Planta
Planta 144, 189-192 (1979)
9 by Springer-Verlag 1979
Genetic Control of Chalcone lsomerase Activity in Anthers of Petunia hybrida G. F o r k m a n n and B. K u h n tnstitut ffir Biologic II, Lehrstuhl ffir Genetik der UniversitS,t, Auf der Morgenstelle 28, D-7400 Ttibingen, Federal Republic of Germany
Abstract. The gene Po in pollen of Petunia hybrida Vilm. controls a discrete step in flavonoid biosynthesis. In recessive genotypes, naringenin-chalcone (4, 2',4',6'-tetrahydroxychalcone) is accumulated, whereas, u n d e r the influence of the wild-type allele flavonols and a n t h o c y a n i n s are formed. E n z y m i c investigations on anthers of four genetically defined lines with different pollen colouration revealed a clear correlation between a c c u m u l a t i o n of naringenin-chalcone and deficiency of chalcone isomerase (EC 5.5.1.6). The results allow the conclusion that chalcone is the first p r o d u c t of the flavanone synthase reaction in anthers o f Petunia hybrida and that chalcone isomerase is essential for the f o r m a t i o n of flavonots and anthocyanins. These results were similar to those previously obtained with Callistephus chinensis (L.) Nees.
Key words: Anthers - Chalcone isomerase - Flavonoid biosynthesis - Genetic block chalcone - Petunia.
Naringenin-
Introduction The chalcone isomerase (CI) was the first enzyme reported to catalyse a discrete step in the actual flavonoid p a t h w a y ( M o u s t a f a and W o n g , 1967). However, up to now, there has been some ambiguity regarding the role o f this enzyme in biosynthesis o f flavonoids ( H a h l b r o c k and Grisebach, 1975; Grisebach, 1975). The c o m b i n a t i o n of enzymic and chemical investigations on defined genotypes should contribute towards an answer to the questions in dispute. Recently, we reported on a recessive m u t a n t in Callistephus chinensis accumulating naringenin-chal-
Abbreviations: EGME : ethylen glycol monomethyl ether; MeOH = methanol; CI = chalcone isomerase; HOAc = acetic acid; T L C thinlayer chromatography
cone-2'-glucoside (isosalipurposide) in blossoms, whereas in genotypes with wild-type alleles, higher oxidized flavonoids and a n t h o c y a n i n s were synthesized ( K u h n et al., 1978). E n z y m i c investigations on different genotypes revealed a clear correlation between a c c u m u l a t i o n of chalcone in recessive genotypes and deficiency of CI activity. These results allowed an u n a m b i g u o u s statement a b o u t the actual role of CI in flavonoid biosynthesis for this plant at least. However, for m o r e detailed knowledge a b o u t the general role o f this enzyme, it is necessary to extend such investigations to other plants. Unfortunately, gene-controlled accumulation of chalcones is a rather rare feature in plants. But one case has been reported by de Vlaming and K h o (1976) on pollen o f genetically defined lines o f Petunia hybrida. In different coloured pollen, they f o u n d a situation chemogenetically c o m p a r a b l e with that in blossoms o f Callistephus chinensis. Their results p r o m p t e d this investigation of the activity of CI in anthers of Petunia hybrida with different pollen colouration.
