Planta (BEE.) 127, 271-279 (1975) 9 by Springer-Verlag 1975
Anthocyanin Synthesis in a White Flowering Mutant of Petunia hybrida by a Complementation Technique K. F. F. Kho and G. J. H. Bennink Department of Plant Physiology, University of Amsterdam, IJdijk 26 Amsterdam, The Netherlands I-I. Wiering Institute of Genetics, University of Amsterdam, Kruislaan 318, Amsterdam, The Netherlands t~eceived 20 l~Iay; accepted 8 August 1975 Summary. In vitro cultured corollas of the white flowering mutant W18 of Petunia hybrida synthesize anthocyanins after administration of a bud extract of the white fl~wering mutant W19. Dihydroquercetin-7-glucoside was identified as the major metabolite from W19, which is converted into anthocyanins in W18. This result supports the genetic evidences with regards to the biosynthetic pathway leading to anthocyanins in this plant. W18 was not able to convert flavanones or flavonols into anthocyanins. This type of experiments may be of value for elucidation of certain aspects of flavonoid biosynthesis. Introduction Genetic studies of colour development in flowers of Petunia hybrida Hort. indicate that at least eight genes are involved in flavonoid biosynthesis, as is shown in Fig. 1 (Wiering, 1974). The anthocyanin genes (Anl, An2 and An3) have a general effect on the biosynthesis of anthocyanins in the flower limb. If one of these genes is present in the homozygous recessive condition, anthocyaniu synthesis is suppressed, resulting in a white or very weakly colourcd flower. Dominant alleles of An3 and F1 are required for the synthesis of flavonols (kaempferol and quercetin, of. Fig. 1). The other genes (Htl, Ht2, Hfl and Hf2 determine the hydroxylation pattern of the aryl side chain of the flavonoid molecule. Anthocyanin and flavonol synthesis are both affected by the genes An3 and H t l , which indicates that both types of compounds are s3mthesized via common precursors. The gene H t l must be dominant to obtain the ortho dihydroxy substitution pattern of the aryl side chain, leading to cyanidin and quercetin. If the gene is homozygous recessive, anthocyanin synthesis is suppressed. I n this case mainly kaempferol is accumulated. Pelargonidin (ef. Fig. 1) has seldom been found in Petunia flouters and than in very minute traces. Its absence can be explained by assuming that only precursors bearing two or three hydroxyl groups in the side chain can be converted into anthocyanins. White flowering mutants may be of value in elucidating of pathways of flavonoid biosynthesis Geissman et al., 1954; Wong and Francis, 1968). When the biosynthetic pathway to anthocyanins is Mocked at a single reaction step, one may expect : (i) that the intermediate formed just before the block is accumulated
272
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W
J
An3 X FI. ~
-
-
H O ~ O H OH O kaempfero[
OH Htl
Ht2 for tube OH H 0 ~ O H
I An1 Y
• Hfl for
Z(OH) [ /~ H O ~ O H OH
OH
HtI pe[argonidin Ht2 for tube OH Z(0H)2 ---* H e . O H
Limb /
OH 0
quercetin
OH
|
OH cyanidin
OH
OH -~ H 0 ~ / 0 ~ 0 H -y -y \ OH 0
myricetin
Z (OH)3---" HO~ O
+'~H
oH OH
~'OH OH de[phinidin
OH
Fig. 1. Relation between 8 genes involved in the biosynthesis of flavonoids in Petunia hybrida ttort. (Wiering, 1974). W, X, Y and Z are precursors of unknown nature, postulated on genetic evidence. Anl, An2 and An3 represent genes involved in the anthocyanin pathway. The genes An3 and F1 participate in the flavonol synthesis (kaempferol, quereetin and myrieetin). Htl, Ht2, I-Ifl and HI2 determine the hydroxylation pattern of the aryl side chain of the flavonoid molecule and (ii) that intermediates normally occurring after this reaction step, when fed to the mutant plant, will be converted into anthoeyanins. This principle is frequently used in studies on primary and secondary metabolism in bacteria and fungi and has proved to be an extremely useful method for elucidating biosynthetic pathways (Wagner and Mitchell, 1964). However, in higher plants this method is not easily applicable, due to technical problems. Reddy and Coe (1962) pressed together pieces of aleurone tissue from anthocyanin-defieient mutants of maize. When pieces of two different blocked mutants were combined only one of them became eoloured and from these complementation experiments the sequence of gene action could be established. The availability of genetically defined mutants of Petunia hybrida with respect to anthocyanin synthesis prompted us to use this technique to elucidate the sequence of reactions involved in anthocyanin biosynthesis. We tried to complement two white flowering mutants, homozygous recessive for different anthocyanin genes and in order to suppress flavonol synthesis homozygous recessive for the flavonol gene FI.
Anthocyanin Synthesis in a M u t a n t of Petunia hybrida
273
Materials and Methods
Plant Material. Clones of the white flowering m u t a n t s W18 (homozygous recessive for An3) a n d W19 (homozygous recessive for An1) of Petunia hybrida were cultivated in the greenhouse.
