Biosynthesis of 8-Substituted Flavonols in Relation to Ontogeny of Flower Colour in Lotus corniculatus

Biochem. Physiol. Pflanzen 181, 199-206 (1986) VEB Gustav Fischer Verlag J ena

Biosynthesis of 8-Substituted Flavonols in Relation to Ontogeny of Flower Colour in Lotus corniculatus MAURICE JAyl) and RAGAI K. IBRAHIM2) 1) Laboratoire de Phytochimie, Universite Claude-Bernard, Lyon I, Villeurbanne, France; 2) Plant Biochemistry Laboratory, Department of Biology, Concordia University, }Iontreal, Quebec, Canada Key Term Index: Leguminosae, 8-substituted flavonols, biosynthesis, O-methylation, flower colour; Lotus corniculatus

Summary 1. The chemical basis for the development of yellow colour in Lotus flower petals involves the presence of flavonoid and carotenoid pigments, both of which increase by 3- and 10-fold, respective!? during flower growth. 2. The flavonoid profile of flower buds consists of the hydroxylated flavonols kaempferol, quercetin and small amounts of gossypetin (8-hydroxyquercetin), as the predominant flavonoids. The first appearance of the yellow colour in flower petals is concomitant with the accumulation of large amounts of gossypetin, corniculatusin (8-methoxyquercetin) as well as much smaller amounts of sexangularetin (8-methoxykaempferol) until anthesis. The two former 8-substituted flavonols are mostly responsible for the intensity of yellow colour during flower development. 3. These results are consistent with the activity of O-methyltransferase observed against 8-hydroxykaempferol and 8-hydroxyquercetin, as well as the efficient incorporation of [2-14 C]-cinnamic acid into the characteristic flavonoids of flower buds and pre-anthesis flowers. 4. A pathway for the biosynthesis and metabolism of 8-substituted flavonols in Lotus flowers is proposed and discussed.

Introduction

The yellow colour which characterizes the flower petals of Lotus corniculatus (Leguminosae) (JAY et al. 1978) and contributes to insect pollination (F AEGRI and VAN DER PIJL 1971; HARBORNE 1977) is due to the presence of two classes of natural pigments: the carotenoids and flavoncids, both of which were found to occur in this material. Previous phytochemical studies (JAY et al. 1978) have indicated that the flavonoids associated with the yellow colour of Lotus flowers consisted of 8-substituted flavonols which are, in decreasing order: gossypetin (8-hydroxyquercetin), corniculatusin (8methoxyquercetin), sexangularetin (8-methoxykaempferol) and trace amounts of limocitrin (8-methoxy-3' -methyl quercetin) (Fig. 1). More recently, we demonstrated the existence, in flower buds of an O-methyltransferase activity which was specific for the 8-position of 8-hydroxyquercetin and

200

M. JAY and R. K. IBRAHIM

H H

Fig. 1. Structural formulae of the flavonoids of Lotus flowers.

Rl

R2

R OR OMe R OR OMe

R R R OR OR OR

Kaempferol Rerbacetin Sexangularetin Quercetin Gossypetin Corniculatusin

8-hydroxykaempferol (JAY et al. 1983). This novel enzyme, 8-O-methyltransferase (EC 6.2.1.-), was purified from flower buds and its properties and kinetic mechanism were studied (JAY et al. 1985). Furthermore, it was observed that the highest 8-O-methyltransferase activity was predominantly associated with the young stages of flower development and even before the appearance of flower colour. It was considered important, therefore, to undertake a comparative study of the phytochemical basis of colour development and 8-O-methyltransferase activity involved in flavonoid biosynthesis during the ontogeny of Lotus flowers. Materials and Methods Plant material Lotus corniculatus flowers, of different growth stage\l (Table 1), were obtained from greenhouse raised plants and the Agricultural Experimental Farm of MacDonald College, McGill University, courtesy of Dr. B. COULMAN. The flowers were collected in liquid nitrogen until brought to the laboratory for analysis. Chemicals S-Adenosyl-L-[14CH3] methionine (60 mCi/mmol) and [2-14 C]-cinnamic acid (3 mCi/mmol) were purchased from Amersham, Oakville, Ontario and Sephadex G-25 from Pharmacia Fine Chemicals, Uppsala, Sweden. 8-Substituted flavonols were from our laboratory collection, except for 8-methoxy3'-methylquercetin which was a gift from Prof. M. No GRADY, Budapest, Hungary. Polyamid DC 6.6 was obtained from Macherey NAGEL, Duren, Germany. All other chemicals and solvents were of analytical grade reagents. Analysis of carotenoid and flavonoid pigments (a) Carotenoids: Fresh flowers of different growth stages were ground in liquid nitrogen, extracted with acetone and centrifuged. The acetone extract was partitioned against petroleum ether

