A novel O-methyltransferase Cp4MP-OMT catalyses the final step in the biosynthesis of the volatile 1,4-dimethoxybenzene in pumpkin (Cucurbita pepo) flowers.

BACKGROUND: Floral scents play a crucial role in attracting insect pollinators. Among the compounds attractive to pollinators is 1,4-dimethoxybenzene (1,4-DMB). It is a significant contributor to the scent profile of plants from various genera, including economically important Cucurbita species. Despite its importance, the biosynthetic pathway for the formation of 1,4-DMB was not elucidated so far. RESULTS: In this study we showed the catalysis of 1,4-DMB in the presence of 4-methoxyphenol (4-MP) by protein extract from Styrian oil pumpkin (Cucurbita pepo) flowers. Based on this finding, we identified a novel O-methyltransferase gene, Cp4MP-OMT, whose expression is highly upregulated in the volatile-producing tissue of pumpkin flowers when compared to vegetative tissues. OMT activity was verified by purified recombinant Cp4MP-OMT, illustrating its ability to catalyse the methylation of 4-MP to 1,4-DMB in the presence of cofactor SAM (S-(5'-adenosyl)-L-methionine). CONCLUSIONS: Cp4MP-OMT is a novel O-methyltransferase from C. pepo, responsible for the final step in the biosynthesis of the floral scent compound 1,4-DMB. Considering the significance of 1,4-DMB in attracting insects for pollination and in the further course fruit formation, enhanced understanding of its biosynthetic pathways holds great promise for both ecological insights and advancements in plant breeding initiatives.

Chemistry, biosynthesis and biology of floral volatiles: roles in pollination and other functions.

Covering: 2010 to 2023Floral volatiles are a chemically diverse group of plant metabolites that serve multiple functions. Their composition is shaped by environmental, ecological and evolutionary factors. This review will summarize recent advances in floral scent research from chemical, molecular and ecological perspectives. It will focus on the major chemical classes of floral volatiles, on notable new structures, and on recent discoveries regarding the biosynthesis and the regulation of volatile emission. Special attention will be devoted to the various functions of floral volatiles, not only as attractants for different types of pollinators, but also as defenses of flowers against enemies. We will also summarize recent findings on how floral volatiles are affected by abiotic stressors, such as increased temperatures and drought, and by other organisms, such as herbivores and flower-dwelling microbes. Finally, this review will indicate current research gaps, such as the very limited knowledge of the isomeric pattern of chiral compounds and its importance in interspecific interactions.

Global warming impairs the olfactory floral signaling in strawberry.

BACKGROUND: Global warming is expected to impact the chemical communication between flowering plants and their pollinators. Surprisingly, it is unknown whether and how temperature-induced changes in scent emission affect pollinator behavior. Strawberry (Fragaria x ananassa) is a plant primarily pollinated by bees and hoverflies, with the former group being particularly attracted to the floral scent they emit. RESULTS: Using chemical analytical, electrophysiological, and behavioral approaches we tested whether temperature-induced shifts in floral scent of strawberry affect chemical communication with its main bee pollinators (Apis mellifera, Bombus terrestris, Osmia bicornis). While strawberry flowers in the optimum scenario released 10.4 ng/flower/hour, mainly p-anisaldehyde (81%) and seven other scent compounds, in the warmer scenario, the flowers did not emit any detectable scent. In the behavioral experiments, the pollinators were attracted by the scents of the optimum scenario. CONCLUSIONS: We predict that the absence of detectable scent emissions from strawberry plants grown under heat stress will reduce the attractiveness of the flowers to the bee pollinators. Our study raises important ecological and agricultural questions, as decreased attractiveness of flowers to pollinators might potentially lead to insufficient bee pollination, with potential negative consequences for ecosystem functioning and crop yields, particularly in regions reliant on bees as primary pollinators. Given that our study centered on bee pollinators, it is needed to conduct further research to evaluate the impact on hoverflies.

