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First published online November 5, 2008; 10.1104/pp.108.128066 Plant Physiology 149:384-394 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Biosynthesis of t-Anethole in Anise: Characterization of t-Anol/Isoeugenol Synthase and an O-Methyltransferase Specific for a C7-C8 Propenyl Side Chain1,[W],[OA]Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048 (T.K., E.P.); and Howard Hughes Medical Institute, Jack H. Skirball Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 (T.J.B., J.P.N.)
The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-L-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 µM for coumaryl acetate and 230 µM for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 µM for isoeugenol and 54.5 µM for S-[methyl-14C]adenosyl-L-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol.
The phenylpropenes are a class of volatile compounds found throughout the gymnosperms and angiosperms. When emitted from flowers, they serve as attractants for pollinators, which detect them through their olfactory systems. In addition, at high concentrations their general toxicity to cells renders them useful as defense compounds, and consequently they are found in vegetative tissues of many plant species, although perhaps due to their toxicity they are typically sequestered in specialized structures or quickly emitted from the plant. For example, eugenol and methylchavicol are synthesized and stored in glandular trichomes on the surface of leaves of sweet basil (Ocimum basilicum, Lamiaceae; Gang et al., 2001  ; Iijima et al., 2004), and eugenol, isoeugenol, and their methylated derivatives are synthesized and emitted from flowers of Clarkia breweri (Onagraceae; Wang et al., 1997). Because of the antimicrobial and aromatic properties of the phenypropenes, humans have historically used spices and herbs containing them as pesticides and food preservatives. t-Anethole, a main component of the essential oils of anise (Pimpinella anisum, Apiaceae) seeds, star anise (Illicium anisatum), and fennel (Foeniculum vulgare), has been used as a fumigant against mosquitoes (Chang and Ahn, 2002; Gross et al., 2002). Clove (Syzygium aromaticum) buds, which are rich in eugenol, have been used as a source of spice for millennia (Jirovetz et al., 2006). The many compounds of the phenylpropene class differ from one another in two aspects: the moieties attached to the phenyl ring and the position of the double bond in the propenyl side chain (Fig. 1 ). For example, t-anol and chavicol both have a para-hydroxy functionality on the phenyl ring, but the double bond in the propenyl side chain in t-anol is between C7 and C8, whereas in chavicol it is between C8 and C9. On the other hand, the position of the double bond in the propenyl side chain is the same (C7 and C8) in t-anethole and methylisoeugenol, but the latter has an additional methoxy functionality at the meta-position of the phenyl ring as compared to t-anethole.
We have recently isolated two distinct NADPH-dependent enzymes, eugenol synthase (EGS) and isoeugenol synthase (IGS), which both utilize the same substrate, coniferyl acetate, and convert it to their respective product via a quinone methide intermediate (Koeduka et al., 2006 , 2008; Louie et al., 2007). EGS and IGS proteins are approximately 50% to 95% identical to each other and are also closely related to several other NADPH-dependent reductases involved in phenylpropanoid biosynthesis in plants, including pinoresinol-lariciresinol reductase (PLR), isoflavone reductase (IFR), and phenylcoumaran benzyl ether reductase (PCBER). Of particular interest is the observation that there are two distinct lineages of EGS enzymes within this large family of reductases, whereas the two IGS enzymes previously characterized, from petunia (Petunia hybrida, Solanaceae) and C. breweri, are more closely related to each other as well as to one of the two lineages of EGS sequences (Koeduka et al., 2008). So far, all natural eugenol and IGSs produce only one product—either eugenol or isoeugenol—but in vitro mutagenesis of a few residues at the active site which crystallographic structures demonstrated to influence the binding position of the substrate resulted in an enzyme that produces a mixture of both phenylpropene regio-isomers (Koeduka et al., 2008).
