The lack of floral synthesis and emission of isoeugenol in Petunia axillaris subsp. parodii is due to a mutation in the isoeugenol synthase gene

Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subsp. axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the artificial hybrid Petunia hybrida, a cross between P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subsp. parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent‐synthesizing and scent‐emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide‐like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame‐shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by an as yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNA interference also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.     

Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol

Petunia flower petals emit large amounts of isoeugenol, which has been shown to be synthesized by isoeugenol synthase (PhIGS1) from an ester of coniferyl alcohol, hypothesized to be coniferyl acetate. This paper describes the identification and characterization of a novel petunia gene encoding an enzyme belonging to the BAHD acyltransferase family whose expression correlates with isoeugenol biosynthesis. RNAi suppression of this gene results in inhibition of isoeugenol biosynthesis. Biochemical characterization of the protein encoded by this gene showed that it has acetyltransferase activity and is most efficient with coniferyl alcohol among the alcohol substrates tested. Overall, these data support the conclusion that coniferyl acetate is the substrate of isoeugenol synthase.     

Floral scent production in Clarkia breweri (Onagraceae). II. Localization and developmental modulation of the enzyme S-adenosyl-L-methionine:(iso)eugenol O-methyltransferase and phenylpropanoid emission.

We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55-67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4'-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step.     

Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.

Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl-l-methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.     

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase,an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate,is not involved in floral scent production in snapdragon flowers

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.     

Structure and reaction mechanism of basil eugenol synthase

Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.     

Floral volatiles fromClarkia breweri andC. concinna (Onagraceae): Recent evolution of floral scent and moth pollination

Clarkia breweri (Onagraceae) is the only species known in its genus to produce strong floral fragrance and to be pollinated by moths. We used gas chromatography-mass spectrometry (GC-MS) to identify 12 abundant compounds in the floral headspace from two inbred lines ofC. breweri. These volatiles are derived from two biochemical pathways, one producing acyclic monoterpenes and their oxides, the other leading from phenylalanine to benzoate and its derivatives. Linalool and linalool oxide (pyran form) were the most abundant monoterpenoids, while linalool oxide (furan form) was present at lower concentrations. Of the aromatic compounds detected, benzyl acetate was most abundant, whereas benzyl benzoate, eugenol, methyl salicylate, and vanillin were present as minor constituents in all floral samples. The two inbredC. breweri lines differed for the presence of the additional benzenoid compounds isoeugenol, methyleugenol, methylisoeugenol, and veratraldehyde. We also analyzed floral headspace fromC. concinna, the likely progenitor ofC. breweri, whose flowers are odorless to the human nose. Ten volatiles (mostly terpenoids) were detected at low concentrations, but only when headspace was collected from 20 or more flowers at a time. Trans--ocimene was the most abundant floral compound identified from this species. Our data are consistent with the hypothesized recent evolution of floral scent production and moth pollination inC. breweri.     

A pinoresinol-lariciresinol reductase homologue from the creosote bush (Larrea tridentata) catalyzes the efficient in vitro conversion of p-coumaryl/coniferyl alcohol esters into the allylphenols chavicol/eugenol, but not the propenylphenols p-anol/isoeugenol

The creosote bush (Larrea tridentata) accumulates a complex mixture of 8-8' regiospecifically linked lignans, of which the potent antioxidant nordihydroguaiaretic acid (NDGA) is the most abundant. Its tetra-O-methyl derivative (M4N) is showing considerable promise in the treatment of refractory (hard-to-treat) brain and central nervous system tumors. NDGA and related 9,9'-deoxygenated lignans are thought to be formed by dimerization of allyl/propenyl phenols, phenylpropanoid compounds that lack C-9 oxygenation, thus differentiating them from the more common monolignol-derived lignans. In our ongoing studies dedicated towards elucidating the biochemical pathway to NDGA and its congeners, a pinoresinol-lariciresinol reductase homologue was isolated from L. tridentata, with the protein obtained in functional recombinant form. This protein efficiently catalyzes the conversion of p-coumaryl and coniferyl alcohol esters into the corresponding allylphenols, chavicol and eugenol; neither of their propenylphenol regioisomers, p-anol and isoeugenol, are formed during this enzyme reaction.     

Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1). This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1). The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

Bioactivity of semisynthetic eugenol derivatives against Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae infesting maize in Colombia

The anti-acetylcholinesterase, larvicidal, antifeedant activities and general toxicity of 15 semisynthetic eugenol derivatives based on clove oil (including the own oil), were evaluated against the maize armyworm, Spodoptera frugiperda (J.E. Smith). Therefore, promising eugenol molecules were classified with larvicidal, anti-acetylcholinesterase and antifeedant activities for controlling this pest. During structure–activity relationship studies and physicochemical profile analysis, it was found that among tested molecules 1–15, eugenol 1, prenyl eugenol 4, isoeugenol 8 and isoeugenol acetate 11 exhibited lethal effects LD₅₀ at concentrations <1 mg/g of insect. On the other hand, eugenol 1, metallyl eugenol 3, isoeugenol 8 and isoeugenol acetate 11 showed a good antifeedant activity (CE₅₀ = 158–209 µg/mL) with a high antifeedant index (70–78%) at concentration 1000 µg/mL, possessing a weak anti-acetylcholinesterase activity (IC₅₀ = 21–31 μg/mL). According to their ecotoxicological profiles (LC₅₀ = 2033.1–6303.8 µg/mL on Artemia salina larvae), isoeugenol 8 and its acetate derivative 11 could be potential used in control of the growth, feeding, or reproduction of S. frugiperda larvae, acting as moderate insecticidal acetylcholinesterase inhibitors and/or antifeedant molecules. Such structure–activity relationship studies could stimulate the identification of lead structures from natural sources for the development of larvicidal and deterrent products against S. frugiperda and related insect pests.     

Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. breweri flower.

Flowers of Clarkia breweri, an annual plant from the coastal range of California, emit a strong sweet scent of which S-linalool, an acyclic monoterpene, is a major component. Chromosomal, chemical, and morphological data, and the species' geographic distribution, suggest that C. breweri evolved from an extant nonscented species, C. concinna. A cDNA of Lis, the gene encoding S-linalool synthase, was isolated from C. breweri. We show that in C. breweri, Lis is highly expressed in cells of the transmitting tract of the stigma and style and in the epidermal cells of petals, as well as in stamens, whereas in the nonscented C. concinna, Lis is expressed only in the stigma and at a relatively low level. In both species, changes in protein levels parallel changes in mRNA levels, and changes in enzyme activity levels parallel changes in protein levels. The results indicate that in C. breweri, the expression of Lis has been upregulated and its range enlarged to include cells not expressing this gene in C. concinna. These results show how scent can evolve in a relatively simple way without the evolution of highly specialized "scent glands" and other specialized structures. Lis encodes a protein that is structurally related to the family of proteins termed terpene synthases. The protein encoded by Lis is the first member of this family found to catalyze the formation of an acyclic monoterpene.     

Evidence for positive selection on the floral scent gene isoeugenol-O-methyltransferase

Isoeugenol-O-methyltransferase (IEMT) is an enzyme involved in the production of the floral volatile compounds methyl eugenol and methyl isoeugenol in Clarkia breweri (Onagraceae). IEMT likely evolved by gene duplication from caffeic acid-O-methyltransferase followed by amino acid divergence, leading to the acquisition of its novel function. To investigate the selective context under which IEMT evolved, maximum likelihood methods that estimate variable d(N)/d(S) ratios among lineages, among sites, and among a combination of both lineages and sites were utilized. Statistically significant support was obtained for a hypothesis of positive selection driving the evolution of IEMT since its origin. Subsequent Bayesian analyses identified several sites in IEMT that have experienced positive selection. Most of these positions are in the active site of IEMT and have been shown by site-directed mutagenesis to have large effects on substrate specificity. Although the selective agent is unknown, the adaptive evolution of this gene may have resulted in increased effectiveness of pollinator attraction or herbivore repellence.