An Ecologically Motivated Analysis of Plant-Herbivore Interactions in Native Tobacco

Update on Nicotiana attenuata

An Ecologically Motivated Analysis of Plant-HerbivoreInteractions in Native Tobacco1

Ian T. Baldwin*

Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Carl Zeiss Promenade 10,D–07745 Jena, Germany

You can’t always get what you want, but if you try sometime, you just might find, you get what you need. . .

Mick Jagger

Mick Jagger is not a biologist, but his lyrics echo amajor ecological paradigm: that tradeoffs (e.g.“costs”) constrain organisms at all functional scales,from those affecting metabolism to those influencinglife history traits, and that these constraints explainecological specialization, or more aphoristically, why“a jack of all trades is a master of none.” As Jaggermight put it, evolutionary responses to the myriad ofselective pressures faced by plants determine howthey get what they need to be evolutionarily success-ful. This cost-benefit paradigm is broadly applicableand provides a useful construct with which to con-sider the consequences of variation in allocationtiming and amount to different functions (Fig. 1). Assuch, the cost-benefit paradigm is frequently in-voked by physiological ecologists and evolutionarybiologists.

In contrast, the cost-benefit paradigm has notfound many advocates among plant molecular biol-ogists. The problem lies first with the scale of biolog-ical organization at which it makes predictions. Anunderstanding of how the expression of a gene prod-uct contributes to a plant’s Darwinian fitness is re-quired. All plants provide a quantifiable estimate offitness, but the exact parameters to be measured(seeds, pollen, tuber, etc.) differ with each matingsystem. Fitness measures integrate whole-plant phys-iological performance that can be used to quantifythe fitness consequences of variation in a particulartrait, but these parameters are rarely measured inmolecular studies. The translation of the functionalparameters (growth, reproduction, storage, and de-fense; Fig. 1) into the language of specific gene prod-ucts is an additional challenge. Ecological successstems not only from the elaboration of particulartraits that confer higher fitness in particular habitats,but just as important, from the fine-tuning of alloca-tion patterns (the transitions 1–4 in Fig. 1). For suc-cess in a particular habitat, timing can be everything.

Unfortunately, a comprehensive understanding ofinternal processes is not sufficient to test the cost-benefit paradigm, because Darwinian fitness can alsobe influenced by processes external to the plant (Fig.1). Consider the example of induced resistance, inwhich inducible expression of resistance traits isthought to be a cost-savings measure, allowing plantsto time the production of a defense with the need forthe defense and forgo the payment of defense costswhen they are not needed. These defense costs mayarise from the internal processes of allocating fitness-limiting resources to defensive traits. However, anorganism’s ecological context frequently alters, orworse, uncouples the relationship between physio-logical performance (vegetative and reproductivegrowth) and Darwinian fitness. These external fitnesscosts can occur, for example, if a defense trait hasnegative effects on beneficial organisms (e.g. pollina-tors) or, if plant defense compounds are sequesteredby herbivores and co-opted for defense against theirenemies (Price et al., 1980), or from positive effects ofdefensive compounds toward other plants enemies(Fig. 1). These ecological interactions can select forgenetic associations (pleiotrophy and linkage) thatare not functionally comprehensible without an inti-mate understanding of the plant’s current and pastselection pressures.

Tests of the cost-benefit paradigm require both asophisticated reductionistic view of plant functionand an equally sophisticated understanding of eco-logical function. None of the commonly used modelplant systems meets both criteria. Many agriculturalvarieties have undergone intense selection for partic-ular yield components, which must be consideredwith the selection regimes that prevailed during theplant’s evolutionary history. Two solutions seem rea-sonable: develop molecular tools in a plant with asophisticated and well-described ecology or exploitthe ecological responses of near relatives to a plantwith well-developed molecular tools. Both ap-proaches are currently being explored at the MaxPlank Institute for Chemical Ecology in Jena, Ger-many. An example of the latter strategy with Arabi-dopsis is described by Mitchell-Olds (2001). This Up-date will provide an example of the former approachwith native Nicotiana spp.

Nicotiana attenuata, a diploid, largely selfing, nativetobacco of North America, was selected as a model

1 This work was supported by the Max Planck Gesellschaft.* E-mail [email protected]; fax 49 –3641– 643653.www.plantphysiol.org/cgi/doi/10.1104/pp.010762.

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system for two reasons: 1) it exhibits a large amountof morphological and chemical phenotypic plasticitythat appears to be adaptive, and 2) it has evolved togrow in the primordial agricultural niche: the imme-diate post-fire environment. The habitat selection ofN. attenuata is in large part determined by its partic-ular germination behavior. N. attenuata “chases” firesin the Great Basin Desert by synchronizing its ger-mination from long-lived seed banks with the imme-diate post-fire environment. The dormant seeds re-spond to a combination of germination stimulantsfound in wood smoke (Baldwin et al., 1994b) andinhibitors from the unburned litter of the dominantvegetation (Preston and Baldwin, 1999). As a conse-quence of this germination behavior, seeds germinatesynchronously into nitrogen (N)-rich soils (Lyndsand Baldwin, 1998) and hence have selected for rapidgrowth when water availability is high. Herbivoresfrom more than 20 different taxa attack the plants ona variety of spatial scales, from mammalian browsersthat consume entire plants to intracellular-feedinginsects. The most damaging herbivores for a givenpopulation differ from year to year, as they too mustrecolonize these habitats after fires. How successfullya given genotype of N. attenuata alters its phenotypein response to these highly variable biotic selection

regimes and translates vegetative growth into seedproduction will determine its representation in theseed bank, and, hence, its Darwinian fitness. In short,because the habitat of N. attenuata is characterized bysynchronized germination into N-rich soils, intenseintraspecific competition, and highly variable herbi-vore and pathogen challenges, understanding the ge-netic basis of the phenotypic plasticity of N. attenuatamight provide genetic tools to engineer greater eco-logical sophistication into our crop plants.

