Floral scent

Floral scent or flower scent is composed of all the volatile organic compounds (VOCs), or aroma compounds, emitted by floral tissue (e.g. flower petals). Floral scent is also referred to as aroma, fragrance, floral odour or perfume. Flower scent of most flowering plant species encompass a diversity of VOCs, sometimes up to several hundred different compounds.^[1][2] The primary functions of floral scent are to deter herbivorous and especially florivorous insects (see Plant defense against herbivory), and to attract pollinators. Floral scent is one of the most important communication channels mediating plant-pollinator interactions, along with visual cues (flower color, shape, etc.).^[3]

Flowers of Lonicera japonica emit a sweet, subtle fragrance mainly composed of linalool.^[4]

Biotic interactions

Perception by flower visitors

Flower visitors such as insects and bats detect floral scent thanks to chemoreceptors of variable specificity to a specific VOC. The fixation of a VOC on a chemoreceptor triggers the activation of an antennal glomerulus, further projecting on an olfactory receptor neuron and finally triggering a behavioral response after processing of the information (see also Olfaction, Insect olfaction). The simultaneous perception of various VOCs may cause the activation of several glomeruli, but the output signal may not be additive due to synergistic or antagonistic mechanisms linked with inter-neuronal activity.^[5] Therefore, the perception of a VOC within a floral blend may trigger a different behavioral response than when perceived isolated. Similarly, the output signal is not proportional to the amount of VOCs, with some VOCs in low amount in the floral blend having major effects on pollinator behavior. A good characterization of floral scent, both qualitative and quantitative, is necessary to understand and potentially predict flower visitors' behaviour.

Flower visitors use floral scent to detect, recognize and locate their host species, and even to discriminate among flowers of the same plant.^[6] This is made possible by the high specificity of floral scent, where both the diversity of VOCs and their relative amount may characterize the flowering species, an individual plant, a flower of the individual plant, and the distance of the plume from the source.

To make the best use of this specific information, flower visitors rely on long-term and short-term memory that allow them to efficiently choose their flower.^[7] They learn to associate the floral scent of a plant to a reward such as nectar and pollen,^[8] and have different behavioral responses to known scents versus unknown ones.^[9] They are also able to react similarly to slightly different odor blends.^[10]

Mediated biotic interactions

Pollinators use both floral scent and floral colour to locate flowers of Antirrhinum majus spp. striatum.^[11]

A major function of floral scent is to attract pollinators, and hence to ensure reproduction of animal-pollinated plants.

Some families of VOCs presented in floral scent have likely evolved as herbivore repellents.^[12] However, these plant defenses are also used by herbivores themselves to locate a plant resource, similarly to pollinators attracted by floral scent.^[13] Therefore, this phenotypic trait is subject to antagonistic selection pressures (positive selection by pollinators and negative selection by herbivores), leading to contrasting evolutionary patterns.

Synthesis of floral VOCs

Most floral VOCs belong to three main chemical classes.^[2][6] VOCs in the same chemical class are synthesized from a shared precursor, but the biochemical pathway is specific for each VOC and often varies from a plant species to the other.

Terpenoids (or isoprenoids) are derived from isoprene and synthesized via the mevalonate pathway or the erythrol phosphate pathway. They represent the majority of floral VOCs, and are often the most abundant compounds in floral scent blends.^[14]

The second chemical class is composed of the fatty acid derivatives, synthesized from acetyl-CoA, of which most of them are also known as green leaf volatiles, because there are also emitted by vegetative parts (ie leaves and stems) of plants, and in sometimes higher abundance than from floral tissue.

The third chemical class is composed of benzenoids/phenylpropanoids, also known as aromatic compounds, they are synthesized from phenylalanine.

