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Although much is known about plant traits that function in nonhost resistance against pathogens, little is known about nonhost resistance against herbivores, despite its agricultural importance. Empoasca leafhoppers, serious agricultural pests, identify host plants by eavesdropping on unknown outputs of jasmonate (JA)–mediated signaling. Forward- and reverse-genetics lines of a native tobacco plant were screened in native habitats with native herbivores using high-throughput genomic, transcriptomic, and metabolomic tools to reveal an Empoasca -elicited JA-JAZi module. This module induces an uncharacterized caffeoylputrescine–green leaf volatile compound, catalyzed by a polyphenol oxidase in a Michael addition reaction, which we reconstitute in vitro; engineer in crop plants, where it requires a berberine bridge enzyme-like 2 (BBL2) for its synthesis; and show that it confers resistance to leafhoppers. Natural history–guided forward genetics reveals a conserved nonhost resistance mechanism useful for crop protection.

Summary Nicotiana attenuata flowers, diurnally open, emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant's native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus floral circadian rhythms may influence a plant's fitness by filtering pollinators leading to altered seed set from outcrossed pollen.

Summary Nutrients are distributed unevenly in the soil. Phenotypic plasticity in root growth and proliferation may enable plants to cope with this variation and effectively forage for essential nutrients. However, how micronutrients shape root architecture of plants in their natural environments is poorly understood. We employed a combination of field and laboratory-based assays to determine the capacity of Nicotiana attenuata to direct root growth towards localized nutrient patches in its native environment. Plants growing in nature displayed a particular root phenotype consisting of a single primary root and a few long, shallow lateral roots. Analysis of bulk soil surrounding the lateral roots revealed a strong positive correlation between lateral root placement and micronutrient gradients, including copper, iron and zinc. In laboratory assays, the application of localized micronutrient salts close to lateral root tips led to roots bending in the direction of copper and iron. This form of chemotropism was absent in ethylene and jasmonic acid deficient lines, suggesting that it is controlled in part by these two hormones. This work demonstrates that directed root growth underlies foraging behavior, and suggests that chemotropism and micronutrient-guided root placement are important factors that shape root architecture in nature.

Benzyl acetone (4-phenylbutan-2-one; BA), the dominant floral fragrance of the wild tobacco Nicotiana attenuata, is known to attract night-time pollinators, such as Manduca sexta hawkmoths. For this pollinator, BA is not only essential for the pollen transport between conspecific plants, but also for the moth’s short-distance handling of flowers at night. The emission of BA from the corolla limb, however, starts up to 4 hr before dusk, peaks at dusk and continues throughout the first half of the night. While the function of the nocturnal part of the emission is well studied, the function of the early part of the peak emission remained unknown. By using plants silenced in the expression of the biosynthetic gene of BA (Nachal1), we investigated the function of this early peak emission of BA in the plants´ native habitat in Southwestern Utah. By emitting BA before dusk, N. attenuata is able to prevent the establishment and resulting floral damage by a florivore, the cucumber beetle (Diabrotica undecimpunctata). Plants lacking BA emissions (CHAL) were not only more often colonized by florivores, but also suffered significantly more damage, than control empty vector plants (EV). Choice assays revealed that D. undecimpunctata feeding required the absence of BA. While feeding damage to CHAL flowers was higher than to EV flowers in the early night hours, the effect disappeared during the second half of the night, when flowers stop emitting BA. The close proximity of BA-emitting flowers to non-emitting CHAL flowers prevented D. undecimpunctata feeding. In the field, the emission of BA at dusk was sufficient to protect plants for the entire night, as beetles search for new hosts during the early evening hours and remain for the duration of the night, once a host is chosen. Floral BA emission before dusk may have evolved as a consequence of antagonistic interactions with florivores. A single floral volatile can thus simultaneously function as an important floral attractant for pollinators and as effective feeding deterrent against florivores in the same plant species. A plain language summary is available for this article.

Nicotiana attenuata flowers, diurnally open, emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant's native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus, floral circadian rhythms may influence a plant's fitness by filtering pollinators leading to altered seed set from outcrossed pollen.