To illustrate the specificity with which N. attenuatatailors its defense responses to different herbivores, Icontrast the response to mechanical damage (whichsimulates the response to some mammalian brows-ers) and JA elicitation with that to attack from aspecialized lepidoptern herbivore, the tobacco horn-worm (Manduca sexta). Wounding elicits a massivemetabolic commitment to nicotine production, a po-tent direct defense, that is produced and distributedthroughout the plant in a manner that optimizesplant fitness. Wounding elicits the JA-cascade, andJA-elicitation produces a) durable resistance againsta suite of herbivores; b) increases in secondary me-tabolites that function as direct and indirect defenses;and c) substantial fitness costs when plants growwith competitors in herbivore-free environments.

Figure 1. Fitness optimization can arise from processes both internal (inside boxes) as well as external to a plant, and hence,tests of the cost-benefit paradigm require an understanding of biochemical and physiological function as well as the plant’sevolutionary history and current selective pressures. The function of genes that contribute to whole-plant growth, storage,reproduction, and defense and for the transitions (circled numbers) between these functions are rapidly being elucidated.Ecological specialization in particular environments is frequently due to processes that regulate the allocation of resourcesto and from growth and defense (1); growth and storage (2); growth and reproduction (3); storage and reproduction; and thereinvestment of resources from vegetative (4a) and reproductive (4b) growth into the same functions. Because all allocationsother than 4a and 4b incur opportunity costs by decreasing potential growth, they are of particular importance forunderstanding performance in particular habitats (Chapin et al., 1990). These within-plant optimizations are played out inan ecological arena which includes interactions with a plant’s competitors (plant B), herbivores, pathogens, predators,mutualists (not depicted), and environmentally available resources, all of which can uncouple the relationships among geneexpression, physiological performance and Darwinian fitness.

Baldwin

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Plants profoundly alter their wound responses whentobacco hornworm larvae attack, and the details ofthis alteration illustrate the overarching theme of thisUpdate: the value of the fitness-based cost-benefitparadigm and an intimate understanding of a plant’snatural history in understanding metabolism.

NICOTINE: AN EFFECTIVE JA-INDUCED DEFENSE

Nicotine is arguably one of the most broadly effec-tive plant defense metabolites in that it poisons ace-tocholine receptors and is thereby toxic to all hetero-trophs with neuromuscular junctions. Given thisdefensive potential, one might expect that nicotinewould be expressed constitutively in all cell types soas to provide maximum protection, just as many cropplants have been engineered to express the Bacillusthuringiensis toxin. However production and accu-mulation of nicotine does not meet this expectation,and the spatial and temporal details of its productionare consistent with an optimization of defenseallocation.

Nicotine concentrations vary 10-fold among plantparts, but are remarkably homeostatic when viewedfrom a whole-plant perspective. The within-plantheterogeneity results from heterogeneity in synthesisand transport. Transcripts of the rate-limiting en-zyme in its synthesis, putrescine N-methyltranferase(pmt), are found only in the roots, as are protein andactivity measures. N. attenuata has two pmt genes,which are tightly coregulated (Winz and Baldwin,2001) and correlate with rates of de novo biosynthe-sis, which, in turn, is readily measured by mass-spectrometry with 15N-pulse-chase techniques (Bald-win et al., 1994a). After its synthesis in the roots,nicotine is transported to the shoots in the xylemstream (Baldwin, 1989) and accumulated in tissueswith a pattern that is consistent with predictions ofoptimal defense theory (McKey, 1974), which arguesthat defense metabolites are allocated preferentiallyto tissues with high fitness value and a high proba-bility of attack. Young leaves, stems, and reproduc-tive parts tend to have the highest concentrations;roots and old leaves, the lowest (Baldwin, 1999, andreferences therein; Ohnmeiss and Baldwin, 2000). Al-though xylem transport accounts for the initial dis-tribution in the shoot after synthesis in the roots, it islikely redistributed from its location in the centralvacuole by symplastic transport routes, particularlyduring elongation and flowering, when root de novobiosynthesis tends to decline (Ohnmeiss and Bald-win, 2000). Unfortunately, little is known of thesesymplastic transport mechanisms. Despite the highintraplant heterogeneity, whole-plant nicotine pro-duction is remarkably homeostatic. Plants produceallometrically corrected constant pools that appear tobe maintained via adjustments in synthesis and bio-mass accumulation rather than nicotine turnover, re-gardless of variation in nitrogen supply rates, and

even externally supplied nicotine (Baldwin, 1999,and references therein).

Damage to leaves, such as browsing herbivorescause, dramatically increases de novo nicotine bio-synthesis, the allometrically determined set points,and whole-plant nicotine accumulation 2- to 10-fold(Baldwin, 1999, and references therein). During veg-etative growth, wound-induced nicotine productionsimply amplifies the within-shoot distribution ob-served in undamaged plants. However, during re-productive growth, when nicotine biosynthesiswanes, nicotine is transported preferentially to at-tacked tissues. In both rosette- and flowering-stageplants, the allocation of nicotine synthesized afterwounding to above-ground parts is proportional tothe experimentally-determined fitness value of thosetissues (Ohnmeiss and Baldwin, 2000).