In Vitro Culture el Corollas. From freshly gathered buds the sepals were removed. The remaining p a r t was sm'face sterilized with 1% calcium hypochlorite for 5 rain and rinsed in sterile distilled water. Subsequently they were cut transversely just above the anthers. The upper p a r t of the corollas, largely existing of the flower limb, was incised lengthwise and unfolded carefully. They were submerged in 70% (v/v) ethanol for 10 s and rinsed twice in sterile distilled water. After this t r e a t m e n t the corollas were incubated b y floating them, with the inner epidermis below, on 3 ml sterile culture medium in petri dishes (60 • 15 ram) to which was added 1 ml of extract. Dishes were placed under constant illumination (Philips TL 33, 5000 lux) a t 24 ~ for 72 h. Culture Medium. One litre of aqueous medium consisted of 1.29 g KH~P04, 0.087 g K2HPO 4, 0.125g Ca(H2PO4)~-H20, 0.125g MgSO4.7H20, 0.010g ferric citrate, 0.500g asparagin, 30 g sucrose and 0.5 ml micronutrient solution. The p H was adjusted to 5.5 + 0.1. Mieronutrient solution consisted of 2.86 g H3BOs, 1.81 g 5[nC12 94H20, 0.220 g ZnSO 4 97H20, 0.080 g CuSO 4 95 H20, 0.070 g Na2MoO 4 92 H20, dissolved in 1 litre water. Extracts o/ Flower Buds. From 100 freshly gathered flower buds the limb parts (fresh weight a b o u t 10 g) were homogenized in 50 ml acetone with a Biihler homogenizer. The homogcnate was centrifuged and the sediment re-extracted with 50 ml acetone. The combined supernatants were filtered and the acetone was removed under reduced pressure a t 30 ~ The residue was t a k e n up in 10 ml methanol, filtered and the filtrate was dried under reduced pressure. The residue was t a k e n up ill 10 ml culture medium and centrifuged at 38000 • g a t 4 ~ for 1 h. The s u p e r n a t a n t was sterilized b y membrane filtration (Sartorius SM 11307, pore size 0.2~zm). Anthocyanin Content el Corollas. After incubation for 72 h the corollas were rinsed in distilled water and dried with filter paper. Pigments were extracted b y shaking each corolla with 3 ml methanol-hydrochloric acid 0.1% for 1 h. Light absorption of the solution was measured in a Zeiss DMR 21 spectrophotometer in 1 cm cuvettcs at 530 nm. Anthocyanin content was expressed as the absorbance at 530 n m of a solution containing the pigments of one corolla in 3 ml solvent (A530/corolla, Klein and Hagen, 1961). Fractionation o/a Bud Extract. The methanol fraction obtained from the acetone extract of 200 flower buds of m u t a n t W19 was concentrated to a few ml a n d fractionated on a column (20• 1.6 cm) of Sephadex LH-20 equilibrated with methanol (Johnston et al., 1968). By eluting with methanol 12 fractions of 9.3 ml were obtained. A 5-ml sample of each fraction was dried under reduced pressure a n d tested for its activity on W18 corollas. The fractionation procedure was repeated several times to obtain sufficient q u a n t i t y of the active compound. The active fractions were purified b y semipreparative chromatography on W h a t m a n 3 paper using propanol-2-water 4:1 (v/v) as solvent. The position of the major compound, showing a reddish brown colour after spraying with the nitrite-tungstatc reagent of Bhatia et al. (1973) was determined a t a smM] strip of the chromatogram. The corresponding p a r t of the rest of this chromatogram was eluted with methanol. Aglycone and Sugar Identi/ication. 1 ml of a methanolic solution containing a few mg of the chromatographically pure compound was hydrolyzed b y heating with 1 ml 2 N HC1 under nitrogen in a fused tube in a boiling water b a t h for 15 min. E t h e r extraction of the hydrolysate yielded the aglycone, which was identified b y co-chromatography with appropriate standards on t h i n layers of cellulose (Merck, Darmstadt, Germany) using as solvents butanol-acetic acid-water (4:1 : 5, v/v/v, upper phase) (BAW) and chloroform-acetic acid-water (50: 45 : 5, v/v/v) (CAW). The extracted water phase was neutralized b y shaking with a solution of 20 % (v/v) di-n octylmethylamine in chloroform and washed with chloroform. After concentrating the solution under reduced pressure the sugar was identified b y co-chromatography with appropriate standards on thin layers of cellulose with the solvent systems b u t a n o l - e t h a n o l - w a t e r (40:10:22, v/v/v), (BEW) and b u t a n o l - b e n z e n e - p y r i d i n e water (50:10:30:24, v/v/v/v), (BBPW). Sugars were made visible b y spraying with anilin p h t a l a t e spray reagent (Merck, Darmstadt, Germany) followed b y heating for 5 min at 110 ~
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Oxydation o/ Dihydro/lavonolglycosides to Flavonolglycosides. A few mg dihydroflavonolglycoside was heated with 1 ml 20% (g/v) aqueous sodium metabisulfite solution for 2 h in a boiling water bath. The flavonolglycoside was isolated by polyamide chromatography according to Birkofer et al. (1962) and identified by co-chromatography with standards on thin layers of cellulose, using as solvent system 15% aqueous acetic acid. Quercetin-7-glucoside was prepared by partial hydrolysis of quereetin-3-sophoroside-7-glucoside, isolated from Petunia hybrida according to Birkofer et al. (1962).
Results 1. Stages o/Flower Bud Development in situ The development of flower buds of Petunia hybrida is presented in Table 1. During the stages II, I I I and IV the corolla elongates rapidly. At the end of stage IV the flower attains its final length and unfolds itself. Anthocyanin synthesis in normally pigmented Petunia flowers is perceptible at the beginning of stage IV.
Table 1. Developmental stages of Petunia flower buds in *itu. Bud length is defined as the distance between the flower receptable and the top of the corolla. The bud stages I, II, III, IV and V are chosen ~rbitrar.dy Bud stage
Bud length (ram)
I II III IV V
0-5 5-15 15-35 35-open flower open flower
2. Organ Culture Dissected corollas of the stages II, I I I and IV grew rapidly when cultured in vitro. The incubation time needed for complete development of corollas in stage IV varied from 12-24 h, for corollas in stage I I and I I I it appeared to be about 72h. The development of corollas from both anthoeyanin containing and white flowering genotypes did not differ significantly from the development in situ. I n vitro anthocyanin synthesis in red and magenta flowers after 72 h was comparable with anthoeyanin synthesis in intact open flowers. Corollas of white flowering mutants cultured in vitro did not synthesize anthoeyanins. In contrast to the experience of Hess (1967), it proved to be essential to work under sterile conditions, since fungal infections prevent further growth of the corollas. Incubations under constant illumination and in complete darkness appeared to be equivalent with regard to anthocyanin synthesis. 3. Complementation Experiments Several attempts were made to achieve anthocyanin synthesis in corollas of white flowering mutants after addition of bud extracts of complementary mutants. A weak reddish pigmentation was observed in corollas of mutant W18 after adding an extract of flower buds of mutant W19. As is shown in Table 2, only the combination of W18 as acceptor and W19 as donor resulted in anthoeyanin
Anthoeyanin Synthesis in a Mutant of Petunia hybrida
275
Table 2. Anthocyanin content in corollas of white flowering mutants W18 and W19 after supplying bud extracts. 1 ml of bud extract wa.s added to 3 mI culture medium. As a control 4 ml culture medium was used. Absorba,nce was measured after an incubation period of 72 h. Values represent the mean of l0 corollas ~ standard deviation Accepter
Donor
Anthocyanin content per corolla (A530)
W18 W18 W18 W19 W19
control W18 W19 control W18
0.09 d_0.02 0.07 j: 0.01 0.42 • 0.09 0.05 • 0.01 0.05•
synthesis. The reverse experiment did not give any pigmel~tation. The red pigments occurring in the corolla of W l 8 after addition of Wt9 extract went into solution by macerating with methanol-tiC1 0.1% and exhibited an absorption maximum at 530 nm, indicating that this pigmentation was a result of anthoeyanin synthesis. Pigment distribution in the corollas was fairly irregular. In the presence of W19 extract eoloured spots of higher intensity were found around places where the tissue had been accidentally damaged, whereas other parts remained practically eolourless. In general the pigmentation was much weaker than in intact flowers. Corollas maturated in absense of the active extract did never show any pigmentation, neither after damage of the tissue. In order to determine the optimal bud stages for donor and accepter, various combinations of bud sizes were investigated. The optimal bud stage for donor material proved to be stage IV and for accepter material stage III. The most active extracts were those made from the limb part of the corolla, extracts of the tube part and of the green parts of the flower being inactive. The pigmentation of the accepter was limited to the inner epidermis of the limb part of the corolla. In pigmented flowers the anthoeyanins are also localized in the same epidermis. From these results it can be concluded that the gene An3 regulates an earlier step in the biosynthetic pathway than the gene Anl.