Nutrients are distributed unevenly in the soil. Phenotypic plasticity in root growth and proliferation may enable plants to cope with this variation and effectively forage for essential nutrients. However, how micronutrients shape root architecture of plants in their natural environments is poorly understood. We used a combination of field and laboratory-based assays to determine the capacity of Nicotiana attenuata to direct root growth towards localized nutrient patches in its native environment. Plants growing in nature displayed a particular root phenotype consisting of a single primary root and a few long, shallow lateral roots. Analysis of bulk soil surrounding the lateral roots revealed a strong positive correlation between lateral root placement and micronutrient gradients, including copper, iron and zinc. In laboratory assays, the application of localized micronutrient salts close to lateral root tips led to roots bending in the direction of copper and iron. This form of chemotropism was absent in ethylene and jasmonic acid deficient lines, suggesting that it is controlled in part by these two hormones. This work demonstrates that directed root growth underlies foraging behavior, and suggests that chemotropism and micronutrient-guided root placement are important factors that shape root architecture in nature.

Summary More than 87% of flowering plant species are animal-pollinated [1] and produce floral scents and other signals to attract pollinators. These floral cues may however also attract antagonistic visitors, including herbivores [2]. The dilemma is exacerbated when adult insects pollinate the same plant that their larvae consume. It remains largely unclear how plants maximize their fitness under these circumstances. Here we show that in the night-flowering wild tobacco Nicotiana attenuata , the emission of a sesquiterpene, ( E )-α-bergamotene, in flowers increases adult Manduca sexta moth-mediated pollination success, while the same compound in leaves is known to mediate indirect defense against M . sexta larvae [3, 4]. Forward and reverse genetic analyses demonstrated that both herbivory-induced and floral ( E )-α-bergamotene are regulated by the expression of a monoterpene-synthase-derived sesquiterpene synthase ( NaTPS38 ). The expression pattern of NaTPS38 also accounts for variation in ( E )-α-bergamotene emission among natural accessions. These results highlight that differential expression of a single gene that results in tissue-specific emission of one compound contributes to resolving the dilemma for plants when their pollinators are also herbivores. Furthermore, this study provides genetic evidence that pollinators and herbivores interactively shape the evolution of floral signals and plant defense.

Ecological interactions between flowers and pollinators are all about timing. Flower opening/closing and scent emissions are largely synchronized with pollinator activity, and a circadian clock regulates these rhythms. However, whether the circadian clock increases a plant's reproductive success by regulating these floral rhythms remains untested. Flowers of Nicotiana attenuata, a wild tobacco, diurnally and rhythmically open, emit scent and move vertically through a 140° arc to interact with nocturnal hawkmoths. We tethered flowers to evaluate the importance of flower positions for Manduca sexta-mediated pollinations; flower position dramatically influenced pollination. We examined the pollination success of phase-shifted flowers, silenced in circadian clock genes, NaZTL, NaLHY, and NaTOC1, by RNAi. Circadian rhythms in N. attenuata flowers are responsible for altered seed set from outcrossed pollen.

Pollination by insects is essential to many ecosystems. Previously, we have shown that floral scent is important to mediate pollen transfer between plants (Kessler et al., 2015). Yet, the mechanisms by which pollinators evaluate volatiles of single flowers remained unclear. Here, Nicotiana attenuata plants, in which floral volatiles have been genetically silenced and its hawkmoth pollinator, Manduca sexta, were used in semi-natural tent and wind-tunnel assays to explore the function of floral scent. We found that floral scent functions to increase the fitness of individual flowers not only by increasing detectability but also by enhancing the pollinator's foraging efforts. Combining proboscis choice tests with neurophysiological, anatomical and molecular analyses we show that this effect is governed by newly discovered olfactory neurons on the tip of the moth's proboscis. With the tip of their tongue, pollinators assess the advertisement of individual flowers, an ability essential for maintaining this important ecosystem service.

