Eran Pichersky. American Scientist. Volume 92, Issue 6. Nov/Dec 2004.
Scents have a way of jolting us into recalling deeply seated memories. The whiff of a plant’s fragrance might bring to mind childhood experiences, evoking a sense of time and place more powerfully than any other stimulus. Our ancestors knew the emotional power of particular smells, which are mentioned in ancient religious texts such as the Egyptian Book of the Dead, the Hindu Vedas and the Judeo-Christian Bible. And aromatics, mostly of plant origin, are still employed to good effect in religious rites, perfumes, cosmetics and home remedies.
As familiar to the naturalist as to the incense maker, plant scents are a subset of the tens of thousands of plant metabolites. Fragrances consist of small organic molecules with high vapor pressures. This physical property means that although a scent compound would be in the liquid or even in the solid phase (most often dissolved in the fluid inside cells) at a plant’s normal growing temperatures, it evaporates easily if exposed to the air. Chemicals with this property are described as volatile. A flower’s perfume is a familiar example of a volatile compound, but characteristic scents can also be released by breaking the vegetative parts of many plants-think of freshly cut grass, herbs or pine needles.
Volatile plant compounds probably evolved to repel herbivores, but they now perform a remarkable range of functions. Most of the animals that interact with plants are insects (not surprising, given their abundance), which detect volatiles through the antennae on their heads and, in some species, certain mouthparts called maxillary palps. On the surface of the antennae, specialized cells each contain a single type of protein receptor that recognizes and binds specific volatile compounds. The array of receptor-decorated cells reports to the brain by way of the nervous system. Although each cell contains only one receptor type, a single compound can be recognized (to a greater or lesser degree) by more than one receptor. As a result, the pattern of neuronal firing that is elicited by a specific compound or mixture will be unique. Furthermore, this system is extremely sensitive-some receptors can detect an airborne volatile at concentrations of a few parts per billion.
Scent Sent Columbus
Unlike “volatile,” which is a physical term, a “scent” is defined by the person who detects it. Human beings perceive many (but certainly not all) volatile substances. For example, we don’t smell water, despite its ubiquity. Our olfactory system has different sensitivity ranges for different chemicals, and some people detect certain compounds more easily because of genetic differences.
Specific words do not exist for scents as they do for colors or tactile sensations. Instead, we classify smells based on subjective experience. One person may say that a particular aroma smells like lemon, but to someone else it is reminiscent of soap. Our emotional associations are usually colored by the context in which we first encountered the smell, and olfactory memories tend to be persistent and quite vivid.
In mammals (including us), the sense of smell requires volatile compounds to travel through the nasal passages to an organ deep inside the nose called the olfactory epithelium. Although the gross anatomy of the olfactory epithelium is quite different from that of insect antennae, the two organs are similar at a cellular level. Like the antennae, the olfactory epithelium contains neuronal cells studded with receptors that bind specific molecules. And because a single compound can be recognized to a varying degree by different receptors, the complex pattern of neuronal activity conveys both qualitative and quantitative information to the brain.
Volatiles can also reach the olfactory epithelium indirectly through the mouth. As food is chewed, mixed with saliva and warmed, volatiles from the food evaporate and ascend through the retro-nasal route. The detection of these aromas is basically instantaneous, giving each food its distinctive flavor. In fact, the tongue only perceives five basic tastes (sweet, sour, salty, bitter and the recently discovered umami), but the number of aroma mixtures is practically unlimited.
Although we generally think of plant aromas as pleasant, many plant volatiles are toxic when eaten. These compounds may be used by plants to protect vulnerable organs (such as sugar-laden fruits) from microbial assault. Humans have recognized and taken advantage of these antimicrobials since antiquity, when they were used to retard spoilage. For example, the spice clove, whose major active ingredient is the compound eugenol, was used in baked goods and prepared meats to prevent mold growth. Indeed, before the development of modern foodpreservation techniques, European civilization was heavily dependent on clove and other tropical spices to ensure a lasting supply of food, particularly during the winter months. Many of these spices could only be obtained by long-distance trade with Asia, making them extremely expensive. The potential for profit was a major reason why Columbus and other explorers began looking for a shorter route to the East Indies. In fact, it can be said that spices-plant volatiles-were one of the ultimate motivations for the discovery of the “new” world.