Material and Methods The investigations included four genetically defined lines (V 10, V 28, V 31 and V 33) of Petunia hybrida using the material originally described by de Vlaming and Kho (1976). The lines differ with regard to the pollen cotour influenced by the two genes Po and An4 (de Vlaming, personal communication). Depending upon the four possible combinations of the two genes, the lines produce anthers with either blue, green, white or yellow pollen (Table 1). The gene An4 controls the anthocyanin synthesis in pollen and in the tube part of the corrolla. In the presence of recessive alleles, the formation of anthocyanins is strongly inhibited but flavonol synthesis can take place (Wiering, 1974). The gene Po also controls a discrete step in the flavonoid biosynthesis. Thus, in recessive genotypes, a yellow pigment was found and identified as naringenin-chalcone (de Vlaming and Kho, 1976). The chalcone is responsible for the yellow colour of the pollen, whe§ under the influence of dominant Po alleles, the colour of pollen is blue or white, depending on the situation at
0032-0935/79/0144/0189/$01.00
190
G. Forkmann and B. Kuhn: Chalcone Isomerase Activity in Anthers
Table 1. Activity of CI in anthers and corrollas of 4 genetically defined lines of Petunia hybrida with different pollen colouration. (Mean values from 3 enzyme preparations per line): Line
Poilen genotype
Pollen colour
Pollen flavonoids
Spec. activity of CI in anthers A A x m i n - I xmg -1 protein ~
Spec. activity of CI in corrollas A Axmin-~ • -1 protein ~
V l0
Po An4
blue
anthocyanin
0.572 • 0.017
9.54 + 0.82
V 28
po An4
green
naringenin-chalcone some anthocyanin
0.026 • 0.014
6.55 _+0.84
V 31
Po an4
white
dihydroflavonoI and flavonol
0.509 • 0.070
9.02 • 0.22
V 33
po an4
yellow
naringenin-chalcone some flavonol and dihydroflavonol
0.023 • 0.020
3.98 _+0.34
a
Protein contents of anthers appr. 5 mg/ml crude extract; protein contents of corrollas appr. 1 mg/ml crude extract
the An4 locus, and no chalcone is detectable. In recessive genotypes, the gene Po actually supresses the formation of higher oxidized flavonoids and anthocyanins but apparently does not interrupt it completely. Thus, in genotype po An4 (line V 28) some anthocyanin is produced in addition to naringenin-chalcone resulting in a green pollen colour. Similarly, our chromatographic analysis of the pollen pigments also revealed appreciable amounts of flavonol and dihydroflavonol beside naringenin-chalcone in yellow pollen (genotype po an4). It is an interesting feature of the material investigated that independent of the pollen colour, all four lines produce high quantities of anthocyanins in their corrollas. We have tested, therefore, the activity of CI not only in the anthers but also in the corrolias of all lines.
Enzyme Preparation To compensate for possible developmental differences in CI activity (Wiermann, 1972), a mixture of anthers of different stages of development was used including all stages from lightly coloured pollen to mature pollen with high colouration. Because of the very low weight, the anthers of 150 flowers (appr. 1 g) were pooled and homogenized at 4~ C, using an Ultra Turrax, together with an equal weight of Dowex 1 x2 and two volumes of 0.1 M sodium phosphate buffer, pH 8.0, containing 1.4 mM mercaptoethanol. After centrifugation, the clear and nearly colourless supernatant (appr. 1.5ml) served as enzyme source. The enzyme preparation from corollas was performed similarly.
Enzyme Assay The reaction mixture (1.65 ml) contained 0.1 M sodium phosphate, pH 8.0, 50 gl enzyme solution and 0.17 mM chalcone (dissolved in EGME). The reaction was started by addition of chalcone. The decrease in absorptivity at the absorption maximum of the chalcone (naringenin-chalcone 385nm) was plotted against time in a registrating spectral photometer at 30 ~ C. The blank contained buffer and enzyme solution. All values were corrected for the rate of the non-enzymic isomerisation.
Identification of the Reaction Produci After 3 rain incubation the reaction mixture (1.65 ml) was extracted with 2 x 3 mI ethyl acetate, the combined ethyl acetate fractions
evaporated to dryness and the solid residue redissolved in 0.1 ml MeOH. The reaction product was identified by spectral analysis and TLC on 0.1 mm cellulose plates in comparison with authentic samples of naringenin and naringenin-chalcone using both 30% HOAc and tert. butanol-acetic acid-water (3 : 1 : 1, by vol.). The preparation of chalcones, the determination of pH optimum and the protein determination was performed as described by Kuhn et al. (1978).