Flavonols and Flower Colour in Lotus corniculatus

201

Table 1. Characteristics of the flowering stages of Lotus corniculatus. Flowering stage

mg dry weight! flower

per flower

per mg dry weight

A B

0.46 1.38

9.2 16.5

31.5 24.4

C

2.40

21.1

8.8

D E

4.50 7.00

31.9 51.1

7.1 7.3

Protein content (pg)

Flower description

Appearance of flowering meristem Flower bud visible, totally enclosed in calyx Emergence of petals, appearance of yellow colour Folded petals emerge fully (pre-anthesis) Open petals (anthesis)

(b.p. 40-60°C). Absorbance of the ether layer was measured in an Unicam SP-8-100 spectrophotometer at 430 nm. The carotenoid content was calculated as pg fJ-carotene!mg dry weight or per flower, using a molar extinction coefficient of 134,000. (b) Total flavonoids: Fresh flowers were hydrolyzed with 2N HCl in a boiling water bath for 40 min. The acid hydrolyzates were rapidly cooled and exhaustively extracted with a mixture of benzene - ethyl acetate (1: 1, v!v). The organic phase was evaporated to dryness, dissolved in a known volume of EtOH and its absorbance was measured at 428 nm in the presence of AlCla• Total flavonoids were calculated as ftg!mg dry weight or per flower, using an average molar extinction coefficient of 21,000. (c) In divid ual fla vonoids: Ethanolic extracts of the acid hydrolyzates of flowers were chromatographed, together with reference compounds, on Polyamid DC 6.6 TLC plates. The latter were developed twice using benzene - methyl ethyl ketone - methanol (70: 15: 15, v!v!v) as solvent system. Flavonoid bands were visualized in UV-light (366 nm), scraped off the plates, then eluted with MeOH. Absorbance of the methanolic extracts was determined at the Ama,. of each compound and the amount of each flavonoid was calculated using its molar extinction coefficient. Analyses were performed in duplicates and all experiments were repeated at least twice.

Labelling experiments Excised flowers from growth stages B, C and D (Table 1) were vacuum infiltrated and floated on an aqueous solution of [2_14C]- cinnamic acid, containing 10 nmol of the cold phenolic precursor for 4 h in the light at room temperature. At the end of the metabolic period, the flowers were thorougly rinsed with water and processed for the isolation and quantitation of individual flavonoids, as described above. After measuring their absorbance, the methanolic extracts were evaporated to dryness, dissolved in 100 pI of benzene - ethyl acetate, and their radioactivity was determined by liquid scintillation in a toluene-based fluid. The specific activities of individual flavonoids were expressed in dpm!pmole. Extraction and assay of 8-0-methyltransferase Crude protein extracts of fresh flowers were prepared as was described by JAY et al. (1983). The latter were stirred with Dowex 1 x 2 for 20 min, then centrifuged. The supernatants were desalted on Sephadex G-25 which was equilibrated with 25 mmol!l imidazole buffer, pH 7.4, containing 14 mmol!l 2-mercaptoethanol and 10 % glycerol. The protein was eluted with the same buffer and was used directly for enzyme assays. The protein content was determined using the Bio-Rad method (BRADFORD 1976). The standard assay misture consisted of 10 ftl of 5 pmol!l solution of the flavonoid substrate (in 50 % DMSO); 10 pI of S-adenosyl-L-methionine (containing 0.05 ftCi); 10 ftl of 5 mmol!l MgCl 2 ; 14 Biochem. PhysioI. Pflanzen, Bd.181

202

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JAY

and R. K.

IBRAHIM

40,al of 0.2 M phosphate buffer, pH 7.5 containing 14 mmol/l 2-mercaptoethanol and 70,al of enzyme protein. The reaction was allowed to proceed for 60 min at 30°C and was terminated by the addition of 10,al of 6 N HCl. The reaction products were extracted with 250,al of 1: 1 mixture fo benzene ethyl acetate and an: aliquot of the organic phase was counted for radioactivity in a toluene-based liquid scintillation fluid.

Results

Chemical basis of yellow colour in Lotus flowers The analysis of both carotenoid and flavonoid pigments was carried out on flowers with first appearance of the yellow colour (stage C, Table 1) and on flowers at anthesis (stage E). The results obtained (Table 2) show that there was a significant increase in the amounts of carotenoid and flavonoid pigments during flower development, especially when calculated at the organ level (fig per flower). There was a 10-fold increase in the amount of carotenoids during flower maturation (stages C to E) as compared with a 3-fold increase in total flavonoids. Of these, 8-hydroxy- and 8-methoxyflavonols exhibited 7- and 4-fold increase during the same flowering period (Table 2). Furthermore when flavonoids were calculated on dry weight basis, only the 8-hydroxyflavonols showed a marked increase. These results indicate that, apart from the presence of carotenoids, 8-substituted flavonols significantly contribute to the development and intensity of the yellow colour in Lotus flowers. Table 2. Analyses of carotenoids and flavonoids in two floral stages of Lotus corniculatus Floral stage1 )

Carotenoids

C E

1.88 18.3

C

0.94 2.61

E

Flavonoids simple

8-hydroxy

8-methoxy

total

,ag per flower 19.0 45.5

5.2 37.8

6.0 23.8

30.2 107.1

,ag per mg dry weight 9.5 2.6 6.5 5.4

3.0 3.4

15.1 15.3

1) As described in Table 1.