If spices are toxic to microbes and insects, aren’t they dangerous for people as well? Actually, yes, they usually are, but toxicity depends on dosage-the amount of toxic compound per total body weight. Some scientists think that pregnant women tend to reject spicy foods during early pregnancy (as part of the nausea and vomiting called morning sickness) to shield the embryo from compounds that might cause deformities or miscarriage.
Although the consumption of spiced food may have been necessary in the past, current refrigeration and foodpreservation techniques have minimized the risk from spoiled food. Yet many people still have a taste for pungent flavors. The reason for this preference is not clear, but it is plausible that people who could detect and enjoy spiced food enjoyed a selective advantage because they were more likely to avoid food-borne illnesses and food poisoning. We have learned to spice our food in amounts that deter other organisms but seem pleasantly piquant to us.
The Measurement of Odor
Biological systems for scent detection depend on the many different receptors for individual compounds. Therefore, each species’ system is understandably specific to its particular environment. To study scent in an objective, general, quantitative way, scientists rely on instruments, primarily the gas Chromatograph-a long, narrow, separation tube, or column, that is coated on the inside with various substances. But before a scent can be analyzed, the volatile or mixture of volatiles that emanate from a plant source first has to be captured. Samples are usually collected by pumping the air around the plant through a filter that lets the air through but retains these organic volatiles. The volatiles are removed from the filter with an organic solvent, usually hexane or pentane. Other volatile compounds inside the plant can be extracted directly from the tissue using the same type of solvent.
A portion of the solution containing the volatiles is placed at the opening of the gas Chromatograph, which is kept at a temperature high enough to immediately vaporize the sample. An inert gas such as helium is pumped through the column to carry the volatile chemicals. Inside the tube, each component has a different affinity to the materials coated on the inside surface, so its movement is retarded to a different extent. The result is that individual compounds emerge from the column at different times. A mass detector is placed at the other end to record the elution of these now-separated volatiles. This type of analysis is often combined with another instrument called a mass spectrometer, which not only detects the mass coming out of the column but can also identify chemical formulas. Volatile analysis by a combined gas chromatograph-mass spectrometer may not always reach the detection levels of insect and animal systems, but it is nonetheless quite sensitive and certainly more uniform.
Floral Fragrance Function
Everyone is familiar with scented flowers, and many people have heard that floral odors help the plant attract pollinators. This common notion is mostly correct, but it is surprising how little scientific proof of it exists. Of course, not all flowers are pollinated by biological agents-for example, many grasses are wind-pollinated-but the flowers of the grasses may still emit volatiles. In fact, plants emit organic molecules all the time, although they may not be obvious to the human nose. As for flower scents that we can detect with our noses, bouquets that attract moths and butterflies generally smell “sweet,” and those that attract certain flies seem “rotten” to us. However, we almost never know the effects of individual compounds on the response of the intended, and unintended, pollinators. Which chemical actually causes the pollinator to decide to visit the flower? Are there compounds with repelling effects? Scientists have hypothesized that some volatiles deter visitation by unwanted insect species or after pollination has already taken place.
Experiments with artificial flowers that combine various scents with a sugar reservoir demonstrate that insects can learn to associate almost any scent with a reward. Entomologists who emphasize this learning ability posit that although insects may have innate preferences for some volatiles, in the long run they will respond to almost any signal that marks a flower worth visiting. In this view, complex floral scents simply provide information that allows insects to distinguish between different types of flowers so that they can decide which one to visit.
This interpretation may understate the coercive ability of plants in this evolving, co-dependent process. An extreme example of plant manipulation of insect behavior is the perfume of orchid flowers of the genus Ophrys, which resembles the female sex pheromones of certain solitary bee species. Male bees detect this scent (a mixture of fatty acid derivatives) in the air and land on the flowers, whose shape and color also resemble the female bee. After landing, the males exhibit copulatory behavior-which does not occur in the absence of scent-causing them to be covered with pollen. As the male bees move from one plant to another in search of the elusive females, they help pollinate the flowers. This system does not seem to offer any advantages to the bee, yet the deception continues.
