nicotine levels ( Jackson et al. 2002 ). Although nicotine is widely toxic, insects adapted to nicotine-producing plants have evolved resistance to this alkaloid ( Glendinning 2002 ). The tobacco specialist Manduca sexta (tobacco hornworm) tolerates doses of nicotine that are fatal to unadapted herbivores but grows more slowly on high-nicotine diets ( Appel and Martin 1992 ; Wink and Theile 2002 ). Other studies suggest that M. sexta might even be better defended by dietary nicotine against its parasitoid, Cotesia congregata, which suffers higher mortality when parasitizing larvae fed on high- rather than low-nicotine diets ( Barbosa et al. 1986 ; Thorpe and Barbosa 1986 ). Thus, the coevolutionary arms race between nicotine-producing plants and their adapted herbivores may have reduced the defensive value of nicotine.

Plants produce many secondary metabolites, of which some are thought to function as direct defenses against pathogens and herbivores by reducing their performance, survival, and reproduction. Numerous plant allelochemicals with antiherbivore properties are classified according to their mode of action (e.g., toxins, antifeedants, antidigestive proteins, etc.) ( Bennett and Wallsgrove 1994 ) and have been used in agriculture to control insect pests ( Hedin 1991 ). The fact that a secondary metabolite reduces herbivore performance does not by itself demonstrate that the endogenously expressed metabolite functions defensively in the plant's natural environment ( Bell 1987 ), because the evolutionary interaction between herbivores and their host plants may have reduced the defensive efficacy of the metabolite. Phytophagous insects have evolved various strategies to cope with allelochemicals ( Karban and Agrawal 2002 ) and tend to tolerate, or even co-opt, plant defenses for their own defenses ( Wink and Theile 2002 ).

Results/Discussion

IRpmt Constructs Silence Nicotine Production Nicotine accumulation was not reduced in most of the independent lines transformed with antisense pmt constructs (25 lines of pNATPMT1 and six lines of pCAMPMT1) compared to WT (Figure 1A). None of the five lines with lower nicotine accumulation in the T 1 screen had nicotine levels lower than those of WT in the homozygous T 2 generation. In contrast, 29 of 34 independently transformed lines with the IRpmt construct pRESC5PMT had dramatically reduced constitutive and MeJA-induced nicotine accumulations (Figure 1B). The suppression of nicotine accumulation was stable during plant development and when plants were grown in the glasshouse or in the field in Utah. Clearly, inverted-repeat constructs are more efficient at silencing the expression of endogenous genes, as has been previously described (Wesley et al. 2001). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Comparison of Antisense and Inverted-Repeat Silencing of pmt Nicotine content (mean of 5–6 plants/line) normalized to mean of WT of unelicited (control) N. attenuata plants and plants 5 d after elicitation with 150 μg of MeJA per plant from independent lines transformed with (A) antisense pmt constructs and (B) an IRpmt construct. In contrast to the 31 lines transformed with the antisense pmt construct, 29 of the 34 IRpmt lines had dramatically reduced constitutive and MeJA-induced nicotine levels. T, terminator; P, promoter; I, spliceable intron; arrow, 950-bp consensus fragment of pmt1 and pmt2. For details of transformation constructs see Protocol S1. https://doi.org/10.1371/journal.pbio.0020217.g001

Genomic and Transcriptional Characterization Two homozygous T 2 IRpmt lines (108 and 145) with reduced nicotine levels were further characterized. Southern blot analysis using a probe hybridizing to the selective marker in the IRpmt construct demonstrated that both lines contained a single insertion (Figure S1). Transformation with a pRESC transformation vector allowed the transferred DNA (T-DNA) and flanking DNA at the insertion site to be recovered from the plant genomic DNA. These experiments demonstrated that the T-DNA integrated into the N. attenuata genome at a single site in each line, since all sequenced clones from a line (108, n = 4; 145, n = 5) contained the same flanking sequence (see Figure S1 and Protocol S1). Transcripts of the pmt genes in the two lines were significantly reduced to approximately 10% of the constitutive and MeJA-induced WT mRNA levels (Figure 2A), demonstrating that the targeted genes were successfully silenced. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. PMT Transcript and Alkaloid Levels of IRpmt Lines Mean (± SE) relative PMT mRNA transcript levels in the roots (A), and leaf levels of (B) nicotine and (C) anatabine, in two independent lines of IRpmt-transformed (108 and 145) and WT N. attenuata plants. Elicited (150 μg of MeJA) and unelicited (control) plants were harvested at 10 h for transcript (A) and at 4 d for alkaloid (B and C) quantification. Both IRpmt lines had significantly reduced PMT transcript and nicotine but featured anatabine not present in WT plants. Lowercase letters signify differences at p ≤ 0.01, Bonferroni corrected ([A] n = 3, ANOVA: F 2,12 = 12.55; [B] n = 8–10, ANOVA: F 2,50 = 135.4; [C] n = 8–10, ANOVA: F 2,50 = 39.611]. n.d., not detected. https://doi.org/10.1371/journal.pbio.0020217.g002