Results F r o m t h e f o u r lines tested, t h e assays w i t h e n z y m e s o l u t i o n p r e p a r e d f r o m a n t h e r s w i t h b l u e or w h i t e p o l l e n (lines V 10 a n d V 31) e x h i b i t e d a clear C I activity. O n t h e c o n t r a r y , e n z y m e p r e p a r a t i o n s f r o m ant h e r s o f line V 28 o r V 33 ( g r e e n or y e l l o w p o l l e n ) w e r e f o u n d to be n e a r l y i n a c t i v e ( T a b l e 1). F o r the l a t t e r t w o lines, n o s i g n i f i c a n t d e c r e a s e in a b s o r p t i v i t y e x c e e d i n g t h o s e o f b o i l e d e x t r a c t s c o u l d be o b s e r v e d w i t h t h e u s u a l q u a n t i t y (50 gl) o f e n z y m e s o l u t i o n . O n l y by i n c r e a s i n g t h e q u a n t i t y o f e n z y m e s o l u t i o n u p to 200 lal c o u l d a c t i v i t y be d e m o n s t r a t e d , a n d t h e n o n l y at a v e r y l o w level (Fig. 1). F o r e n z y m e s o l u t i o n w i t h a c t i v e e n z y m e , t h e react i o n r a t e was p r o p o r t i o n a l to p r o t e i n c o n c e n t r a t i o n . Mixtures containing enzyme preparations with high a c t i v i t y a n d t h o s e w i t h l o w a c t i v i t y b e h a v e d in a n a d d i t i v e f a s h i o n (Fig. 1). F o r assays w i t h a c t i v e e n z y m e , t h e s p e c t r a l a n d chromatographic analysis of the reaction mixture ( K u h n et al., 1978) s h o w e d a c o m p l e t e c o n v e r s i o n o f n a r i n g e n i n - c h a l c o n e to n a r i n g e n i n a f t e r 3 m i n incub a t i o n . I n t h e case o f e n z y m e p r e p a r a t i o n s f r o m anthers with yellow or green pollen, however, a part o f t h e a p p l i e d n a r i n g e n i n - c h a l c o n e was still d e t e c t e d , in a d d i t i o n t o s o m e n a r i n g e n i n f o r m e d m a i n l y by non-enzymic isomerisation.
G. Forkmann and B. Kuhn: Chalcone Isomerase Activity in Anthers
03~ 0.6-
< 0.50.40.30203-'
0}1
~
a 013
0!4
015
46
0.~/
O:B
ol 2'5
5;
7'5
1;0
1;0~
b) +75
+ 50
+25
+100
+ 50/d
0[9
1:0mgprotein
al., 1978). The shortage of enzyme solution which could be prepared from anthers precluded more detailed investigations of the substrate specificity and of further enzyme activators and inhibitors. Independent of the situation found in enzyme preparations from anthers of Petunia hybrida, a clear CI activity could be demonstrated in corrolla extracts of each of the four lines. But these measurements also revealed significant differences in CI activity between the two lines V 10 and V 31 with dominant Po alleles and the two lines V 28 and V 33 with recessive po alleles (Table 1). Discussion
Fig. 1. Dependence of the enzymic isomerisation of naringeni.nchalcone on protein concentration, x - - x line V 10; :~--D line V28; o Mixtures containing enzyme extract of line V10 (a) and line V28 (b) in ~tl per assay. The values were plotted against protein contents of two different V 10 enzyme extracts (a). Lines V31 and V33 behaved similarly
Table 2. The effect of some substrates and several additions upon
the enzymic conversion of chalcone to flavanone Additions
191
Substrate
Activity of the enzyme i n %
None
naringeniu-chalcone
100
None
naringenin-chalcone-2"glucoside (isosalipurposide)
0
None
isoliquiritigenin
0
1 mM KCN
naringenin-chalcone
100
10 m M K C N
naringenin-chalcone
100
0.02 M MgClz
naringenin-chalcone
104
0.1 mM p-hydro- naringenin-chalcone xymercuribenzoate
22.2
0.5mM p-hydro- naringenin-chalcone xymercuribenzoate
12,5
The enzyme from anthers of Petunia hybrida has a pH optimum of about pH 8.6. This value corresponds to the values found for chalcone isomerases from other plants (Halbrock et al., 1970; Kuhn et al., 1978). In accordance with the data given by Moustafa and Wong (1967) for CI, the enzymic conversion of naringenin-chalcone to naringenin is not affected by chelating reagents but is strongly inhibited by low concentrations of p-hydroxymercuribenzoate (Table 2). Moreover, the enzyme from anthers of Petunia hybrida showed the same high substrate specificity (Table 2) as the chalcone isomerases from other plants (Hahlbrock et al., 1970; Wiermann, 1972; Kuhn et
The results obtained from chemogenetical investigations of a yellow pigment in pollen of some lines of Petunia hybrida (de Vlaming and Kho, 1976) correspond largely with the results obtained from similar investigations of the flavonoid compounds in yellow flowering lines of Callistephus chinensis (Kuhn et al., 1978). In both plants, a definite gene (Po in Petunia and Ch in Callistephus) controls a discrete step in flavonoid biosynthesis localized between chalcones and higher oxidized flavonoids. An accumulation of chalcones only takes place in recessive genotypes while in genotypes with wild-type alleles flavonols and anthocyanins are synthesized. Contrary to the temporary accumulation of chalcones in anthers of Tulipa reported by Quast and Wiermann (1973), the accumulation of naringenin-chalcone in pollen of Petunia hybrida is not restricted to a specific developmental stage. The recessive alleles of the genes Po and Ch also agree with regard to their