Flavonoid 3´-O-Methylation by a Zea mays L. Preparation1

Biochem. Physiol. Pflanzen 184, 453-460 (1989) VEB Gustav Fischer Verlag Jena

Flavonoid 3'-O-Methylation by a Zea mays L. Preparation 1 RUSSELL

L.

LARSON

United States Department of Agriculture, Agricultural Research Service, Agronomy and Biochemistry Departments, University of Missouri, Columbia, U.S.A. Key Term Index: Flavonoid O-methyltransferase, flavonoid biosynthesis; Zea mays L.

Summary The methylated flavonoids, peonidin and isorhamnetin, found in pigment extracts of maize (Zea mays L.) tissues are the products of methylation by the S-adenosylmethionine-flavonoid 3'-0methyltransferase described herein. The transferase is found in seedlings, leaf sheaths, and immature aleurone tissues and requires S-Adenosylmethionine as the methyl donor. S-adenosylhomocysteine inhibited the enzyme 50% at the 27 IlM level whereas the same inhibition was observed with sinefungin present at al IlM level. The transferase had no apparent metal ion requirement, an optimum temperature of 40°C and an optimum pH range between 8.0 and 8.5. The enzyme utilized either eriodictyol, a flavanone ; luteolin, a flavone; or quercetin a flavonol as the substrate but would not methylate dihydroquercetin, a partiaily reduced flavonol or quercetin 3-glucoside. This suggests that methylation occurs sometime prior to the glucosylation reaction which is thought to occur near the end of the biosynthetic sequence for the flavonoid compounds in maize. Identification of the biochemical-genetic interrelationships of the transferase with the genes known to be involved in flavonoid biosynthesis in maize remains to be determined.

Introduction As a dass of compounds in the plant kingdom, the flavonoids are ubiquitous and possess wide variation in chemical structure. Although the first chemical analysis of these compounds was carried out in 1849 (MOROT), there has been little interest in how they are synthesized or the basic reason for their existence in the plant kingdom until recent times. The implication of these compounds in disease resistance (KIRAL Y 1986) and the utilization of the genes involved in this system in molecular biological efforts (FEDOROFF et al. 1984; SCHWARZ-SOMMER et al. 1987) have focussed attention on the need to know more about their chemistry and function in the plant. One of the many structural variations observed in this dass of compounds involves 0methylation, which would appear to lend stability to these oxygen-rich, potentially reactive compounds. Two of the types of compounds found within this dass are the flavonols and the anthocyanidins, O-methylated examples of which are isorhamnetin (3'-0-methylquercetin) and peonidin (3'-0-methylanthocyanidin), respectively. Although these O-methylated Abbreviations: DTE, dithioerythritol; Mes, 2-(N-morpholino)ethanesulfonic acid; Tes, N-tris(hydroxymethyl )meth y1-2-aminoethanesulfonic acid; Hepes, N-2-h ydrox yethylpiperazine-N' -2-ethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine I Cooperative investigations, Agricultural Research Service, United States Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia, MO 65211. Journal Series No. 9361.

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flavonoids are known to be widely distributed in the plantkingdom (HARBORNE 1967; GOTTLlEB 1975), the O-methyltransferases involved in their synthesis have been studied in a limited number of species. The distribution and properties of these enzymes have been summarized by POUL TON (1981) and SCHÜTTE (1985). The most extensive studies of the plant flavonoid methyltransferases have been those of JONSSON et al. (1984) on Petunia hybrida and IBRAHIM et al. (1987) on Chrysosplenium americanum. JONSSON et al. (1984) were able to identify 4 methyltransferases which had been separated by isoelectrofocussing, by their enzymatic properties. The structural genes for these 4 methyltransferases had been identified previously (JONSSON et al. 1983). IBRAHIM et al . (1987) describe a multienzyme system in Chrysosplenium americanum which catalyzed the stepwise methylation and also the glucosylation offlavonoids. Isorhamnetin and peonidin have been identified in the flavonoid fraction of maize (Zea mays L.) (CHEN 1973; WIERMANN 1968; STYLES and CESKA ]98] a and 1981 b). However, liUle is known about the O-methyltransferases involved in their synthesis , e.g. where the methylation reaction fits into the overall biosynthetic scheme or how it is influenced by the several genes identified with f1avonoid biosynthesis in maize (COE et al. 1988). As part of a continuing biochemical-genetic study concemed with isolation and characterization of the enzymes and identification of the controlling genes involved in flavonoid biosynthesis in maize, it was of interest to identify the methyltransferase that catalyzes methylation of quercetin and also anthocyanidin to form isorhamnetin and peonidin, respectively. This report is concemed with the isolation and partial characterization of f1avonoid 3' -O-methyltransferase from maize leaf sheats, seedlings and aleurone tissue.