The synthesis of some of the phenylpropenes found in plants, such as t-anethole, methylchavicol, methyleugenol, and methylisoeugenol, requires methylation of the para-hydroxyl group of their phenyl rings (Fig. 1). It has been previously reported that in basil glands, two closely related (90% identical) enzymes chavicol O-methyltransferase (CVOMT) and eugenol O-methyltransferase (EOMT) catalyze the formation of methylchavicol and methyleugenol from chavicol and eugenol, respectively (Gang et al., 2002
Previous studies showed that the essential oil of the seeds and leaves of anise consists of 90% t-anethole as well as some methylchavicol, anisaldehyde, and β-caryophyllene (Orav et al., 2008
Distribution of Phenylpropenes in Different Parts of the Anise Plant
It was previously reported that t-anethole is present in anise seeds (Rodrigues et al., 2003
Isolation and Characterization of Anise AIS1 To isolate cDNAs encoding t-anol synthase from anise, we designed redundant oligonucleotides based on conserved amino acid sequences in phenylpropene synthases (see Supplemental Table S1) and used them in a reverse transcription (RT)-PCR experiment with leaf tissue mRNA. The RT-PCR reaction yielded one fragment encoding a protein with high similarity to petunia isoeugenol synthase 1 (PhIGS1). A full-length cDNA of the gene encoding this fragment was obtained by 3'- and 5'-RACE. This cDNA encodes a protein, designated PaAIS1, with 323 amino acid residues. A phylogenetic analysis indicated that PaAIS1 is 58.8% identical to PhIGS1 and also closely related to basil EGS1 and Clarkia EGS1 and IGS1 with 52% to 54% identities (Fig. 4 ). PaAIS1 is less similar to petunia EGS1 and Clarkia EGS2. The later two enzymes reside in a separate clade together with the previously characterized PCBER enzymes.
Purified PaAIS1, obtained by expression in Escherichia coli, was able to catalyze the formation of t-anol from coumaryl acetate and of isoeugenol from coniferyl acetate (Fig. 5 ). An apparent Km value of 229.9 ± 46.2 µM for coniferyl acetate was measured with an apparent kcat value of 1.02 ± 0.17 per s, whereas the apparent Km value for coumaryl acetate was 134.9 ± 36.4 µM with an apparent kcat value of 0.07 ± 0.01 per second (Table II). We note however that in the assays containing high concentration of the coumaryl acetate substrate (>100 µM), some precipitation was observed, perhaps due to the low solubility of the product or the substrate.
Detailed characterization of PaAIS1 showed that the enzyme was active in the pH range of 6.0 to 7.5 with optimal activity at pH values between 6.0 and 6.5 with Bis/Tris buffer. At the pH range of 6.5 to 7.5, a small amount of eugenol was detected. PaAIS1 activity was strongly inhibited by 2.5 mM Zn2+, Cu2+, and Fe2+ (63%–100% inhibition) and moderately inhibited at 2.5 mM of K+, Mn2+, and Ca2+ (10%–18% inhibition). In contrast, no inhibitory effect on the activity was observed by incubation with 2.5 mM Na+ and Mg2+. PaAIS1 was stable at 20°C for 30 min and retained 85% activity after incubation for 30 min at 37°C. However, it was almost completely inactivated when incubated for 30 min at 50°C (less than 4% activity remaining).
To isolate anise cDNAs encoding an MT capable of methylating t-anol to produce t-anethole, we designed redundant oligonucleotides based on conserved amino acid sequences in phenylpropene MTs (see Supplemental Table S1) and used them in an RT-PCR experiment with leaf tissue mRNA. After nested PCR, clear PCR products (approximately 180 bp) were obtained and sequenced. Three MT-like sequences were identified and 5'- and 3'-RACE experiments led to the isolation of three complete and distinct cDNAs. One of the full-length cDNAs contained an open reading frame encoding a protein of 358 amino acids. The protein encoded by this cDNA, produced in E. coli and purified by nickel-nitrilotriacetic acid agarose affinity chromatography, catalyzed the formation of t-anethole from t-anol (Fig. 6 ) as well as the formation of methylisoeugenol from isoeugenol (see below), and therefore this cDNA was designated as PaAIMT1. The other two MT-like cDNAs encoded proteins that did not show appreciable levels of activities against t-anol. Sequence analysis indicated that PaAIMT1 was 43.2% and 41.4% identical to Medicago sativa COMT and C. breweri IEMT, respectively, and only 28.2% and 27.3% identical to basil CVOMT and EOMT, respectively (Fig. 7 ).