Stems, Roots, and Leaves
The release of volatiles from vegetative parts (leaf, stem or root) of the plant is also familiar, although until recently the physiological functions of these chemicals were less clear and had received much less attention from scientists. When the trunk of a pine tree is injured-for example, when a beetle tries to burrow into it-it exudes a very smelly resin. This resin consists mostly of terpenes-hydrocarbons with a backbone of 10, 15 or 20 carbons that may also contain atoms of oxygen. The heavier C20 terpenes, called diterpenes, are gluelike and can cover and immobilize insects as they plug the hole. This defense mechanism is as ancient as it is effective: Many samples of fossilized resin, or amber, contain the remains of insects trapped inside. The lighter C15 sesquiterpenes and the C10 monoterpenes are both volatile and toxic. Some resin terpenes are synthesized in the tree before any attack has occurred and are stored in specialized structures known as resin ducts. But Rod Croteau and his colleagues at Washington State University have found that once the plant is attacked, it revs up the synthesis of this family of defensive chemicals, and some specific compounds are made at particularly high levels. Whether these terpenes function simply as toxins against the invading insects or whether their dispersal into the atmosphere serves additional functions is not yet clear.
Many other plants emit volatiles when injured, and in some cases the emitted signal helps defend the plant. For example, (Z)-3-hexenyl acetate, which is known as a “green leaf volatile” because it is emitted by many plants upon injury, deters females of the moth Heliothis virescens from laying eggs on injured tobacco plants. Interestingly, the profile of emitted tobacco volatiles is different at night than during the day, and it is the nocturnal blend, rich in several (Z)-3-hexen-1-ol esters, that is most effective in repelling the night-active H. virescens moths.
The pioneering efforts of the teams led by Marcel Dicke at Wageningen University in the Netherlands, Jim Tumlinson at the U.S. Department of Agriculture (and now Pennsylvania State University) and several other groups have established that herbivore-induced volatiles often serve as indirect defenses. These bulwarks exist in a variety of plant species, including corn, beans, and the model plant species Arabidopsis thaliana. Plants not only emit volatiles acutely, at the site where caterpillars, mites, aphids or similar insects are eating them, but also generally from nondamaged parts of the plant. These signals attract a variety of predatory insects that prey on the plant-eaters. For example, some parasitic wasps can detect the volatile signature of a damaged plant and will lay their eggs inside the offending caterpillar; eventually the wasp eggs hatch, and the emerging larvae feed on the caterpillar from the inside out. The growth of infected caterpillars is retarded considerably, to the benefit of the plant. Similarly, volatiles released by plants in response to herbivore egg laying can attract parasites of the eggs, thereby preventing them from hatching and avoiding the onslaught of hungry herbivores that would have emerged.
Some fascinating details about the molecular triggers for a plant’s defensive responses have recently come to light with the identification of specific compounds in the oral regurgitant of Lepidoptera herbivores. One such substance, known as volititin, was isolated from maize-browsing larvae Spodoptera exigua, the beet armyworm. This compound, with the chemical name N-(17hydroxylmolenoyl)-L-glutamine, works in plant cells to activate genes involved in the biosynthesis of toxic indole and sesquiterpene compounds. Other fatty acid conjugates with similar effects on plant-gene expression are found in other butterfly and moth larvae.
Plant volatiles can also be used as a kind of currency in some very indirect defensive schemes. In the rainforest understory tree Leonardoxa africana, ants of the species Petalomyrmex phylax patrol young leaves and attack any herbivorous insects that they encounter. The young leaves emit high levels of the volatile compound methyl salicylate (the main ingredient in oil of wintergreen), a compound that the ants use either as a pheromone or as an antiseptic in their nests. It appears that methyl salicylate is both an attractant and a reward offered by the tree to get the ants to perform this valuable deterrent role.
Plants also emit volatile compounds through their roots. For example, parasitic plants such as striga grow their roots in the direction of the host root by following the release of volatile cues related to sesquiterpenes. A recent study showed that Arabidopsis roots secrete the monoterpene 1,8-cineole. However, whether such signals propagate as volatiles through air inside the soil or whether they move around as solutes in solution is unknown.
As evidence has accumulated that volatiles from vegetative parts of plants mediate direct and indirect defenses, questions about their evolutionary origin and present value to the plant have come to the fore. Whatever the original reasons for general and induced emission of volatiles, this type of broadcast provides lots of information to a variety of organisms. Clearly, some insects can identify specific plants by their emissions, even from a distance or in the dark, and such insects may or may not harbor good intentions toward the plant. On the other hand, if the release of specific volatiles became closely associated with herbivore damage during the course of evolution, the signal might serve a number of functions. The emissions could cue herbivore predators about the location of prey, or they might indicate to other plant eaters the presence of competitors, enemies or poor food quality caused by the induction of the plant’s direct defenses, such the production of nonvolatile toxins. The volatile release could conceivably alert neighboring plants about the nature and timing of a nearby herbivore attack.