Chemical Attraction of Gall Midge Pollinators (Cecidomyiidae: Cecidomyiinae) to Anthurium acutangulum (Araceae).

Flowering plants often use chemical signals to attract their pollinators, and compounds that elicit attraction are known for several groups of pollinators. For other pollinators such as gall midges, however, compounds responsible for their attraction to flowers are largely unknown. Here, we describe the pollination biology of Anthurium acutangulum, a Neotropical aroid species found to be attractive to gall midges. We collected and analyzed its floral scent by dynamic headspace collections and gas chromatography coupled to mass spectrometry, and identified compounds responsible for pollinator attraction. The inflorescences were almost exclusively visited by gall midges (females; Cecidomyiidae: Cecidomyiinae) and released a strong scent reminiscent of freshly cut cucumber, mainly (5S,7S)-trans-conophthorin, (E2,Z6)-2,6-nonadienal, and cis-conophthorin. Behavioral assays with the two most abundant compounds identified (E2,Z6)-2,6-nonadienal as being highly attractive to the female gall midge pollinators, whereas (5S,7S)-trans-conophthorin was not attractive. Overall, we introduce a new specialized gall midge pollination system and identify the chemical mediating communication between the pollinators and their host plants.

Neural and behavioural responses of the pollen-specialist bee Andrena vaga to Salix odours.

An effective means of finding food is crucial for organisms. Whereas specialized animals select a small number of potentially available food sources, generalists use a broader range. Specialist (oligolectic) bees forage on a small range of flowering plants for pollen and use primarily olfactory and visual cues to locate their host flowers. So far, however, little is known about the specific cues oligoleges use to discriminate between hosts and non-hosts and how floral scent compounds of hosts and non-hosts are processed in the bees' olfactory system. In this study, we recorded physiological responses of the antennae (electroantennographic detection coupled to gas chromatography; GC-EAD) and in the brain (optical imaging; GC imaging), and studied host-finding behaviour of oligolectic Andrena vaga bees, a specialist on Salix plants. In total, we detected 37 physiologically active compounds in host and non-host scents. 4-Oxoisophorone, a common constituent in the scent of many Salix species, evoked strong responses in the antennal lobe glomeruli of A. vaga, but not the generalist honeybee Apis mellifera. The specific glomerular responses to 4-oxoisophorone in natural Salix scents reveals a high degree of specialization in A. vaga for this typical Salix odorant component. In behavioural experiments, we found olfactory cues to be the key attractants for A. vaga to Salix hosts, which are also used to discriminate between hosts and non-hosts, and A. vaga demonstrated a behavioural activity for 4-oxoisophorone. A high sensitivity to floral scents enables the specialized bees to effectively find flowers and it appears that A. vaga bees are highly tuned to 4-oxoisophorone at a very low concentration.

Synthesis, Absolute Configurations, and Biological Activities of Floral Scent Compounds from Night-Blooming Araceae.

The uncommon jasmone derivatives dehydrojasmone, isojasmol, and isojasmyl acetate, floral scent compounds from night-blooming Araceae, were synthesized in a scalable synthesis employing conjugate addition with a selenoacetal as the key step. The stereoselective strategy with subsequent enzymatic kinetic resolution allowed determining the absolute configuration of the natural compounds by GC on a chiral phase. The homoterpene (E)-4,8-dimethyl-1,3,7-nonatrien-5-yl acetate, another uncommon scent compound, was obtained by α-regioselective aldehyde prenylation. The biological activities of dehydrojasmone and isojasmol were investigated in field assays, showing that these unique volatiles are able to selectively attract specific cyclocephaline scarab beetle pollinators.

Succession of Dung-Inhabiting Beetles and Flies Reflects the Succession of Dung-Emitted Volatile Compounds.