performance. Both suppress the synthesis of other flavonoids and anthocyanins but apparently do not interrupt it completely. The close agreement between the genes Po and Ch on chemogenetical basis suggested that, both in the blossoms of Callistephus chinensis and in the pollen of Petunia hybrida, a deficiency of an enzyme catalysing the conversion of chalcones to other classes of flavonoids should be responsible for the chalcone accumulation. In Callistephus chinensis, the enzyme concerned was found to be the chalcone isomerase (Kuhn et al., 1978). The results of the present paper demonstrate that the accumulation of chalcone in pollen of Petunia hybrida is also caused by a nearly complete deficiency of this enzyme. Because of the extremely small amounts of material, the enzymic tests were performed with crude extracts. Nevertheless, CI could be demonstrated in anthers with blue or white pollen (Po dominant) in relatively high activity. In this connection it should be mentioned that compared to the high values of the specific activity of CI found in the corrollas, the
192 substantially lower values in the anthers are partly caused by the very high protein concentration of anther extracts in comparison to those of the corrolla extracts. The very low activity of CI found in anthers with yellow or green pollen (Po recessive) is obviously not sufficient for a complete conversion of naringeninchalcone to naringenin in vivo. But this low enzyme activity is probably responsible for the presence of appreciable amounts of dihydroflavonol and flavonol in yellow pollen and anthocyanin in green pollen in addition to naringenin-chalcone. Due to the largely additive behaviour of the mixed extracts from anthers with high enzyme activity and those with very low activity, it is unlikely that an inhibitor is responsible for the deficiency of CI activity in pollen which accumulate naringenin-chalcone. Thus, the Po gene is more likely to be concerned with the synthesis of the enzyme. As far as is known, in pollen of Petunia hybrida only flavonoids with a phloroglucinol type of A-ring occur. Therefore, the substrate specificity of the CI in pollen corresponds completely to expectations. Furthermore, chalcone glucosides were also not isomerized by chalcone-isomerases from other plants (Hahlbrock et al., 1970; G r a m b o w and Grisebach, 1971). Application of 10 mM K C N caused no decrease in reaction velocity. This proved that the reaction rate measured is due only to CI and not partly caused by peroxidase (Boland and Wong, 1975). Both the chemogenetic and enzymologic evidence allows a clear statement regarding the role of CI in the biosynthesis of flavonoids in pollen of Petunia hybrida. In blue and white pollen, the CI catalyses the conversion of naringenin-chalcone to naringenin and yields by this reaction the substrate for the formation of flavonols and anthocyanins. In yellow and green pollen, however, the synthesis of flavonols and anthocyanins is largely interrupted by reason of the nearly complete deficiency of CI activity. Since, in this case, naringenin-chalcone is accumulated, the first reaction product of the flavanone synthase in pollen of Petunia hybrida is more likely to be chalcone rather than flavanone, as found by Kreuzaler and Hahlbrock (1975) in Petroselinum. Thus, in pollen of Petunia hybrida, CI plays the same important role as in blossoms of Callistephus chinensis. The situation found in Callistephus chinensis, therefore, seems not to be an exceptional case. This is also supported by a further correlation between chalcone accumulation and deficiency of CI activity demonstrated on chemogenetic and enzymic investigations of yellow flowering lines of Dianthus caryophyllus (Forkmann and Kuhn, in preparation). Moreover, similar conclusions could be drawn from
G. Forkmann and B. Kuhn: Chalcone Isomerase Activity in Anthers the results of investigations on anthers of Tulipa and Lilium (Wiermann, 1972) although in this case the accumulation of chalcone and the activity of CI were not measured in relation to a definite gene but to different developmental stages of the anthers. The clear CI activity demonstrated in the corrollas of each line of Petunia hybrida investigated can not be explained by activation and inactivation of the gene Po in different plant tissues alone; it seems preferable to postulate that another gene is mainly responsible for the enzyme activity measured in the corrollas. Of course, this postulated gene has to be inactivated in pollen. The gene Po, however, could be active in both pollen and corrollas. Possibly, the significantly higher activities found in corrollas of the lines V 10 and V 31 with dominant Po alleles could be caused by two genes, the postulated isomerase gene and the Po gene. These investigations were supported by a grant from the Deutsche Forschungsgemeinschaft. The authors are very grateful to Dr. de Vlaming, Institute of Genetics, University of Amsterdam for her generous gift of seeds of the four genetically defined lines of Petunia hybrida and to Prof. H. Grisebach for his critical reading of the manuscript and valuable suggestions.
