Further observations on rhythmic emission of fragrance in flowers

Observations regarding floral fragrance and the rhythmicity of its emission in four plant species are reported. In the case of flowers of Hoya carnosa R. Br. which are characterized by circadian rhythmicity of scentedness (R. Altenburger and P. Matile, 1988, Planta 174, 248–252), temperature compensation of the free-running period as well as persistence of oscillations in permanent darkness have been demonstrated. A hitherto unidentified component of fragrance turned out to be identical to an unusual sesquiterpene recently discovered in cardamom oil (B. Maurer et al., 1986, Tetrahedron Lett. 27, 2111–2112). In Stephanotis floribunda Brongs. the rhythmic emission of fragrance is circadian in nature, but in a constant environment the oscillations of individual components are increasingly asynchronous. In excised flowers of Odontoglossum constrictum Lindl. the diurnal oscillations observed in a natural photoperiod are abolished under constant environmental conditions. They are resumed upon the return to a 1212-h photoperiod. The absence of circadian control could also be demonstrated in excised flowers of Citrus medica L. In this species, too, the daily maxima of scent emission reappear upon the transfer of flowers to a 1212-h light/ dark cycle. Results obtained upon the comparative analysis of volatiles in the headspace above the flowers and in petal extract indicate that the relative abundance of an individual compound in the floral fragrance is not a function of differential volatility.Abbreviations DL 1212 h photoperiod - DD continuous darkness - GLC gas-liquid chromatography - LL continuous illumination The authors are indebted to R. Kaiser, Givaudon Corp.for kindly carrying out the identifikation of volatiles by gas-liquid chromalography/mass spectrometry.     

Circadian rhythmicity of fragrance emission in flowers of Hoya carnosa R. Br.

In flowers of Hoya carnosa R. Br. the nocturnal emission of fragrance occurs according to an endogenous circadian rhythmicity. This was demonstrated by the continuation of rhythmical emission for two to three cycles under conditions of permanent illumination. The free-running period was approx. 29 h under these conditions. The inversion of light/dark cycles caused the entrainment of the next peak of emission by approx. 12 h whether the inversion was started with a light period or a with dark period prolonged for 12 h. When only the flower used for fragrance analysis was subjected to an inverted photoperiod, the rest of the plant remaining under the original light/dark cycle, the synchronization of fragrance emission occurred according to the Zeitgeber perceived by the individual flower.     

Pollen advertisement: chemical contrasts between whole-flower and pollen odors

Odors of pollen and whole flowers were compared in taxonomically unrelated species that offer pollen as the only food reward to pollinators. Volatiles were collected using headspace adsorption and analyzed by gas chromatography and mass spectrometry. The odor of pollen was found to be chemically distinct from the total flower odor, and this pollen-odor distinctness varied among the three species. In Papaver rhoeas (Papaveraceae), the contrast between pollen and whole-flower odors was most subtle, with differences observed only in the proportions of individual volatiles (almost exclusively aliphatic hydrocarbons). In Filipendula vulgaris (Rosaceae), pollen volatiles were fewer than in the flowers (comprising mainly benzenoids and fatty-acid derivatives) and their relative proportions produced an odor dominated by 2-heptadecanone that contrasted strikingly with the flower odor dominated by 2-phenyl ethanol. In Lupinus polyphyllus (Fabaceae), the pollen odor contained fewer volatiles and in differing proportions than the flower fragrance (comprising almost exclusively isoprenoids). The findings add to earlier chemical evidence of odor contrasts between pollen and other flower parts in two other species. Drawing on information from pollination studies of these various species, it is suggested that pollen odor is used by pollen-foraging insects both to discriminate between plant species and to assess reward availability in individual flowers, and that it might in addition serve a protective function against destructive flower-feeding insects and pathogens.     

Olfactory versus visual cues in a floral mimicry system

 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. Received: 3 January 1996 / Accepted: 13 August 1996     

Influence of temperature on the rhythmic emission of volatiles from Ribes nigrum flowers in situ

The floral volatiles of blackcurrant (Ribes nigrum) were collected in situ using the headspace technique. Eleven compounds were identified, including monoterpenes hydrocarbons and monoterpene ethers. The fragrance was emitted in a rhythmic manner, the maximum being in the middle of the photoperiod. Emission at 20°C was significantly higher than at 10°C. The rhythmic nature of emission seemed to cease under conditions of constant light. However, a slight rise in emission during the second period in continuous light may indicate a circadian control of emission. The significance of flower volatiles in relation to pollination is discussed.     