4. Isolation and Identi/ication o/an active Precursor ]rein Flower Buds o/Mutant W19 The active extract from W19 buds was subjected to gel chromatography on Sephadex LH-20. The fractions were examined for their effect on anthoeyanin synthesis in corollas of WlS. The results are given in Fig. 2. The combined activity of fractions 6 and 7 accounted for the total activity before fraetionation. Fraction 7 was further separated by paper chromatography. A biologically active compound (RI = 0.40), giving a reddish bream eolour with Bhatia's reagent was eluted from the paper. Acid hydrolysis proved that the compound was a glycoside. The aglycone was extracted with ether and identified as dihydroquercetin (3,3',4',5,7pentahydroxyflavanone) by thin-layer chromatography (Table 3). Table 3 also shows that the remaining water phase contained only glucose. Therefore the active compound was a glucoside of dihydroquercetin. The methanol spectrum of this compound (2max = 287 nm) did not shift after addition of sodium acetate. This indicates that a glucose residue is attached to dihydroqnercetin at position 7
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K.F.F.
A 530 TOTAL CONTROL EXTRACT
0.300
0.200
0.100
0 ~
2
3
4
5
6
7
8
9
10
11
fraction number
:Fig. 2. Column c h r o m a t o g r a p h y on S e p h a d e x LH-20 of e x t r a c t of flower b u d s of m u t a n t W19. A n t h o c y a n i n synthesis occurring in corollas of m u t a n t W18 after adding different chromatographic fractions oi e x t r a c t f r o m W19 was expressed as A530/corolla. The figure also shows the activity of t h e e x t r a c t before fractionation (total extract) a n d the a c t i v i t y of the contro], which was j u s t culture m e d i u m
Table 3. Identification of an active precursor for a n t h o c y a n i n biosynthesis, isolated f r o m flower b u d s of m u t a n t W19 Compound
R f values on cellulose TLC plates
Active c o m p o u n d Aglycone ~ Dihydrokaempferol Dihydroquercetin Sugar a Glucose Galactose Quercetin glucoside b Quercetin-7-glucoside Quercetin-3-glucoside Quercetin-4'-glucoside
BAW
CAW
15 % acetic acid
BEW
BBPW
0.55 0.87 0.93 0.87 ---0.27 0.26 0.69 0.58
0.06 0.24 0.49 0.26 ---0.08 0.08 0.23 0.11
-------0.07 0.07 0.33 0.09
----0.23 0.23 0.20 -----
----0.18 0.18 0.15 -----
a After hydrolysis of the active compound. b O b t a i n e d b y bisulfate o x y d a t i o n of the active c o m p o u n d .
(Mabry
et al.,
1970).
Bisulfite
oxydation
of t he
active
compound
flavonolglucoside, which was identified as quercetin-7-glucoside
yielded
t h e s e r e s u l t s i t c a n b e c o n c l u d e d t h a t t h e a c t i v e c o m p o u n d is d i h y d r o q u e r c e t i n - 7 glucoside.
a
( T a b l e 3). F r o m
Anthocyanin Synthesis in a Mutant of Petunia hybrida
277
5. E]fect o/various Flavonoids on the Anthoeyanin Synthesis in the Mutants W18 and W19 In order to determine the specificity of the induced anthocyanin synthesis corollas of W18 were maturated in culture medium after addition of pure flavonoids. Flavanones (naringenin, naringin) and flavonols (kaempferol, quercetin) exhibited no activity. From the dihydroflavonols we investigated (dihydrokaempferol, dihydroquercetin and dihydroquercetin-7-glucoside) only dihydroquercetin and dihydroquercetin-7-glucoside were active (cf. Table 4.). None of these compounds were able to induce any pigmentation in corollas of W19. Table 4. Anthocyanin content, expressed as A530 in corollas of W18 after incubation for 72 h with various flavonoids. Concentration of the compounds in the culture medium (4 ml) was 0.5 • 10-a 2VI.As a control 4 ml culture medium was used. Values represent the mean of 10 corollas -~ standard deviation Compound
Anthocyanin content per corolla (A530)
Dihydroquercetin-7-glucosidea Dihydroquercetin Dihydrokaempferol Naringenin b Naringin Kaempferol b Quercetin b Control
0.24 ~ 0.03 0.36 :t:0.06 0.07 ~ 0.02 0.05 -l-0.01 0.07 =]=0.02 0.05 ~0.01 0.03 ~=0.01 0.05 =~=0.01
a Isolated from W19. b Saturated solution.
