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First published online April 30, 2004; 10.1104/pp.103.037051 Plant Physiology 135:95-102 (2004) © 2004 American Society of Plant Biologists The Key Role of Phloroglucinol O-Methyltransferase in the Biosynthesis of Rosa chinensis Volatile 1,3,5-Trimethoxybenzene1Department of Applied Biological Chemistry, Faculty of Agriculture (S.W., N.W.), and Institute for Genetic Research and Biotechnology (S.M., H.D.), Shizuoka University, Shizuoka 422–8529, Japan; Center for Environment, Health and Field Science, Chiba University, Kashiwa, Chiba 277–0882, Japan (Y.U.); andDepartment of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyoku, Tokyo 113–0033, Japan (M.S., Y.E.)
1,3,5-Trimethoxybenzene is a key component of the Chinese rose odor. This compound is synthesized in three successive methylation steps from phloroglucinol, the initial precursor. A novel, to our knowledge, phloroglucinol O-methyltransferase (POMT) characterized here methylates the first step to produce the intermediate 3,5-dihydroxyanisole, while two previously described orcinol O-methyltransferases catalyze the subsequent steps. We isolated POMT from rose petals and determined partial amino acid sequences of the purified enzyme. The full-length POMT cDNA was isolated and expressed in Escherichia coli. Both the native and recombinant POMT exhibited substrate specificity for phloroglucinol. POMT was expressed specifically in floral organs, in accordance with its role as a key enzyme in the synthesis of rose floral scent compounds.
Roses have been called the queen of flowers and are one of the economically most important groups of ornamental plants (Krussmann, 1981  ). More than 150 rose species and 20,000 cultivars have been registered (Cairns, 2000), most of which belong to the lineage of Chinese roses. These lines originated around 1752, when Chinese roses were first introduced to Europe and were bred for traits such as flower shape, recurrent flowering, and scent (Wylie, 1954). However, most modern roses lost their fragrance when breeding efforts focused on floral color and shape (Zuker et al., 1998).
Studies on rose floral scent have concentrated on its chemical composition (Watanabe et al., 1998
Rosa chinensis, the ancestor of modern roses (Rougetel, 1988
Floral scent plays a crucial role in both the attraction of pollinators and in the repellence of herbivores (Dobson et al., 1996
In this study, we purified and characterized a novel, to our knowledge, POMT from rose petals that encodes a protein which specifically methylates PLG to produce DHA, and characterized its biological role.
Purification of POMT from Rose Petals
In a previous study, we had established that crude enzyme extracts from rose petals contained high levels of PLG-directed O-methyltransferase (OMT) activity (Wu et al., 2003
We monitored OMT activities acting on the three TMB precursors (PLG, DHA, and DMP; Fig. 1). Two classes of OMT activity were detected (Fig. 2 ). The first one (marked by arrow 1 in Fig. 2) showed a high specificity for PLG (4.1 pkat/mL) and a much smaller DHA-directed activity (0.066 pkat/mL). The second one (arrow 2) did not act on PLG, but on DHA and DMP (0.066 and 0.076 pkat/mL, respectively). We designated the PLG-methylating OMT activity, which produced DHA as POMT. SDS-PAGE of the purified POMT protein yielded one major (designated as P) and two weak bands (L for larger size band and S for smaller size band; Fig. 3A ). We attempted further purification using S-adenosyl-L-Met (SAM) and PLG substrate affinity chromatography but were unable to obtain POMT as a single band.
The major POMT band (P) identified by SDS-PAGE had a molecular mass of about 43 kD (Fig. 3A). On the other hand, molecular mass determination of the native protein by HPLC gel filtration suggested a molecular mass of 86 kD (data not shown), indicating that native POMT is a dimeric enzyme. We gel-purified the three bands obtained from SDS-PAGE and subjected them to tryptic digestion prior to matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. Interestingly, digestion products of all bands gave very similar spectra (Fig. 3B). The spectra of band L and P were almost identical with major ion peaks at m/z 1,056.55, m/z 1,235.62, m/z 1,779.73, and m/z 1,795.90. Band S gave these peaks as well as an additional one at m/z 2,050.03. A mixture of all three bands yielded similar results, but m/z 1,779.73 became predominant while another major peak occurred at m/z 2,592.29. The similarity of the spectra suggested that the proteins of the L, P, and S bands were probably derived from one protein and differed only slightly in their degrees of hydrolysis or posttranslational modification.