Changes in flavonoid composition during flower development Flavonoid compounds were analyzed during different stages of flower development and their content was expressed at the organ (Fig. 2 A) and dry weight (Fig. 2 B) level. Except for kaempferol, there exists an orderly increase of total and individual flavonoids during flower ontogeny (Fig. 2A). Quercetin, gossypetin and its 8-methoxy derivatives represent the major flavonoid compounds in stages C to D. A similar behavior was observed for the same floral stages when flavonoids were expressed on dry weight basis (Fig. 2 B), though with a marked decline in stage E. Kaempferol, on the other hand, was found to accumulate in significantly large amounts in stages A and B, then

203

Flavonols and Flower Colour in Lotus corniculatus C

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Fig. 2. Flavonoid composition of Lotus flowers during development, as determineed per flower (A) and per mg dry weight (B). Total flavonoids (-.-); kaempferol (--e- -); 8-methoxykaempferol (- -0 - -); quercetin (-e-); 8-hydroquercetin (8-methoxyquercetin (- - 0- -).

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Fig. 3. O-Methyltrans(erase activity during flower development as determined per flower (A) and per mg protein/min (B), using quercetin (-e-), 8-hydroxyquercetin ( - e-) and 8-hydroxykaempferol

(- -e- -) as substrates.

it declined rapidly thereafter (Fig. 2 B). In contrast with 8-methoxyquercetin, the kaempferol analog was found to occur in small quantities during all stages of flower development (Fig. 2A, B).

O-Methylation of flavonoids during flower development The O-methylating activity of flowers at different stages of development was tested against quercetin, 8-hydroxykaempferol and 8-hydroxyquercetin as substrates, and was expressed as dpm per mg protein (Fig. 3A) or per flower (Fig. 3B). The results show that, in contrast with quercetin, both 8-hydroxy flavonols undergo active O-methylation, mostly at position 8, which increased consistently during flower development (Fig.3A, B). O-Methylation of quercetin, on the other hand, occurs at positions 3 and/or 3' (JAY et al. 1983) and does not seem to contribute to flower colour. While the 14*

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Table 3. Incorporation of [2- 14 CJ-cinnamic acid into flavonoids of different floral stages1 ) Flavonoid compound

Gossypetin Quercetin 8-Methoxyquercetin Kaempferol 8-Methoxykaempferol

Specific activity (1()4 . dpm/,umol) B

C

D

n.d. 2 ) 8.7 9.9 2.0 31.7

4.4 2.0 3.2 1.5 15.6

2.1 6.3 8.4 3.1 20.8

1) See Table 1 for description of floral stages B to D 2) Not determined

phytochemical analysis indicates the accumulation of 8-methoxyquercetin in substantial amounts in the flowering stages C to E (Fig. 2 A, B), there is no corresponding accumulation of its kaempferol analog, which may be due to its rapid turnover.

Biosynthesis of Lotus flower flavonoids The biosynthesis of flavonoids from [2-14C]-cinnamic acid was studied in three floral stages which accumulate high levels of flavonoids, namely stages B, C and D (Fig. 2A, B). The results obtained (Table 3) show that the label of the phenolic precursor was efficiently incorporated into all flavonoid compounds. This indicates that flower buds continue to synthesize their characteristic flavonoids until pre-anthesis. It is interesting to note that the specific activities of most flavonoid compounds were higher in flower buds than in either of the floral stages C or D (Table 3). This may be due to the fairly large pool of flavonoids in the flowers of both stages (Fig. 2) which results in the dilution of label. However, a relationship seems to exist between the sequential biosynthesis of the major flavonoids and flower ontogeny; so that quercetin reaches its highest specific activity at stage B, gossypetin at C and corniculatusin at D. A comparison between the specific activities of 8-methoxykaempferol and those of the quercetin analog, during flower development, indicates that 8-0-methylation is in favor of herb acetin (8-hydroxykaempferol) than gossypetin; although 8-methoxyquercetin represents the major methylated flavonol in Lotus flowers. Discussion