Materials and Methods Plant Materials Maize seedlings, leaf sheaths and aleurone tissue were used as sources of the flavonoid 3'-0methyltransferase. The 7 to 10 day old seedlings that were used were grown under natural light on a greenhouse sandbench . Leaf sheaths were obtained from mature field grown plants at or near anthesis. Aleurone tissue was obtained from immature ears harvested 20 to 27 days after pollination or from mature ears . Aleurone tissue was obtained by peeling the pericarp and scraping the aleurone layer free of the endosperm. All tissues utilized were obtained from seed having all the genetic factors necessary to produce anthocyanidin or the analogous flavonol compounds. Ch emieals Chemicals used in this study were those available commercially with the exception of eriodictyol, luteolin, chrysoeriol and isorhamnetin , which were obtained from Extrasynthese, Z. 1. La Rechassiere, F -697 30 Genay, France. Romoeriodictyol was a gift from Dr. T. J. MABRY, Univ. of Texas-Austin. Buffer So/utions The following buffers were used in these studies: Buffer A: 0.05 M Hepes (N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid) , pH 8.0 containing 2 mM dithioerythritol (DTE) . Buffer B: 0.01 M Repes buffer, pR 8.0 containing 2 mM DTE . Buffer C: 0.01 M Hepes buffer, pR 8.0 containing 2 mM DTE + 10 % glycerin. Additional buffers used in determining the optimum pR for the methyltransferase were as follows: 2-(N-morpholino)ethanesulfonic acid (Mes), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Tes) and N, N-bis(2-hydroxyethyl)glycine (Bicine), each at a 50 mM level with 2 mM DTE added.

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Enzyme Preparation Seedlings minus the roots, or mature leaf sheaths which had been washed in distilled water were homogenized in a Waring Blendor 1 ) in buffer A (ca. 2 ml/g tissue) with added Polyc1ar AT (1 g/3 g tissue) for 1 min. The mixture was filtered through Pelon and the filtrate was centrifuged for 20 min at 31,000 X g. The supematant (crude fraction) was then fractionated by addition of solid ammonium sulfate while the pH was maintained at 8.0. That fraction precipitating between 30 and 45% saturation was collected by centrifugation for 30 min at 31,000 X g (ammonium sulfate fraction). This precipitate (ca. 2 mg protein) was suspended in buffer A and applied to a Sephadex G-50 column, previously equilibrated with buffer B. The column was then eluted with buffer Band the eluant collected in 3 ml fractions and assayed for methyltransferase activity. Active fractions (ca. 2 mg protein) were pooled (G-50 fraction) and applied to a DEAE Sephadex A-50 column previously equilibrated with buffer e. The DEAE column was washed with buffer C for 45 min and eluted (10 ml h- I ) with a linear 0-0.5 M KCI gradient in buffer e. Fractions (3 ml) were collected and assayed for methyltransferase activity. The active fractions, which comprised the DEAE fraction, were pooled (ca. 0.5 mg protein) and added to a Sephadex G-200 column and the column eluted with 0.01 M Hepes buffer, pH 8.0. The G-200 column had been equilibrated with the elution buffer prior to addition of the DEAE fraction. 5 ml fractions were collected, assayed and the active fractions pooled as the "G-200 fraction". The Sephadex and DEAE columns were prepared to have a bed volume of 35 ml. All fractions were stored at - 50°C, whereas the purification procedures were carried ou1 at 4 oe. Transferase was extracted from the aleurone tissue by grinding the tissue with a cold mortar and pestle with added sand, Polyc1ar AT (1 g/4 g tissue) and buffer A. The resulting mixture was centrifuged for 30 min at 31,000 X g and the supematant purified by the procedure described above for seedling and sheath methyltransferase. Methyltransferase Assay