Antennal and behavioural responses of the spruce seed moth,Cydia strobilella,to floral volatiles of Norway spruce,Picea abies,and temporal variation in emission of active compounds

We investigated whether spruce seed moth, Cydia strobilella L. (Lepidoptera: Tortricidae, Grapholitini), one of the most damaging seed predators on Norway spruce, Picea abies (L.) H. Karst (Pinaceae), uses olfactory cues during host search. Analyses with coupled gas chromatography and electroantennography revealed that antennae of both sexes of moths responded consistently to three compounds in the headspace from female spruce flowers, i.e., α‐pinene, β‐pinene, and myrcene, but not to limonene as has been previously reported for this species. The amounts of these active volatiles released from flowers and cones of P. abies were quantified, and their diurnal and seasonal variation was monitored. The total release of the active volatiles correlated well with the diurnal and seasonal flight activity of C. strobilella as revealed by catches of males in pheromone‐baited traps. In field trapping experiments, where baits were loaded with proportions and enantiomeric ratios of α‐pinene, β‐pinene, and myrcene matching those of the female P. abies floral headspace, substantial catches of male C. strobilella were achieved, whereas few females were captured. These surprising results suggest that male C. strobilella make use of host volatiles to aid them in their search for females.     

Dynamic trajectories of volatile and non‐volatile specialised metabolites in ‘overnight’ fragrant flowers of Murraya paniculata

• Ephemeral flowers, especially nocturnal ones, usually emit characteristic scent profiles within their post‐anthesis lifespans of a few hours. Whether these flowers exhibit temporal variability in the composition and profile of volatile and non‐volatile specialised metabolites has received little attention.
• Flowers of Murraya paniculata bloom in the evenings during the summer and monsoon, and their sweet, intense fragrance enhances the plant's value as an ornamental. We aimed to investigate profiles of both volatile and non‐volatile endogenous specialised metabolites (ESM) in nocturnal ephemeral flowers of M. paniculata to examine whether any biochemically diverse groups of ESM follow distinct patterns of accumulation while maintaining synchrony with defensive physiological functions.
• Targeted ESM contents of M. paniculata flowers were profiled at ten time points at 2‐h intervals, starting from late bud stage (afternoon) up to the start of petal senescence (mid‐morning). Emitted volatiles were monitored continuously within the whole 20‐h period using headspace sampling. The ESM contents were mapped by time point to obtain a highly dynamic and biochemically diverse profile. Relative temporal patterns of ESM accumulation indicated that the active fragrance‐emitting period might be divided into ‘early bloom’, ‘mid‐bloom’ and ‘late bloom’ phases. Early and late bloom phases were characterised by high free radical generation, with immediate enhancement of antioxidant enzymes and phenolic compounds. The mid‐bloom phase was relatively stable and dedicated to maximum fragrance emission, with provision for strong terpenoid‐mediated defence against herbivores. The late bloom phase merged into senescence with the start of daylight; however, even the senescent petals continued to emit fragrance to attract diurnal pollinators.
• Our study suggests that dynamic relations between the different ESM groups regulate the short‐term requirements of floral advertisement and phytochemical defence in this ephemeral flower. This study also provided fundamental information on the temporal occurrence of emitted volatiles and internal pools of specialised metabolites in M. paniculata flowers, which could serve as an important model for pollination biology of Rutaceae, which includes many important fruit crops.

     

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.     

Circadian rhythmicity in emission of volatile compounds by flowers of Rosa hybrida L. cv. Honesty

Quantitative headspace analyses of rose flowers showed no significant differences in composition of emitted volatile compounds between flowers on the intact plant and cut flowers placed in vase water containing sucrose at 0, 15 or 30 g · l⁻¹. Volatile components emitted were geraniol, nerol, citronellol, E-citral, Z-citral, methylgeranylate, trans-caryophyllene, β-cubebene, dihydro-β-ionone, 2-phenylethanol, 2-phenylethylacetate, 3,5-dimethoxytoluene and hexylacetate. When exposed to a 12-h photoperiod these components showed maximum emission during the light period and a rhythmicity which differed for the individual compounds. The circadian nature of the rhythmicity was confirmed by the continuation of rhythmicity during continued darkness or light, and was characterized by `transient' and `free running' periods of 27 and 24 h, respectively, and a phase shift of 12 h in rhythmicity when a 24-h period of continuous darkness was followed by re-exposure to a 12-h photoperiod. Rhythmicity in emission was not observed when flowers were kept in darkness before flower bud opening, but started immediately upon exposure to a 12-h photoperiod. Received: 14 April 1998 / Accepted: 28 May 1998     