References Boland, M.J., Wong, E.: Purification and kinetic properties of chalcone-flavanone-isomerase from Soya bean. Eur. J. Biochem. 50, 383-389 (1975) Grambow, H.J., Grisebach, H.: Further studies on biosynthesis of flavonoids in Datisea cannabina. Phytochemistry 10, 789 (1971) Grisebach, H. : Enzymologieder Flavonoidbiosynthese. Ber. dtsch. Bot. Ges. 88, 61-69 (1975) Hahlbrock, K., Wong, E., Schill, L., Grisebach, H.: Comparison of chalcone-flavanone isomerase heteroenzymes and isoenzymes. Phytochemistry9, 949-958 (1970) Hahlbrock, K., Grisebach, H. : Biosynthesis of flavonoids. In : The flavonoids. Harborne, J.B. Mabry, T.J., Mabry, H., ed., p. 866 915. London: Chapman and Hail I975 Kreuzaler, F., Hahlbrock, K.: Enzymic synthesis of an aromatic ring from acetate units. Eur. J. Biochem. 56, 205-213 (1975) Kuhn, B., Forkmann, G., Seyffert, W.: Genetic control of chalcone-flavanone isomerase activity in Callisthephus chinensis. Planta 138, 199 203 (1978) Moustafa, E., Wong, E.: Purification and properties of chalconeflavanone isomerase from Soya bean seed. Phytochemistry 6, 625 632 (1967) Quast, L., Wiermann, R. : Uber das Vorkommen verschieden substituierter Chalkone wS,hrend der Mikrosporogenese. Experienta 29, 1165 (1973) de u P_, Kho, F.F.K.: 4,T,4',C-Tetrahydroxychalconein pollen of Petunia hybrida. Phytochemistry 15, 348 349 (1976) Wiering, H.: Genetics of flower colour in Petunia hybrida hort. Gen. Phaenen. 17, 117-134 (1974) Wiermann, R.: Aktivitfit der Chalkon-Flavanon Isomerase und Akkumulation yon phenylpropanoiden Verbindungen in Antheren. Planta 102, 55-60 (1972) Received 10 July ; accepted 6 September 1978
Planta 144, 189-192 (1979)
9 by Springer-Verlag 1979
Genetic Control of Chalcone lsomerase Activity in Anthers of Petunia hybrida G. F o r k m a n n and B. K u h n tnstitut ffir Biologic II, Lehrstuhl ffir Genetik der UniversitS,t, Auf der Morgenstelle 28, D-7400 Ttibingen, Federal Republic of Germany
Abstract. The gene Po in pollen of Petunia hybrida Vilm. controls a discrete step in flavonoid biosynthesis. In recessive genotypes, naringenin-chalcone (4, 2',4',6'-tetrahydroxychalcone) is accumulated, whereas, u n d e r the influence of the wild-type allele flavonols and a n t h o c y a n i n s are formed. E n z y m i c investigations on anthers of four genetically defined lines with different pollen colouration revealed a clear correlation between a c c u m u l a t i o n of naringenin-chalcone and deficiency of chalcone isomerase (EC 5.5.1.6). The results allow the conclusion that chalcone is the first p r o d u c t of the flavanone synthase reaction in anthers o f Petunia hybrida and that chalcone isomerase is essential for the f o r m a t i o n of flavonots and anthocyanins. These results were similar to those previously obtained with Callistephus chinensis (L.) Nees.