The assay contained Hepes buffer (50 mM, pH 8.4) + 2 mM DTE, 0.29 mM S-adenosylmethionine, 30 ~M quercetin and enzyme in 3 ml total volume. Controls minus either the substrate or the enzyme were run in conjunction with the sampIes and all were incubated at 40°C for 20 min. Assays were terminated by the addition of 0.2 ml of 3 N HCI after which the product was extracted in two 5 ml volumes of ethyl acetate. The ethyl acetate extract was then taken to dryness under nitrogen and suspended in 0.1 ml of methanol. Product formation was determined by HPLC ofthe methanol suspensions on a ~Bondapak C I8 column using methanol: acetic acid: H 20 (30: 10: 60, v/v/v) as the eluant with a flow rate of 2 ml min- 1 . The column eluant was monitored at 365 nm for isorhamnetin and chrysoeriol and at 280 nm for homoeriodictyol at a sensitivity of 0.01 absorbance units full scale. The concentration of either reaction product could be determined by comparison of peak height data with that obtained for the particular standard chromatographed at different concentrations. Substrate specificity was assayed by substituting other flavonoids for quercetin on an equimolar basis. The effects of inhibitors and metal ions were assayed by addition in the 2 ml re action volume. Protein was determined by the method of BRADFORD (1976) using bovine serum albumin, fraction V as the standard. A ~katal of methyltransferase is defined as that amount of enzyme needed to transform one ~mol Si of substrate and specific activity is ~katals/mg protein. This assay was used to determine KM values for quercetin, eriodictyol, luteolin, caffeic acid and S-adenosylmethionine, using the Sephadex G-50 fraction as the source of the enzyme. The pH optimum for the transferase was determined using a mixture of Mes, Tes, Bicine and Hepes buffers over a pH range of 6.0 to 9.0.

Results

Flavonoid 3'-O-methyltransferase was extraeted in a soluble form from maize seedlings, leaf sheaths and aleurone tissue as deseribed in the previous seetion. The resulting erude I) Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warranty by the Uni ted States Department of Agriculture or the U niversity of Missouri and does not imply approval to the exc1usion of other products or vendors that mayaIso be suitab1e.

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extracts were then subjected to purification as described and assayed for transferase activity. Specific activity in the crude fraction from the leaf sheath was much high er than that in either the aleurone or the seedlings and remained so throughout the purification procedure (Table 1). Table I . Purification offlavonoid 3' -O-methyltransferase Activi ty 1.2

Fraction

Seedling

Specific (lJ.kat mg prot- I)

Total IJ.kat

(x 10- 5 )

(x 10- 5 )

Enrichment (-fold)

Yield

(%)

erude Ammonium Sulfate Sephadex G-50 DEAE

0.19 0.59 0.61 4.27

40.53 25.93 7.09 4.95

1.0 3.1 3.1 22.1

100 63 .9 17 .5 12.2

LeafSheath erude Ammonium Sulfate Sephadex G-50 DEAE Sephadex G-200

1.88 4 .14 5.03 15 .44 53 .36

204.35 123.25 105.37 105.02 48.56

1.0 2.2 2.7 8.2 23.8

100 60.3 51.6 51.4 23.8

0.74 3.41 5.41 82.08

1.96 1.57 1.31 1.03

1.0 4 .6 7.3 110.8

100 80.3 66 .8 52 .8

Aleurone erude Ammonium Sulfate Sephadex G-50 DEAE

I See Materials and Methods section for details of assay. Activity : a IJ.katal is that amount of enzyme that converts 1 IJ.mol of substrate S- I to product.