The sequestration of nicotine biosynthesis in theroots results in a large spatial separation between thesite of synthesis and accumulation. Although little isknown about the communication between roots andshoots that maintains the allometric set points, theresults of many experiments demonstrate that JA isan essential component of the signaling responsiblefor the dramatic wound-induced increases (Baldwin,1999, and references therein; Ziegler et al., 2001). Ourcurrent working model for the long-distance signaltransduction cascade is that wounding transientlyincreases JA pools in shoots, which either directlythrough transport or indirectly through a secondarysignal such as systemin (Pearce et al., 2001) increasesJA pools in roots; these, in turn, stimulate nicotinesynthesis in the roots and increase nicotine poolsthroughout the plant.

Given the defensive effectiveness of nicotine, whydo plants wait for herbivore attack to up-regulatetheir production? The answer may well lie in themetabolic demands of production. The N investmentin nicotine can be substantial. After wounding, 8% ofwhole-plant N is in this alkaloid alone, and thisfigure does not include the N used in biosynthesis,transport, and storage (Baldwin et al., 1994a, 1998). Alarge fraction of the increase in nicotine production isderived from N assimilated after attack, but plantsare capable of mobilizing endogenous N pools whenplants are grown in N-free conditions. Endogenouslysynthesized nicotine is not appreciably metabolizedbeyond demethylation to nornicotine and dehydra-tion to anatabine (Baldwin and Ohnmeiss, 1994).Consequently, the plant is not able recoup the sub-stantial N investments it makes in this defense andreutilize this fitness-limiting element for growth andreproduction (Fig. 1, transition 1). In addition to thelarge N requirements, nicotine biosynthesis costs Glc,the magnitude of which depends on the oxidationstate of N used for its synthesis; nicotine synthesizedfrom the NH4 costs 2.86 g of Glc/g of nicotine,whereas synthesis from NO3 costs 26% more (Ger-shenzon, 1994). Not surprisingly, plants preferen-

The Ecological Sophistication of Nicotiana attenuata

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tially use NH4 for nicotine biosynthesis over NO3when given a choice (Lynds and Baldwin, 1998). Thisenergy savings may represent an added benefit of the“fire-chasing” germination behavior of N. attenuata,which ensures germination and growth in NH4-richsoils for the first growing season after a fire (Lyndsand Baldwin, 1998).

Although the resource costs of nicotine productionmay account for its inducible expression, the seques-tration of nicotine biosynthesis in roots adds a 10-hdelay to the transcriptional activation of this induceddefense (Winz and Baldwin, 2001). Plants exhibit a“memory” in so far as they increase their rate ofnicotine biosynthesis and accumulation more rapidlyin response to subsequent elicitations (Baldwin andSchmelz, 1996). However, a biologically significantdelay in defense activation remains and suggests thatbelow-ground nicotine production may have benefitsthat offset this obvious disadvantage. Below-groundsequestration may protect the induced response: abrowser may remove leaves but not the biosyntheticability to produce the alkaloid. Protection of the bio-synthetic capacity to launch a defense response islikely important during regrowth after browsingwhen a plant’s photosynthetic capacity is severelycompromised. Plants can launch a full allometricallycorrected nicotine response even after a browser re-moves 88% of the shoot (Baldwin and Schmelz, 1994).In summary, the intraplant details of nicotine biosyn-thesis and accumulation are consistent with an opti-mization of the costs and benefits of a metabolicallydemanding metabolite and underscore the need tounderstand secondary metabolites in a whole-plantcontext.

JA ELICITS DIRECT AND INDIRECT DEFENSESAND DURABLE RESISTANCE

The fitness benefits of JA elicitation for plants un-der attack are readily seen in field and laboratorystudies. N. attenuata plants growing in native popu-lations induced with a root JA treatment early in thegrowing season had higher nicotine concentrationsfor the duration of the growing season, lost less leafarea to mammalian browsers, had a lower mortalityrate, and produced more viable seed than size-matched controls (Fig. 2, left panel, inset). Similarly,in laboratory studies, survivorship and growth of thetobacco hornworm on JA-treated plants is dramati-cally lower than on untreated control plants, andwhen larvae have the opportunity, they move frominduced plants to feed on neighboring controls (vanDam et al., 1999, 2001a). In these experiments, JAelicitation clearly increased a plant’s direct defenses,which could account for the increase in resistance.

JA-elicited nicotine production is likely to accountfor some of the observed JA-induced resistance. To-bacco hornworm larvae, despite their nicotine resis-tant physiology, grow faster on low nicotine leaves

compared with leaves cultured in xylem solutionswith induced nicotine concentrations (Baldwin,1988b) and on plants with constitutive and inducednicotine production suppressed by the anti-sense ex-pression of a pmt transcripts (Voelckel et al., 2001a).However, many other secondary metabolites are in-duced by JA elicitation of N. attenuata (includingphenolics, flavonoids, phenolic putrescine conju-gates, and diterpene sugar esters; Keinanen et al.,2001), and some of these are known to influenceherbivore performance. Proteinase inhibitors (PI), forexample, are up-regulated by herbivore attack andJA treatment (van Dam et al., 2001b) and are power-ful anti-feedants. Moreover, a study that incorpo-rated leaf material from plants flash-frozen at differ-ent times after JA elicitation into artificial diets to“freeze” the JA-induced chemical dynamics and ex-amine their effects on tobacco hornworm larvae per-formance found that rapidly induced but uncharac-terized changes in direct defenses were as importantas the induced changes in PIs and nicotine (Pohlonand Baldwin, 2001). Hence, although a number of thechemical changes responsible for induced resistancehave been identified, many additional ones clearlyremain to be identified. A major challenge will be tounderstand how induced resistance emerges from allof the chemical changes brought about by elicitation.