The major fragments identified by MALDI-TOF-MS were subjected to de novo sequence determination. Three peptide sequences (Pep1, corresponding to m/z 1,056.55, was obtained from band L; Pep2 and Pep3, corresponding to m/z 1,779.73 and m/z 2,592.29, respectively, were obtained from the mixture of all bands) could be determined (Table II).
Isolation of cDNA Encoding POMT We designed a degenerate primer based on the sequence of Pep2 (Table II) for cDNA cloning, because this peptide was represented by a major peak in the MALDI-TOF-MS spectra of all three bands (Fig. 3B). The first partial clones were obtained by reverse transcription (RT)-PCR using this primer together with an oligo(dT) primer. The full-length cDNA was completed by 5'-RACE using specific primers designed on the basis of the obtained partial sequences. As a result, a sequence of 1,393 bp was determined that predicted 371 amino acids. All three partial sequences derived from de novo sequence determination (Table II) were included in the predicted protein sequence (Fig. 4 ).
A database search revealed that POMT had 61% identity with caffeic acid 3-O-methyltransferase (COMT) of Prunus amygdalus (Suelves and Puigdomenech, 1998 ). An amino acid sequence-based phylogenetic tree of POMT and related OMTs showed a division into two major groups (Fig. 5
). Group I mainly included flavonoid OMTs, while group II consisted mostly of COMTs. POMT was classified into the latter group.
RT-PCR analysis showed that POMT was specifically expressed in the floral organs, particularly in the petals. POMT transcripts were barely detectable in stamina and sepals and seemed absent from leaves (Fig. 6 ).
Functional Analysis of POMT The POMT coding region was inserted into pET-32 and overexpressed in Escherichia coli. The fusion POMT purified on a nickel His-binding affinity column gave a single band with a molecular mass of about 60 kD (data not shown). After removing the 109-amino acid Trix-Tag by partial digestion with enterokinase, two bands with molecular masses of approximately 44 and 60 kD were obtained (data not shown). Further digestion caused hydrolysis of POMT. We therefore used the fusion POMT to investigate OMT activity. As shown in Figure 7 , POMT specifically methylated PLG to yield DHA. Further methylation to form DMP was not observed.
Recombinant and native POMTs had similar substrate preferences (Table III). Both methylated only the meta-position hydroxyl group of polyphenol compounds. POMT specifically methylated PLG to produce DHA, while methylation rates of DHA and DMP were negligible and not detectable, respectively. POMT also methylated resorcinol and, to a lesser extent, orcinol, which is the precursor of the rose volatile dimethoxytoluene (Lavid et al., 2002 ; Scalliet et al., 2002). Nevertheless, the Km values of recombinant POMT with respect to PLG and SAM (94.5 and 16.2 µm, respectively) were significantly higher than those of the native POMT (14.4 and 1.9 µm, respectively; Table IV).
Analysis of the Intracellular Contents of TMB Precursors We determined the intracellular contents of volatile TMB and its precursors (PLG, DHA, and DMP) in petals at different flowering stages (S4–7), and in different organs at flowering stage 5 (Fig. 8 ). TMB was detected at high levels (up to a maximum of 1,451.5 µg g–1 fresh weight in petals at stage 5) in flower organs at all flowering stages examined. The three precursors were present at much lower levels. In petals, their variation with flowering stages showed no obvious correlation with the changes of TMB content (Fig. 8).