Flowers recruit floral visitors for pollination services by emitting fragrances. These scent signals can be intercepted by antagonists such as florivores to locate host plants. Hence, as a consequence of interactions with both mutualists and antagonists, floral bouquets likely consist of both attractive and defensive components. While the attractive functions of floral bouquets have been studied, their defensive function has not, and field-based evidence for the deterrence of floral-scent constituents is lacking. In field and glasshouse experiments with five lines of transgenic Petunia x hybrida plants specifically silenced in their ability to release particular components of their floral volatile bouquet, we demonstrate that the emission of single floral-scent compounds can dramatically decrease damage from generalist florivores. While some compounds are used in host location, others prevent florivory. We conclude that the complex blends that comprise floral scents are likely sculpted by the selective pressures of both pollinators and herbivores.

SUMMARYMany plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotianaattenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds,increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show thatthese important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show nosigns of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, areunpredictably variable among flowers, not only among populations, but also within populations, and evenamong flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrationsincrease outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. atten-uata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic genefor nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized andtransplanted into native populations. When only exposed to hummingbird pollinators, seeds produced byflowers with nicotine in their nectar had a greater number of genetically different sires, compared to seedsfrom nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescencedecreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, smallRNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.Keywords: nectar toxins, Nicotiana attenuata, nicotine, floral visitors, hummingbirds, pollinators, malereproductive success, outcrossing, Archilochus alexandri, putrescine N-methyl transferase 1, Dicer-like genes.INTRODUCTIONFloral nectar frequently contains toxic compounds (Kernervon Marilaun, 1879; Adler, 2000), which have mainly deter-rent functions, and either repel unwanted flower visitorssuch as nectar robbers and nectar thieves (Stephenson,1981; Kessler et al., 2008) and inefficient pollinators (John-sonet al.,2006),orincreasenectarshelf-lifebyantimicrobialproperties (Carter and Thornburg, 2004). The existence ofnectar toxins was long thought to be an unavoidable con-sequence of selection for resistance traits in response tofolivorous herbivores and pathogens, and the accumulationof toxins in nectar and their repellent effects on pollinatorsmay be an example of collateral damage resulting fromdeploying chemical defenses, commonly referred to as the‘pleiotrophy hypothesis’ (Adler, 2000). However, recentstudies suggest that nectar toxins may also increase polli-nator fidelity. For example, high levels of nectar alkaloidswere suggested to benefit plants via increased pollen export(Irwin and Adler, 2008).Flowers of the native tobacco, Nicotiana attenuata, accu-mulate the alkaloid nicotine, which is synthesized in theroots and transported throughout the plant, where itprovides resistance against herbivores (Steppuhn et al.,2004; Steppuhn and Baldwin, 2007). Nectar laced withnicotine protects flowers against nectar robbing and flori-vores, and changes the behavior of an important pollinator,hummingbirds (Archilochus alexandri) (Kessler et al., 2008).Although nicotine is a repellant that decreases nectarremoval by floral visitors (Kessler and Baldwin, 2007), thepresence of this toxin increases plant fitness through bothmaternal and paternal means (Kessler et al., 2008). Theproposed mechanism for the increase in plant fitness isthrough a change in the nectaring behavior of humming-birds, which visit more flowers per plant if a plant producesnicotine (Kessler et al., 2008). Why hummingbirds continueto visit flowers that contain repellent nectar and whethertoxic nectar benefits the plant by increasing outcrossing

Manipulations of the interactions between plants and their floral visitors remain the most successful path to an understanding of floral traits, which may have been shaped by both herbivores and pollinators. By using genetic tools in combination with old-fashioned field work the dual protective/advertisement functions of floral traits are being realized. The distinction between wanted and unwanted floral visitors is blurring, and plants with specialized pollination systems are being found capable of using alternative pollinators if the specialized pollinators fail to perform.

Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C6 aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant’s apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata’s fitness.

