PVs serve multiple functions in both floral and vegetative organs, and these roles are not always related to their volatility (1). Most PVs are restricted to specific lineages and are involved in species-specific ecological interactions, leading to their designation as specialized, or secondary, metabolites (4). To humans, pollinator-attracting floral scents have been a source of olfactory pleasure since antiquity, and we also use a large number of aromatic plants as flavorings, preservatives, and herbal remedies (5). Flower and herb aromas may contain many individual chemicals, sometimes with very little overlap in the PV profiles of even closely related species (6). It is unlikely that the observed differences are due entirely to differential gene expression. That most PVs are restricted to a few lineages, both ancient and derived, argues for frequent changes in enzymatic profiles through evolution. Considerable diversity and fast rates of change have also been observed in nonvolatile specialized metabolites in plants, raising similar questions regarding the underlying evolutionary mechanisms (7).

Improvements in analytical techniques and molecular and biochemical methods have made PVs one of the best-studied groups of plant secondary metabolites. Here we describe insights into molecular mechanisms involved in the biosynthesis of PVs and their implications for our understanding of plant metabolic diversity.

Plants also have enzymes called terpene synthases (TPSs), which catalyze the formation of diverse hemi-, mono-, sesqui-, and diterpene PVs from DMAPP, GPP, FPP, and GGPP,respectively (9). Many distinct TPSs that synthesize monoterpenes and sesquiterpenes (the bulk of terpenoid PVs) have been characterized from various plants (1, 2, 10). Each species contains a number of these mechanistically and structurally related TPSs. For example, the Arabidopsis genome contains 32 genes that appear to encode functional TPSs, including six proven monoterpene synthases and at least two proven sesquiterpene synthases (11).

The first step in the reaction catalyzed by TPSs is removal of the pyrophosphate group, leading to a carbocation (Fig. 1A). This highly unstable intermediate can then undergo a number of multistep transformations. The active site of each TPS channels and directs the intermediates through regioand stereochemical rearrangements to produce often one or two major products and a few (or even scores of) minor “derailment” products (9, 11, 12). For example, four residues lining the active-site cavities of two highly related maize TPSs, designated TPS4 and TPS5, control the proportions of sesquiterpenes produced in these enzymes (Fig. 1) (12). Changing only these four residues in TPS5 to those found in TPS4 produced a volatile profile and catalytic efficiency identical to that of native TPS4, even though differences elsewhere in the proteins remained (12).

The second largest class of PVs consists of compounds containing an aromatic ring. Although not all reactions leading to the synthesis of the basic skeletons have been determined, most are derived from intermediates in the pathway that leads from shikimate to phenylalanine and then to an array of primary (such as lignin) and secondary nonvolatile compounds (2). Eugenol (clove essence) is a reduced version of coniferyl alcohol, a lignin precursor (13). Phenylacetaldehyde, a compound present in tomato fruit (14), is derived from phenylalanine by decarboxylation and oxidative removal of the amino group (15). Shortening of the three-carbon side chain of phenylalanine-derived hydroxycinnamates to one carbon also leads to aromatic building blocks such as benzoic acid and benzaldehyde (16). Still other PVs are produced by type III polyketide synthases (PKSs) that use cinnamoyl- and malonyl–coenzyme As (CoAs) as starting material (17).

In these protein families, little correlation is observed between the level of sequence similarity among enzymes and the structural similarity of their substrates. An illustrative example involves a group of O-methyltransferases that catalyze the formation of scent compounds in rose, basil, and C. breweri (Fig. 2). In rose flowers, 1,3,5-trimethoxybenzene is produced from phloroglucinol (1,3,5-trihydroxybenzene) through three successive methylations. An enzyme designated phloroglucinol O-methyltransferase (POMT) monomethylates phloroglucinol but lacks iterative activity with subsequent methylated intermediates (31). The second and third reactions are catalyzed by two highly similar proteins (96% amino acid sequence identity), OOMT1 (orcinol O-methyltransferase 1) and OOMT2 (32). OOMT1 slightly prefers the first intermediate (1-methoxy-3,5-dihydroxybenzene) and OOMT2 prefers the second intermediate (1,3-dimethoxy-5-hydroxybenzene). The high degree of sequence identity between OOMT1 and OOMT2 indicates recent gene duplication and incipient gene evolution. However, neither of these two enzymes methylates phloroglucinol. POMT is highly divergent from OOMT1 and OOMT2 (30% identity), having higher sequence similarity (54% identity) to IEMT (isoeugenol/eugenol methyltransferase), the enzyme that methylates eugenol (and isoeugenol) in C. breweri. Moreover, POMT and IEMT have higher sequence identity (>60%) with COMT (caffeic acid O-methyltransferase), a methyltransferase involved in lignin biosynthesis (31, 33). On the other hand, OOMT1 and OOMT2 are more similar (50% identical) to EOMT (eugenol O-methyltransferase), the basil enzyme that methylates eugenol, as well as to enzymes involved in isoflavone biosynthesis (34).

The rates of biosynthesis of leaf PVs are typically highest when leaves are young and not fully expanded and need the most protection, which PVs provide directly because of their toxicity or indirectly through the summoning of herbivores’ predators (1, 2, 41). High rates are also observed when flowers are ready for pollination, and they decrease drastically after fertilization (2, 23). Biosynthetic rates are correlated with levels of transcripts of genes encoding the final biosynthetic enzymes, or the concentration of the substrates of these enzymes, or both (2, 42).