TMB and its immediate precursor DMP reached similar concentrations in stamina as in petals, while sepals contained much lower levels; only traces could be detected in leaves. DHA showed a similar pattern: relatively high levels in stamina and petals, but much lower concentrations in leaves and sepals. In contrast, the PLG contents of sepals and leaves were slightly higher than in petals; sepals actually contained the highest amounts of PLG per tissue fresh weight found in all organs tested.
Genomic approaches have facilitated the discovery of novel floral fragrance-related genes in rose (Guterman et al., 2002 ). For example, several homologous OOMTs involved in the biosynthesis of 3,5-dimethoxytoluene, a common volatile compound in rose, have been isolated and characterized (Lavid et al., 2002; Scalliet et al., 2002). OOMTs were also implicated in the final two methylation steps of the biosynthesis of TMB. However, the OMT responsible for the first methylation in this pathway remained unknown (Wu et al., 2003). We here describe the purification of this enzyme (POMT) and the isolation of the cDNA of its gene. This represents the first, to our knowledge, identification of an enzyme involved in the biosynthesis of rose floral scent compounds using protein purification methods.
POMT specifically methylated PLG to produce DHA (Table III; Fig. 7). Thus, two functionally distinct classes of OMTs cooperate in the synthesis of TMB: POMT methylates the initial precursor PLG to form the intermediate DHA, while the two previously known OOMTs catalyze the subsequent methylation steps. The origin of PLG in rose flower organs is not yet clear. It may be derived from cyclic polyketides (Bangera and Thomashow, 1999
We found the highest levels of TMB in petals (Fig. 8), which are the main source of volatile fragrance compounds in rose (Dobson et al., 1990
POMT shares approximately 60% identity with COMT from P. amygdalus. Both enzymes catalyze the methylation of meta-position hydroxyl groups (Table III). However, POMT does not act on substrates such as ortho-catechol and 2-methoxyphenol whereas COMT does. Thus, the activity of POMT appears to be limited to the methylation of very small molecules such as PLG. Comparison of the amino acid sequences of rose POMT and Medicago sativa COMT, the crystal structure of which is known (Zubieta et al., 2001 Native and recombinant POMTs showed similar relative activities with a variety of substrates (Table III). This indicated that we had cloned the genuine POMT gene. Intriguingly, the recombinant POMT had a 6-fold higher Km with respect to the substrate PLG than native POMT (Table IV). Since the recombinant fusion POMT was used in the kinetic assays, we suspect that the fused 109-amino acid Trix-Tag may have affected POMT structure and reduced its activity. Another ambiguity comes from the fact that the purified POMT activity appeared as three protein bands on SDS-PAGE gels (Fig. 3A), while the MS analysis suggested that all three bands represented the same protein. It is unclear whether the native POMT protein exists in several differentially modified versions, or whether there are three highly homologous isoenzymes. Our finding of only one single POMT cDNA clone supports the former explanation.
Plant Material
Rosa chinensis Jacq. var. spontanea (Rehd. & Wils.) Yu & Ku red was grown at the experimental farm of Keisei Rose Nursery (Chiba, Japan) under natural conditions. R. chinensis var. spontanea produces flowers from mid-April until mid-May in Chiba. Flower stages were defined as described before (Wu et al., 2003
Extraction and purification procedures were carried out at 4°C, except as noted. Ten grams of rose petals in stage 5 were frozen in liquid nitrogen and ground using a mortar and pestle. The ground powder was mixed with 50 mL buffer A (100 mM potassium-phosphate buffer, pH 7.0, 10% (w/v) glycerol, 1 mM dithiothreitol) and 0.1% (v/v) mercaptoethanol containing 200 mg sodium ascorbate and 2 g polyvinylpyrrolidone (PVP-40). The resulting slurry was mixed and then centrifuged at 20,000g for 20 min. The pellet was discarded, and the supernatant was used as a crude enzyme extract.