Discussion Feeding extraneous substances to higher plants is facilitated by organ culture. Klein and Hagen (1961) succeeded in maturating very young petals of Impatiens balsamina in vitro. However, they found that anthocyanin production in vitro was considerably different from in vivo. Also light proved to be essental for their development. Mansell and Hagen (1965) administered pelargonidin-3-glucoside to petals of a white flowering genotype and observed the conversion to higher substituted anthocyanins. I n vitro culture of corollas of Petunia hybrida has been reported earlier by Hess (1967). He observed no differences in anthocyanin composition between corollas in vitro and in situ. The in vitro culture method we used was essentially an improved version of that described by Hess. Our culture medium differs from the nutrient solutions employed by Klein and Hagen (1961) and Hess (1967). I t gives a more vigorous growth of both detached corollas and complete flower buds. Using this culture method we were able to detect anthocyanin synthesis in a white flower (W18) after addition of bud extract from a complementary white flowering mutant (W19). W18 is homozygous recessive for the gene An3 and W19 for the gene An1 (cf. Fig. 1). The biosynthetic sequence of these two genes is in agreement with the scheme of Fig. 1. The assumption that the observed effect is really a complcmentation phenomenon is further supported by the occurrence of dihydroquercetin-7-glucoside in W19 as the active precursor.
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Dihydroflavonols are well known precursors, both of anthocyanins and of flavonols (Patsehke and Grisebaeh, 1968). I n m u t a n t W i 9 , dihydroquereetin-7glueoside (compound X in Fig. 1) is accumulated, due to blocking of the step leading to the intermediate u Mutant W18 is able to metabolize it to anthoeyanins. This m u t a n t is also able to metabolize dihydroquercetin into anthocyanins, suggesting t h a t this aglycone is likewise an intermediate in the biosynthetic pathway. I n W18, flavanones and flavonols cannot be metabolized into a n t h o e y a n i n s As flavanones are well-known intermediates in anthocyanin and flavonol biosynthesis the blocked step in W18 seems to be localized after flavanone formation. The observed effect proved to be light-independent. Flavonoid biosynthesis however is known to be light mediated b y the enzyme phenylManine-ammonialyase (PAL) (Amrhein and Zenk, 1970; Engelsma, 1970) and by other enzymes involved in the biosynthetic p a t h w a y (Wellman and Baron, 1974). However, in some eases a light independent anthoeyanin synthesis m a y occur (Sehmitz and Seitz, 1972). As in Petunia hybrida a n t h o c y a n i n synthesis is k n o w n to be light dependent (Steiner 1972), we assume t h a t the induction of the enzymes involved in our experiments occurs at a much earlier stage of bud development. The irregular pigmentation of W t 8 corollas and the accumulation of higher pigment levels around damaged spots suggests t h a t the epidermis is relatively impermeable to the active substance. As anthoeyanin synthesis appears to be restricted to the corolla, there is no evidence for a n y transport of precursors from other parts of the flower bud to the epidermal cells. The identification of anthoeyanins synthesized in W18 corollas after addition of precursors will be reported in a separate paper. We thank Professor T. J. Mabry, University of Texas at Austin for a sample of dihydrokaempferol and Professor H. Wagner, Universitgt Miinchen, for a gitt of quercetin-3-glueoside and quercetin-4'-glueoside.
References Amrhein, N., Zenk, M. H.: Untersuchungen zur Rolle der PhenylManin-Ammonium-Lyase (PAL) bei der l~egulation der Flavonoidsynthese im Buehweizen (Fagopyrum eseuIentum Moench). Z. Pflanzenphysiol. 64, 145-168 (1971) Bhatia, I. S., Singh, J., Bajaj, K. L. : A new chromogenic reagent for the detection of phenolic compounds on thin layer plates. J. Chromat. 79, 350-352 (1973) Birkofer, L., Kaiser, C.: Neue Flavonglycoside aus Petunia hybrida. Z. Naturforsch. 17b, 359-368 (1962) Engelsma, G. : A comparative investigation of the control of phenylManine ammonia lyase activity in gherkin and red cabbage seedlings. Acta Bot. Neerl. 19,403-414 (1970) Geissman, T. A., Jorgensen, E. C., Lcnnart Johnson, B. : The chemistry of flower pigmentation in Antirrhinum majus color genotypes I. The flavonoid components of the homozygous P, M, Y color types. Arch. Biochem. Biophys. 49, 368-388 (1954) Hess, D. : Die Wirkung yon Zimtsiiuren auf die Anthocyansynthese in isolierten Petalen yon Petunia hybrida. Z. Pflanzenphysiol. 56, 12-19 (1967) Johnston, K. 3/i., Stern, D. J., Waiss, A. C. Jr.: Separation of flavonoid compounds on Sephadex LH-20. J. Chromat. 33, 539-541 (1968) Klein, A. O., Hagen, C.W. Jr. : Anthoeyanin production in detached petMs of Impatiens balsamina L. Plant Physiol. 36, 1-9 (1961) Mabry, T. J., Markham, K. R., Thomas, M. B.: The systematic identification of flavonoids. Berlin-Heidelberg-New York: Springer 1970
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Mansell, R. L., Hagen, C. W. Jr. : Metabolism of some exogeneous ~nthoeyanins by detached petals of Impatiens balsamina L. Amer. J. Bot. 53, 875-882 (1966) Patschke, L., Grisebach, H.: Dihydrokaempferol and dihydroquercetin as precursors of kaempferol and quercetin in Pisum sativum. Phytochem. 7, 235-237 (1968) Reddy, G. M., Coe, E. H. Jr.: Inter-tissue complementation: a simple technique for direct analysis of gene action sequence. Science 138, 149 (1962). Schmitz, M., Seitz, U. : Hemmung der Anthocyansynthese durch Gibberellinsiiure bei Kalluskulturen von Daucus carota. Z. Pflanzenphysiol. 68, 259-265 (1972) Steiner, A. M. : Der Einflul~ der Lichtintensit~L auf die Akkumulation einzelner Anthocyane in isolierten Petalen yon Petunia hybrida. Z. Pflanzenphysiol. 68, 266-271 (1972) Wagner, R. P., Mitchell, H. K. : Genetics and metabolism. 2nd ed. p. 286. New York: Wiley 1964 Wellman, E., Baron, D. : Durch Phytoehrom kontrollierte Enzyme der Flavonoidsynthese in Zellsuspensionskulturen yon Petersilie (Petroselinum hortense Hoffm.). Planta (Berl.) 119,