Enzymatic Characterization of PaAIMT1 We tested the activities of PaAIMT1 with a number of potential substrates at a final concentration of 10 µM, since some of the substrates, including t-anol, are not completely soluble in aqueous solution at higher concentrations (Fig. 8 ; Table III). The results of these assays indicated that PaAIMT1 methylated isoeugenol at the highest rate in an S-[methyl-14C]adenosyl-L-Met (SAM)-dependent fashion, followed by dihydroeugenol and t-anol. Neither chavicol, eugenol, nor 5-methoxyeugenol, which all possess an allylic propenyl side chain, served as efficient substrates. In addition, compounds with either no alkene side chain (guaiacol), with hydrophilic functionalities on the end of the side chain (e.g. caffeic acid), or with an ortho-hydroxy group on the phenyl ring (2-propylphenol) were not substrates for PaAIMT1. Interestingly, the activity of PaAIMT1 with dihydrochavicol was approximately half of that with t-anol, and a similar ratio of activity was observed with dihydroeugenol versus isoeugenol. CbIEMT, on the other hand, showed the opposite behavior for these substrates and preferred dihydroeugenol over isoeugenol (and eugenol) by a factor of 2 (t-anol, chavicol, and dihydrochavicol could not be methylated by CbIEMT).
Detailed characterization of PaAIMT1 showed that the enzyme was active in the pH range of 6.0 to 9.0 with optimal activity at pH values between 7.5 and 8.0 with Tris buffer. PaAIMT1 activity was strongly inhibited by 2.5 mM Zn2+ and Cu2+ (97% inhibition, respectively) and moderately inhibited at 2.5 mM of Mn2+ and Fe2+ (35% and 47% inhibition, respectively). In contrast, no inhibitory effect on the activity was observed by incubation with 2.5 mM K+, Na+, Ca2+, and Mg2+. PaAIMT1 was stable at 20°C to 37°C for 30 min and retained 88% activity after incubation for 30 min at 50°C. However, it was almost completely inactivated when incubated for 30 min at 65°C (less than 5% activity remaining). The apparent Km values of 19.3 ± 2.9 and 54.5 ± 6.5 µM (n = 3) for isoeugenol and SAM, respectively, were calculated with an apparent kcat value of 0.015 ± 0.001 per second (n = 3) for isoeugenol (Table II). Kinetic parameters for t-anol could not be obtained because of the lack of solubility of the substrate at concentrations above 75 µM.
Crude protein extracts from different organs of anise plants and during different stages of development were incubated with t-anol and [14C]SAM to measure t-anol MT activity. The results indicated that the highest levels of such activity were found in mature buds, followed by flowers and developing fruits (Fig. 9 ). Similar measurements for t-anol-forming activity could not be obtained because of the lack of radiochemical substrate to follow newly synthesized product; nonradioactive assays were not sensitive enough because of the low solubility of the substrate and because any t-anol formed was quickly converted to t-anethole, which was already present in some tissues at high concentrations.
Expression Patterns of PaAIS1 and PaAIMT1 We determined the steady-state levels of PaAIS1 and PaAIMT1 transcripts in different organs of anise plants and during different stages of the development by quantitative RT-PCR analysis (Fig. 10 ). The patterns of steady-state transcript levels for the two genes were very similar to each other in all organs tested, but the levels of PaAIMT1 transcripts were 15-fold higher than those of PaAIS1 throughout the plant. Transcript levels of both PaAIS1 and PaAIMT1 were lower in mature leaves than in young leaves. On the other hand, transcript levels of both genes increase as buds matured into flowers and were highest at the stage of fruit development. Intermediate levels of PaAIMT1 transcripts were detected in roots.
Synthesis of t-Anol Is Catalyzed by an Enzyme Most Closely Related to PhIGS1
We previously examined the biosynthesis of eugenol and isoeugenol from coniferyl acetate in basil, petunia, and C. breweri (Koeduka et al., 2006
The sequence of PaAIMT1 indicates that it is not closely related to either C. breweri IEMT or basil EOMT and CVOMT (Fig. 7), suggesting that its phenylpropene-methylating activity evolved independently of these former enzymes. PaAIMT1's substrate preference is also quite different from these former enzymes, having a 10-fold greater preference for substrates with a C7-C8 double bond including t-anol and isoeugenol. Its decreased activity with dihydrochavicol and dihydroeugenol, which lack a C7-C8 double bond, also suggests that this double bond is important for either enzyme binding or methyl group transfer by AIMT1. To identify the amino acids or regions that are responsible for the substrate specificities of AIMT1, additional structural studies of AIMT1 along with other phenylpropene MTs such as CbIEMT, are currently being carried out.