2

All attempts to identify the transferase in a particulate fonn were unsuccessful. The degree of purification achieved varies with a I11-fold purification for the aleurone enzyme down to a 8-fold purification for the leaf sheath enzyme through the same purification steps. The added Sephadex G-200 step increased enrichment of the leaf sheath enyzme 3-fold over the results of the DEAE step . The elution patterns for the Sephadex G-50 and G-200 purification steps showed symmetrical peaks for the enyzme activity. The final yield for the seedling and leaf sheath enzymes through the DEAE fraction ranged from 45 to 51 % whereas the DEAE fraction from the aleurone had a 53 % yield through the same step with the biggest 10ss of activity occurring at the ammonium sulfate step. The transferase catalyzed methylation of either eriodictyol to homoeriodictyol, luteolin to chrysoeriol or quercetin to isorhamnetin (Table 2) but not dihydroquercetin to the 3' -0methylated product of dihydroquercetin nor quercetin-3-0-glucoside to isorhamnetin-3-0glucoside. Neither could peonidin be detected by our methods as a product of the enzymatic methylation of cyanidin . Michaelis-Menten (KM) values were obtained by the replot method described by MAHLER and CORDES (1966). Initially aseries of curves were obtained by plotting l/v vs. l!(substrate a) at different, fixed concentrations of substrate b. A second plot of the resulting y-intercepts vs. lI(substrate b) will yield the true KM for substrate b. A similar process 456

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Table 2. Substrate specijicity of the jlavonoid 3' -O-methyltrallsferase in extracts of maize. The assay contained Hepes buffer (50 Mm, pH 8.4) + 2 mM DTE, 0.29 mM S-adenosylmethionine, 30 f.lM substrate and enzyme in 3 ml, incubation time 20 min, temperature 40 oe.

0 Substrate

Hydroxylation

KM

Ymax (f.lmoIH - l)

9.2 NR NR 5.9 62.9 NR NR 212.0 7.3

13.44

(f.lM) Quercetin Dihydroquercetin 1 Quercetin-3-glucoside Luteolin Eriodictyoll Apigenin Chrysin Caffeic Acid S-adenosylmethionine 1

3,3',4' 3,3' , 4' 3'.4' 3' , 4' 3',4' 4'

48.41 46.63

0.06 31.68

Dihydroquercetin and eriodictyol are saturated across the 2 - 3 bond.

will yield the true KM for substrate a. KM values were obtained by the same method for S-adenosylmethionine for each of the flavonoid substrates resulting in a range of values from 5 !lM for quercetin to 10 !lM for eridictyol, and the average for the three of 7.3 !lM (Table 2) . A KM of 212 !lM and a Vmax of 0 .06 !lmol h -1 for caffeic acid are also given in Table 2 along with the basic structure for the flavonoid compounds. The KM values observed for the separate substrates were quite similar for the ammonium sulfate precipitate and the Sephadex G-50 fractions. Assay of the effect of divalent metal ions of cobalt, copper , mercury, zinc, manganese and ferrous ion at 2.0 mM resulted in nearly total inhibition ofthe transferase while Ca 2 +, Mg2+, Ba2+, Ag+. and NR4 +, all at2 mMhad essentially no effecton activity. The sulfhydryl binding agents, iodoacetate and p-cbloromercuribenzoate at 2 mM, bad no effect on the transferase. Several metal chelators incIuding diethyldithiocarbamate, EDTA, cis-oxaloacetate and ex,ex'dipyridyl at 2 mM were assayed for inhibition but had no effect on the transferase. Addition of Sadenosylhomocysteine (26.9 !lM) or sinefungin (J !lM) resulted in approximately 50% inhibition of the methyltransferase reaction . Optimum temperature for the transferase reaction was determined to be 40 °C and optimum pR to be in the range of 8.0 to 8.5. Discussion

Flavonoid 3'-O-methyltransferase hase been extracted in soluble form and partially cbaracterized from maize aleurone , mature leaf sheaths and seedlings. Efforts to identify transferase activity in a particulate form met with failure. Whereas the methyltransferase utilized 30