161-164 (1974) Wiering, H. : Genetics of flower cotour in Petunia hybrida Hort. Genen Phaenen 17, 117-134 (1974) Wong, E., Francis, C. M. : Flavonoids in genotypes of Tri/olium subterraneum II. : Mutants of the geraldton variety. Phytoehem. 7, 2131-2137 (1968)
Anthocyanin Synthesis in a White Flowering Mutant of Petunia hybrida by a Complementation Technique K. F. F. Kho and G. J. H. Bennink Department of Plant Physiology, University of Amsterdam, IJdijk 26 Amsterdam, The Netherlands I-I. Wiering Institute of Genetics, University of Amsterdam, Kruislaan 318, Amsterdam, The Netherlands t~eceived 20 l~Iay; accepted 8 August 1975 Summary. In vitro cultured corollas of the white flowering mutant W18 of Petunia hybrida synthesize anthocyanins after administration of a bud extract of the white fl~wering mutant W19. Dihydroquercetin-7-glucoside was identified as the major metabolite from W19, which is converted into anthocyanins in W18. This result supports the genetic evidences with regards to the biosynthetic pathway leading to anthocyanins in this plant. W18 was not able to convert flavanones or flavonols into anthocyanins. This type of experiments may be of value for elucidation of certain aspects of flavonoid biosynthesis. Introduction Genetic studies of colour development in flowers of Petunia hybrida Hort. indicate that at least eight genes are involved in flavonoid biosynthesis, as is shown in Fig. 1 (Wiering, 1974). The anthocyanin genes (Anl, An2 and An3) have a general effect on the biosynthesis of anthocyanins in the flower limb. If one of these genes is present in the homozygous recessive condition, anthocyaniu synthesis is suppressed, resulting in a white or very weakly colourcd flower. Dominant alleles of An3 and F1 are required for the synthesis of flavonols (kaempferol and quercetin, of. Fig. 1). The other genes (Htl, Ht2, Hfl and Hf2 determine the hydroxylation pattern of the aryl side chain of the flavonoid molecule. Anthocyanin and flavonol synthesis are both affected by the genes An3 and H t l , which indicates that both types of compounds are s3mthesized via common precursors. The gene H t l must be dominant to obtain the ortho dihydroxy substitution pattern of the aryl side chain, leading to cyanidin and quercetin. If the gene is homozygous recessive, anthocyanin synthesis is suppressed. I n this case mainly kaempferol is accumulated. Pelargonidin (ef. Fig. 1) has seldom been found in Petunia flouters and than in very minute traces. Its absence can be explained by assuming that only precursors bearing two or three hydroxyl groups in the side chain can be converted into anthocyanins. White flowering mutants may be of value in elucidating of pathways of flavonoid biosynthesis Geissman et al., 1954; Wong and Francis, 1968). When the biosynthetic pathway to anthocyanins is Mocked at a single reaction step, one may expect : (i) that the intermediate formed just before the block is accumulated
272
K . F . F . Kho et al.
W
J
An3 X FI. ~
-
-
H O ~ O H OH O kaempfero[
OH Htl
Ht2 for tube OH H 0 ~ O H
I An1 Y
• Hfl for
Z(OH) [ /~ H O ~ O H OH
OH
HtI pe[argonidin Ht2 for tube OH Z(0H)2 ---* H e . O H
Limb /
OH 0
quercetin
OH
|
OH cyanidin
OH
OH -~ H 0 ~ / 0 ~ 0 H -y -y \ OH 0
myricetin
Z (OH)3---" HO~ O
+'~H
oH OH
~'OH OH de[phinidin
OH
Fig. 1. Relation between 8 genes involved in the biosynthesis of flavonoids in Petunia hybrida ttort. (Wiering, 1974). W, X, Y and Z are precursors of unknown nature, postulated on genetic evidence. Anl, An2 and An3 represent genes involved in the anthocyanin pathway. The genes An3 and F1 participate in the flavonol synthesis (kaempferol, quereetin and myrieetin). Htl, Ht2, I-Ifl and HI2 determine the hydroxylation pattern of the aryl side chain of the flavonoid molecule and (ii) that intermediates normally occurring after this reaction step, when fed to the mutant plant, will be converted into anthoeyanins. This principle is frequently used in studies on primary and secondary metabolism in bacteria and fungi and has proved to be an extremely useful method for elucidating biosynthetic pathways (Wagner and Mitchell, 1964). However, in higher plants this method is not easily applicable, due to technical problems. Reddy and Coe (1962) pressed together pieces of aleurone tissue from anthocyanin-defieient mutants of maize. When pieces of two different blocked mutants were combined only one of them became eoloured and from these complementation experiments the sequence of gene action could be established. The availability of genetically defined mutants of Petunia hybrida with respect to anthocyanin synthesis prompted us to use this technique to elucidate the sequence of reactions involved in anthocyanin biosynthesis. We tried to complement two white flowering mutants, homozygous recessive for different anthocyanin genes and in order to suppress flavonol synthesis homozygous recessive for the flavonol gene FI.
Anthocyanin Synthesis in a M u t a n t of Petunia hybrida
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Materials and Methods
Plant Material. Clones of the white flowering m u t a n t s W18 (homozygous recessive for An3) a n d W19 (homozygous recessive for An1) of Petunia hybrida were cultivated in the greenhouse.