Summary Flowers produce a plethora of secondary metabolites but only nectar sugars, floral pigments and headspace volatiles have been examined in the context of pollinator behavior. We identify secondary metabolites in the headspace and nectar of glasshouse- and field-grown Nicotiana attenuata plants, infer within-flower origins by analyzing six flower parts, and compare the attractiveness of 16 constituents in standardized choice tests with two guilds of natural pollinators (Manducasexta moths and Archilochus alexandri and Selasphorus rufus hummingbirds) and one nectar thief (Solenopsis xyloni ants) to determine whether nectar metabolites can ‘filter’ flower visitors: only two could. Moths responded more strongly than did hummingbirds to headspace presentation of nicotine and benzylacetone, the most abundant repellent and attractant compounds, respectively. For both pollinators, nectar repellents decreased nectaring time and nectar volume removed, but increased visitation number, particularly for hummingbirds. Fewer ants visited if the nectar contained repellents. To determine whether nicotine reduced nectar removal rates in nature, we planted transformed, nicotine-silenced plants into native populations in Utah over 2 years. Plants completely lacking nicotine in their nectar had 68–70% more nectar removed per night by the native community of floral visitors than did wild-type plants. We hypothesize that nectar repellents optimize the number of flower visitors per volume of nectar produced, allowing plants to keep their nectar volumes small.

Many plants attract and reward pollinators with floral scents and nectar, respectively, but these traits can also incur fitness costs as they also attract herbivores. This dilemma, common to most flowering plants, could be solved by not producing nectar and/or scent, thereby cheating pollinators. Both nectar and scent are highly variable in native populations of coyote tobacco, Nicotiana attenuata, with some producing no nectar at all, uncorrelated with the tobacco's main floral attractant, benzylacetone. By silencing benzylacetone biosynthesis and nectar production in all combinations by RNAi, we experimentally uncouple these floral rewards/attractrants and measure their costs/benefits in the plant's native habitat and experimental tents. Both scent and nectar increase outcrossing rates for three, separately tested, pollinators and both traits increase oviposition by a hawkmoth herbivore, with nectar being more influential than scent. These results underscore that it makes little sense to study floral traits as if they only mediated pollination services. DOI: http://dx.doi.org/10.7554/eLife.07641.001, eLife digest Flowering plants have evolved a number of different approaches to reproduction. Some use their own pollen and self-fertilize, while others use pollen from other nearby plants. This fertilization by other plants is called ‘outcrossing’ and introduces new genetic variation into each generation, which is extremely important for the evolutionary process. Some flowering plants rely on animals to help with pollination, attracting visitors with floral scents and rewarding the visitors with sugar-rich nectar. But scent and nectar also attract herbivores that damage the plants. This causes a dilemma for flowering plants, which has led some to evolve to not produce scent or to offer no nectar while masquerading as a plant that does. Previous studies into the costs and benefits of such strategies have looked at the effects of either floral scent or nectar, but no-one has uncoupled the effects of these two traits on both pollination and herbivore attack. Kessler et al. have addressed this issue in wild tobacco plants, which can both self-fertilize and outcross, and which produce varying amounts of scent and nectar. The experiments were conducted under mesh tents and in field trials in the plant's natural habitat: the Great Basin Desert in Utah. Kessler et al. used a gene-silencing technique called ‘RNA interference’ to inhibit the production of scent or nectar, either separately or together. When grown in field trials, under conditions that prevent self-fertilization, these tobacco plants are frequently visited by a hummingbird and three species of hawkmoth. All four of these animals pollinate the tobacco plants, but one of the moths also lays eggs that hatch into caterpillars, which damage the plant. Kessler et al. monitored the effects that the loss of scent, nectar or both had on visits by each pollinator and on outcrossing. These experiments revealed that scent is essential to attract one hawkmoth species but not for another (called Hyles lineata). Furthermore, while, the hummingbird needs nectar, the H. lineata moth does not; but this moth won't visit flowers that lack both scent and nectar. The experiments also show that, for the moth that lays its eggs on the tobacco plants, both scent and nectar increase pollination and egg laying, but nectar has a stronger effect. Thus reducing nectar, as this tobacco plant does in the wild, is one strategy that can be used to reduce herbivore attack by caterpillars. Together, these findings highlight that it is important to study both herbivores and pollinators when attempting to understand the evolution of floral traits. DOI: http://dx.doi.org/10.7554/eLife.07641.002