The results presented here clearly indicate that both carotenoid and flavonoid pigments contribute to the yellow colour during the ontogeny of Lotus flowers. Both total and individual flavonoids increase in amount during flower development. Whereas kaempferol and quercetin predominate in flower buds (stages A and B) and continue to increase in amount thereafter, both 8-hydroxyquercetin and its methyl derivative and, to a lesser extent, the kaempferol analog accumulate in large amounts in the floral stages C to E. The accumulation of the two major flavonoids coincides with the first appearance of the yellow colour in flower petals (Table 1). These results are in agreement with the high

Flavonols and Flower Colour in Lotus corniculatus

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K . .1.0(> 8-0H-K 2..,.. 1---18-0Me-K I _____ 1 Fig. 4. A proposed pathway for the biosynthesis of 8-substituted flavonols in Lotus flowers. K, kaempferol; Q, quercetin; 1, 8-hydroxylation; 2, 8-0-methylation; 3, 3'-hydroxylation. Com· pounds inside rectangles accumulate in large (solid lines) or small (dotted lines) amounts.

O-methylating activity observed with 8-hydroxykaempferol and 8-hydroxyquercetin as substrates (Fig. 3), as well as the efficient incorporation of label of cinnamic acid into their O-methylated products (Table 3), as compared gossypetin occurs as a natural flower pigment that is subsequently O-methylated to corniculatusin, its kaempferol analog, herb acetin was notable by its absence. We were not able to detect the latter compound at any stage of flower development, despite the accumulation of its precursor, kaempferol in large amount in flower buds (Fig. 2 B). This seems to suggest a two-fold metabolic pathway (Fig. 4) of kaempferol during flower ontogeny: (1) its hydroxylation at position 8 to form herbacetin, which seems to be tightly coupled with an active 8-0methyltransferase to give sexangularetin (Fig. 3). The fact that the latter accumulates only in small amounts during flower development (Fig. 2), despite its efficient labelling from cinnamic acid (Table 3), suggests its further metabolism by 3' -hydroxylation to corniculatusin, one of the major flavonoid constituents responsible for the yellow colour of Lotus petals; (2) dihydrokaempferol and kaempferol may also undergo 3' -hydroxylation (ROBERTS and VAUGHAN 1971; BRITSCH et al. 1981) to quercetin, which is then further hydroxylated and O-methylated as is shown in Fig. 4. Both of these metabolic pathways may result in diverting the kaempferol pool towards the accumulation of gossypetin and corniculatusin which are the dominant flavonoids during flower ontogeny, and are responsible for the intensity of yellow colour. So far, nothing is known of the enzymology of 8-hydroxylation of either kaempferol or quercetin, nor whether the 3' -hydroxylase accepts any of the substituted derivatives of these two flavonols. Acknowledgements This work was supported in part by operating grants from the Natural Sciences and Engineering Research Counsil of Canada and the Department of Higher Education, Government of Quebec, as well as a NATO travel grant to M.J.

References BRADFORD, M. M.: 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 (1976). BRITSCH, L., HELLER, W., and GRISEBACH, H.: Conversion of flavanone to flavone, dihydroflavonol and flavonol with an enzyme system from cell cultures of parsley. Z. Naturforsch. SSe, 742-750 (1981).

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M. JAY and R. K. IBRAHIM, Flavonols and Flower Colour in Lotus corniculatus

FAEGRI, K., and VAN DER PIJL, L.: Principles of Pollination Ecology. 2nd edition, Pergamon Press, London 1971. HARBORNE, J. B.: Introduction to Ecological Biochemistry. Academic Press, London 1977. JAY, M., HASAN, A., VOIRIN, B., and VIRICEL, M. R.: Les flavonoides du Lotus corniculatus. Phytochemistry 17, 827-829 (1978). JAY, M., DE LUCA, V., and IBRAHIM, R. K.: Meta-Methylation of flavonol rings A (8-) and B (3'-) is catalyzed by two distinct O-methyltransferases in Lotus corniculatus. Z. Naturforsch. asc, 413 -417 (1983). JAY, M., DE LUCA, V., and IBRAHIM, R. K.: Purification, properties and kinetic mechanism of Sadenosyl-L-methionine: 8-hydroxyflavonol 8-0-methyltransferase from Lotus corniculatus. Eur. J. Biochem. (in press). ROBERTS, R. J., and VAUGHAN, P.F. T.: Hydroxylation of kaempferol, dihydrokaempferol and naringenin by a phenolase preparation from spinach beet. Phytochemistry 10, 2649-2652 (1971).

Received October 8,1985; accepted November 4, 1985 Author's addresses: MAURICE JAY, Laboratoire de Phytochimie, Universite Claude-Bernard, Lyon I, F - 69622 Villeurbanne, France; RAGAI K. IBRAHIM, Plant Biochemistry Laboratory, Department of Biology, Concordia University, Montreal, Quebec, Canada H3G 1M8.

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