**6. Functions of Orchid Volatile Compounds**

Previous reviews provide a good summary of floral emissions and the involvement of biochemical processes in the interactions of flowers with their flower visitors [20], their action over pollinator behavior [21], and the ecological processes that drive their evolution [22], wherein they mediate intra- and interspecific interactions. The role of vegetative VOCs has been extensively reviewed [20]. Here, we present the functions of the floral VOCs, which are especially involved in the attraction of pollinators [20]. Floral volatiles have a role in many multifaceted functions that contribute to pollinator attraction, plant defense, plant reproduction, and plant diversity (Figure 2).

**Figure 2.** Functions of floral volatiles in orchid flowers.

#### *6.1. Flower Defense*

Generally, flowers have effective physical barriers comprising highly lignified cell walls, although the generation of these cell walls renders flowers highly vulnerable to pathogens and florivores. Plants constitutively emit VOCs from flowers, leaves, and roots. Emission usually increases when plants are attacked by antagonists such as insect herbivores or pathogens [51].

Many VOCs were shown to exhibit antimicrobial and antifungal activities in vitro [74] or inferred to have these antimicrobial activities based on tissue-specific expression patterns [75]. However, only a few VOCs have been explored for their role in defense against pathogens. (*E*)-β-caryophyllene, emitted from stigmas of *Arabidopsis* flowers, was shown to limit bacterial growth; furthermore, *Arabidopsis* plants lacking (*E*)-β-caryophyllene emission displayed denser bacterial populations on their stigmas and reduced seed weight than wild-type plants, indicating that (*E*)-β-caryophyllene acts in the defense against pathogenic bacteria and is also important for plant fitness [76]. VOCs emitted by petals of *Saponaria o*ffi*cinalis* were also shown to inhibit bacterial growth, supporting their roles in controlling bacterial community diversity in petals [77].

#### *6.2. Pollinator Attraction*

In many flowering plant species, the emission of volatile scents from the flower is important for attracting insect pollinators. Orchid flowers exhibit visual, chemical, and morphological advertisements to guide their pollinators, and may offer rewards such as nectar, pollen, fragrance, or oil [78]. Over the past few years, evidence has supported the role of floral volatiles in pollinator attraction. In fact, a high occurrence of non-rewarding flowers has been noted in orchids compared to other plant families [79]. Floral volatile profiles are specific to each species depending on the type of pollinator [80]. However, the selection of pollinator has played a key role affecting the pattern of floral VOC profile across angiosperms.

Approximately one-third of all orchid species reach pollination over food deception, whereby flowers contain no nectar or other rewards but resemble or mimic floral signals of rewarding plants to attract pollinators [78]. Subsequently, intraspecific variation in floral traits is estimated to be high in food-deceptive orchids, since flowers must delay the avoidance learning of pollinators [79]. Flowers of the fly-pollinated *Satyrium pumilum* orchids emit a cocktail of six compounds containing sulfurous oligo sulfides such as dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). Secretion of these volatiles is also tissue-specific, which is anticipated to be the key olfactory cue for attracting flesh-eating fly pollinators [80] and lepidopteran pollinators [81]. Floral scent can be distinguished even among closely related taxa when species differ in pollination systems, such as lepidopteran vs. bee fly pollination in

*Narcissus* species [80] and bee vs. hummingbird pollination in two *Mimulus* species [80], suggesting that differences in the dominating functional group of pollinators drive divergence in floral scent.

#### *6.3. Plant Reproduction*

Pollinator attraction is often intermediated by multimodal signaling mechanisms including floral morphology, color, and scent [81]. In deceptive species, attractiveness is very important for ensuring reproductive success. For instance, *Ophrys* and *Neotinea* species are known to produce complex bouquets of volatiles typically consisting of more than 100 chemical compounds [82]. The species belonging to these genera are all deceptive, but *Ophrys* species use a sexual deception strategy, while *Neotinea* is a food-deceptive genus [83]. Various orchid volatiles play a key role in plant reproduction. In the Dracula orchid, *Dracula lafleurii*, the labellum acts as both a visual and an olfactory mimic of mushrooms that often grow alongside these orchids [84]. The labellum emits an unusual floral volatile blend of mushroom alcohols, especially (R)-1-octen-3-ol [85]. *Cypripedium calceolus* is pollinated by bees. Scent profile consists mainly of aliphatics, terpenoids, and aromatics. In this context, orchids are highly pertinent models for studying plant reproduction, as they present a great variety of floral traits and trait associations. This is mirrored by the great diversity of reproductive strategies in orchids. One of the most intriguing strategies is deceptive pollination (i.e., nectarless flowers), which is found in about one-third of orchid species. Floral scent analyses in *Ophrys orchids* [81] showed that their flowers emit attractive blends of VOCs. Moreover, different *Ophrys* species, which mainly use alkenes with certain double-bond positions as key signals for plant reproduction (e.g., non-hydrocarbons with low molecular weight), were found as "long-range" attractants [85].

Though by no means exclusive to orchids, deceptive pollination approaches are particularly well-developed in the Orchidaceae, with an estimated one-third of the family (around 10,000 species) using such strategies [85].

#### *6.4. Evolution*

Evolution of angiosperms has resulted in an immense diversity of flower traits such as shape, size, color, and scent. Remarkably, the quality and quantity of emitted volatiles are species-specific and vary among different populations within a species [20]. While much effort has so far been invested in describing scent composition in various flowering species, the mechanisms driving the evolution and diversification of floral scent remain underexplored. Analysis of the genetic basis for differences in scent profiles between these two species revealed that only two quantitative trait loci are responsible for the distinct scent phenotypes [86]. One of these locus maps to the MYB TF ODO1, which controls flux over the shikimate pathway and, therefore, the amount of precursors available for benzenoid biosynthesis [87], while the genetic identity of the second locus is presently unknown. *Ophrys* may rely on species-specific alkene emission profiles that are distinct in enzyme activity and on the gene expression of a few stearoyl acyl carrier protein desaturases of the *Ophrys* genus, and only limited genetic variation among species and populations was observed with microsatellite markers. These findings suggest that divergent pollinator-mediated selection rather than genetic drift explains the strong differences in volatile profiles. Taken together, the above examples demonstrate that small genetic variations can have large effects on floral scent chemistry and interactions with pollinators.
