Robert A. Raguso

Synergy between visual and olfactory cues in nectar feeding by wild hawkmoths, Manduca sexta

We performed field experiments to measure the relative importance of olfactory and visual cues in nectar foraging by wild tobacco hornworm moths, Manduca sexta (Lepidoptera, Sphingidae) in the Sonoran Desert of Arizona, U.S.A. We manipulated flowers of sacred Datura (Datura wrightii; Solanaceae) to experimentally decouple floral scent from visual display and presented these cues to free-flying moths in mixed and homogeneous arrays. Moths did not feed from cloth-bagged fragrant flowers lacking strong visual contrast, nor did they feed from paper model flowers lacking plant odours. Unexpectedly, moths fed from paper model flowers that were associated solely with vegetative odours, albeit at lower levels than when floral scent was present. Subsequent experiments revealed that the combination of floral and vegetative odours did not incrementally increase nectar feeding and that floral scent without vegetation was sufficient to elicit feeding when paper flowers were present. Thus, wild M. sexta in our study did not show generalized feeding responses to natural or artificial flowers with single sensory stimuli; like naive laboratory-reared moths, they required a combination of visual and olfactory cues. However, given the prior foraging experience of our study population on Datura flowers, their lack of generalized feeding responses may reflect a learned preference for the full complement of floral cues, rather than the persistence of innate sensory constraints. Future efforts to distinguish between these hypotheses should focus on whether M. sexta can be conditioned to associate nectar with visual cues alone, and whether moths that feed ad libitum from nectar-rich plants learn floral attributes as a search image or generalize to single-modality stimuli.

Fragrance chemistry, nocturnal rhythms and pollination ''syndromes'' in Nicotiana

GC-MS analyses of nocturnal and diurnal floral volatiles from nine tobacco species (Nicotiana; Solanaceae) resulted in the identification of 125 volatiles, including mono- and sesquiterpenoids, benzenoid and aliphatic alcohols, aldehydes and esters. Fragrance chemistry was species-specific during nocturnal emissions, whereas odors emitted diurnally were less distinct. All species emitted greater amounts of fragrance at night, regardless of pollinator affinity. However, these species differed markedly in odor complexity and emission rates, even among close relatives. Species-specific differences in emission rates per flower and per unit fresh or dry flower mass were significantly correlated; fragrance differences between species were not greatly affected by different forms of standardization. Flowers of hawkmoth-pollinated species emitted nitrogenous aldoximes and benzenoid esters on nocturnal rhythms. Four Nicotiana species in section Alatae sensu strictu have flowers that emit large amounts of 1,8 cineole, with smaller amounts of monoterpene hydrocarbons and alpha-terpineol on a nocturnal rhythm. This pattern suggests the activity of a single biosynthetic enzyme (1,8 cineole synthase) with major and minor products; however, several terpene synthase enzymes could contribute to total monoterpene emissions. Our analyses, combined with other studies of tobacco volatiles, suggest that phenotypic fragrance variation in Nicotiana is shaped by pollinator- and herbivore-mediated selection, biosynthetic pathway dynamics and shared evolutionary history.

WHY ARE SOME FLORAL NECTARS SCENTED

Despite recent interest in the non-sugar components of floral nectar, nearly nothing is known about the ecological importance and phylogenetic distribution of scented nectar. If present, the scent of nectar would provide an honest signal to nectar-feeding animals. Nectar odors may directly impact plant reproductive fitness, through pollinator attraction or deterrence of nectar robbers and florivores. In addition, nectar odors may indirectly impact plant fitness through antimicrobial activity, pleiotropic interactions with plant defense, and communication with predators and parasitoids. The literature provides only circumstantial evidence for scented nectar, through the study of bee honey odors. Here I confirm the presence of scent in the nectar of four out of seven angiosperm species sampled with solid-phase micro-extraction and gas chromatography–mass spectrometry. In Abelia x grandiflora and Hedychium coronarium, nectar odors are a hydrophilic subset of the compounds emitted by surrounding floral tissues, suggesting passive absorption by the nectar standing crop. Sucrose solution applied to the petals of a nectarless flower, Magnolia grandiflora, absorbed a hydrophilic subset of scent compounds after one hour, lending support to this hypothesis. Nectar from Oenothera primiveris and Agave palmeri contained unique scent compounds compared to the floral tissues. The presence of fermentation volatiles in A. palmeri nectar suggests a dynamic role for yeast in its floral biology. These data highlight the need for systematic studies on the distribution and mechanistic importance of scented floral nectar to plant–animal interactions.