In Vitro Culture el Corollas. From freshly gathered buds the sepals were removed. The remaining p a r t was sm'face sterilized with 1% calcium hypochlorite for 5 rain and rinsed in sterile distilled water. Subsequently they were cut transversely just above the anthers. The upper p a r t of the corollas, largely existing of the flower limb, was incised lengthwise and unfolded carefully. They were submerged in 70% (v/v) ethanol for 10 s and rinsed twice in sterile distilled water. After this t r e a t m e n t the corollas were incubated b y floating them, with the inner epidermis below, on 3 ml sterile culture medium in petri dishes (60 • 15 ram) to which was added 1 ml of extract. Dishes were placed under constant illumination (Philips TL 33, 5000 lux) a t 24 ~ for 72 h. Culture Medium. One litre of aqueous medium consisted of 1.29 g KH~P04, 0.087 g K2HPO 4, 0.125g Ca(H2PO4)~-H20, 0.125g MgSO4.7H20, 0.010g ferric citrate, 0.500g asparagin, 30 g sucrose and 0.5 ml micronutrient solution. The p H was adjusted to 5.5 + 0.1. Mieronutrient solution consisted of 2.86 g H3BOs, 1.81 g 5[nC12 94H20, 0.220 g ZnSO 4 97H20, 0.080 g CuSO 4 95 H20, 0.070 g Na2MoO 4 92 H20, dissolved in 1 litre water. Extracts o/ Flower Buds. From 100 freshly gathered flower buds the limb parts (fresh weight a b o u t 10 g) were homogenized in 50 ml acetone with a Biihler homogenizer. The homogcnate was centrifuged and the sediment re-extracted with 50 ml acetone. The combined supernatants were filtered and the acetone was removed under reduced pressure a t 30 ~ The residue was t a k e n up in 10 ml methanol, filtered and the filtrate was dried under reduced pressure. The residue was t a k e n up ill 10 ml culture medium and centrifuged at 38000 • g a t 4 ~ for 1 h. The s u p e r n a t a n t was sterilized b y membrane filtration (Sartorius SM 11307, pore size 0.2~zm). Anthocyanin Content el Corollas. After incubation for 72 h the corollas were rinsed in distilled water and dried with filter paper. Pigments were extracted b y shaking each corolla with 3 ml methanol-hydrochloric acid 0.1% for 1 h. Light absorption of the solution was measured in a Zeiss DMR 21 spectrophotometer in 1 cm cuvettcs at 530 nm. Anthocyanin content was expressed as the absorbance at 530 n m of a solution containing the pigments of one corolla in 3 ml solvent (A530/corolla, Klein and Hagen, 1961). Fractionation o/a Bud Extract. The methanol fraction obtained from the acetone extract of 200 flower buds of m u t a n t W19 was concentrated to a few ml a n d fractionated on a column (20• 1.6 cm) of Sephadex LH-20 equilibrated with methanol (Johnston et al., 1968). By eluting with methanol 12 fractions of 9.3 ml were obtained. A 5-ml sample of each fraction was dried under reduced pressure a n d tested for its activity on W18 corollas. The fractionation procedure was repeated several times to obtain sufficient q u a n t i t y of the active compound. The active fractions were purified b y semipreparative chromatography on W h a t m a n 3 paper using propanol-2-water 4:1 (v/v) as solvent. The position of the major compound, showing a reddish brown colour after spraying with the nitrite-tungstatc reagent of Bhatia et al. (1973) was determined a t a smM] strip of the chromatogram. The corresponding p a r t of the rest of this chromatogram was eluted with methanol. Aglycone and Sugar Identi/ication. 1 ml of a methanolic solution containing a few mg of the chromatographically pure compound was hydrolyzed b y heating with 1 ml 2 N HC1 under nitrogen in a fused tube in a boiling water b a t h for 15 min. E t h e r extraction of the hydrolysate yielded the aglycone, which was identified b y co-chromatography with appropriate standards on t h i n layers of cellulose (Merck, Darmstadt, Germany) using as solvents butanol-acetic acid-water (4:1 : 5, v/v/v, upper phase) (BAW) and chloroform-acetic acid-water (50: 45 : 5, v/v/v) (CAW). The extracted water phase was neutralized b y shaking with a solution of 20 % (v/v) di-n octylmethylamine in chloroform and washed with chloroform. After concentrating the solution under reduced pressure the sugar was identified b y co-chromatography with appropriate standards on thin layers of cellulose with the solvent systems b u t a n o l - e t h a n o l - w a t e r (40:10:22, v/v/v), (BEW) and b u t a n o l - b e n z e n e - p y r i d i n e water (50:10:30:24, v/v/v/v), (BBPW). Sugars were made visible b y spraying with anilin p h t a l a t e spray reagent (Merck, Darmstadt, Germany) followed b y heating for 5 min at 110 ~
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Oxydation o/ Dihydro/lavonolglycosides to Flavonolglycosides. A few mg dihydroflavonolglycoside was heated with 1 ml 20% (g/v) aqueous sodium metabisulfite solution for 2 h in a boiling water bath. The flavonolglycoside was isolated by polyamide chromatography according to Birkofer et al. (1962) and identified by co-chromatography with standards on thin layers of cellulose, using as solvent system 15% aqueous acetic acid. Quercetin-7-glucoside was prepared by partial hydrolysis of quereetin-3-sophoroside-7-glucoside, isolated from Petunia hybrida according to Birkofer et al. (1962).
Results 1. Stages o/Flower Bud Development in situ The development of flower buds of Petunia hybrida is presented in Table 1. During the stages II, I I I and IV the corolla elongates rapidly. At the end of stage IV the flower attains its final length and unfolds itself. Anthocyanin synthesis in normally pigmented Petunia flowers is perceptible at the beginning of stage IV.
Table 1. Developmental stages of Petunia flower buds in *itu. Bud length is defined as the distance between the flower receptable and the top of the corolla. The bud stages I, II, III, IV and V are chosen ~rbitrar.dy Bud stage
Bud length (ram)
I II III IV V
0-5 5-15 15-35 35-open flower open flower
2. Organ Culture Dissected corollas of the stages II, I I I and IV grew rapidly when cultured in vitro. The incubation time needed for complete development of corollas in stage IV varied from 12-24 h, for corollas in stage I I and I I I it appeared to be about 72h. The development of corollas from both anthoeyanin containing and white flowering genotypes did not differ significantly from the development in situ. I n vitro anthocyanin synthesis in red and magenta flowers after 72 h was comparable with anthoeyanin synthesis in intact open flowers. Corollas of white flowering mutants cultured in vitro did not synthesize anthoeyanins. In contrast to the experience of Hess (1967), it proved to be essential to work under sterile conditions, since fungal infections prevent further growth of the corollas. Incubations under constant illumination and in complete darkness appeared to be equivalent with regard to anthocyanin synthesis. 3. Complementation Experiments Several attempts were made to achieve anthocyanin synthesis in corollas of white flowering mutants after addition of bud extracts of complementary mutants. A weak reddish pigmentation was observed in corollas of mutant W18 after adding an extract of flower buds of mutant W19. As is shown in Table 2, only the combination of W18 as acceptor and W19 as donor resulted in anthoeyanin
Anthoeyanin Synthesis in a Mutant of Petunia hybrida
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Table 2. Anthocyanin content in corollas of white flowering mutants W18 and W19 after supplying bud extracts. 1 ml of bud extract wa.s added to 3 mI culture medium. As a control 4 ml culture medium was used. Absorba,nce was measured after an incubation period of 72 h. Values represent the mean of l0 corollas ~ standard deviation Accepter
Donor
Anthocyanin content per corolla (A530)
W18 W18 W18 W19 W19
control W18 W19 control W18
0.09 d_0.02 0.07 j: 0.01 0.42 • 0.09 0.05 • 0.01 0.05•
synthesis. The reverse experiment did not give any pigmel~tation. The red pigments occurring in the corolla of W l 8 after addition of Wt9 extract went into solution by macerating with methanol-tiC1 0.1% and exhibited an absorption maximum at 530 nm, indicating that this pigmentation was a result of anthoeyanin synthesis. Pigment distribution in the corollas was fairly irregular. In the presence of W19 extract eoloured spots of higher intensity were found around places where the tissue had been accidentally damaged, whereas other parts remained practically eolourless. In general the pigmentation was much weaker than in intact flowers. Corollas maturated in absense of the active extract did never show any pigmentation, neither after damage of the tissue. In order to determine the optimal bud stages for donor and accepter, various combinations of bud sizes were investigated. The optimal bud stage for donor material proved to be stage IV and for accepter material stage III. The most active extracts were those made from the limb part of the corolla, extracts of the tube part and of the green parts of the flower being inactive. The pigmentation of the accepter was limited to the inner epidermis of the limb part of the corolla. In pigmented flowers the anthoeyanins are also localized in the same epidermis. From these results it can be concluded that the gene An3 regulates an earlier step in the biosynthetic pathway than the gene Anl.