This web of interactions among many potential parties can become so complicated that it would be very difficult to predict what the outcome for a specific plant would be. Yet plants continue to emit volatiles. It may be that in this arms race, the plants are locked into a physiological response-volatile emissionthat is constantly being co-opted by certain insects to the detriment of the plant. But it is also clear that the system does serve a defensive purpose, as many experiments have shown that the deactivation of a plant’s volatile-emission system makes it more vulnerable to herbivores.
How to Make a Plant Volatile
Although the chemical structures of many plant volatiles were identified years ago, knowledge of how plants synthesize them has, until recently, lagged far behind. One major reason for this delay was the fact that many of the enzymes that make plant volatiles are only present in minute amounts (because only minute amounts of volatiles need to be made) and are therefore difficult to identify, isolate, and characterize. However, advances in biochemical and biomolecular techniques, particularly the isolation of plant genes and large-scale production of plant proteins, have recently led to major advances in this area. Several biochemical pathways that synthesize plant volatiles, including many catalytic enzymes, have been identified and characterized at length. These results have led to some fascinating conclusions that have addressed long-standing mysteries.
Scientists who study plant volatiles have known for many years that although some compounds are common to most plant species, many other volatiles are only found in one or a few species. Often, even closely related species that diverged from a common ancestor only a few million years ago (at most) can have substantially different bouquets. Given this variation, and keeping in mind that the responsible enzymes are encoded by genes, how could these interspecies differences in volatile compounds have evolved in such a (relatively) short time? As it turns out, the genetic evidence suggests that the mechanisms for this evolution, although varied, do follow some basic principles.
The simplest cause of variation in volatile production among related plant species stems from differences in how a given gene is activated. For example, flowers of Clarkia breweri, an annual from California’s coastal range, synthesize and emit easily detectable amounts of a monoterpene called linalool. A specific gene, encoding the enzyme linalool synthase, is turned on in the petals and stigma just when the flowers open. This enzyme catalyzes the formation of linalool in these parts of the flower. By contrast, the same gene is expressed at very low levels in the stigma, and not at all in the petals, of the closely related species Clarkia concinna. Consequently, much less enzyme is made, therefore much less linalool is synthesized; none is emitted from C. concinna flowers.
Genetic changes can also cause the resultant proteins to have slightly different catalytic activity in related species. For example, C. breweri flowers emit methylsalicylate, and snapdragon flowers emit methylbenzoate. The addition of methyl groups to very similar precursors-salicylic acid in Clarkia and benzoic acid in snapdragons-produces both of these compounds. The enzymes that catalyze these reactions are called methyltransferases, and their amino acid sequences and overall three-dimensional structures are so similar that they must have evolved from a common ancestor. In the intervening time since the snapdragon and Clarkia lineages diverged, the two methyltransferase proteins accrued mutations so that their catalytic sites now accommodate different compounds and produce different volatiles.
Some of the enzymes of volatile biosynthesis have the unusual feature of an active site that can accommodate more than one substrate. For example, although the snapdragon methyltransferase can only act on benzoic acid, a similar enzyme in the flowers of the tropical plant Stephanotis floribunda can methylate either benzoic acid or salicylic acid, producing methylbenzoate or methylsalicylate, respectively. Although the enzyme is inherently more active with salicylic acid, the flowers synthesize and emit more methylbenzoate because the overall pathway for benzoic acid synthesis is more active than that of salicylic acid. In this case, the availability of the substrate dictates how much of each product is made.
Sometimes the variation between similar enzymes causes them to produce different products from the same starting compound. For example, the linalool synthase enzyme in C. breweri flowers acts on the substrate geranyl diphosphate to make linalool, but a similar enzyme in basil leaves acts on geranyl diphosphate to produce the monoterpene geraniol, an isomer of linalool that has a very different smell.