In contrast to the situation with direct defenses, thechemical basis of indirect defense function is betterunderstood. JA elicitation and herbivore attack fromfour different species of insects—but not mechanicalwounding—cause plants to systemically release abouquet of mono- and sesquiterpenes, in addition tothe green leaf volatiles that are primarily releasedfrom the wounded leaves (Halitschke et al., 2000;Kessler and Baldwin, 2001). This herbivore-inducedvolatile release occurs principally during the day andcannot be inhibited by treating attacked tissues withlypoxygenase inhibitors (Halitschke et al., 2000). Thevolatile release has been verified in plants grown innative populations, where it functions as an indirectdefense in two distinct ways. First, the volatile re-lease attracts predatory bugs to tobacco hornwormeggs and feeding larvae and dramatically increasespredation rates. Second, the volatile release decreasesoviposition rates from adult moths (Kessler and Bald-win, 2001). These ovipositing adults are likely usingthe volatile release to identify host plants lackingcompetitors (for a single tobacco hornworm larvaerequires many host plants to complete development)and to avoid plants on which predators are likelypresent. This indirect defense can be profoundly ef-fective, and in a field study, the volatile release wasestimated to decrease herbivore loads by 90%(Kessler and Baldwin, 2001). By synthesizing andapplying single components of the herbivore-induced volatile bouquet to unattacked plants inquantities naturally emitted by the plant, it was dem-onstrated that individual components from all three

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major biosynthetic pathways contributing to the vol-atile bouquet, namely a monoterpene (linalool), asesquiterpene (bergamotene), and a green leaf vola-tile (cis-3 hexenanol), were each active in attractingpredatory bugs. The observation that enhancing therelease of single components of the complex blendwas sufficient to attract predators in nature makesthe engineering of this type of indirect defense incrop plants, perhaps in conjunction with direct de-fenses, a tractable proposition.

JA-ELICITED RESISTANCE IS ASSOCIATED WITHA FITNESS COST

Although JA-elicited plants realize a higher fitnesswhen they are under attack, this resistance comes ata substantial fitness cost if plants are not attacked. Inthe same field experiment in which JA-treated plantsrealized a fitness benefit (Fig. 2, left panel), JA elici-tation reduced lifetime viable seed production by26% (1,550 seeds) in plants protected from herbivores

Figure 2. The fitness consequences of jasmonate (JA) elicitation in N. attenuata depend on selective pressure fromherbivores and competitors. Treating plants with methyl ester of JA (MeJA) provides a convenient method of quantitativelysimulating wound- and herbivore-induced changes in endogenous JA pools and secondary metabolites. In natural popula-tions that germinate from long-lived seed banks after fires in the sage-juniper habitats of the Great Basin desert (left panels,Field), an early-season MeJA root treatment increases nicotine concentrations and decreases leaf loss from mammalianbrowsers (inset, Top right control plant, lower left MeJA treated plant) and increases lifetime seed production. When matchedpairs of control and MeJA-treated plants were attacked, treated plants produced an average of 537 seeds and eight capsulesmore than were produced by the controls members of the pair (middle left panel). However, when plant pairs were notattacked (lower left panel), MeJA treatment decreased lifetime seed production by an average of 1,476 seeds and 13 capsulescompared with untreated controls. Left panels depict frequency distribution of differences in capsules produced per plantbetween treated and control plants in each pair. Negative values reflect a cost of MeJA treatment (control member producingmore capsules than its treated counterpart) and positive values reflect a benefit. Right panels depict mean (�SE) lifetimeviable seed production and statistical analysis from paired tdf-tests (data from Baldwin, 1998). In laboratory experiments(right panels, Laboratory), elicitation of plants growing in competition with untreated controls in the same pots reduces seedproduction (middle right panel, Different letters indicate significant differences at P � 0.05) and the ability to compete forsoil 15NO3 (lower right panel) and allocate the acquired N to seed production (percent values above bars), thereby providing alarge fitness benefit for neighboring controls. However, these fitness costs were not found when plants are growing withoutcompetitors (Baldwin et al., 1998) or when plants compete with similarly treated plants (data from van Dam and Baldwin, 2001).





by insecticide spraying and fencing (Baldwin et al.,1998) or by 20% (1,476 seeds) if plants were simplynot attacked (Fig. 2, left panel). These JA-inducedfitness costs mirror the costs of wound inductionobserved in a plantation experiment with the siblingspecies, Nicotiana sylvestris (Baldwin et al., 1990). Inthis experiment, plants were wounded using a stan-dardized mechanical damage technique and hadtheir wound-induced nicotine response suppressedwith IAA applications to the wound site, a procedurethat inhibits wound-induced JA production (Baldwinet al., 1997). The lifetime seed production ofwounded plants that exhibited the normal wound-induced nicotine response was 32% less than that ofsimilarly wounded plants that had had their wound-induced nicotine suppressed with IAA. The similar-ity between the two estimates of fitness costs sup-ports the contention that JA-elicitation simulated theresponses to wounding and that wound-induced re-sponses (those that include nicotine production) ex-act a large cost from seed production.