Chemical cues, such as volatile organic compounds (VOCs), are often essential for insects to locate food. Relative to the volume of studies on the role of VOCs in insect-plant relationships, the role of VOCs emitted by dung and carrion in mediating the behavior of insect decomposers is understudied. Such relationships may provide a mechanistic understanding of the temporal axis of community assembly processes in decomposing insect communities. We focused on the temporal succession of volatiles released by cow dung pats and the potential influence on dung-inhabiting insects. Using gas chromatography/mass spectrometry we identified and quantified VOCs released from dung 1-h, and 1, 2 3, 5, and 7 d-old. We then related changes in VOCs to successional patterns of dung-inhabiting beetles and flies. We detected 54 VOCs which could be assigned to two successional groups, with chemical turnover in dung changing around day 2. The early successional group consisted primarily of aliphatic alcohols and phenols, and the late one of aliphatic esters, nitrogen- and sulfur-bearing compounds. Flies were predominately associated with the early successional group, mainly with 1-butanol. Beetles were associated predominately with the late-successional group, mainly with dimethyl trisulfide. This association between insect and chemical successional patterns supports the idea that habitat filtering drives the community assembly of dung-inhabiting insects on an aging resource. Moreover, the affinity of both insect groups to specific VOC groups provides a mechanistic explanation for the predictability of successional patterns found in dung-inhabiting insect communities.

Distinct Roles of Cuticular Aldehydes as Pheromonal Cues in Two Cotesia Parasitoids.

Cuticular compounds (CCs) that cover the surface of insects primarily serve as protection against entomopathogens, harmful substances, and desiccation. However, CCs may also have secondary signaling functions. By studying the role of CCs in intraspecific interactions, we may advance our understanding of the evolution of pheromonal communication in insects. We previously found that the gregarious parasitoid, Cotesia glomerata (L.), uses heptanal as a repellent pheromone to help avoid mate competition among sibling males, whereas another cuticular aldehyde, nonanal, is part of the female-produced attractive sex pheromone. Here, we show that the same aldehydes have different pheromonal functions in a related solitary parasitoid, Cotesia marginiventris (Cresson). Heptanal enhances the attractiveness of the female's sex pheromone, whereas nonanal does not affect a female's attractiveness. Hence, these common aldehydes are differentially used by the two Cotesia species to mediate, synergistically, the attractiveness of the main constituents of their respective sex pheromones. The specificity of the complete sex pheromone blend is apparently regulated by two specific, less volatile compounds, which evoke strong electroantennographic (EAG) responses. This is the first demonstration that volatile CCs have evolved distinct pheromonal functions to aid divergent mating strategies in closely related species. We discuss the possibility that additional compounds are involved in attraction and that, like the aldehydes, they are likely oxidative products of unsaturated cuticular hydrocarbons.

A Novel Class of Defensive Compounds in Harvestmen: Hydroxy-γ-Lactones from the Phalangiid Egaenus convexus.

When threatened, the harvestman Egaenus convexus (Opiliones: Phalangiidae) ejects a secretion against offenders. The secretion originates from large prosomal scent glands and is mainly composed of two isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1), a ß-hydroxy-γ-lactone. The compounds were characterized by GC-MS of their microreaction derivatives, HRMS, and NMR. After the synthesis of all four possible stereoisomers of 1, followed by their separation by chiral-phase GC, the absolute configurations of the lactones in the Egaenus secretion was found to be (4S,5R)-1 (90%) and (4S,5S)-1 (10%). Hydroxy-γ-lactones represent a new class of exocrine defense compounds in harvestmen.

Bimodal Pollination Systems in Andean Melastomataceae Involving Birds, Bats, and Rodents.

Floral adaptation to a single most effective functional pollinator group leads to specialized pollination syndromes. However, adaptations allowing for pollination by two functional groups (bimodal pollination systems) remain a rarely investigated conundrum. We tested whether floral scent and nectar traits of species visited by two functional pollinator groups indicate specialization on either of the two pollinator groups or adaptations of both (bimodal systems). We studied pollination biology in four species of Meriania (Melastomataceae) in the Ecuadorian Andes. Pollinator observations and exclusion experiments showed that each species was effectively pollinated by two functional groups (hummingbirds/bats, hummingbirds/rodents, flowerpiercers/rodents), nectar composition followed known bird preferences, and scent profiles gave mixed support for specialization on bats and rodents. Our results suggest that nectar-rewarding Meriania species have evolved stable bimodal pollination strategies with parallel adaptations to two functional pollinator groups. The discovery of rodent pollination is particularly important given its rarity outside of South Africa.