Circadian rhythm of volatile emission from flowers of Nicotiana sylvestris and N. suaveolens

The volatile profiles from flowers of Nicotiana sylvestris and N. suaveolens were investigated by means of dynamic headspace sampling and capillary gas chromatography. Under conditions of light/dark entrainment both species emitted phenylpropanoid-derived volatiles (e.g. benzyl alcohol, methyl benzoate) with maximum emission occurring during the dark period. Emission of these compounds was demonstrated to be circadian by continuance of rhythmicity under conditions of constant light and subsequent re-entrainment to a new light/dark cycle. In contrast, emission of the sesquiterpene hydrocarbon, caryophyllene, from N. sylvestris followed no apparent pattern. The emission of monoterpene hydrocarbons from flowers of N. suaveolens showed diurnal differences only under conditions of light/dark entrainment.     

Floral and insect-induced volatile formation in <Emphasis Type="Italic">Arabidopsis lyrata</Emphasis> ssp.<Emphasis Type="Italic"> petraea</Emphasis>, a perennial,outcrossing relative of <Emphasis Type="Italic">A. thaliana</Emphasis>

Volatile organic compounds have been reported to serve some important roles in plant communication with other organisms, but little is known about the biological functions of most of these substances. To gain insight into this problem, we have compared differences in floral and vegetative volatiles between two closely related plant species with different life histories. The self-pollinating annual, Arabidopsis thaliana, and its relative, the outcrossing perennial, Arabidopsis lyrata, have markedly divergent life cycles and breeding systems. We show that these differences are in part reflected in the formation of distinct volatile mixtures in flowers and foliage. Volatiles emitted from flowers of a German A. lyrata ssp. petraea population are dominated by benzenoid compounds in contrast to the previously described sesquiterpene-dominated emissions of A. thaliana flowers. Flowers of A. lyrata ssp. petraea release benzenoid volatiles in a diurnal rhythm with highest emission rates at midday coinciding with observed visitations of pollinating insects. Insect feeding on leaves of A. lyrata ssp. petraea causes a variable release of the volatiles methyl salicylate, C₁₁- and C₁₆-homoterpenes, nerolidol, plus the sesquiterpene (E)-β-caryophyllene, which in A. thaliana is emitted exclusively from flowers. An insect-induced gene (AlCarS) with high sequence similarity to the florally expressed (E)-β-caryophyllene synthase (AtTPS21) from A. thaliana was identified from individuals of a German A. lyrata ssp. petraea population. Recombinant AlCarS converts the sesquiterpene precursor, farnesyl diphosphate, into (E)-β-caryophyllene with α-humulene and α-copaene as minor products indicating its close functional relationship to the A. thaliana AtTPS21. Differential regulation of these genes in flowers and foliage is consistent with the different functions of volatiles in the two Arabidopsis species.     

CAM-idling in Hoya carnosa (Asclepiadaceae)

In the leaf succulent Asclepiad Hoya carnosa (L.) R. Br., CAM photosynthesis occurred under well-watered conditions, as characterized by diurnal gas exchange and changes in titratable acidity. Following 10–12 days of severe water stress, the plants shifted from CAM to a modified CAM-idling mode of metabolism. CAM-idling was characterized by complete or almost complete stomatal closure accompanied by CAM-like diurnal changes in titratable acidity. H. carnosa plants maintained this CAM-idling mode of photosynthesis for at least 8 weeks. Upon reirrigation, the plants returned to the original CAM mode within 1 week. These results suggested that CAM-idling is a reversible, intermediate form of sustained metabolism which enables plant survival under conditions of extended drought.This work was supported in part by NSF Grant PCM 8200366 and in part by the Science and Education Administration of the United States Department of Agriculture under Competitive Grant 5901-0420-8-0018-0.     