Floral Scent Production in Clarkia (Onagraceae) (I. Localization and Developmental Modulation of Monoterpene Emission and Linalool Synthase Activity)

The flowers of many plants emit volatile compounds as a means of attracting pollinators. We have previously shown that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of approximately 8 to 12 volatile compounds[mdash]three monoterpenes and nine benzoate derivatives (R.A. Raguso and E. Pichersky [1994] Plant Syst Evol [in press]). Here we report that the monoterpene alcohol linalool is synthesized and emitted mostly by petals but to a lesser extent also by the pistil and stamens. Two linalool oxides are produced and emitted almost exclusively by the pistil. These three monoterpenes are first discernible in mature unopened buds, and their tissue levels are highest during the first 2 to 3 d after anthesis. Levels of emission by the different floral parts throughout the life span of the flower were correlated with levels of these monoterpenes in the respective tissues, suggesting that these monoterpenes are emitted soon after their synthesis. Activity of linalool synthase, an enzyme that converts the ubiquitous C10 isoprenoid intermediate geranyl pyrophosphate to linalool, was highest in petals, the organ that emits most of the linalool. However, linalool synthase activity on a fresh weight basis was highest in stigma and style (i.e. the pistil). Most of the linalool produced in the pistil is apparently converted into linalool oxides. Lower levels (0.1%) of monoterpene emission and linalool synthase activity are found in the stigma of Clarkia concinna, a nonscented relative of C. breweri, suggesting that monoterpenes may have other functions in the flower in addition to attracting pollinators.

Floral volatiles fromClarkia breweri andC. concinna (Onagraceae): Recent evolution of floral scent and moth pollination

Clarkia breweri (Onagraceae) is the only species known in its genus to produce strong floral fragrance and to be pollinated by moths. We used gas chromatography-mass spectrometry (GC-MS) to identify 12 abundant compounds in the floral headspace from two inbred lines ofC. breweri. These volatiles are derived from two biochemical pathways, one producing acyclic monoterpenes and their oxides, the other leading from phenylalanine to benzoate and its derivatives. Linalool and linalool oxide (pyran form) were the most abundant monoterpenoids, while linalool oxide (furan form) was present at lower concentrations. Of the aromatic compounds detected, benzyl acetate was most abundant, whereas benzyl benzoate, eugenol, methyl salicylate, and vanillin were present as minor constituents in all floral samples. The two inbredC. breweri lines differed for the presence of the additional benzenoid compounds isoeugenol, methyleugenol, methylisoeugenol, and veratraldehyde. We also analyzed floral headspace fromC. concinna, the likely progenitor ofC. breweri, whose flowers are odorless to the human nose. Ten volatiles (mostly terpenoids) were detected at low concentrations, but only when headspace was collected from 20 or more flowers at a time. Trans-β-ocimene was the most abundant floral compound identified from this species. Our data are consistent with the hypothesized recent evolution of floral scent production and moth pollination inC. breweri.

Floral Scent Production in Clarkia breweri (Onagraceae) (II. Localization and Developmental Modulation of the Enzyme S-Adenosyl-L-Methionine:(Iso)Eugenol O-Methyltransferase and Phenylpropanoid Emission)

We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55–67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4[prime]-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step.

