The Para-O-Methylation of Apigenin to Acacetin by Cell-Free Extracts of Robinia pseudoacacia L. Gary Kuroki and Jonathan E. Poulton Department of Botany, University of Iowa, Iowa City, Iowa 52242 Z. Naturforsch. 36 c, 916-920 (1981); received July 6, 1981 Robinia pseudoacacia , Biosynthesis, Flavonoids, Para-O-Methyltransferase, Acacetin Crude extracts from young Robinia pseudoacacia seedlings, shoots, and callus tissue catalyze the />ara-0-methylation of apigenin to acacetin using S-adenosyl-L-methionine as methyl donor. Optimum activity was exhibited at pH 9.0, and Mg2+ was not required for maximum activity. EDTA (10 m M ) did not affect the reaction rate, but 47% inhibition was observed with SAH (100 u m ). /?-Mercaptoethanol (5 m M ) was required in the homogenization medium for optimum O-methyltransferase activity. Apigenin ( K m, 50 hm) was the best substrate, but significant activity was shown towards caffeic acid, 5-hydroxyferulic acid, naringenin, and quercetin. Para- coumaric, ferulic, and sinapic acids were not methylated. The Km for S-adenosyl-L-methionine was 31 hm. Our demonstration of a /wra-O-methyltransferase activity methylating apigenin, but not /rara-coumaric acid, strongly supports the conclusion that the B-ring methylation pattern of acacetin is determined at the C15-level in Robinia pseudoacacia. Introduction Materials and Methods Flavonoids possessing methoxyl groups at the 3'- Chemicals and/or 5'-positions of the B-ring are widespread in Nature, and their enzymatic biosynthesis is well S-Adenosyl-L-[14CH3]methionine (57.9 mCi/mmol) was purchased from New England Nuclear, Boston, documented [1]. By contrast, flavonoids bearing a 4'-methoxyl group are relatively rare, and virtually Mass., and diluted with unlabelled SAM to a nothing is known about the enzymes involved in the specific activity of 1.1 mCi/mmol. S-Adenosyl-L- homcysteine was purchased from Sigma Chemical biosynthesis of these pigments [2]. Co., St. Louis, Mo. Acacetin glycosides were iso­ Since black locust ( Robinia pseudoacacia L.) seed­ lated from Robinia seedlings and hydrolyzed using lings rapidly accumulate acacetin (4'-methoxy-5,7- dihydroxyflavone) glycosides in the first week follow­ 1 m HC1 at 90 °C to obtain acacetin, which was ing germination, they provide an excellent system identified by standard spectral methods [4], Phenolic with which to investigate the biosynthesis of 4'- substrates were available from our laboratory col­ lection. Clorox was purchased from the Clorox Co., methoxylated flavonoids. We wish to report a novel Oakland, Ca. /wa-O-methyltransferase activity in crude Robinia extracts, which methylates apigenin at the 4'-posi- tion, yielding acacetin. This activity is discussed in Plant Materials relation to earlier isotopic studies designed to estab­ Black locust (Robinia pseudoacacia L.) seeds were lish whether the B-ring substitution pattern of collected from the University of Iowa campus. The acacetin is determined at the C9- or C15-level [3]. seeds were sterilized in 10% Clorox for 0.5 h, washed three times with sterile water, and scarified with a razor blade. The seeds were placed in sterile aerated water for 48 h and sown in sterile soil. The germinated seeds were placed in a growth chamber Abbreviations: SAM, S-adenosyl-L-methionine; EGME, ethylene glycol monomethyl ether; PPO, 2,5-diphenyl- maintained at 24 °C and under a 16:8 h (light: dark) oxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)] benzene; photoperiod. Cell cultures were established from EDTA, ethylenediaminetetraacetic acid; SAH, S-adenosyl- Robinia cotyledons by placing the sterile explants on L-homocysteine. Murashige and Skoog media [5] containing 10 mg/1 Reprint requests to J. E. Poulton. naphthalene acetic acid, and 0.1 mg/1 kinetin. Cal­ 0341-0382/81/1100-0916 $01.00/0 lus tissue was maintained at 25 °C under a 16:8 h G. Kuroki and J. E. Poulton • Para-O-Methylation of Apigenin in Robinia 917 (light:dark) photoperiod and subcultured every 4 of Andersons scintillation fluid (0.3% PPO and weeks. 0.02% POPOP in xylene-Triton X-l 14 (3:1, by vol.)) and the radioactivity measured in a Beckman LS- Enzyme Purification 100C scintillation counter. Control reaction vessels, in which the phenolic substrates were omitted, were Unless otherwise indicated, all stages were carried included in all assays performed. out at 4°C. Robinia seedlings (10-12 days old; weighing approximately 5.5 g) were homogenized Chromatographic identification of reaction product with 30 ml of buffer I and 2.5 g of quartz sand in a mortar. The homogenate was filtered through 2 Acacetin was identified as the product of api­ layers of cheesecloth and centrifuged at 12000x# genin methylation by co-chromatography with an for 15 min. The supernatant liquid was brought to authentic sample on Whatman 3 