J. Japan. Soc. 1-Iort. Sci. 57(3) : •162-466. 1988.

Two Genes Controlling the Conversion from Flavanonol to Anthocyanidin in Sepals of Genus Aquilegia

Masao BESS110 Faculty of Agrictcltare, Ta»tagazca t',riversity, Mac/tiida, Tokyo 194

Summary In order to make clear the conversion process from flavanonol to aiithocyanidin, flavonoid components in sepals of .lguilegia flabellata with white flowers (Fw) and .1. hybrida cv. McKana's Giant with creamy white flowers (Mw) were analysed. Isovitextn and populin were identified in both Fw and Mw while leucopelargonidin existed only in Mw. F1 hybrids between Fw and \,Iw showed blue-violet sepals and F, progeny showed two-gene segregation of sepal coloration. Thus the conversion process is controlled by two genes. One recessive gene of Fw controls the reduction of flavanonol and the other recessive gene of Mw controls the dehydration of lcuco- anthocyanidin. Complementation of these two genes enables the synthesis of anthocyani- din in F1 hybrids.

anthocyanidin was synthesized. Introduction Materials and Methods Genes controlling flower color expression and the enzymes related to flavonoid metabolism Two strains of /iquilegia flabellata, one of have been studied by many researchers and which had white flowers (Fw) and the other the pathway of anthocyanidin synthesis have had blue-violet ordinal flowers (Fb), and a been well explained by Grisebach(3) and Wong strain with creamy white flowers (Mw) of .l. (11). However, the pathway from flavanonol hybricla cv. McKana's Giant were used for to anthocyanidin is still obscure, although the experiments. Ft and F2 progeny between there have been a few reports on the conver- Fw and Mw were also used for investigation sion process(4, 6, 9, 10). of heredity of flavonoids. For the analysis of Recently, from feeding experiments of 3H- flavonoids, only sepals of the flowers were used. labelled leucopelargonidin to petals of two The coloration of sepals was compared by a white mutant lines of alatthiola incana, Heller color difference meter (ND-101 DP, Nippon et al. (5) suppose(] that there were two steps in Denshoku Kohgyoh Co.). Absorption spectra the conversion of flavanonol to anthocyanidin of fresh sepals were measured by an integrat- and the two steps should he controlled by two ing sphere attachment to Shimazu UV-2-10 different genes, one controlling an enzyme apparatus. which reduces flavanonol and the other con- The sepals of Few, Mw and the F1 hybrid, trolling a dehydration of leucoanthocyanidin 40 g fresh weight each, were collected and into anthocyanidin. extracted with ca. 400 ml of boiling methanol. In order to make clear the conversion steps The methanolic extract was evaporated at re- from flavanonol to anthocyanidin, the author duced pressure, the residue was extracted with analysed flavonoid components in sepals of a hot water. The extract was shaken in a strain of Aquilc g-ia fabellata Sieb. et Zucc. separating funnel with ether and subsequently with white sepals and a strain of .1. hybricla with ethyl acetate. The ethyl acetate extract was Hort. cv. McKana's Giant with creamy white evaporated to dryness, dissolved in a small sepals, because those strains produced F1 hy- volume of methanol and stored in a refrigerator brids having blue-violet colored sepals in which at 4°C.The UV absorption spectrum in a part of Received for publication August 15, 1986. the last methanolic solution was measured. The