Key words: Anthers - Chalcone isomerase - Flavonoid biosynthesis - Genetic block chalcone - Petunia.
Naringenin-
Introduction The chalcone isomerase (CI) was the first enzyme reported to catalyse a discrete step in the actual flavonoid p a t h w a y ( M o u s t a f a and W o n g , 1967). However, up to now, there has been some ambiguity regarding the role o f this enzyme in biosynthesis o f flavonoids ( H a h l b r o c k and Grisebach, 1975; Grisebach, 1975). The c o m b i n a t i o n of enzymic and chemical investigations on defined genotypes should contribute towards an answer to the questions in dispute. Recently, we reported on a recessive m u t a n t in Callistephus chinensis accumulating naringenin-chal-
Abbreviations: EGME : ethylen glycol monomethyl ether; MeOH = methanol; CI = chalcone isomerase; HOAc = acetic acid; T L C thinlayer chromatography
cone-2'-glucoside (isosalipurposide) in blossoms, whereas in genotypes with wild-type alleles, higher oxidized flavonoids and a n t h o c y a n i n s were synthesized ( K u h n et al., 1978). E n z y m i c investigations on different genotypes revealed a clear correlation between a c c u m u l a t i o n of chalcone in recessive genotypes and deficiency of CI activity. These results allowed an u n a m b i g u o u s statement a b o u t the actual role of CI in flavonoid biosynthesis for this plant at least. However, for m o r e detailed knowledge a b o u t the general role o f this enzyme, it is necessary to extend such investigations to other plants. Unfortunately, gene-controlled accumulation of chalcones is a rather rare feature in plants. But one case has been reported by de Vlaming and K h o (1976) on pollen o f genetically defined lines o f Petunia hybrida. In different coloured pollen, they f o u n d a situation chemogenetically c o m p a r a b l e with that in blossoms o f Callistephus chinensis. Their results p r o m p t e d this investigation of the activity of CI in anthers of Petunia hybrida with different pollen colouration.
Material and Methods The investigations included four genetically defined lines (V 10, V 28, V 31 and V 33) of Petunia hybrida using the material originally described by de Vlaming and Kho (1976). The lines differ with regard to the pollen cotour influenced by the two genes Po and An4 (de Vlaming, personal communication). Depending upon the four possible combinations of the two genes, the lines produce anthers with either blue, green, white or yellow pollen (Table 1). The gene An4 controls the anthocyanin synthesis in pollen and in the tube part of the corrolla. In the presence of recessive alleles, the formation of anthocyanins is strongly inhibited but flavonol synthesis can take place (Wiering, 1974). The gene Po also controls a discrete step in the flavonoid biosynthesis. Thus, in recessive genotypes, a yellow pigment was found and identified as naringenin-chalcone (de Vlaming and Kho, 1976). The chalcone is responsible for the yellow colour of the pollen, whe§ under the influence of dominant Po alleles, the colour of pollen is blue or white, depending on the situation at
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G. Forkmann and B. Kuhn: Chalcone Isomerase Activity in Anthers
Table 1. Activity of CI in anthers and corrollas of 4 genetically defined lines of Petunia hybrida with different pollen colouration. (Mean values from 3 enzyme preparations per line): Line
Poilen genotype
Pollen colour
Pollen flavonoids
Spec. activity of CI in anthers A A x m i n - I xmg -1 protein ~
Spec. activity of CI in corrollas A Axmin-~ • -1 protein ~
V l0
Po An4
blue
anthocyanin
0.572 • 0.017
9.54 + 0.82
V 28
po An4
green
naringenin-chalcone some anthocyanin
0.026 • 0.014
6.55 _+0.84
V 31
Po an4
white
dihydroflavonoI and flavonol
0.509 • 0.070
9.02 • 0.22
V 33
po an4
yellow
naringenin-chalcone some flavonol and dihydroflavonol
0.023 • 0.020
3.98 _+0.34
a
Protein contents of anthers appr. 5 mg/ml crude extract; protein contents of corrollas appr. 1 mg/ml crude extract
the An4 locus, and no chalcone is detectable. In recessive genotypes, the gene Po actually supresses the formation of higher oxidized flavonoids and anthocyanins but apparently does not interrupt it completely. Thus, in genotype po An4 (line V 28) some anthocyanin is produced in addition to naringenin-chalcone resulting in a green pollen colour. Similarly, our chromatographic analysis of the pollen pigments also revealed appreciable amounts of flavonol and dihydroflavonol beside naringenin-chalcone in yellow pollen (genotype po an4). It is an interesting feature of the material investigated that independent of the pollen colour, all four lines produce high quantities of anthocyanins in their corrollas. We have tested, therefore, the activity of CI not only in the anthers but also in the corrolias of all lines.