4. Isolation and Identi/ication o/an active Precursor ]rein Flower Buds o/Mutant W19 The active extract from W19 buds was subjected to gel chromatography on Sephadex LH-20. The fractions were examined for their effect on anthoeyanin synthesis in corollas of WlS. The results are given in Fig. 2. The combined activity of fractions 6 and 7 accounted for the total activity before fraetionation. Fraction 7 was further separated by paper chromatography. A biologically active compound (RI = 0.40), giving a reddish bream eolour with Bhatia's reagent was eluted from the paper. Acid hydrolysis proved that the compound was a glycoside. The aglycone was extracted with ether and identified as dihydroquercetin (3,3',4',5,7pentahydroxyflavanone) by thin-layer chromatography (Table 3). Table 3 also shows that the remaining water phase contained only glucose. Therefore the active compound was a glucoside of dihydroquercetin. The methanol spectrum of this compound (2max = 287 nm) did not shift after addition of sodium acetate. This indicates that a glucose residue is attached to dihydroqnercetin at position 7
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K.F.F.
A 530 TOTAL CONTROL EXTRACT
0.300
0.200
0.100
0 ~
2
3
4
5
6
7
8
9
10
11
fraction number
:Fig. 2. Column c h r o m a t o g r a p h y on S e p h a d e x LH-20 of e x t r a c t of flower b u d s of m u t a n t W19. A n t h o c y a n i n synthesis occurring in corollas of m u t a n t W18 after adding different chromatographic fractions oi e x t r a c t f r o m W19 was expressed as A530/corolla. The figure also shows the activity of t h e e x t r a c t before fractionation (total extract) a n d the a c t i v i t y of the contro], which was j u s t culture m e d i u m
Table 3. Identification of an active precursor for a n t h o c y a n i n biosynthesis, isolated f r o m flower b u d s of m u t a n t W19 Compound
R f values on cellulose TLC plates
Active c o m p o u n d Aglycone ~ Dihydrokaempferol Dihydroquercetin Sugar a Glucose Galactose Quercetin glucoside b Quercetin-7-glucoside Quercetin-3-glucoside Quercetin-4'-glucoside
BAW
CAW
15 % acetic acid
BEW
BBPW
0.55 0.87 0.93 0.87 ---0.27 0.26 0.69 0.58
0.06 0.24 0.49 0.26 ---0.08 0.08 0.23 0.11
-------0.07 0.07 0.33 0.09
----0.23 0.23 0.20 -----
----0.18 0.18 0.15 -----
a After hydrolysis of the active compound. b O b t a i n e d b y bisulfate o x y d a t i o n of the active c o m p o u n d .
(Mabry
et al.,
1970).
Bisulfite
oxydation
of t he
active
compound
flavonolglucoside, which was identified as quercetin-7-glucoside
yielded
t h e s e r e s u l t s i t c a n b e c o n c l u d e d t h a t t h e a c t i v e c o m p o u n d is d i h y d r o q u e r c e t i n - 7 glucoside.
a
( T a b l e 3). F r o m
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5. E]fect o/various Flavonoids on the Anthoeyanin Synthesis in the Mutants W18 and W19 In order to determine the specificity of the induced anthocyanin synthesis corollas of W18 were maturated in culture medium after addition of pure flavonoids. Flavanones (naringenin, naringin) and flavonols (kaempferol, quercetin) exhibited no activity. From the dihydroflavonols we investigated (dihydrokaempferol, dihydroquercetin and dihydroquercetin-7-glucoside) only dihydroquercetin and dihydroquercetin-7-glucoside were active (cf. Table 4.). None of these compounds were able to induce any pigmentation in corollas of W19. Table 4. Anthocyanin content, expressed as A530 in corollas of W18 after incubation for 72 h with various flavonoids. Concentration of the compounds in the culture medium (4 ml) was 0.5 • 10-a 2VI.As a control 4 ml culture medium was used. Values represent the mean of 10 corollas -~ standard deviation Compound
Anthocyanin content per corolla (A530)
Dihydroquercetin-7-glucosidea Dihydroquercetin Dihydrokaempferol Naringenin b Naringin Kaempferol b Quercetin b Control
0.24 ~ 0.03 0.36 :t:0.06 0.07 ~ 0.02 0.05 -l-0.01 0.07 =]=0.02 0.05 ~0.01 0.03 ~=0.01 0.05 =~=0.01
a Isolated from W19. b Saturated solution.