Enzyme assays were performed in order to monitor POMT elution during purification. The standard reaction mixture consisted of 30 µL of any given fraction, 5 µL of 1 M Tris-HCl (pH 7.4), 0.5 µL of 1 M DTT, 5 µL glycerol, 1 µL S-adenosyl-L-[methyl-14C]Met (40–60 mCi/mmol), 2 µL of 5 mM substrate (each of PLG, DHA, or DMP), and 6.5 µL water to bring the assay volume to 50 µL. The reaction mixture was incubated at 30°C for 30 min, and 5 µL of 50% acetic acid were added to stop the reaction. The radiolabeled product was extracted by addition of 80 µL of ethyl acetate. A 40-µL aliquot of the organic phase was applied to a thin-layer chromatography plate precoated with silica gel 60 F254 (Merck, Darmstadt, Germany), and the chromatography was run with 1:1 (v/v) CHCl3:EtOAc. An x-ray imaging plate (BAS-MS2025, Fuji Photo Film, Tokyo) was exposed to the thin-layer chromatography plate, and the radioactive products were visualized using a molecular imaging FX system (Bio-Rad Laboratories, Hercules, CA). Enzyme activities were independently checked by gas chromatography (GC)-MS; reaction conditions were as described except that 5 µL of 5 mM nonlabeled SAM was used instead of the 14C-labeled compound. The extracted organic phase was concentrated to a volume of 20 µL under a stream of nitrogen before being analyzed by a QP5000 GC-MS system (Shimadzu, Kyoto).
Fifty milliliters of crude extract were loaded onto a Sephadex column (G-25, 30 cm x 5 cm) equilibrated with buffer B (buffer A with 0.15 M NaCl) and were eluted with buffer B. In total, 50 elution fractions of 2 mL each were collected. All fractions with significant PLG-methylation OMT activity (approximately 75 mL in total) were combined and subjected to ammonium sulfate precipitation (between 30% and 80% saturation). The precipitate was collected by centrifugation. The pellet was resuspended in buffer C (20 mM Tris-HCl, pH 7.4, 10% glycerol, 1 mM DTT), dialyzed in buffer C and concentrated to a volume of 3 mL. The samples (1 mL for each chromatography run; three runs in total) were applied to a Poros HQ column (1.7 mL bed volume; PerSeptive Biosystems, Tokyo) preequilibrated with buffer C, which was attached to an FPLC apparatus (Perseptive Biosystems, Tokyo). The flow rate was set to 6 mL min–1. After washing with 15 bed volumes (25.5 mL) of buffer D (20 mM Tris HCl, pH 7.4), bound proteins were eluted with an NaCl concentration gradient (0–500 mM) in 15 bed volumes of buffer D. The column was finally washed with 1.5 M NaCl in 5 bed volumes of buffer D. One-milliliter elution fractions were collected, and a total of 51 fractions were obtained. Each fraction was analyzed for OMT activity with three different substrates (PLG, DHA, and DMP). Three fractions with particularly high PLG-methylation activity were combined and dialyzed in buffer C and were then subjected to another round of Poros HQ column chromatography as described above. The resulting active fractions were dialyzed in buffer C and concentrated to a volume of 1 mL.
The molecular mass of the native POMT protein was determined by HPLC gel filtration on a TSK-gel G3000SW column (TOSOH, Tokyo). Glutamate dehydroxygenase (290 kD), lactate dehydroxygenase (142 kD), enolase (67 kD), myokinase (32 kD), and cytochrome c (12.4 kD; Oriental Yeast, Tokyo) were used as markers.
