Biosynthesis of t-anethole in anise: characterization of t-anol/isoeugenol synthase and an O-methyltransferase specific for a C7-C8 propenyl side chain

Abstract

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 microm for coumaryl acetate and 230 microm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 microm for isoeugenol and 54.5 microm 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.

Figure 1.

Biochemical reactions leading to phenylpropenes in plants. The reactions catalyzed by known enzymes are indicated. The carbon numbering system used in the text is shown.

Figure 2.

Aerial organs of anise are covered with trichomes. A, Adaxial surface of mature leaves. B, Side view of mature buds. C, Single flower of anise. The inset shows a closer view of the hairy trichomes on the abaxial side of petals. D, Surface of stems.

Figure 3.

Accumulation of phenylpropenes in anise during development. Tissues were ground in liquid nitrogen and the compounds were extracted with hexane and analyzed by GC-MS. Each value in the figure is an average of three replicates from three independent experiments.

Figure 4.

Phylogenetic analysis showing the relatedness of PaAIS1 to other representative NADPH-dependent PIP reductases. An unrooted tree was constructed by using the neighbor-joining method with EGS/IGS proteins and the PIP family of NADPH-dependent reductases. Bootstrap values from a minimum of 1,000 trials are shown. Cb, C. breweri; Du, Desmodium uncinatum; Fi, Forsythia intermedia; Lj, Lotus japonicus; Md, Malus domes; Ms, M. sativa; Ob, O. basilicum; Pa, P. anisum; Ph, P. hybrida; Ps, Pisum sativum; Pt, Populus trichocarpa; Ptd, Pinus taeda; Th, Tsuga heterophylla; Tp, Thuja plicata; Vv, Vitis vinifera; At, A. thaliana; Ca, Cicer arietinum; Lt, L. tridentata. In addition to IGS/EGS-like proteins including AIS1 and chavicol/EGS, the PIP family includes PLRs, pinoresinol reductases, IFRs, PCBER, leucocyanidin reductase, and pterocarpan reductase. Clones biochemically characterized in C. breweri, basil, and petunia are underlined. Anise AIS1 cloned in this study is indicated with an oval. The accession numbers of the sequences analyzed in this figure are given in “Materials and Methods.”

Figure 5.

Product analysis by GC-MS of the reaction catalyzed by PaAIS1. A, The reaction product of anise AIS1 using coumaryl acetate as the substrate (and NADPH). B, The reaction product of anise AIS1 using coniferyl acetate as the substrate (and NADPH).

Figure 6.

Product analysis by GC-MS of the reaction catalyzed by PaAIMT1. A, An authentic t-anethole standard. B, The reaction product of anise AIMT1 using t-anol and SAM.

Figure 7.

A phylogenetic tree showing the relatedness of PaAIMT1 to selected MTs. An unrooted tree was constructed by using the neighbor-joining method. Bootstrap values from a minimum of 1,000 trials are shown. MpF8OMT, Mentha piperita flavonoid 8-OMT; RcOMT1, Rosa chinensis OMT1; ObCVOMT, O. basilicum chavicol OMT; ObEOMT, O. basilicum eugenol OMT; CrF4OMT, Catharanthus roseus flavonoid 4-OMT; MsIOMT, M. sativa isoflavone OMT; HvF7OMT, Hordeum vulgare flavonoid 7-OMT; HlOMT1, Humulus lupulus OMT1; HlOMT2, Humulus lupulus OMT2; EcRT7OMT, Eschscholzia californica reticuline 7-OMT; PsRT7OMT, Papaver somniferum (R,S)-reticuline 7-OMT; PaAIMT1, P. anisum t-anol/isoeugenol MT; McI4OMT, Mesembryanthemum crystallinum inositol 4-OMT; CbIEMT, C. breweri isoeugenol/eugenol MT; CbCOMT, C. breweri caffeic acid OMT; MsCOMT, M. sativa caffeic acid OMT; CaCOMT, Chrysosplenium americanum caffeic acid OMT; PamCOMT, Prunus armeniaca caffeic acid OMT; ObCOMT, O. basilicum caffeic acid OMT; ZeCOMT, Zinnia elegans caffeic acid OMT; RhOOMT1, Rosa hybrida orcinol OMT1. Clones biochemically characterized in C. breweri, basil, and rose (Rosa spp.) are underlined. Anise AIMT1 cloned in this study is indicated with an oval. The accession numbers of the sequences analyzed in this figure are given in “Materials and Methods.”

Figure 8.

Substrates used to measure the relative specific activities of anise AIMT1 and Clarkia IEMT. A, Compounds that served as substrates for anise AIMT1 enzyme. B, Compounds that did not serve or showed less than 5% relative activities for anise AIMT1 enzyme as substrates.

Figure 9.

Levels of t-anol MT activities during the lifespan of anise plants. The specific activity with t-anol in buds is set at 100%.

Figure 10.

Relative expression levels of PaAIMT1 and PaAIS1 transcripts in the different stages of anise development. A, Transcript levels of PaAIMT1. B, Transcripts levels of PaAIS1. The level of expression of PaAIMT1 in developing fruit was set as 100%. Results for each transcript in each sample were standardized with 18S rRNA transcripts levels. Results represent the average of three replications. Bar indicates se.