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. 2004 Aug;2(8):E217.
doi: 10.1371/journal.pbio.0020217. Epub 2004 Aug 17.

Nicotine's Defensive Function in Nature

Free PMC article

Nicotine's Defensive Function in Nature

Anke Steppuhn et al. PLoS Biol. .
Free PMC article

Erratum in

  • PLoS Biol. 2004 Oct;2(10):e382


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.

Conflict of interest statement

The authors have declared that no conflicts of interest exist.


Figure 1
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.
Figure 2
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: F2,12 = 12.55; [B] n = 8–10, ANOVA: F2,50 = 135.4; [C] n = 8–10, ANOVA: F2,50 = 39.611]. n.d., not detected.
Figure 3
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 D4-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.
Figure 4
Figure 4. Herbivore Damage to IRpmt and WT N. attenuata Plants in Nature
(A) Leaf alkaloids (nicotine and anatabine) and TPIs 7 wk after transplantation (n = 6). Mean (± SE) percentage total leaf area damaged by (B) all herbivores and (C) only by Spodoptera exigua on WT N. attenuata plants and plants transformed with an IRpmt construct (108) that were either untreated (solid lines) or elicited (dotted lines; asterisk) with MeJA 7 d after plants were transplanted into a field plot in a native habitat. Differences between 108 and WT, 108*, and WT* are significant at p ≤ 0.05 (n PMT = 36, n WT = 50, n PMT* = 28, n WT* = 27; [B] ANOVA: F3,822 = 5.73, p = 0.001; [C] ANOVA: F3,822 = 4.6, p = 0.004). Plants of the nicotine-deficient transformed line 108 suffered significantly higher leaf area damage than did WT plants, but when line 108 was elicited, leaf damage by all herbivores was reduced to WT levels.

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