462 TWO GENES CONTROLLING THE CONVERSION FROM FLAVANONOL TO ANTHOCYANIDIN 463

remaining methanolic solution was examined by two-dimensional thin layer chromatography (Merck DC-Alufolien cellulose Art. 5552)using two kinds of solvents, n-butanol : acetic acid : water (6: 1 : 2, v/v) and 15 °o acetic acid. The flavonoid compounds were separated by large scale cellulose thin layer chromatography (20X20 cm) using the above solvents. The method of Mabry et al. (8) was used for iden- tification of flavonoids. Authentic samples were supplied by Dr. M. Hasegawa. The sample of leucopelargonidin used in this experiment was prepared by reduction of aromadendrin with sodium borohydride. Results and Discussion The L. a. b. value of Mw was closer to y axis than that of Fw (Fig. 1). This means that the coloration of Mw is more yellowish than that of Fw. The F, hybrid apd Fb were plotted in the fourth quadrant and showed Fig. 1. L. a. b. values of sepals measured by color ,fl : Mw, blue-violet sepals. difference meter. 0 : F•w , • : 1~b, A : Fi hybrids. The absorption spectra of fresh sepals of Fw, Mw and F, hybrids were compared in Fig. 2. Fw had a maximum absorption at 327 nm and shown in Fig. 3. Fw had two absorption peaks Mw had a maximum at 336 nm. The spectrum at 293 and 328 nm which were similar to that of Mw showed a slight shoulder at 360-370 rim of chlorogenic acid (296 and 327 rim), while which was not observed in the Fw spectrum ; Mw had similar absorption maxima to a group this shoulder might be caused by the presence of flavones (270 and 333 rim). of flavonol. F, hybrids and Fb had three ab- From the above results, it is clear that two sorption maxima (533, 575 and 628 rim) in the kinds of white flowers of Fw and Mw differ visible region, which were caused by antho- each other in tint, and the soluble compounds cyanins. in hot methanol extracts of Fw differ qualita- The absorption spectra of ethyl acetate tively and quantitatively from those of Mw. soluble portions in hot methanol extracts are In the next experiment, ethyl acetate soluble

k rim) Fig. 2. Absorption spectra of sepals measured by integrating sphere. 464 MASAO BESSHO

Fig. 4. Two dimensional chromatograms of ethyl acetate soluble portion in Mw. The spots were numbered for convenience. Fig. 3. Absorption spectra of ethyl acetate soluble portions. tive derivatives of the above hydroxy-cinnamic compounds of Few, Mw and F1 hybrids were acids. From the area of each spot using auto- separated by two-dimensional chromatography matic area meter, Fw had a low content of (Fig. 4), and the main spots on the chromato- flavonoids but a high content of organic acid grams were identified by comparison with esters. On the contrary, Mw had a high con- sample substances (Table 1). Consequently, tent of flavonoids but a low content of organic apigenin-6-C-glucoside (isovitexin) belonging acid esters. It has been reported in garden to a group of flavones, and kaempferol-7-0- snapdragon (Antirrhinum majus) that the glucoside (populin) belonging to a group of albino flowers with semitransparent white petals flavonols, were identified as flavonoids in either did not have flavonoids, although the petals Few, Mw or F1 hybrids. Caffeic acid, p-couma- had accumulated a lot of esters of p-coumaric ric acid and ferulic acid were confirmed by acid and caffeic acid(2). The white sepals of thin layer chromatography after hydrolysis, Fw had a low content of flavonoids and a high and compounds of spot Nos. 35, 40, 42, 43, content of organic acid esters like the albino 53, 54, 56 and 57 were presumed to be respec- flowers of garden snapdragon. On the contrary,