MM paper using 80% saturation by addition of solid (NH4)2S04 over the following solvent systems: (I) 1% HCl, (II) 15% a period of 20 min. Small volumes of dilute aqueous acetic acid, (III) 30% methanol, (IV) ethyl acetate- KOH were added, where necessary, to maintain the pyridine-H20 (12:5:4, by vol.). Additional confir­ pH at 7.3. The mixture was allowed to stand for an mation was provided by co-chromatography on additional 40 min at 0°C and was then centrifuged silica gel IB-F thin layer sheets (J. T. Baker Chem. at 17600xg for 15 min. The pellet was resus­ Co., N.J.) with the following solvent systems: (V) pended in 3 ml of buffer II and centrifuged for benzene-dioxane-acetic acid (90:25:4, by vol.), (VI) 5 min at 17600xgr to remove undissolved material. toluene-ethyl acetate-acetic acid (9:2:0.5, by vol.). An aliquot (2.5 ml) of the supernatant liquid was chromatographed on a Sephadex G-25 column Protein estimation (1.5 x 8.3 cm), which had been pre-equilibrated with Protein was determined by the Lowry method, as buffer II. Elution was carried out with this buffer, modified by Leggett Bailey [7], after precipitation and the eluate was collected for assay of methyl- from solution by 5% (w/v) trichloroacetic acid; transferase activity. Aliquots (0.5 ml) of this en­ crystalline bovine serum albumin was used as the zyme preparation were stored at — 20 °C and standard. thawed as required. Buffer solutions Results and Discussion The following buffer solutions were used: (I) A central problem in flavonoid biochemistry which still remains unresolved is to establish when 0.1 M potassium phosphate buffer, pH 7.5, contain­ the B-ring substitution pattern is determined. Black ing 5 m M /?-mercaptoethanol; (II) 20 m M potassium locust (Robinia pseudoacacia ) shoots contain gly­ phosphate buffer, pH 7.5, containing 5 m M /?-mer- captoethanol. cosides not only of the common flavonoids apigenin, kaempferol and quercetin but also of acacetin, which exhibits the more unusual 4'-methoxylated O-Methyltransferase assay B-ring [3]. The predominant acacetin glycosides are The standard assay mixture for O-methyltrans- acaciin (acacetin-7-rutinoside) [8] and acacetin 7- ferase activity contained 150 nmol phenolic substrate xylosylrhamnosylglucoside [9]. The time at which (dissolved in 10|il EGME), 26 nmol S-adenosyl-L- the B-ring substitution pattern of acacetin is estab­ [14CH3]methionine (containing 41 nCi), 50 nmol lished was investigated by Ebel, Barz and Grisebach MgCl2, 10 nmol Tris-HCl buffer, pH 9.0, and up to [3] using radiotracer techniques. When older Robinia 0.88 mg protein in a total volume of 0.15 ml. After leaves were incubated for 64 h with /?-methoxycin- incubation at 30 °C for 45 or 60 min, the reaction namic acid-[/?-14C-methyl-14C-methyl-T], the T /14C was terminated by adding 50 |il EGME and placing ratio in acacetin was only 23% of that of the the reaction mixture on ice. After addition of 20 |il precursor, suggesting active demethylation of p- glacial acetic acid, the radioactive product was methoxycinnamate in such leaves. By contrast, when extracted into 0.5 ml of ethyl acetate [6]. Aliquots younger leaves were fed /?-methoxycinnamic acid- (0.3 ml) of the organic layer were mixed with 5 ml [/?-14C-methyl-T] for shorter periods of time, the 918 G. Kuroki and J. E. Poulton • Para-O-Methylation of Apigenin in Robinia T /14C ratio in acacetin was almost the same as that of the precursor. Although this constitutes strong 2 4 ■ evidence for the incorporation of /7-methoxycin- namic acid as an intact unit into acacetin, the extent of incorporation was only 0.01 -0.02%. 18 - j\ We wished to complement these radiotracer data CO £ \ o A / \ by enzymological studies, especially since little is X " c \ NJ known about the pöra-0-methylation of either o t e i i t y \ / /i A . \ ® \ /7-coumaric acid [10] or flavonoids [11]. As climatic .> Q_ u U) conditions preclude the full-year availability of < E / \ y ^ 6 ■ Robinia shoots, additional experimental systems II y S 'V 'Oy Pc were investigated. Robinia seeds possess no acacetin CL ci­ t o glycosides. However, in the first week following t------1------ 1------1------1------1— germination, the young seedlings rapidly accumu­ 6 7 8 9 10 11 late 4 acacetin glycosides (nature of sugar moieties pH as yet unidentified) and therefore provide an excel­ Fig. 1. Effect of pH on the O-methyltransferase activity towards apigenin (II) and caffeic acid (I). The phenolic lent system for identifying enzymes involved in the substrate and SAM were incubated with 0.69 mg of protein biosynthesis of the flavonoids. In addition, callus as described under Materials and Methods, using 67 mM concentrations of the following buffers: K2HP04-KH2P0 4 cultures were established from Robinia seedling ( • - • ) , Tris-HCl (▲-▲), and glycine-NaOH (M l).