Regulation of emissions

Floral scent emissions of most flowering plants vary predictably throughout the day, following a circadian rhythm. This variation is controlled by light intensity.^[15] Maximal emissions coincide with peaks of highest activity of visiting pollinators. For instance, snapdragon flowers, mostly pollinated by bees, have highest emissions at noon, whereas nocturnally-visited tobacco plants have highest emissions at night.^[16]

Floral scent emissions also vary along floral development, with highest emissions at anthesis,^[17] i.e. when the flower is fecund, and reduced emissions after pollination, probably due to mechanisms linked with fecundation.^[18]

Abiotic factors, such as temperature, atmospheric CO2 concentration, hydric stress and soil nutrient status also impact the regulation of floral scent.^[19]

Finally, biotic interactions may affect floral scent. Plant leaves attacked by herbivores emit new VOCs in response to the attack, the so-called herbivore-induced plant volatiles (HIPVs).^[20] Similarly, damaged flowers have a modified floral scent compared to undamaged ones. Micro-organisms present in nectar may alter floral scent emissions as well.^[21]

Measurement

Measuring floral scent both qualitatively (identification of VOCs) and quantitatively (absolute and/or relative emission of VOCs) requires the use of analytical chemistry techniques. It requires to collect floral VOCs, and then to analyze them.

VOCs sampling

Most used methods rely on adsorbing floral VOCs on an adsorbent material such as SPME fibers or cartridges by pumping air sampled around inflorescences through the adsorbent material.

It is also possible to extract chemicals stocked in petals by immersing them into a solvent, and then analyze the liquid residue. This is more adapted to the study of heavier organic compounds, and/or VOCs that are stored in floral tissue before being emitted in air.

Sample analysis

Desorption

• thermodesorption: the adsorbent material is flash-heated so that all adsorbed VOCs are carried away from the adsorbent and injected into the separation system. This is how work injectors in gas chromatography machines, which literally volatilize introduced samples. For VOCs adsorbed on bigger amount of adsorbent material such as cartridges, thermodesorption may require the use of a specific machine, a thermodesorber, connected to the separation system.
• desorption by solvent: VOCs adsorbed on the adsorbent material are carried away by a small quantity of solvent which is then volatilized in injected in the separation system. Most commonly used solvents are very volatile molecules, such as methanol, so as to avoid co-elution with slightly heavier VOCs

Separation

Gas chromatography (GC) is ideal to separate volatilized VOCs due to their low molecular weight. VOCs are carried by a gas vector (helium) through a chromatographic column (the solid phase) on which they have different affinities, which allows to separate them.

Liquid chromatography may be used for liquid extractions of floral tissue.

Detection and identification

Separation systems are coupled with a detector, that allows the detection and identification of VOCs based on their molecular weight and chemical properties. The most used system for the analysis of floral scent samples is GC-MS (gas chromatography coupled with mass spectrometry).

Quantification

Quantification of VOCs is based on the peak area measured on the chromatogram and compared to the peak area of a chemical standard:^[22]

• internal calibration: a known quantity of a specific chemical standard is injected together with the VOCs, the measured area on the chromatogram is proportional to the injected quantity. Because the chemical properties of VOCs alter their affinity to the solid phase (the chromatographic column) and subsequently the peak area on the chromatogram, it is best to use several standards that best reflect the chemical diversity of the floral scent sample. This method allows a more robust comparison among samples.
• external calibration: calibration curves (quantity vs. peak area) are established independently by the injection of a range of quantities of chemical standard. This method is best when the relative and absolute amount of VOCs in floral scent samples varies from sample to sample and from VOC to VOC and when the chemical diversity of VOCs in the sample is high. However, it is more time consuming and may be source of errors (e.g. matrix effects due to solvent or very abundant VOCs compared to trace VOCs^[23]).

Specificity of floral scent analysis

Floral scent is often composed of hundreds of VOCs, in very variable proportions. The method used is a tradeoff between accurately detecting quantifying minor compounds and avoiding detector saturation by major compounds. For most analysis methods routinely used, the detection threshold of many VOCs is still higher than the perception threshold of insects,^[24] which reduces our capacity to understand plant-insect interactions mediated by floral scent.

Further, the chemical diversity in floral scent samples is challenging. The time of analysis is proportional to the range in molecular weight of VOCs present in the sample, hence a high diversity will increase analysis time. Floral scent may also be composed of very similar molecules, such as isomers and especially enantiomers, which tend to co-elute and then to be very hardly separated. Unambiguously detecting and quantifying them is of importance though, as enantiomers may trigger very different responses in pollinators.^[25]

References