Flower scent of Ceropegia stenantha: electrophysiological activity and synthesis of novel components.

In specialized pollination systems, floral scents are crucial for flower-pollinator communication, but key volatiles that attract pollinators are unknown for most systems. Deceptive Ceropegia trap flowers are famous for their elaborate mechanisms to trap flies. Recent studies revealed species-specific floral chemistry suggesting highly specialized mimicry strategies. However, volatiles involved in fly attraction were until now identified in C. dolichophylla and C. sandersonii, only. We here present data on C. stenantha for which flower scent and pollinators were recently described, but volatiles involved in flower-fly communication stayed unknown. We performed electrophysiological measurements with scatopsid fly pollinators (Coboldia fuscipes) and identified 12 out of 13 biologically active floral components. Among these volatiles some were never described from any organism but C. stenantha. We synthesized these components, tested them on antennae of male and female flies, and confirmed their biological activity. Overall, our data show that half of the volatiles emitted from C. stenantha flowers are perceived by male and female fly pollinators and are potentially important for flower-fly communication in this pollination system. Further studies are needed to clarify the role of the electrophysiologically active components in the life of scatopsid fly pollinators, and to fully understand the pollination strategy of C. stenantha.

The Combined Use of an Attractive and a Repellent Sex Pheromonal Component by a Gregarious Parasitoid.

Gregarious parasitoids usually clump their cocoons together and the adults emerge in a synchronized fashion. This makes it easy for them to find mating partners and most copulations indeed take place at the natal patch. Yet, males should leave such sites when females are no longer receptive. As yet, this decision-making process and the possible involvement of pheromones were poorly understood. Here we report on a remarkable use of attractive and repellent pheromones of the well-studied gregarious parasitoid species Cotesia glomerata (L.) (Hymenoptera: Braconidae). Virgin C. glomerata females were found to release an attractive as well as a repellent compound, which in combination arrest males on the natal patch, but after mating the females stop the production of the attractant and the males are repelled. The repellent compound was identified as heptanal, which was also released by males, probably reducing male-male competition on the natal patch. We also confirmed that the sex ratio of the emerging wasps can vary considerably among patches, depending on the relative quality of hosts and the number of females that parasitize a host. The newly revealed use of attractive and repellent pheromone compounds by C. glomerata possibly helps maximize mating success under these variable conditions.

Novel Floral Scent Compounds from Night-Blooming Araceae Pollinated by Cyclocephaline Scarabs (Melolonthidae, Cyclocephalini).

Nocturnal flowering plants often release strong scents to attract their pollinators. Among night active flower visitors are cyclocephaline scarab beetles, which have been demonstrated to respond to uncommon volatile organic compounds released in high amounts by their host plants. In Araceae, the molecular structure of several such compounds is yet to be unveiled. We investigated headspace floral scent samples of Philodendron squamiferum, Thaumatophyllum mello-baretoanum, and Xanthosoma hylaeae by a variety of approaches, leading to the identification of novel compounds. Dehydrojasmone, (Z)-4-methylene-5-(pent-2-en-1-yl)cyclopent-2-en-1-one (1), (Z)-3-methylene-2-(pent-2-en-1-yl)cyclopentyl acetate (isojasmyl acetate, 3), and (E)-4,8-dimethylnona-1,3,7-trien-5-yl acetate (4) had not been previously reported, while full analytical data of the recently described (Z)-3-methylene-2-(pent-2-en-1-yl)cyclopentan-1-ol (isojasmol, 2) are presented here. All these compounds are derived from more common precursors, (Z)-jasmone and (E)-4,8-dimethyl-1,3,7-nonatriene, likely through biosynthetic "post-processing".