HQ-column-purified fractions were subjected to 10% SDS-PAGE. Three silver-stained bands were excised for TOF-MS analysis. In-gel purification and digestion with trypsin (Roche Diagnostics, Mannheim, Germany) were carried out using the protocols of Shevchenko et al. (1996)
RNA extraction and cDNA amplification were conducted using rose petals in flower stage 5, as previously described (Wu et al., 2003
For expression in Escherichia coli, the protein coding regions were amplified with primers corresponding to the N and C termini in which a restriction enzyme site was included. These primers were: POMT-N (5'AGTCGTGGATCCAGGAGCACAAGTCCCCAGA-3', sense primer; BamHI site in italics) and POMT-C (5'-AGGATTAAGCTTTTTGTGGAACTCCATGAC-3', anti-sense primer; HindIII site in italics). The BamHI/HindIII fragments were inserted into a pET-32 (a) vector (Invitrogen) and transformed into E. coli BL21(DE3). The crude overexpressed proteins were extracted from cells with an ultrasonic disruptor (UD-201, Tomy, Tokyo) and were purified with a Chelating Sepharose Fast Flow column (Amersham Biosciences, Piscataway, NJ). The protein concentration was determined by the Bradford method using bovine serum albumin as a standard (Bradford, 1976
Substrate preference and relative activity toward various phenolic compounds including the three TMB precursors (PLG, DHA, and DMP) was investigated for both native and recombinant POMTs (Table III). Conditions were as described above except that the enzyme concentration and incubation time were adjusted to ensure a linear reaction velocity during the reaction period. Three independent experiments were performed. Km and Kcat/Km were determined as described previously (Wu et al., 2003
The nonradioisotopic quantitative RT-PCR method was used to determine mRNA levels in different rose organs (Yokoi et al., 1993
Petals (100 mg) at different flowering stages and of different organs at flowering stage 5 were ground in liquid nitrogen and extracted with EtOAc. The extracts were concentrated and analyzed as previously described (Oka et al., 1999 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AB121046.
We thank Satoi Kasai of Bruker Daltonics K.K. (Tokyo) for amino acid sequence determination. We also thank Prof. Eran Pichersky of the University of Michigan (Ann Arbor, MI) for discussion and for providing 3-methoxy, 5-hydroxytoluene. We are grateful to Shunsuke Takeuchi of Keisei Rose Nursery for providing the plant materials used in this study. Received November 30, 2003; returned for revision February 13, 2004; accepted February 13, 2004.
1 This work was supported by the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific Research (grant no. 14360016).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037051. * Corresponding author; e-mail acnwata{at}shizuoka.ac.jp; fax 81–54–238–4870.
Bangera MG, Thomashow LS (1999) Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87. J Bacteriol 181: 3155–3163 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][Web of Science][Medline] Cairns T (2000) Modern Roses XI. Academic Press, San Diego Channeliere S, Riviere S, Scalliet G, Jullien F, Szecsi J, Dolle C, Vergne P, Dumas C, Bendahmane M, Hugueney P, et al. (2002) Analysis of gene expression in rose petals using expressed sequence tags. FEBS Lett 515: 35–38[CrossRef][Web of Science][Medline] Dewick PM (2001) Aromatic polyketides. In Medicinal Natural Products. A Biosynthetic Approach. John Wiley & Sons, Chichester, UK, pp 61–62 Dobson HEM, Bergstroem G, Groth I (1990) Differences in fragrance chemistry between flower parts of Rosa rugosa Thunb. (Rosaceae). Isr J Bot 39: 143–156 Dobson HEM, Groth I, Bergstrom G (1996) Pollen advertisement: chemical contrasts between whole flower and pollen odors. Am J Bot 83: 877–885 Flament I Debonneville C, Furrer A (1993) Volatile constituents of roses: characterization of cultivars based on the headspace analysis of living flower emissions. In R Teranishi, RG Buttery, H Sugisawa, eds, Bioactive Volatile Compounds from Plants. American Chemical Society, Washington, DC, pp 269–281
Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002
Guterman I, Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev G, Bar E, Davydov O, Ovadis M, Emanuel M, et al. (2002) Rose scent: genomics approach to discovering novel floral fragrance-related genes. Plant Cell 14: 2325–2338
Haddock JD, Ferry JG (1989) Purification and properties of phloroglucinol reductase from Eubacterium oxidoreducens G-41. J Biol Chem 264: 4423–4427 Krussmann G (1981) The history of the modern garden rose. In The Complete Book of Roses. Timber Press, Portland, OR, pp 67–105
Laempe D, Jahn M, Breese K, Schagger H, Fuchs G (2001) Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica. J Bacteriol 183: 968–969
Lavid N, Wang J, Shalit M, Guterman I, Bar E, Beuerle T, Menda N, Shafir S, Zamir D, Adam Z, et al. (2002) O-methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiol 129: 1899–1907 Oka N, Ohishi H, Hatano T, Hornberger M, Sakata K, Watanabe N (1999) Aroma evolution during flower opening in Rosa damascena Mill. Z Naturforsch Sect C Biosci 54c: 889–895 Rougetel HL (1988) Chinese ancestors and their descendants. In A Heritage of Roses. Unwin Hyman, London, pp 42–50 Scalliet G, Journot N, Jullien F, Baudino S, Magnard JL, Channeliere S, Vergne P, Durmas C, Bendahmane M, Cock JM, et al. (2002) Biosynthesis of the major scent components 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose O-methyltransferases. FEBS Lett 523: 113–118[Medline]
Shalit M, Guterman I, Volpin H, Bar E, Tamari T, Menda N, Adama Z, Zamir D, Adam Z, Vainstein A, et al. (2003) Volatile ester formation in roses. Identification of an acetyl-coenzyme A. Geraniol/citronellol acetyltransferase in developing rose petals. Plant Physiol 131: 1868–1876 Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68: 850–858[Medline] Shoji K, Takeshi M, Joichi A, Yomogida K (2000) Sedative for vaporizing inhalation and sedative perfume composition containing the same as active ingredient. Japan Patent Gazette, JP,2000-086478,A. Japan Patent Office, Tokyo Suelves M, Puigdomenech P (1998) Specific mRNA accumulation of a gene coding for an O-methyltransferase in almond (Prunus amygdalus, Batsch) flower tissues. Plant Sci 134: 79–88[CrossRef]
Vainstein A, Lewinsohn E, Pichersky E, Weiss D (2001) Floral fragrance: new inroads into an old commodity. Plant Physiol 127: 1383–1389 Watanabe N, Washio H, Straubinger M, Knapp H, Winterhalter P (1998) Occurrence of a glucosidic progenitor of rose oxide in rose flowers, Rosa damascena Mill. Nat Prod Lett 12: 5–10 Wu S, Ueda Y, He H, Nishihara S, Matsumoto S (2000) Phylogenetic analysis of Japanese Rosa species using matK sequences. Breed Sci 50: 275–281 Wu S, Watanabe N, Mita S, Ueda Y, Shibuya M, Ebizuka Y (2003) Two O-methyltransferases isolated from flower petals of Rosa chinensis var. spontanea involved in scent biosynthesis. J Biosci Bioeng 96: 119–128[Medline] Wylie AP (1954) The history of garden roses (Part I). J R Hortic Soc 79: 555–571 Yokoi H, Natsuyama S, Iwai M, Noda Y, Mori T, Mori KJ, Fujita K, Nakayama H, Fujita J (1993) Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development. Biochem Biophys Res Commun 195: 769–775[CrossRef][Web of Science][Medline]
Yokoya K, Roberts AV, Mottley J, Lewis R, Brandham PE (2000) Nuclear DNA amounts in roses. Ann Bot (Lond) 85: 557–561 Yomogida K (1992) Scent of modern roses. Koryo 175: 65–89 Zubieta C, He XZ, Dixon RA, Noel JP (2001) Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferase. Nat Struct Biol 8: 271–279[CrossRef][Web of Science][Medline]
Zubieta C, Kota P, Ferrer J, Dixon RA, Noel JP (2002) Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase. Plant Cell 14: 1265–1277 Zuker A, Tzfira T, Vainstein A (1998) Cut-flower improvement using genetic engineering. Biotechnol Adv 16: 33–79 This article has been cited by other articles:
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