The mechanisms responsible for these large fitnesscosts were explored in laboratory experiments de-signed to determine whether the fitness reductionscould be attributed to the large investments of N intonicotine production that otherwise could not be usedfor growth and reproduction (Fig. 1). It is surprisingthat when plants were grown in individual hydro-ponic chambers in which the uptake and use of Ncould be carefully quantified, no decrements in seedproduction were found, even when plants were elic-ited so that 8% of their whole-plant N pool was tiedup in nicotine (Baldwin et al., 1998). Because both ofthe experiments that had found fitness costs of JAelicitation were conducted under field conditionswith intraspecific competitors, the fitness conse-quences of elicitation were then examined in thelaboratory, in soil and hydroponic culture in whichelicited plants were grown in competition in variouscombinations of elicited and control plants withvarying N supply rates (van Dam and Baldwin, 1998,2001; Baldwin and Hamilton, 2000). In these experi-ments, the lifetime seed production of JA-elicitedplants competing with other elicited plants did notdiffer from that of control plants competing withcontrol plants, although the time required for repro-ductive maturation was longer. The situation wasdramatically different when controls competed withJA-elicited plants. In these unbalanced competitionsituations (Fig. 2, right panel), controls realized anopportunity benefit with a large increase in lifetimeseed production at the expense of the seed produc-tion of the neighboring JA-elicited plants. The fitnesscost of JA elicitation increased with N supply rate,and was associated with both a greater ability tocompete for below-ground N resources, measuredwith 15N pulse-chase techniques, as well as an in-crease in allocation of acquired 15N to seed produc-tion (Fig. 2, right panel). Therefore, the reductions in

seed production associated with JA-induced re-sponses could not be attributed directly to resourceallocations associated with the production of resis-tance traits. Rather, the costly component of JA elic-itation appears to be diminished competitive abilityresulting from a temporary slowing of growth.

It remains open as to whether the slowing ofgrowth, with its concomitant loss of competitive abil-ity, is required for JA-elicited induced resistance. JAelicitation decreases transcripts of a number ofphotosynthetic-related genes (lhb C1, chl H, and rbc S;Hermsmeier et al., 2001) and this down-regulationmay be required to free up resources for defense-related processes. However, these pleitrophic effectsof JA elicitation may not be strictly a result of “allo-cation costs.” Many other metabolic processes maybe responsible for these coordinated changes in me-tabolism, such as the autotoxicity of metabolite pro-duction (Baldwin and Callahan, 1993) or the historyof JA-inducible elements recruited in the response.Direct genetic manipulations of particular resistancetraits (PIs, nicotine, etc.) will allow researchers todetermine precisely whether defense traits are intrin-sically costly.

WHY DOES N. ATTENUATA ALTER ITS WOUND-AND JA-ELICITED RESPONSES AFTER TOBACCOHORNWORM ATTACK?

When attacked by the nicotine-tolerant tobaccospecialist tobacco hornworm, N. attenuata “recog-nizes” the attack, as evidenced by alterations in anumber of its wound- and JA-elicited responses. Theinduced increase in JA levels that are normally pro-portional to the amount of mechanical woundingerupts into a JA burst that increases concentrationstwo to 10 times wound-induced levels (McCloud andBaldwin, 1997; Kahl et al., 2000; Ziegler et al., 2001)and is propagated throughout the damaged leafahead of the rapidly foraging herbivore (Schittko etal., 1999). Wounding and JA-elicitation do not resultin ethylene emissions, but tobacco hornworm attackproduces a rapid ethylene burst, which is sustainedduring larval feeding (Kahl et al., 2000). The ethyleneburst suppresses the wound- and JA- induced accu-mulation of nicotine biosynthetic genes, NaPMT1 and-2, and the associated nicotine accumulations (Winzand Baldwin, 2001). The ethylene burst does not,however, suppress the volatile release (Kahl et al.,2000). Tobacco hornworm attack, therefore, down-regulates a major direct defense, nicotine, while up-regulating an indirect defense, the volatile release(Fig. 3, left).

All of the tobacco hornworm-induced changes inthe wound responses of N. attenuata can be mimickedby applying larval oral secretions and regurgitants tomechanical wounds (Halitschke et al., 2001). A suiteof eight fatty acid amino acid conjugates (FACs; Fig.3) in the oral secretions are necessary and sufficient

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for not only the transcriptional changes mentionedbelow but also the JA burst and the volatile release. Ifthese FACs are removed from the oral secretions byion-exchange chromatography, eliciting activity islost and regained when synthetic FACs are addedback to the ion-exchanged, inactive, oral secretions(Halitschke et al., 2001).

In addition to the changes in defense phenotypeafter tobacco hornworm attack, N. attenuata also un-dergoes a major transcriptional re-organization.mRNA differential display reverse transcriptase-PCRwas used to gain an unbiased view of the transcrip-tional changes and from this study, it was estimatedthat more than 500 genes respond to herbivore attack(Hermsmeier et al., 2001). The herbivore-regulatedgenes could be crudely classified as being related tophotosynthesis, electron transport, cytoskeleton, car-bon and nitrogen metabolism, signaling, and a groupresponding to stress, wounding, or invasion ofpathogens. Overall, transcripts involved in photo-synthesis were strongly down-regulated, whereasthose responding to stress, wounding, and pathogensand involved in shifting carbon and nitrogen to de-fense were strongly up-regulated. These coordinated

changes point to the existence of central herbivore-activated regulators of metabolism, which in turn areactivated by minute amounts of FACs in tobaccohornworm’s oral secretions (Schittko et al., 2001).Although the overall patterns of transcriptionalchanges agree generally with the observed pheno-typic alterations, the transcriptional basis for theknown phenotypic alterations remains obscure.Clearly, a functional understanding of the alterationswould help generate predictions about the nature ofthe herbivore-elicited trans-acting factors. In otherwords, if we knew “why” the secondary metabolitephenotype and transcriptome of N. attenuata was sostrongly altered after tobacco hornworm attack, wewould be in a stronger position to understand howthe changes come about. We are currently exploringthree hypotheses.