Table 1. Characteristics of compounds isolated from sepals of Fw, Mw and F1 hybrids. TWO GENES CONTROLLING THE CONVERSION FROM FLAVANONOL TO ANTHOCYANIDIN 465 creamy white sepals of Mw showed slight Table 2. Segregation of F2 progenies in sepal color. yellow coloration due to accumulation of flavone and flavonol. As populin was produced, it is certain that two kinds of white flowers had the ability to synthesize flavanonol, an intermediate of antho- cyanidin (Fig. 5). But, because of absence of Heller et al. (5) suggested that the process from anthocyanin, it is presumed that a genetic flavanonol to anthocyanidin consisted of two block exists in the process from flavanonol to steps and was controlled by two genes. anthocyanidin. Compounds in the ethyl acetate The flower color segregation in F2 progenies soluble portion were developed on cellulose from Few times Mw and the goodness of fit in TLC. The spots on this TLC were reduced 72-test are shown in Table 2. The F2 indivi- by sodium borohydride and then the plate was duals were separated into two groups by their exposed to HCl gas. Consequently, spot No. sepal colors, viz. colored and white, demon- 61 (Fig. 4) which was specific only for extract strating a good fit to a ratio of two-gene of Mw changed color to red. The reaction segregation (9: 7). suggests that spot No. 61 contains a kind of It can be concluded that two genes partici- flavanonol or leucoanthocyanidin(1). After pate in two kind of blocks in Fw and Mw. purification, the compound in spot No. 61 was One gene controlling the reduction of -CO- determined as leucopelargonidin (Table 1). radical in flavanonol to leucoanthocyanidin Thus Mw has the ability to reduce flavanonol exists in Mw, while the other gene controlling to leucoanthocyanidin. Leucoanthocyanidin the dehydration of leucoanthocyanidin exists can be chemically changed to anthocyanidin in Fw as shown in Fig. 5. The complement by dehydration. In the feeding experiment to of these two genes prompts the synthesis of petals of Matthiola incana, 3H-labelled leuco- anthocyanidin in Fl hybrids and then the pelargonidin was converted to 3H-labelled sepals of F, hybrids express blue-violet color. pelargonidin(5). The F, hybrid of Mw with Thus, the presumption of Heller et al. (5) was Few produced anthocyanidin. Therefore, it confirmed in the present experiments. appears that Fw is able to convert leucoantho- Acknowledgement cyanidin into anthocyanidin. The present re- sults mean that the blocking parts of Few and The author wishes to express gratitude to Mw are the process from flavanonol to leuco- Dr. Masao Hasegawa and Prof. Tetsuo Naka- anthocyanidin and that from leucoanthocyani- jima of Tamagawa University for their pro- din, respectively. fitable guidance. The author also thanks Dr. Sticklands and IIarrison(10) in Anti 7-7-hinu7n. M. Hasegawa for his kind supply of authentic inajus, Kho and Bennink(6) and Kho et al. samples. (7) in Petunia hybrida supposed that the con- version from flavanonol to anthocyanidin was Literature Cited controlled by one gene named Pal and Ant, 1. EIGEN, E., M. BLITZ and E. GUNSBERG. respectively. However, this process consisted 1957. The detection of same naturally occur- of two steps, one being reduction and the ing flavanone compounds on paper chromato- other, dehydration. As mentioned earlier, grams. Arch. Biochem. Biophys. 68: 501-

Fig. 5. The pathway of flavonoi d biosynth esis. 466 MASAO BESSHO

502. 7. KHO, K. F. F., A. C. BOLSMAN-LoNWEN, J. 2 GEISSINMAN,T. A. and J. B. IIARBORNE. C. VUIK and G. J. 14. BENNINK. 1977. 1955. The chemistry of flower pigmentation Anthocyanin synthesis in a white flowering in Antirrhinum inajus. The albino (--mm mutant of Petunia hybrida. Planta 135: --nn) form . Arch. Biochem. Biophys. 55: 109-118. 447-454. 8 MABRY, T. J., K. R. MARKHAM and M. B. 3 GRISEBACH, H. 1967. Biosynthesis patterns THOMAS. 1970. The synthernatic identifi- in microorganisms and higher plants. p. 1- cation of flavonoids. Springer-Verlag, New 31, John Wiley and Sons, Inc., New York. York. p. 354. 41 HARRISON, B. J. and R. G. STICKLAND. 9 SPRIBILLE, R. and G. FORKMANN. 1982. 1974. Precursors and genetic control of pig- Genetic control of chalcone synthase activity mentation. 2. Genotype analysis of pigment in flower of Antirrhinunt inajus. Phytochem. controlling genes in acyanic phenotypes in istry 21: 2231-2234. Antrrihinuna nnrjus. Heredity 33: 112-115. 10. STICKLAND, R. G. and B. J. HARRISON. T-TELLER, W., L. BRITSCII, G. FORKMANN 1974. Precursors and genetic control of pig- and 11. GRISEBACH. 1985. Leucoanthocyani- mentation. 1. Induced biosynthesis of pel- dins as intermediates in anthocyanidin bio- argonidin, cyanidin and delphinidin in Antir- synthesis in flowers of Matthiola incana R. rhinwn inajus. Heredity 33: 108-112. Br., Planta 163: 191-196. 11. WONG, E. 1976. Biosynthesis of flavonoids. 6 KHO, K. F. F. and G. J. H. BENNINK. 1975. p. 464-526. In: T.W. Goodwin(ed.) Chemistry Anthocyanin synthesis in a white flowering and biochemistry of plant pigments (2nd ed. mutant of Petuniaa hybrida by a complementa- Vol. 1) Academic Press, London. tion technique. Planta 127: 271-279.

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