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eitherthe flavanone, eriodictyol; the flavone, luteolin; or the flavonol, quercetin as a substrate, it was highly specific forthe 3' -position since no other reaction products could be detected. This is in agreement with the identification of only peonidin and isorhamnetin, 3'-O-methylated compounds, in organic extracts of maize. A specific effort to get methylation at the 4'-position using apigenin as the substrate and in the A-ring using chrysin, which lacks any hydroxylation in the B-ring, as the substrate met with failure. In addition the transferase would not catalyze methylation of either dihydroquercetin or anthocyanidin. Dihydroquercetin is saturated across the 2 - 3 positions similar to eriodictyol and has a hydroxyl group at the 3-position similar to quercetin. Although enzyme preparations from maize will 3-g1ucosylate a 3' -methylated flavonol, the methyltransferase discussed here would not 3'-methylate a 3-g1ucosylated flavonol. Thus in the biosynthetic sequence for flavonoids in maize, methylation would appear to occur before glucosylation in contrast to what is found in Petunia where glucosylation occurs prior to methylation (JONSSON et al. 1982). Flavonoid 3'-O-methyltransferase has been identified in a number of plant species (POUL TON 1981 ; MACHEIX and IBRAHIM 1984; JONSSON et al. 1984) and characterized to varying degrees. However, it is difficult to make comparisons of the separate properties because of the different degrees of purification reported, the different techniques utilized and different substrates tested for activity. POUL TON (1981) has compiled and compared the properties of methyltransferase activities isolated from cell cultures of parsley (P etroselinum hortense) and soybeans (Glycine max) and Tulipa anthers. The pH optima for the separate enzyme ranged from 7.6 to 9.7 with the maize enzyme falling in between at 8.0 to 8.5. Sinefungin (FERENZ et al. 1986) proved to be a much more potent inhibitor of the methyltransferase than S-adenosylhomocysteine, but in neither case was it possible to determine whether inhibition was competitive or noncompetitive after several experiments. The homocysteine in S-adenosylhomocysteine is replaced with omithine in the sinefungin structure. The KM values obtained for the separate substrates would suggest that the 15 carbon flavonoid compounds were more favorable as substrates than the cinnamic acid derivatives were. Further, flavone or the flavonol compounds were preferred over flavanone type compounds as substrates in the maize system. In addition, substrate inhibition ofthe enzyme was observed when the concentration of quercetin exceeded 50 [!M whereas such was not observed with either luteolin or eriodictyol. The maize methyltransferase described here is highly specific for the 3' -position of the flavonoid compounds, a position for which the monooxygenase from maize that catalyzes hydroxylation was recently described (LARSON and BUSSARD 1986). The structural gene for the monooxygenase was identified and reported (LARSON et al. 1986), but such information remains to be determined for the maize methyltransferase.

Acknowledgements The author expresses appreciation to Dr. E. H. COE Jr. for plant materials and to JAMES B. BUSSARD for skillful technical assistance in this study, and to Mrs. SHIRLEY KOWALEWSKI for typing this manuscript. Reading and helpful criticism of the manuscript by the aforementioned and Dr. BRIAN BAILEY are also appreciated.

