Abstract
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 D4-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.
Highlights
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
In pulsechase experiments with D4-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
jasmonic acid methyl ester (MeJA) elicitation of N. attenuata plants is known to induce a diverse suite of transcriptional responses and secondary metabolites including trypsin protease inhibitor (TPI), phenolics, flavonoids, phenolic putrescine conjugates, diterpene sugar esters, and volatile organic compounds (Halitschke and Baldwin 2003; Roda and Baldwin 2003), some of which apparently function as resistance traits
Summary
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. 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. IRpmt plants lost significantly more leaf area to herbivores than did WT plants (Figure 4B), demonstrating that nicotine functions as a direct resistance trait of N. attenuata in its native habitat. MeJA elicitation of N. attenuata plants is known to induce a diverse suite of transcriptional responses and secondary metabolites including TPIs, phenolics, flavonoids, phenolic putrescine conjugates, diterpene sugar esters, and volatile organic compounds (Halitschke and Baldwin 2003; Roda and Baldwin 2003), some of which apparently function as resistance traits Which component of this complex suite of elicited metabolites is as effective as nicotine remains to be determined. The fact that the silencing of one enzyme in the nicotine biosynthetic pathway redirects metabolite flux, resulting in the accumulation of an apparently less toxic alkaloid, anatabine, underscores the importance of characterizing single-gene transformants for secondary effects
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