Inverse dynamics is a standard analysis in biomechanics to reconstruct time histories of internal driving forces and torques from measured external forces and segmental kinematics. The main sources of inconsistency leading to analytical artefacts in this process are skin marker and soft tissue motion. These potentially artificial high frequency fluctuations in the joint torques may serve as an erroneous basis of (misleading) assumptions with respect to muscular activity. Here we suggest techniques to reduce these errors. In both parts of this study, high-speed video and force platform data were acquired. In one part, 69 sequences of human barefoot running were sampled followed by an inverse dynamic analysis of the stance leg. The time history of the hip joint torque in the sagittal plane served as a sensitive "detector" of dynamic analysis artefacts. We show that the most important error — the relative skin to bone motion especially of the knee marker — can be reduced significantly by processing kinematic data using bone rigidity (constant segment lengths) and bony contour (frontal knee edge) information. Further on, neglecting significantly initiated soft tissue dynamics in the inverse dynamic model introduces another inconsistency in the analytical process. Therefore, in a second part of this study, soft tissue kinematics from 14 jumping sequences were identified. These data provided a set of coupling parameters of wobbling masses to the bone that were ready to be implemented in the inverse dynamic model. Using realistic bone kinematics mainly avoids phase shifts in the acceleration scenario within the leg, and thus artifical hip torque fluctuations within the whole contact period. In human running, accounting for soft tissue dynamics mainly affects the calculated timing of the hip joint torque during the impact phase.

Big-eyed bugs (Geocoris spp. Fallén, Hemiptera: Lygaeidae) are ubiquitous, omnivorous insect predators whose plant feeding behavior raises the question of whether they benefit or harm plants. However, several studies have investigated both the potential of Geocoris spp. to serve as biological control agents in agriculture and their importance as agents of plant indirect defense in nature. These studies have demonstrated that Geocoris spp. effectively reduce herbivore populations and increase plant yield. Previous work has also indicated that Geocoris spp. respond to visual and olfactory cues when foraging and choosing their prey and that associative learning of prey and plant cues informs their foraging strategies. For these reasons, Geocoris spp. have become models for the study of tritrophic plant-herbivore-predator interactions. Here, we present detailed images and ecological observations of G. pallens Stål and G. punctipes (Say) native to the Great Basin Desert of southwestern Utah, including observations of their life histories and color morphs, dynamics of their predatory feeding behavior and prey choice over space and time, and novel aspects of Geocoris spp.’s relationships to their host plants. These observations open up new areas to be explored regarding the behavior of Geocoris spp. and their interactions with plant and herbivore populations.

Although nectar is known to be important, for example in plant–insect interactions, little has been known about the mechanism of its secretion; sucrose phosphate synthases are now reported to be essential for the synthesis of the sucrose component of nectar and the transporter protein SWEET9 is shown to mediate sucrose export into the extracellular space of the nectary. Nectar is an important factor in interactions between plants and insects, mediating both pollination and defensive mutualisms. The function and composition of floral nectaries have been well characterized, but the mechanism of nectar secretion has remained elusive. In a study of three flowering plant species, Wolf Frommer and colleagues show that sucrose phosphate synthases are highly expressed in floral nectaries and are essential for the synthesis of the sucrose component of nectar. The transporter protein SWEET9 mediates sucrose export from the site of production, in the nectary parenchyma, to the extracellular space of the nectary. Angiosperms developed floral nectaries that reward pollinating insects1. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear2. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

Background Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata. Results We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary. Conclusions Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.

(Abstract) In this protocol, the determination of the nicotine concentration in nectar of Nicotiana attenuata is described. This method is applicable for the investigation of small amounts of nectar (above 1 µl). It is a high-throughput protocol optimized and streamlined for one skilled person to process approximately 100 nectar samples per day.

Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.

Plants use many means to attract pollinators, including visual cues and odor. We investigated how nonpigment floral chemistry influences nectar removal, floral visitation, florivory, rates of outcrossing, and fitness through both male and female functions. We blocked expression of biosynthetic genes of the dominant floral attractant [benzyl acetone ( Nachal1 )] and nectar repellent [nicotine ( Napmt1/2 )] in all combinations in the native tobacco Nicotiana attenuata and measured their effects on plants in their native habitat. Both repellent and attractant were required to maximize capsule production and seed siring in emasculated flowers and flower visitation by native pollinators, whereas nicotine reduced florivory and nectar robbing.