Related proteins even exist within the cells of the same plant. For example, special glands on basil leaves not only contain geraniol synthase to make geraniol, but also linalool synthase to make linalool. Such similarities are found when, at some point in the distant past, a single gene became duplicated in the genome. Eventually, the two copies diverged so that they now encode different enzymes. This process can repeat itself over and over, leading to what are termed “gene families” that encode “protein families.” Although scientists usually discover scent genes in a piecemeal fashion as they explore the synthesis pathways of specific volatiles, discovery proceeds differently when all the genes in a species are known, as the case is for Arabidopsis and rice. With such genomic data, investigators can use bioinformatic tools to search for genes encoding proteins that provide evidence of gene duplication and divergence. In the case of Arabidopsis, this analysis indicates that the genome contains about 30 genes with similarity to terpene synthases such as linalool and geraniol synthases. Several of these genes encode enzymes that catalyze the formation of familiar monoterpenes (including linalool, but not geraniol) and sesquiterpenes.
One noteworthy property of many of the enzymes of the terpene synthase family, and one that is very rarely found among enzymes of either primary or secondary metabolism, is an ability to produce many products from a single substrate. For example, one Arabidopsis enzyme catalyzes the formation of ten different monoterpenes from the same geranyl pyrophosphate precursor. The enzyme can manufacture such variety because it produces a kind of unstable intermediate called a carbocation, which can then be transformed into a stable final product in several different ways. This conversion occurs within the active site of the enzyme, and therefore is guided by the topology of the active site, but the process is essentially stochastic so that a certain percentage of catalytic events produces one final product, another percent ends up giving a second product, et cetera. The result is a mixture of monoterpenes that always shows the same ratio of its components. The trick of creating multiple products with a single protein catalyst is not only valuable for its economy. A more important benefit may be the ability to transfer, by inheritance of a single gene, the means of synthesizing a complex and easily recognized signature for insect pollinators or, alternately, a complex arsenal of toxins that pathogens and herbivores find difficult to overcome.
A Palette of Scents?
Floral scent has a strong impact on the economic success of many agricultural crops that rely on insect pollinators, including fruit trees such as the bee-pollinated cherry, apple, apricot and peach, as well as vegetables and tropical plants such as papaya. Pollination not only affects crop yield, but also the quality and efficiency of crop production. Many crops (notably apples, berries and watermelons) require most, if not all, ovules to be fertilized for optimum fruit size and shape. A decrease in fragrance emission reduces the ability of flowers to attract pollinators and results in considerable losses for growers, particularly for introduced species that had a specialized pollinator in their place of origin. This problem has been exacerbated by recent disease epidemics that have killed many honeybees, the major insect pollinators in the United States.
One means by which plant breeders circumvent the pollination problem is by breeding self-compatible, or apomictic, varieties that do not require fertilization. Although this solution is (at times) adequate, its drawbacks include near genetic uniformity and consequent susceptibility to pathogens. Some growers have attempted to enhance honeybee foraging by spraying scent compounds on orchard trees, but this approach was costly, had to be repeated, had potentially toxic effects on the soil or local biota, and, in the end, proved to be inefficient. The poor effectiveness of this strategy probably reflects inherent limitations of the artificial, topically applied compounds, which clearly fail to convey the appropriate message to the bees. For example, general spraying of the volatile mixture cannot tell the insects where exactly the blossoms are. Clearly, a more refined strategy is needed. The ability to enhance existing floral scent, create scent de novo or change the characteristics of the scent, which could all be accomplished by genetic engineering, would allow us to manipulate the types of insect pollinators and the frequency of their visits. Moreover, the metabolic engineering of fragrance could increase crop protection against pathogens and pests.
Genetic manipulation of scent will also benefit the floriculture industry. Ornamentals, including cut flowers, foliage and potted plants, play an important aesthetic role in human life. Unfortunately, traditional breeding has often produced cultivars with improved vase life, shipping characteristics, color and shape while sacrificing desirable perfumes. The loss of scent among ornamentals, which have a worldwide value of more than $30 billion, makes them important targets for the genetic manipulation of flower fragrance. Some work has already begun in this area, as several groups have created petunia and carnation plants that express the linalool synthase gene from C. breweri. These experiments are still preliminary: For technical reasons, the gene was expressed everywhere in the plant, and although the transgenic plants did create small amounts of linalool (not normally made by carnations and petunias), the level was below the threshold of detection for the human nose. Similar experiments in tobacco used genes for other monoterpene synthases, such as the one that produces limonene (a citrus smell), but gave similar results. The next generation of experiments, already in progress, includes sophisticated schemes that target the expression of scent genes specifically to flowers or other organs-such as special glands that can store antimicrobial or herbivore-repellent compounds.