First, these adapted larvae appear to be feeding ina “stealthy” fashion, reducing their dietary intake ofnicotine by suppressing the nicotine responses belowthat of plants suffering comparable tissue loss (Bald-win, 1988a; McCloud and Baldwin, 1997). Tobaccohornworm larvae clearly pay a growth penalty todetoxify nicotine, as is evidenced by their higher

Figure 3. Two examples of the down-regulation of a direct defense (nicotine) with the release of volatiles that function toattract beneficial insects in N. attenuata. A, Attack by tobacco hornworm larvae is distinguished from simple wounding bythe introduction of FACs at the wound site (the most abundant FAC, n-linolenoyl-L-Glu, is depicted) and results in JA andethylene bursts and a systemic release of volatiles, which functions as an indirect defense by attracting predatory bugs anddecreasing the oviposition rate of adult moths. The ethylene burst suppresses wound-induced nicotine production bydecreasing the accumulation of putrescine N-methyl transferase (pmt) transcripts, the rate limiting step in nicotinebiosynthesis and inhibits the decrease in competitive ability that results from JA-elicitation. B, Benzyl acetone (BA;4-phenyl-2-butanone), found only in the outer lip of the corolla where pollinators come in contact with the flower, isreleased from the corolla in the evening. Diurnal changes in the size of the corolla BA pool closely parallel the amountemitted by flowers. Nicotine can be detected in the headspace of flowers and is localized in the basal parts of the corolla,below the attachment of the filaments and the nectar reward. The corolla pools of nicotine are stable throughout the dayexcept during the period of peak BA production and emission when nicotine pools decrease significantly (Euler and Baldwin,1996).





growth rates on plants with nicotine levels sup-pressed by the anti-sense expression of pmt (Voelckelet al., 2001a).

Second, N. attenuata may be optimizing the defen-sive function of its volatile release by suppressingnicotine production, which could be sequestered bythe herbivore and used against predators attracted bythe volatile release. Plant defense compounds arecommonly sequestered by adapted herbivores fortheir own defense and tobacco hornworm’s larvaeare thought to use dietary nicotine against the larvalparasitoid, Cotesia congregata (Barbosa et al., 1991).Thus, induced nicotine production may wreak havocwith the plant’s ability to use “top-down” processesas a defense. A similar process occurs when N. at-tenuata plants attract pollinators by releasing volatilefloral scents (Fig. 3, right panel). Consistent with thedefense optimization hypothesis is the observationthat tobacco hornworm attack does not suppress thePI elicitation (van Dam et al., 2001b). PIs are notknown to be sequestered by larvae for defense andtend to slow herbivore growth, thereby prolongingthe time that predators could be attracted to plants bythe volatile release and attack larvae. The main pred-ator attracted by the volatile release in nature of N.attenuata, Geocoris pallens, is relatively small and ef-fectively kills only eggs and first and second instarlarvae (Kessler and Baldwin, 2001). Direct defensesthat extend the time during which larvae remain inthese instars would likely increase the effectivenessof the indirect defense. Hence, the changes in thewound response may represent an optimization ofdefense, in which the plant assembles a suite of directand indirect defenses particularly effective against aparticular herbivore species.

Third, the plant’s altered wound response may alsoreflect the fitness consequences of intraspecific com-petition and function to manipulate herbivore behav-ior to maximize fitness in response to selection fromboth herbivory and competition. N. attenuata plantsmass germinate from long-lived seed banks afterfires and hence are commonly competing with con-specifics. Because these competitive interactions mayprofoundly determine an individual plant’s fitness(Fig. 2), they may influence the fitness consequencesof anti-herbivore defense. JA-elicitation and the up-regulation of the associated resistance traits tran-siently slows growth and makes JA-elicited and re-sistant plants inferior to neighboring susceptiblecontrols (Fig. 2). Tobacco hornworm attack results ina dramatic ethylene burst that transcriptionallydown-regulates nicotine production, and this ethyl-ene burst also inhibits the reduction in competitiveability associated with JA elicitation (Voelckel et al.,2001b). That the herbivore-induced ethylene burstinhibits both nicotine production and the reductionin competitive ability is consistent with the hypoth-esis that the large N demands of nicotine biosynthe-sis contribute to the transient slowing of growth dur-

ing JA elicitation. The ethylene burst was not foundto influence the accumulation of transcripts fromseven other tobacco hornworm-regulated genes(Schittko et al., 2001), but it remains to be determinedwhether the effect of the ethylene burst on JA-elicitedgrowth is mediated by responses other than the sup-pression of pmt transcript accumulation and subse-quent nicotine production. Although the mechanismsremain to be elucidated, the fact that N. attenuatasuppresses a potent defense metabolite and does notdown-regulate its growth as part of its herbivore“recognition” response, suggests that competitive in-teractions may have influenced plant-herbivore inter-actions and focuses attention to larval movementbehavior on induced and uninduced plants.