Subtle Chemical Variations with Strong Ecological Significance: Stereoselective Responses of Male Orchid Bees to Stereoisomers of Carvone Epoxide.

Different enantiomers of chiral compounds within floral perfumes usually trigger distinct responses in insects; however, this has frequently been neglected in studies investigating semiochemicals in plant-pollinator interactions. Approximately 1000 neotropical plants produce floral perfumes as the only reward for pollinators, i.e. male euglossine bees. The chiral compound carvone epoxide is a key component of the scent bouquet of many perfume-rewarding plants that are pollinated by males of Eulaema. Here, we tested the biological activity of the four carvone epoxide stereoisomers to four Eulaema species occurring in the Atlantic Rainforest of NE-Brazil. We determined the stereochemistry of carvone epoxide in the floral scent of several Catasetum species, tested whether the antennae of bees respond differentially to these stereoisomers and investigated if there is a behavioural preference for any of the stereoisomers. We found that 1) Catasetum species emit only the (-)-trans-stereoisomer of carvone epoxide, 2) for E. atleticana and E. niveofasciata antennal responses to the (-)-trans-carvone epoxide were significantly stronger than those to (-)-cis-carvone epoxide, 3) the strength and pattern of antennal responses to all 4 stereoisomers (separately tested) did not differ among Eulaema species, and 4) there were significant differences in attractiveness of the four stereoisomers to the bees species with the (-)-trans-stereoisomer being particularly attractive. We assume (-)-trans-carvone epoxide to be the dominant isomer in perfume-rewarding plants pollinated by Eulaema. The universal occurrence of carvone epoxide in Catasetum species pollinated by Eulaema, suggests that this compound has evolved in perfume-rewarding as a specific attractant for Eulaema bees as pollinators.

Correction to: Novel floral scent compounds from night-blooming Araceae pollinated by cyclocephaline scarabs (Melolonthidae, Cyclocephalini).

The original version of this article unfortunately contained a mistake. The description of subheadings of Figs. 4c and d have to be interchanged.

Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals.

Unveiling the osmophores of Philodendron adamantinum (Araceae) as a means to understanding interactions with pollinators.

Background and Aims: Araceae species pollinated by nocturnal Cyclocephalini beetles attract their pollinators by inflorescence scents. In Philodendron , despite the intense odour, the osmophores exhibit no definite morphological identity, making them difficult to locate. This may explain why structural studies of the scent-releasing tissue are not available so far. Methods: Several approaches were employed for locating and understanding the osmophores of Philodendron adamantinum . A sensory test allowed other analyses to be restricted to fertile and sterile stamens as odour production sites. Stamens were studied under light and electron microscopy. Dynamic headspace and gas chromatography-mass spectrometry were used to collect and analyse scents from different zones of the inflorescence. Key Results: The epidermal cells of the distal portion of fertile stamens and staminodes are papillose and, similar to the parenchyma cells of this region, have dense cytoplasm and large nuclei. In these cells, the composition of organelles is compatible with secretory activity, especially the great number of mitochondria and plastids. In this portion, lipid droplets that are consumed concomitantly with the release of odour were observed. Quantitative scent analyses revealed that the scent, with a predominance of dihydro-ß-ionone, is mainly emitted by the fertile and sterile staminate zones of the spadix. An amorphous substance in the stomata pores indicates that the components are secreted and volatilized outside of the osmophore under thermogenic heat. Conclusions: Despite the difficulty in locating osmophores in the absence of morphological identity and inefficiency of neutral red staining, the osmophores of P. adamantinum have some features expected for these structures. The results indicate a functional link between thermogenesis and volatilization of osmophore secretions to produce olfactory signals for attracting specialized beetle pollinators. These first experimental data about the precise location of osmophores in Philodendron will stimulate studies in related species that will allow future comparison and the establishment of patterns of functional morphology.