Day–night fluctuations in floral scent and their effects on reproductive success in <Emphasis Type="Italic">Lilium auratum</Emphasis>

We examined the contribution of diurnal and nocturnal pollination to male and female reproductive success in Lilium auratum. Plants were bagged for either 12 h during the day or at night to allow either only nocturnal or only diurnal visitors to forage throughout the flowering period. We found that there was no significant difference in the seed:ovule ratio among diurnally pollinated, nocturnally pollinated, or control flowers. However, in terms of male reproductive success, it was more advantageous for the plants to be pollinated both diurnally and nocturnally: the numbers of pollen grains remaining in diurnally pollinated or nocturnally pollinated flowers were significantly greater than those in control flowers. The total amount of floral volatiles of L. auratum was significantly higher at night than during the day. The constituents of floral scent of all time series examined were mostly monoterpenoids, many of which serve as attractants for nocturnal hawkmoths. Such nocturnally biased floral scent emission of L. auratum might achieve male reproductive success by attracting nocturnal visitors, which may suggest that the relative contribution of floral scent in this species is biased towards male reproductive success.     

CAM-idling in Hoya carnosa (Asclepiadaceae)

In the leaf sueculent Asclepial Hoya carnosa (L.) R. Br., CAM photosynthesis occurred under well-watered conditions, as characterized by diurnal gas exchange and changes in titratable acidity. Following 10–12 days of severe water stress, the plants shifted from CAM to a modified CAM-idling mode of metabolism. CAM-idling was characterized by complete or almost complete stomatal closure accompanied by CAM-like diurnal changes in titratable acidity. H. carnosa plants maintained this CAM-idling mode of photosynthesis for at least 8 weeks. Upon reirrigation, the plants returned to the original CAM mode within 1 week. These results suggested that CAM-idling is a reversible, intermediate form of sustained metabolism which enables plant survival under conditions of extended drought.This work was supported in part by NSF Grant PCM 8200366 and in part by the Science and Education Administration of the United States Department of Agriculture under Competitive Grant 5901-0420-8-0018-0.     

Scent glands in legume flowers

Scent glands, or osmophores, are predominantly floral secretory structures that secrete volatile substances during anthesis, and therefore act in interactions with pollinators. The Leguminosae family, despite being the third largest angiosperm family, with a wide geographical distribution and diversity of habits, morphology and pollinators, has been ignored with respect to these glands. Thus, we localised and characterised the sites of fragrance production and release in flowers of legumes, in which scent plays an important role in pollination, and also tested whether there are relationships between the structure of the scent gland and the pollinator habit: diurnal or nocturnal. Flowers in pre‐anthesis and anthesis of 12 legume species were collected and analysed using immersion in neutral red, olfactory tests and anatomical studies (light and scanning electron microscopy). The main production site of floral scent is the perianth, especially the petals. The scent glands are distributed in a restricted way in Caesalpinia pulcherrima, Anadenanthera peregrina, Inga edulis and Parkia pendula, constituting mesophilic osmophores, and in a diffuse way in Bauhinia rufa, Hymenaea courbaril, Erythrostemon gilliesii, Poincianella pluviosa, Pterodon pubescens, Platycyamus regnellii, Mucuna urens and Tipuana tipu. The glands are comprised of cells of the epidermis and mesophyll that secrete mainly terpenes, nitrogen compounds and phenols. Relationships between the presence of osmophores and type of anthesis (diurnal and nocturnal) and the pollinator were not found. Our data on scent glands in Leguminosae are original and detail the type of diffuse release, which has been very poorly studied.     

Specialized pollination by spider-hunting wasps in the African orchid Disa sankeyi

Disa sankeyi Rolfe was found to be pollinated almost exclusively by Hemipepsis wasps (Pompilidae) at sites across its range in the Drakensberg mountains of South Africa. Wasps of both sexes appear to locate the cryptic yellow-green flowers primarily by using scent cues, as they show a classic zigzag flight pattern when approaching inflorescences. The sweet-spicy fragrance emitted by the flowers is strong enough to be discernable by a human from several metres away and consists of a blend of at least 65 volatile compounds, with (E)- cinnamic aldehyde and eugenol as the major constituents. On settling, wasps lick the exposed droplet of nectar that is secreted from the mouth of the reduced floral spur on each flower. Pollinaria become attached to the front feet of the wasps as they grasp flowers adjacent to those on which they are feeding. Pollen transfer to stigmas similarly takes place on flowers adjacent to those that are used as a nectar source. Almost 100% of flowers in the study populations were found to have been pollinated. Specialized pollination by pompilid wasps appears to be a rare phenomenon that has been reported only in southern African plant species. It appears to be associated with cryptic green-yellow flowers and highly accessible nectar. Behavioural assays are required to determine whether the basis of the specialization in this pollination system lies in a particular chemical composition of the floral fragrance.     