Many plants synthesize and accumulate high levels of volatiles, including phenylpropenes, in their vegetative tissue for defense (Pichersky and Gershenzon, 2002
The concentration of t-anethole in the aerial parts of the anise plant increases as the plant matures, and reaches a maximum in developing fruits (Fig. 3). The higher levels of t-anethole, a fungicide, in the reproductive tissues of the plant is a likely adaptation for increased protection of these structures, and its accumulation in the seeds allows for protection during germination and at the early stages of seedling growth. Comparisons of the changes in t-anethole levels, AIMT1 activity levels (PaAIS1 activity levels could not be measured), and PaAIMT1 and PaAIS1 transcript levels from young leaves to mature leaves and from buds to flowers to developing fruit suggest that product accumulation is regulated at multiple levels. The levels of t-anol methylating activity (Fig. 9) are higher in young leaves than in older leaves and transcript levels for both genes are also higher in young leaves, but t-anethole levels are higher in mature leaves (Fig. 3). This is consistent with observations in basil leaves (Gang et al., 2002
Plant Materials and Growth Conditions Anise (Pimpinella anisum) plants were grown in a regular soil in a growth chamber under a 14-h light/10-h dark photoperiod. Temperature was set to 22°C during the light period and 18°C during the dark period. The root tissues for measuring metabolites, crude enzyme activities, and total RNA extraction were grown with vermiculite instead of soil.
All chemicals were from Sigma, unless otherwise indicated. Coniferyl acetate was synthesized as previously described (Koeduka et al., 2008
The leaf epidermal tissue was peeled out by the forceps and immediately incubated in hexane for the extraction of t-anethole. The area of leaf epidermis was calculated by weighing the footprint, where it was peeled off, and the sequential comparison of the weight for 1.1 x 1.1 cm (1.2 cm2) area as a control. In other tissues, t-anethole was extracted with hexane, as described in the text. GC-MS analyses of the extracts were preformed as described in Koeduka et al. (2008)
Crude protein extracts were prepared by homogenizing freshly excised plant tissues using a mortar and pestle in the presence of ice-cold extraction buffer (10:1 [v/w], buffer:tissue) containing 50 mM Tris-HCl, pH 7.0, 1 mM 2-mercaptoethanol, 5 mM Na2S2O5, 1% (w/v) polyvinylpyrrolidone (PVP-40; Sigma), 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride. The slurry was centrifuged at 12,000g for 10 min. The supernatant was used for enzyme assays immediately after clarification.
The standard radiochemical assay mixture for the recombinant proteins consisted of 5 µL of 0.5 M Tris-HCl (pH 7.5), 3 or 5 µL of purified proteins (AIMT1; 0.8 µg or IEMT; 4.7 µg, respectively), 1 µL of 0.5 mM substrate dissolved in methanol (final concentration of substrates was 10 µM), and 0.25 µL of SAM (specific activity 48.8 mCi/mmol [Perkin Elmer Instruments]), and 40.75 or 38.75 µL of water to bring the assay volume to 50 µL. Reactions were incubated at 25°C for 30 min and stopped by the addition of 2 N HCl. The products were extracted with 200 µL ethyl acetate and counted in a scintillation counter as previously described (Wang et al., 1997
Single-strand cDNAs were synthesized from poly-A RNA isolated from 3-week-old anise leaves with Oligo-dT primer and Superscript II reverse transcriptase (Invitrogen). These cDNAs were used as the template for the amplification of MT and phenylpropene synthase cDNA fragments. Degenerate primers (Supplemental Table S1) were designed to correspond to peptide sequences within phenylpropene synthases and MTs that are highly conserved in different proteins belonging to each group as identified by protein sequence comparisons. The identified conserved sequences included the regions encoding the catalytic domain