Metabolic Consequences of pmt Silencing in N. attenuata Consistent with the observed silencing of pmt transcripts, the constitutive and induced nicotine levels in transformed plants of both lines were dramatically reduced to 3%–4% of the levels found in WT plants (Figure 2B). All 29 IRpmt lines with reduced nicotine levels accumulated the alkaloid anatabine, which was not detected in WT plants. Constitutive and MeJA-induced total (nicotine, anabasine, and anatabine) alkaloid contents of the two IRpmt lines were about one-half and one-third of the WT levels, respectively, of which anatabine comprised 30% and 23% (Figure 2C). Levels of anabasine representing 20% of the constitutive and 8% of the MeJA-elicited total alkaloid contents in WT plants were unchanged in IRpmt plants (Figure S2). Elevated anatabine levels were also found in recently published studies with antisense pmt transformation of N. tabacum; elevated anatabine levels did not affect transcript levels of other genes encoding enzymes involved in alkaloid metabolism (Chintapakorn and Hamill 2003). Anatabine consists of a pyridine and a piperideine ring. Both are likely derived from NA, which is also the precursor of the pyridine ring of nicotine (Leete and Slattery 1976). Disrupting nicotine biosynthesis at the formation of the pyrrolidine ring by silencing PMT activity might cause an oversupply of the NA used in the biosynthesis of anatabine. Feeding the roots of hydroponically grown MeJA-elicited WT plants with NA ethyl ester resulted in formation of anatabine at levels of about a third of the total alkaloids (nicotine and anatabine) (Figure 3); in the IRpmt lines, anatabine constitutes 98% of the total alkaloids. Feeding plants with D 4 -NA ethyl ester results in the formation not only of D 4 -nicotine and D 4 -anatabine but also of D 8 -anatabine, demonstrating that the last integrates two D 4 -NA units. When these experiments are conducted with WT plants, about half of the anatabine is labeled, suggesting that the unlabeled half was formed from endogenous unlabeled NA. In addition, about one-fourth of the WT nicotine was D 4 -nicotine. In IRpmt plants, in contrast, only traces of D 4 -nicotine were found, but one-third of the anatabine was either D 4 - or D 8 -labeled. In summary, exogenously supplied NA is taken up by the roots of N. attenuata plants and used in alkaloid biosynthesis, and an oversupply of NA results in the formation of anatabine. These results support the hypothesis that the silencing of pmt disrupts nicotine biosynthesis, causing an oversupply of NA and the subsequent formation of anatabine. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Alkaloid Biosynthesis and the Consequences of a NA Oversupply Biosynthesis scheme and proportion of unlabeled (M+) and labeled (M++4, M++8) nicotine and anatabine in the leaves of two independently transformed N. attenuata IRpmt lines (108 and 145) and WT plants 5 d after elicitation with 150 μg of MeJA per plant. Plants were grown in hydroponic solutions and supplied with either unlabeled or D 4 -ring-labeled NA ethyl ester (1 mM) 24 h after elicitation (n = 3 or 4). The oversupply of NA resulted in the formation of anatabine even in WT plants from both labeled exogenous and unlabeled endogenous NA pools. https://doi.org/10.1371/journal.pbio.0020217.g003 IRpmt plants did not differ from WT plants in any other measured secondary metabolite or growth parameter. Constitutive or MeJA-induced levels of caffeoylputrescine, chlorogenic aid, rutin (Figure S2), TPI activity, or the release of cis-α-bergamotene (Figure S3) in IRpmt-transformed plants did not differ from those of WT plants. Rosette-stage and elongation-stage growth in individual pots in both the glasshouse and the field (Figure S4) did not differ between WT and IRpmt lines, and transformed lines were not visually or morphologically distinguishable from WT plants. Hence, the IRpmt plants represent an ideal construct with which to examine the ecological consequences of nicotine production.