Epiphytic Bacteria Alter Floral Scent Emissions.

Floral scents are key mediators of biotic interactions between flowers and various organisms such as pollinators, antagonistic animals and bacteria. It has been shown that emissions of floral volatiles are influenced by interactions with other organisms at the levels of roots, leaves and flowers. However, it is largely unknown whether and how epiphytic bacteria associated with flowers affect the composition of floral scent. By comparing volatiles of sterile and inoculated plants we found that bacteria may add components, induce or reduce the emission of compounds, and potentially catabolize others. These mechanisms collectively altered the floral scent emission and led to clearly different compositions. Our results confirm that bacteria have the potential to interfere with flower-animal interactions with consequences for pollination and plant reproduction.

Flower Visitors of Campanula: Are Oligoleges More Sensitive to Host-Specific Floral Scents Than Polyleges?

The pollen diet provided by adult bees to their offspring varies immensely. While some species collect pollen on several plants irrespective of their phylogenetic relatedness (polyleges), others collect only on plants within a genus or family (oligoleges). Floral scents play a central role in bee-plant interactions. To locate flowers, polyleges are assumed to rely on compounds commonly found as floral scent constituents, whereas oligoleges rely on unusual compounds to recognize host flowers unambiguously. Campanula flowers are visited by both polylectic and oligolectic species, and their scent bouquets consist of common and unusual (e.g., spiroacetals) volatiles. In a comparative approach, we performed electroantennographic analyses to investigate the antennal responses of three polyleges and three oligoleges to three common volatiles and four spiroacetals. We hypothesized that: 1) oligoleges and polyleges should respond similarly to common flower volatiles, and 2) Campanula oligoleges should be more sensitive to spiroacetals than are polyleges. In corroboration, we found that antennal sensitivity to common volatiles was similar among bees irrespective of pollen diet, whereas oligoleges of Campanula were more sensitive to spiroacetals than polyleges. Newly emerged bees of the Campanula oligolege Chelostoma rapunculi rely on spiroacetals for recognizing host-flowers, and our results suggest that this might also be true for other Campanula oligoleges, since Chelostoma campanularum and Hoplitis mitis also were able to perceive these specific volatiles at very low concentrations. Together, our results provide interesting insights into the significance of olfactory adaptations in oligolectic and polylectic bee species.

Circadian rhythm of a Silene species favours nocturnal pollination and constrains diurnal visitation.

Background and Aims Traits related to flower advertisement and reward sometimes vary in a circadian way, reflecting phenotypic specialization. However, specialized flowers are not necessarily restricted to specialized pollinators. This is the case of most Silene species, typically associated with diurnal or nocturnal syndromes of pollination but usually showing complex suites of pollinators. Methods A Silene species with mixed floral features between diurnal and nocturnal syndromes was used to test how petal opening, nectar production, scent emission and pollination success correlate in a circadian rhythm, and whether this is influenced by environmental conditions. The effect of diurnal and nocturnal visitation rates on plant reproductive success is also explored in three populations, including the effect of the pollinating seed predator Hadena sancta. Key Results The result showed that repeated petal opening at dusk was correlated with nectar secretion and higher scent production during the night. However, depending on environmental conditions, petals remain opened for a while in the morning, when nectar and pollen still were available. Pollen deposition was similarly effective at night and in the morning, but less effective in the afternoon. These results were consistent with field studies. Conclusions The circadian rhythm regulating floral attractiveness and reward in S. colorata is predominantly adapted to nocturnal flower visitors. However, favourable environmental conditions lengthen the optimal daily period of flower attraction and pollination towards morning. This allows the complementarity of day and night pollination. Diurnal pollination may help to compensate the plant reproductive success when nocturnal pollinators are scarce and when the net outcome of H. sancta shifts from mutualism to parasitism. These results suggest a functional mechanism explaining why the supposed nocturnal syndrome of many Silene species does not successfully predict their pollinator guilds.