Regulation of Floral Terpenoid Emission and Biosynthesis in Sweet Basil (<Emphasis Type="Italic">Ocimum basilicum</Emphasis>)

Past studies have focused on the composition of essential oil of Ocimum basilicum leaves, but data on composition and regulation of its aerial emissions, especially floral volatile emissions, are scarce. We studied the chemical profile, within-flower spatial distribution (sepals, petals, pistils with stamina, and pedicels), diurnal emission kinetics and effects of exogenous methyl jasmonate (MeJA) application on the emission of floral volatiles by dynamic headspace collection, and identification using gas chromatography-mass spectrometry (GC–MS) and proton-transfer reaction mass spectrometry. We observed more abundant floral emissions from flowers compared with leaves. Sepals were the main emitters of floral volatiles among the flower parts studied. The emissions of lipoxygenase compounds and monoterpenoids, but not sesquiterpene emissions, displayed a diurnal variation driven by light. Response to exogenous MeJA treatment of flowers consisted of a rapid stress response and a longer-term acclimation response. The initial response was associated with enhanced emissions of fatty acid derivatives, monoterpenoids, and sesquiterpenoids without variation of the composition of individual compounds. The longer-term response was associated with enhanced monoterpenoid and sesquiterpenoid emissions with profound changes in the emission spectrum. According to correlated patterns of terpenoid emission changes upon stress, highlighted by a hierarchical cluster analysis, candidate terpenoid synthases responsible for observed diversity and complexity of released terpenoid blends were postulated. We conclude that flower volatile emissions differ quantitatively and qualitatively from leaf emissions, and overall contribute importantly to O. basilicum flavor, especially under stress conditions.     

Yeasts associated with pollinating bees and flower nectar

A study of the yeast flora of 328 honey stomachs from 7 different pollinating bee species, and 342 flower nectar samples of 9 different flower species yielded 766 yeast isolates composed of 16 genera and 47 species. Most of the yeast species from both the sources belonged to the genusCandida, while the most frequently isolated yeasts wereDekkera intermedia from honey stomach andCandida blankii from flower nectar. Among the honey bees,Xylocopa sp., and among flowers,Citrus medica, yielded the highest number of yeast species. Nineteen species of yeasts belonging to 9 genera were common to both the sources.     

Chemical cues involved in the attraction of the oligolectic bee Hoplitis adunca to its host plant Echium vulgare

Host recognition is a key process in oligolectic bees but the mechanisms through which they find and recognize appropriate pollen host plant are not entirely clear. Hoplitis adunca is a monolectic bee collecting pollen only from Echium spp. (Boraginaceae). We aimed to test whether Echium vulgare floral scent plays a major role in the attraction of H. adunca females, and to identify components of E. vulgare scent that may be involved in this specific attraction. We used a combination of behavioral and chemical (GC/GC–MS, PTR-MS) analyses. In order to identify the chemical cues likely to be involved in the specific attraction of H. adunca, we compared the scent of fresh flowers, nectar, pollen, and whole plants of E. vulgare and Anchusa officinalis, another Boraginaceae, which does not attract H. adunca. H. adunca females were attracted to the scent of E. vulgare flowers when offered against a blank or against the scent of A. officinalis flowers. However, H. adunca females were not attracted to the scent of A. officinalis flowers when offered against a blank. The emission spectra of the two plant species differed markedly, as did the emission spectra of various flower components (pollen, nectar and whole flowers) within a species. Pollen presented a low volatile release, but emitted significantly higher amounts of mass 55 (butanal, 1,3-butadiene, or other volatiles of molecular mass 54), and mass 83 (hexanal, hexenols, hexenyl acetate, or other volatiles of molecular mass 82) in E. vulgare than in A. officinalis. Nectar produced a particular emission spectrum with high emission rates of masses 109 and 123. Mass 109 may likely correspond to 1,4-benzoquinone, a volatile specifically measured in E. vulgare in parallel studies to this one. The flower emission spectrum was mainly a combination of the pollen and the nectar scents, although it also contained additional volatile compounds such as those of mass 63 or mass 81. As for terpenes, E. vulgare emitted limonene, longicyclene, junipene, trans-caryophyllene and α-humulene, that were not detected in A. officinalis, and the most emitted monoterpenes were α-pinene, junipene and limonene whereas the most emitted terpenoid by A. officinalis was α-pinene. After identifying these chemical cues, olfactory/behavioural assays with specific volatiles and combinations of volatiles are necessary to understand the chemical interactions of the H. adunca-E. vulgare system.