Discussion Feeding extraneous substances to higher plants is facilitated by organ culture. Klein and Hagen (1961) succeeded in maturating very young petals of Impatiens balsamina in vitro. However, they found that anthocyanin production in vitro was considerably different from in vivo. Also light proved to be essental for their development. Mansell and Hagen (1965) administered pelargonidin-3-glucoside to petals of a white flowering genotype and observed the conversion to higher substituted anthocyanins. I n vitro culture of corollas of Petunia hybrida has been reported earlier by Hess (1967). He observed no differences in anthocyanin composition between corollas in vitro and in situ. The in vitro culture method we used was essentially an improved version of that described by Hess. Our culture medium differs from the nutrient solutions employed by Klein and Hagen (1961) and Hess (1967). I t gives a more vigorous growth of both detached corollas and complete flower buds. Using this culture method we were able to detect anthocyanin synthesis in a white flower (W18) after addition of bud extract from a complementary white flowering mutant (W19). W18 is homozygous recessive for the gene An3 and W19 for the gene An1 (cf. Fig. 1). The biosynthetic sequence of these two genes is in agreement with the scheme of Fig. 1. The assumption that the observed effect is really a complcmentation phenomenon is further supported by the occurrence of dihydroquercetin-7-glucoside in W19 as the active precursor.
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Dihydroflavonols are well known precursors, both of anthocyanins and of flavonols (Patsehke and Grisebaeh, 1968). I n m u t a n t W i 9 , dihydroquereetin-7glueoside (compound X in Fig. 1) is accumulated, due to blocking of the step leading to the intermediate u Mutant W18 is able to metabolize it to anthoeyanins. This m u t a n t is also able to metabolize dihydroquercetin into anthocyanins, suggesting t h a t this aglycone is likewise an intermediate in the biosynthetic pathway. I n W18, flavanones and flavonols cannot be metabolized into a n t h o e y a n i n s As flavanones are well-known intermediates in anthocyanin and flavonol biosynthesis the blocked step in W18 seems to be localized after flavanone formation. The observed effect proved to be light-independent. Flavonoid biosynthesis however is known to be light mediated b y the enzyme phenylManine-ammonialyase (PAL) (Amrhein and Zenk, 1970; Engelsma, 1970) and by other enzymes involved in the biosynthetic p a t h w a y (Wellman and Baron, 1974). However, in some eases a light independent anthoeyanin synthesis m a y occur (Sehmitz and Seitz, 1972). As in Petunia hybrida a n t h o c y a n i n synthesis is k n o w n to be light dependent (Steiner 1972), we assume t h a t the induction of the enzymes involved in our experiments occurs at a much earlier stage of bud development. The irregular pigmentation of W t 8 corollas and the accumulation of higher pigment levels around damaged spots suggests t h a t the epidermis is relatively impermeable to the active substance. As anthoeyanin synthesis appears to be restricted to the corolla, there is no evidence for a n y transport of precursors from other parts of the flower bud to the epidermal cells. The identification of anthoeyanins synthesized in W18 corollas after addition of precursors will be reported in a separate paper. We thank Professor T. J. Mabry, University of Texas at Austin for a sample of dihydrokaempferol and Professor H. Wagner, Universitgt Miinchen, for a gitt of quercetin-3-glueoside and quercetin-4'-glueoside.
References Amrhein, N., Zenk, M. H.: Untersuchungen zur Rolle der PhenylManin-Ammonium-Lyase (PAL) bei der l~egulation der Flavonoidsynthese im Buehweizen (Fagopyrum eseuIentum Moench). Z. Pflanzenphysiol. 64, 145-168 (1971) Bhatia, I. S., Singh, J., Bajaj, K. L. : A new chromogenic reagent for the detection of phenolic compounds on thin layer plates. J. Chromat. 79, 350-352 (1973) Birkofer, L., Kaiser, C.: Neue Flavonglycoside aus Petunia hybrida. Z. Naturforsch. 17b, 359-368 (1962) Engelsma, G. : A comparative investigation of the control of phenylManine ammonia lyase activity in gherkin and red cabbage seedlings. Acta Bot. Neerl. 19,403-414 (1970) Geissman, T. A., Jorgensen, E. C., Lcnnart Johnson, B. : The chemistry of flower pigmentation in Antirrhinum majus color genotypes I. The flavonoid components of the homozygous P, M, Y color types. Arch. Biochem. Biophys. 49, 368-388 (1954) Hess, D. : Die Wirkung yon Zimtsiiuren auf die Anthocyansynthese in isolierten Petalen yon Petunia hybrida. Z. Pflanzenphysiol. 56, 12-19 (1967) Johnston, K. 3/i., Stern, D. J., Waiss, A. C. Jr.: Separation of flavonoid compounds on Sephadex LH-20. J. Chromat. 33, 539-541 (1968) Klein, A. O., Hagen, C.W. Jr. : Anthoeyanin production in detached petMs of Impatiens balsamina L. Plant Physiol. 36, 1-9 (1961) Mabry, T. J., Markham, K. R., Thomas, M. B.: The systematic identification of flavonoids. Berlin-Heidelberg-New York: Springer 1970
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Mansell, R. L., Hagen, C. W. Jr. : Metabolism of some exogeneous ~nthoeyanins by detached petals of Impatiens balsamina L. Amer. J. Bot. 53, 875-882 (1966) Patschke, L., Grisebach, H.: Dihydrokaempferol and dihydroquercetin as precursors of kaempferol and quercetin in Pisum sativum. Phytochem. 7, 235-237 (1968) Reddy, G. M., Coe, E. H. Jr.: Inter-tissue complementation: a simple technique for direct analysis of gene action sequence. Science 138, 149 (1962). Schmitz, M., Seitz, U. : Hemmung der Anthocyansynthese durch Gibberellinsiiure bei Kalluskulturen von Daucus carota. Z. Pflanzenphysiol. 68, 259-265 (1972) Steiner, A. M. : Der Einflul~ der Lichtintensit~L auf die Akkumulation einzelner Anthocyane in isolierten Petalen yon Petunia hybrida. Z. Pflanzenphysiol. 68, 266-271 (1972) Wagner, R. P., Mitchell, H. K. : Genetics and metabolism. 2nd ed. p. 286. New York: Wiley 1964 Wellman, E., Baron, D. : Durch Phytoehrom kontrollierte Enzyme der Flavonoidsynthese in Zellsuspensionskulturen yon Petersilie (Petroselinum hortense Hoffm.). Planta (Berl.) 119,
161-164 (1974) Wiering, H. : Genetics of flower cotour in Petunia hybrida Hort. Genen Phaenen 17, 117-134 (1974) Wong, E., Francis, C. M. : Flavonoids in genotypes of Tri/olium subterraneum II. : Mutants of the geraldton variety. Phytoehem. 7, 2131-2137 (1968)