and the SAM and NADPH binding sites, respectively. In the first PCR, the degenerated primers were used to amplify phenylpropene synthases and MTs partial cDNAs under the following PCR conditions: denaturation, 2 min at 94°C; five cycles, 15 s at 94°C, 1 min at 37°C, 1 min at 40°C, and 1 min at 72°C; five cycles, 15 s at 94°C, 1 min at 40°C, 1 min at 45°C, and 1 min at 72°C; five cycles, 15 s at 94°C, 2 min at 45°C, and 1 min at 72°C; five cycles, 15 s at 94°C, 1 min at 50°C, and 1 min at 72°C; 20 cycles, 15 s at 94°C, 1 min at 55°C, and 1 min at 72°C. Amplified PCR products at about 550 bp and 270 bp for phenylpropene synthases and MTs, respectively, were used as the template for the second PCR under the same PCR conditions as in the first PCR. The resulting PCR products at about 350 bp and 150 bp for phenylpropene synthases and MTs, respectively, were cloned into the pGEM-T easy vector (Promega) and sequenced. 5'- and 3'-RACE transcripts were performed to obtain the complete coding sequence of PaAIS1 and PaAIMT1 using SMART RACE cDNA amplification kit (CLONTECH) following the manufacturer's instructions with internal gene-specific primers. The resultant PCR products were subcloned into pGEM-T easy vector and their nucleotides were determined completely. The sequenced clones of 5'- and 3'-RACE covered the desired missing sequences. Based on these additional sequences, full-length cDNAs were cloned into the expression vector pEXP5-CT/TOPO (Invitrogen) to encode C-terminally His-tagged proteins. Resulting constructs were expressed in Escherichia coli. E. coli cells harboring PaAIS1 or PaAIMT1 genes were induced with isopropylthio-β-galactoside (final concentration; 0.5 mM), and the purification of induced proteins were performed using previously described methods (Koeduka et al., 2006
Total RNA was isolated from each tissue with an RNeasy Plant Mini kit (Qiagen). The RNA was subjected to DNase treatment using the DNA-free kit (Ambion), and first-strand cDNA was synthesized by Superscript II transcriptase (Invitrogen) with poly-T primer in parallel with a negative control reaction in which no Superscript II reverse transcriptase was added. The qPCR reactions utilizing SYBR-Green I dye (Molecular Probes), Taq polymerase (New England Biolabs), fluorescein (Bio-Rad), the specific primers (Supplemental Table S2), and a dilution series of each cDNA as standards, were performed as previously described (Varbanova et al., 2007 Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: CbIGS1, EF467238; CbEGS1, EF467239; CbEGS2, EF467240; ObEGS1, DQ372812; PhIGS1, DQ372813; PhEGS1, EF467241; FiPLR, AAC49608; TpPLR, AAF63507; ThPLR, AAF64184; ThPCBER, AAF64178; PtdPCBER, AAC32591; FiPCBER, AAF64174; PtPCBER, CAA06707; PsPCBER, ABF39004; CaIFR, Q00016; PsIFR, P52576; MsIFR, AAC48976; LjPTR, BAF34841; VvLAR, CAI26309; MdLAR, AAZ79364; DuLAR, Q84V83; AtPrR1, At1g32100; AtPrR2, At4g13660; PaAIS1, EU925388; MpF8OMT, AAR09600; RcOMT1, CAD29458; ObCVOMT, AAL30423; ObEOMT, AF435008; CrF4OMT, AAR02420; MsIOMT, T09254; HvF7OMT, S52015; HlOMT1, EU309725; HlOMT2, EU309726; EcRT7OMT, BAE79723; PsRT7OMT, AAQ01668; RcOMT1, BAC78826; McI4OMT, P45986; CbIEMT, AAC01533; CbCOMT, AAB71141; MsCOMT, P28002; CaCOMT, AAA86982; PamCOMT, AAB71213; ObCOMT, AAD38189; ZeCOMT, AAA86718; RhOOMT1, AAM23004; and PaAIMT1, EU925389.
The following materials are available in the online version of this article.
We thank Dr. Efraim Lewinsohn for his kind gift of t-anol. Received August 13, 2008; accepted October 30, 2008; published November 5, 2008.
1 This work was supported by the National Science Foundation (grant nos. MCB–0718152 [to E.P.] and MCB–0718064 [to J.P.N.]). T.K. was supported by a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eran Pichersky (lelx{at}umich.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128066 * Corresponding author; e-mail lelx{at}umich.edu.
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