When tobacco hornworm larvae are small (first andsecond instars), the costs of movement betweenplants are larger than the costs of remaining on JA-elicited plants (van Dam et al., 2001a). However,when they reach the third instar, they readily leaveJA-elicited plants to feed on neighboring controlplants. More than 98% of the lifetime N. attenuata leafmass consumed by a tobacco hornworm larva is con-sumed during the fourth and fifth instars. Thus ifherbivores are motivated by a plant’s induced re-sponses to move to neighboring plants when they aremost voracious, a delayed or suppressed activationof defense might provide plants with an effectiveresponse to the combined selective pressures of her-bivore attack and intraspecific competition. Specifi-cally, plants may tolerate herbivore attack when lar-vae are small and move them to neighboring andcompeting conspecifics when they are most vora-cious (van Dam et al., 2001a). Plants clearly “recog-nize” attack from first instar larvae and have thecapability of launching a lethal, however costly, de-fense response against first instar larvae, as is evidentfrom larval performance studies on JA-elicited plants(van Dam et al., 1999). However, plants appear not toavail themselves of this ability. As such, the suppres-sion of costly defense responses during herbivore“recognition” may represent a higher-level optimiza-tion of a plant’s defense responses.

CONCLUSION

Native plants have clearly evolved sophisticatedmeans of coping with the myriad of selection pres-sures with which they are faced. Only by measuringplant fitness attributes can one understand theplant’s solutions to these selective pressures. Themechanisms responsible for this ecological sophisti-cation are likely lurking in the details of the regula-tion of the “transcriptome” but without an intimateunderstanding of a plant’s natural history, the tran-scriptional responses will remain functionally ob-scure. The cost-benefit paradigm is a useful heuristictool to generate testable hypotheses about the func-tion of a trait, but tools are needed to manipulate the

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traits and test the fitness consequences of the manip-ulations in ecologically complex environments. Assuch, the development of gene disruption, silencingand over-expression techniques that can be used withplants growing in natural habitats may provide thefastest way forward toward a functional understand-ing of metabolism.

ACKNOWLEDGMENTS

I thank E. Claussen and E. Wheeler for assistance withthe figures and editing, respectively, and my coworkers for16 exciting years of research on Nicotiana spp. Space con-straints prevented citing the vast amount of excellent re-search from cultivated tobacco, Nicotiana tabacum, whichinspired much of this work.

Received September 14, 2001; accepted October 1, 2001.

LITERATURE CITED

Baldwin IT (1988a) The alkaloidal responses of wild to-bacco to real and simulated herbivory. Oecologia378–381

Baldwin IT (1988b) Short-term damage-induced increasesin tobacco alkaloids protect plants. Oecologia 75: 367–370

Baldwin IT (1989) Mechanism of damaged-induced alka-loids in wild tobacco. J Chem Ecol 15: 1661–1680

Baldwin IT (1999) Inducible nicotine production in nativeNicotiana as an example of adaptive phenotypic plastic-ity. J Chem Ecol 25: 3–30

Baldwin IT, Callahan P (1993) Autotoxicity and chemicaldefense: nicotine accumulation and carbon gain in so-lanaceous plants. Oecologia 94: 534–541

Baldwin IT, Gorham D, Schmelz EA, Lewandowski C,Lynds GY (1998) Allocation of nitrogen to an inducibledefense and seed production in Nicotiana attenuata. Oe-cologia 115: 541–552

Baldwin IT, Hamilton W (2000) Jasmonate-induced re-sponses of Nicotiana sylvestris results in fitness costs dueto impaired competitive ability for nitrogen. J Chem Ecol26: 915–952

Baldwin IT, Karb MJ, Ohnmeiss TE (1994a) Allocation of15N from nitrate to nicotine: production and turnover ofa damage-induced mobile defense. Ecology 75: 1703–1713

Baldwin IT, Ohnmeiss TE (1994) Swords into plowshares?Nicotiana sylvestris does not use nicotine as a nitrogensource under nitrogen-limited growth. Oecologia 98:385–392

Baldwin IT, Schmelz EA (1994) Constraints on an induceddefense: the role of canopy area. Oecologia 97: 424–430

Baldwin IT, Schmelz EA (1996) Immunological “memory”in the induced accumulation of nicotine in wild tobacco.Ecology 77: 236–246

Baldwin IT, Sims CL, Kean SE (1990) The reproductiveconsequences associated with inducible alkaloidal re-sponses in wild tobacco. Ecology 71: 252–262

Baldwin IT, Staszak-Kozinski L, Davidson R (1994b) Upin smoke: I. smoke-derived germination cues for the

post-fire annual Nicotiana attenuata Torr. Ex. Watson.J Chem Ecol 20: 2345–2371

Baldwin IT, Zhang Z-P, Diab N, Ohnmeiss TE, McCloudES, Lynds GY, Schmelz EA (1997) Quantification, corre-lations and manipulations of wound-induced changes injasmonic acid and nicotine in Nicotiana sylvestris. Planta201: 397–404

Barbosa P, Gross P, Kemper J (1991) Influence of plantallelochemicals on the tobacco hornworm and its parasi-toid, Cotesia congregata. Ecology 72: 1567–1575

Chapin FS, Schulze E-D, Mooney HA (1990) The ecologyand economics of storage in plants. Annu Rev Ecol Syst21: 423–447

Euler M, Baldwin IT (1996) The chemistry of defense andapparency in the corollas of Nicotiana attenuata. Oecolo-gia 107: 102–112

Gershenzon J (1994) The cost of plant chemical defenseagainst herbivory: a biochemical perspective. In EA Ber-nays, ed, Insect-Plant Interactions. CRC Press, Boca Ra-ton, FL, pp 105–173

Halitschke R, Kessler A, Kahl J, Lorenz A, Baldwin IT(2000) Ecophysiological comparison of direct and indi-rect defenses in Nicotiana attenuata. Oecologia 124:408–417