Dynamic headspace analysis of floral volatiles: A comparison of methods

Lack of standardization of floral scent analysis using dynamic headspace collection and GC-MS has prevented meaningful biological comparisons of results from different studies. We explored technique-related sources of variation by comparing the relative performances of different trapping sorbents, elution solvents and vacuum pump flow rates in the collection of known volatiles from (a) synthetic blends on filter papers and (b) living flowers of Clarkia breweri (Onagraceae). The greatest amounts of volatiles were collected when Porapak Q sorbent and hexane solvent were used, but most analyses were qualitatively similar. Low and high pump flow rates yield a tradeoff, with higher rates trapping low volatility compounds but also raising ambient contamination levels. Significant variation was associated with the use of different sorbents and solvents, but detailed statistical comparisons were precluded by significant interaction effects involving other experimental variables. Given the tightly controlled experimental conditions and use of inbred plant lines, this unexpected result suggests that analyses of small samples carry a high risk of experimental artifact. We recommend that floral scent analyses incorporate experimental and statistical approaches that address as many method-related variables as possible, that samples of 10 or more individuals be used, and that GC-MS parameters be held constant for all comparative analyses.

Fragrance chemistry and pollinator affinities in Nyctaginaceae

We present results of dynamic head-space collections and GC-MS analyses of floral and vegetative fragrances for 20 species in three genera of Nyctaginaceae: Acleisanthes, Mirabilis and Selinocarpus. Most of the species included in this study are either hawkmoth or noctuid moth-pollinated. A wide variety of compounds were observed, including mono- and sesquiterpenoids, aromatics (both benzenoids and phenylpropanoids), aliphatic compounds, lactones, and nitrogen-bearing compounds. Intraspecific variation in fragrance profiles was significantly lower than interspecific variation. Each species had a unique blend of volatiles, and the fragrance of many species contained species-specific compounds. The fragrance profiles presented here are generally consistent with previous studies of fragrance in a variety of moth-pollinated angiosperms.

Olfactory versus visual cues in a floral mimicry system

Abstract We used arrays of artificial flowers with and without fragrance to determine the importance of olfactory and visual cues in attracting insects to a floral mimic. The mimic is a fungus, Puccinia monoica Arth., which causes its crucifer hosts (here, Arabis drummondii Gray) to form pseudoflowers that mimic co-occurring flowers such as the buttercup, Ranunculus inamoenus Greene. Although pseudoflowers are visually similar to buttercups, their sweet fragrance is distinct. To determine whether visitors to pseudoflowers were responding to fragrance we performed an experiment in which we removed the visual cues, but allowed fragrance to still be perceived. In this experiment we found that pseudoflower fragrance can attract visitors by itself. In other experiments we found that the relative importance of olfactory and visual cues depended on the species of visitor. Halictid bees (Dialictus sp.) had a somewhat greater visual than olfactory response, whereas flies (muscids and anthomyiids) were more dependent on olfactory cues. We also used bioassays to determine which of the many compounds present in the natural fragrance were responsible for attraction. We found that halictid bees were equally attracted to pseudoflowers and to a blend containing phenylacetaldehyde, 2-phenylethanol, benzaldehyde and methylbenzoate in the same relative concentrations as in pseudoflowers. Flies, on the other hand, only responded to pseudoflower scent, indicating that we have not yet identified the compound(s) present in pseudoflowers that are attracting them. The ability of insects to differentiate pseudoflowers from true flowers by their fragrance may be important in the evolution of the mimicry system. Different fragrances may facilitate proper transfer of both fungal spermatia and pollen, and thus make it possible for the visual mimicry to evolve.

THE SCENT OF A MALE: THE ROLE OF FLORAL VOLATILES IN POLLINATION OF A GENDER DIMORPHIC PLANT

Most flowering plants rely on animal pollinators to transfer male gametes between individuals, and thus a significant problem for gender dimorphic plants is that pollinators often avoid female flowers. Here we show for the first time that one important reason pollinators shun female flowers is because they do not smell like males. We compared emission rates and floral scent composition in a gynodioecious wild strawberry (Fragaria virginiana) where females receive half as many visits by generalist pollinators as conspecific hermaphrodites. We used floral extracts to determine the source of sexually dimorphic odor and pollinator responses. Specifically, we used extracts of whole flowers and specific floral parts in choice tests to determine that pollinators preferred the scent of hermaphrodite flowers over those of females and that this discrimination was due primarily to the scent of hermaphrodite anthers. These data conclusively show that scent can be a major driver of pollinator behavior in gender dimorphic plants. Our results also indicate that scent is an important modulator of pollinator behavior even in a small flowered, weakly scented species visited by generalist pollinators, and not just peculiar to intensely scented, deceptive, or specialized pollination systems.