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: (i) an Agrobacterium-based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

The attraction of natural enemies of herbivores by volatile organic compounds as an induced indirect defence has been studied in several plant systems. The evidence for their defensive function originates mainly from laboratory studies with trained parasitoids and predators; the defensive function of these emissions for plants in natural settings has been rarely demonstrated. In native populations and laboratory Y-tube choice experiments with transgenic Nicotiana attenuata plants unable to release particular volatiles, we demonstrate that predatory bugs use terpenoids and green leaf volatiles (GLVs) to locate their prey on herbivore-attacked plants. By attracting predators with volatile signals, this native plant reduces its herbivore load - demonstrating the defensive function of herbivore-induced volatile emissions. However, plants producing GLVs are also damaged more by flea beetles. The implications of these conflicting ecological effects for the evolution of induced volatile emissions and for the development of sustainable agricultural practices are discussed.

SummaryAll animal-pollinated plants must solve the problem of attracting pollinators while remaining inconspicuous to herbivores, a dilemma exacerbated when voracious larval-stage herbivores mature into important pollinators for a plant [1]. Herbivory is known to alter pollination rates, by altering flower number [2], size [3, 4], nectar production [5], seasonal timing of flowering [6], or pollinator behavior [7]. Nicotiana attenuata, a night-flowering tobacco that germinates after fires in the Southwestern United States, normally produces flowers that open at night and release benzyl acetone (BA) to attract night-active hawkmoth pollinators (Manduca quinquemaculata and M. sexta), which are both herbivores and pollinators. When plants are attacked by hawkmoth larvae, the plants produce flowers with reduced BA emissions that open in the morning and are preferentially pollinated by day-active hummingbirds. This dramatic change in flower phenology, which is elicited by oral secretions (OSs) from feeding hawkmoth larvae and requires jasmonate (JA) signal transduction, causes the majority of outcrossed seeds to be produced by pollinations from day-active hummingbirds rather than night-active hawkmoths. Because oviposition and nectaring are frequently coupled behaviors in hawkmoths, we propose that this OS-elicited, JA-mediated change in flower phenology complements similarly elicited responses to herbivore attack (direct defenses, indirect defenses, and tolerance responses) that reduce the risk and fitness consequences of herbivory to plants.

Big-eyed bugs (Geocorisspp. Fallén, Hemiptera: Lygaeidae) are ubiquitous, omnivorous insect predators whose plant feeding behavior raises the question of whether they benefit or harm plants. However, several studies have investigated both the potential ofGeocorisspp. to serve as biological control agents in agriculture and their importance as agents of plant indirect defense in nature. These studies have demonstrated thatGeocorisspp. effectively reduce herbivore populations and increase plant yield. Previous work has also indicated thatGeocorisspp. respond to visual and olfactory cues when foraging and choosing their prey and that associative learning of prey and plant cues informs their foraging strategies. For these reasons,Geocorisspp. have become models for the study of tritrophic plant-herbivore-predator interactions. Here, we present detailed images and ecological observations ofG. pallensStål andG. punctipes(Say) native to the Great Basin Desert of southwestern Utah, including observations of their life histories and color morphs, dynamics of their predatory feeding behavior and prey choice over space and time, and novel aspects ofGeocorisspp.’s relationships to their host plants. These observations open up new areas to be explored regarding the behavior ofGeocorisspp. and their interactions with plant and herbivore populations.

To investigate the role of secondary metabolites in the feeding behavior of Manduca sexta larvae feeding on Nicotiana attenuata, an aqueous acetone extract of the aerial parts of the plant was subjected to feeding-performance bioassay-guided fractionation. We isolated three 20- hydroxygeranyllinalool glycosides from the leaves of N. attenuata, which acted as mild deterrents to the feeding herbivore M. sexta. One of the diterpenoid glycosides, attenoside (3), is a novel natural product. The structures of the compounds were determined using APCI mass spectrometry and 1- and 2D-NMR spectroscopy.