Halitschke R, Schittko U, Pohnert G, Boland W, BaldwinIT (2001) Molecular interactions between the specialistherbivore Manduca sexta (Lepidoptera, Sphingidae) andits natural host Nicotiana attenuata: III. Fatty acid-aminoacid conjugates in herbivore oral secretions are necessaryand sufficient for herbivore-specific plant responses.Plant Physiol 125: 711–717

Hermsmeier D, Schittko U, Baldwin IT (2001) Molecularinteractions between the specialist herbivore Manducasexta (Lepidoptera, Sphingidae) and its natural host Nico-tiana attenuata: I. Large-scale changes in the accumulationof growth- and defense-related plant mRNAs. PlantPhysiol 125: 683–700

Kahl J, Siemens DH, Aerts RJ, Gabler R, Kuhnemann F,Preston CA, Baldwin IT (2000) Herbivore-induced eth-ylene suppresses a direct defense but not a indirect de-fense against an adapted herbivore. Planta 210: 336–342

Keinanen M, Oldham NJ, Baldwin IT (2001) Rapid HPLCscreening of jasmonate-induced increases in tobacco al-kaloids, phenolics and diterpene glycosides in Nicotianaattenuata. J Agric Food Chem 49: 3553–3558

Kessler A, Baldwin IT (2001) Defensive function ofherbivore-induced plant volatile emissions in nature. Sci-ence 291: 2141–2144

Lynds GY, Baldwin IT (1998) Fire, nitrogen, and defensiveplasticity in Nicotiana attenuata. Oecologia 115: 531–540

McCloud ES, Baldwin IT (1997) Herbivory and caterpillarregurgitants amplify the wound-induced increases in jas-monic acid but not nicotine in Nicotiana sylvestris. Planta203: 430–435

McKey D (1974) Adaptive patterns in alkaloid physiology.Am Nat 108: 305–320

Mitchell-Olds T (2001) Arabidopsis thaliana and its wildrelatives: a model system for ecology and evolution.Trends Ecol Evol (in press)





Ohnmeiss TE, Baldwin IT (2000) Optimal defense theorypredicts the ontogeny of an induced nicotine defense.Ecology 81: 1765–1783

Pearce G, Moura DS, Stratmann J, Ryan CA (2001) Pro-duction of multiple plant hormones from a singlepolyprotein precursor. Nature 411: 817–820

Pohlon E, Baldwin IT (2001) Artificial diets “capture” thedynamics of jasmonate-induced defenses in plants. En-tomol Exp Appl 100: 127–130

Preston CA, Baldwin IT (1999) Positive and negative sig-nals regulate germination in the post-fire annual, Nicoti-ana attenuata. Ecology 80: 481–494

Price PW, Bouton CE, Gross P, McPheron BA, ThompsonJN, Weis AE (1980) Interactions among three trophiclevels: influence of plants on interactions between insectherbivores and natural enemies. Annu Rev Ecol Syst 11:41–65

Schittko U, Hermsmeier D, Baldwin IT (2001) Molecularinteractions between the specialist herbivore Manducasexta (Lepidoptera, Sphingidae) and its natural host Nico-tiana attenuata: II. Accumulation of plant mRNAs in re-sponse to insect-derived cues. Plant Physiol 125: 701–710

Schittko U, Preston CA, Baldwin IT (1999) Eating theevidence? Manduca sexta larvae can not disrupt jas-monate induction in Nicotiana attenuata through rapidconsumption. Planta 210: 343–346

van Dam NM, Baldwin IT (1998) Costs of jasmonate-induced responses in plants competing for limited re-sources. Ecol Lett 1: 30–33

van Dam NM, Baldwin IT (2001) Competition mediatescosts of jasmonate-induced defenses, N acquisition and

transgenerational plasticity in Nicotiana attenuata. FunctEcol 15: 406–415

van Dam NM, Hadwich K, Baldwin IT (1999) Inducedresponses in Nicotiana attenuata affect behavior andgrowth of the specialist herbivore Manduca sexta. Oeco-logia 122: 371–379

van Dam NM, Hermenau U, Baldwin IT (2001a) Instar-specific sensitivity of specialist Manduca sexta larvae toinduced defenses in their host plant Nicotiana attenuata.Ecol Entomol (in press)

van Dam NM, Horn M, Mares M, Baldwin IT (2001b)Ontogeny constrains systemic protease inhibitor re-sponse in Nicotiana attenuata. J Chem Ecol 27: 547–568

Voelckel C, Krugel T, Gase K, Heidrich N, van Dam NM,Winz R, Baldwin IT (2001a) Anti-sense expression ofputrescine N-methyltransferase confirms defensive roleof nicotine in Nicotiana sylvestris against Manduca sexta.Chemoecology 11: 121–126

Voelckel C, Schittko U, Baldwin IT (2001b) Herbivore-induced ethylene burst reduces fitness costs ofjasmonate-oral secretion-induced defenses in Nicotianaattenuata. Oecologia 127: 274–280

Winz RA, Baldwin IT (2001) Molecular interactions be-tween the specialist herbivore Manduca sexta (Lepidop-tera, Sphingidae) and its natural host Nicotiana attenuata:IV. Insect-induced ethylene reduces jasmonate-inducednicotine by regulating putrescine N-methyltransferasetranscripts. Plant Physiol 125: 2189–2202

Ziegler J, Keinanen M, Baldwin IT (2001) Herbivore-induced allene oxide synthase transcripts and jasmonicacid in Nicotiana attenuata. Phytochemistry (in press)

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