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References BRADFORD, M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Bioehern. 72, 248-254 (1976). CHEN, S.-M.: Anthocyanins and their control by the C locus in maize. Ph.D. Thesis, University of Missouri-Columbia 1973. COE, E. H., Jr., NEUFFER, M. G., and HOISINGTON, D. A.: The genetics of corno In: Corn and Corn Improvement, (SPRAGUE, G. F., and DUDLY, J. W., eds.) Am. SOC. Agron., Madison 1988. FEDOROFF, N. V., FURTEK, D. B., and NELSON, O. E.: Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable controlling element activator (Ac). Proc. Natl. Acad. Sei. USA 81,3825-3829 (1984). FERENZ, H.-J., PETER, M. G., and BERG, D.: Inhibition of famesoic acid methyltransferase by sinefungin. Agric. Biol. Chem. 50, 1003-1008 (1986). GOTTLlEB, O. R.: Flavonols. In: Flavonoids. (HARBoRNE, J. B., MABRY, T. J., and MABRY, H., eds.) pp. 296-375. Academic Press, New York and London 1975. HARBoRNE, J. B.: Comparative Biochemistry of the Flavonoids. pp. 127, 186,234. Academic Press, New York and London 1967. IBRAHIM. R. K., DE LUCA, V., KHOURI, H., LATCHINIAN, L., BRISSON, L., and CHAREST, P. M.: Enzymology and compartmentation of polymethylated flavonol glucosides in Chrysosplenium americanum. Phytochem. 26, 1237-1245 (1987). JONSSON, L. M. V., AARSMAN, M. E. G., SCHRAM, A. W., and BENNINK, G. J. H.: Methylation of anthocyanins by cell-free extracts of flower buds of Petunia hybrida. Phytochem. 21, 2457 - 2459 (1982). JONSSON, L. M. V., DE VLAMING, P., WIERING, H., AARSMAN, M. E. G., and SCHRAM, A. W.: Genetic control of anthocyanin-O-methyltransferase activity in flowers of Petunia hybrida. Theor. Appl. Genet. 66, 349-355 (1983). JONSSON, L. M. V., AARSMAN, M. E. G., DE VLAMING, P., and SCHRAM, A. W.: On the origin of methyltransferase isozymes of Petunia hybrida and their role in regulation of anthocyanin methylation. Theor. Appl. Genet. 68,459-466 (1984). KIRALY, K.: Secondary metabolites. In: The Biochemistry Plant Disease (GOODMAN, R. N., KIRALY, Z., and WOOD, K. R., eds.) pp. 211-244, Univ. Mo. Press, Columbia 1986. LARSON, R. L., and BUSSARD, J. B.: Microsomal flavonoid 3'-monooxygenase from maize seedlings. Plant Physiology 80,483-486 (1986). LARSON, R. L., BUSSARD, J. B., and COE, Jr., E. H.: Gene-dependent flavonoid 3' -hydroxylation in maize. Bioehern. Genet. 24, 615-624 (1986). MACHEIX, 1.-J., and IBRAHIM, R. K.: The O-methyltransferase system of apple fmit cell suspension culture. Bioehern. Physiol. Pflanzen 179, 659-665 (1984). MAHLER, H. R., and CORDES, E. H.: Enzyme kinetics. In: Biological Chemistry, pp. 219-277, Harper and Row, New York 1966. MOROT, G. S.: Recherehes sur la coloration des vegetaux. Ann. Sei. Nat. (Bot), Paris, 3, XIII: 160-235, (1849). POULTON, J. E.: Transmethylation and demethylation reactions in the metabolism of secondary plant products. In: The Biochemistry of Plants (CONN, E. E., ed.) vol. 7, pp. 667-723, Academic Press, New York 1981. SCHÜTTE, H.-R.: V. Secondary plant substances special topics of the flavonoid metabolism. In: Progress in Botany, vol. 47, pp. 118-141, Springer-Verlag, Berlin-Heidelberg 1985. SCHWARZ-SOMMER, Z., SHEPHERD, N., TACKE, E., GIERL, A., ROHDE, W., LECLERCQ, L., MATTES, M., BERNDTGEN, R., PETERSON, P. A., and SAEDLER, H.: Influence of transposable elements on the stmcture and function of the Al gene of Zea mays. EMBO J. 6, 287-294 (1987). STYLES, E. D., and CESKA, 0.: P and R control of flavonoids in Bronze coleoptiles of maize. Can. J. Genet. Cytol. 23, 691-704 (1981a). STYLES, E. D., and CESKA, 0.: Genotypes affecting the flavonoid constituents of maize pollen. Maydica 26, 141-152 (1981 b). 30*

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Untersuchungen zum Phenylpropanstoffwechsel des Pollen. Ber. Dtsch. Bot. Ges. 81, 3-16 (1968).

WIERMANN, R.:

Received February 15, 1988; revisedform accepted October 28, 1988

Author's address: Dr. RUSSELL L. USA, 65211.

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LARSON,

304 Curtis Hall, University ofMissouri, Columbia, MO,