Enzyme Preparation To compensate for possible developmental differences in CI activity (Wiermann, 1972), a mixture of anthers of different stages of development was used including all stages from lightly coloured pollen to mature pollen with high colouration. Because of the very low weight, the anthers of 150 flowers (appr. 1 g) were pooled and homogenized at 4~ C, using an Ultra Turrax, together with an equal weight of Dowex 1 x2 and two volumes of 0.1 M sodium phosphate buffer, pH 8.0, containing 1.4 mM mercaptoethanol. After centrifugation, the clear and nearly colourless supernatant (appr. 1.5ml) served as enzyme source. The enzyme preparation from corollas was performed similarly.
Enzyme Assay The reaction mixture (1.65 ml) contained 0.1 M sodium phosphate, pH 8.0, 50 gl enzyme solution and 0.17 mM chalcone (dissolved in EGME). The reaction was started by addition of chalcone. The decrease in absorptivity at the absorption maximum of the chalcone (naringenin-chalcone 385nm) was plotted against time in a registrating spectral photometer at 30 ~ C. The blank contained buffer and enzyme solution. All values were corrected for the rate of the non-enzymic isomerisation.
Identification of the Reaction Produci After 3 rain incubation the reaction mixture (1.65 ml) was extracted with 2 x 3 mI ethyl acetate, the combined ethyl acetate fractions
evaporated to dryness and the solid residue redissolved in 0.1 ml MeOH. The reaction product was identified by spectral analysis and TLC on 0.1 mm cellulose plates in comparison with authentic samples of naringenin and naringenin-chalcone using both 30% HOAc and tert. butanol-acetic acid-water (3 : 1 : 1, by vol.). The preparation of chalcones, the determination of pH optimum and the protein determination was performed as described by Kuhn et al. (1978).
Results F r o m t h e f o u r lines tested, t h e assays w i t h e n z y m e s o l u t i o n p r e p a r e d f r o m a n t h e r s w i t h b l u e or w h i t e p o l l e n (lines V 10 a n d V 31) e x h i b i t e d a clear C I activity. O n t h e c o n t r a r y , e n z y m e p r e p a r a t i o n s f r o m ant h e r s o f line V 28 o r V 33 ( g r e e n or y e l l o w p o l l e n ) w e r e f o u n d to be n e a r l y i n a c t i v e ( T a b l e 1). F o r the l a t t e r t w o lines, n o s i g n i f i c a n t d e c r e a s e in a b s o r p t i v i t y e x c e e d i n g t h o s e o f b o i l e d e x t r a c t s c o u l d be o b s e r v e d w i t h t h e u s u a l q u a n t i t y (50 gl) o f e n z y m e s o l u t i o n . O n l y by i n c r e a s i n g t h e q u a n t i t y o f e n z y m e s o l u t i o n u p to 200 lal c o u l d a c t i v i t y be d e m o n s t r a t e d , a n d t h e n o n l y at a v e r y l o w level (Fig. 1). F o r e n z y m e s o l u t i o n w i t h a c t i v e e n z y m e , t h e react i o n r a t e was p r o p o r t i o n a l to p r o t e i n c o n c e n t r a t i o n . Mixtures containing enzyme preparations with high a c t i v i t y a n d t h o s e w i t h l o w a c t i v i t y b e h a v e d in a n a d d i t i v e f a s h i o n (Fig. 1). F o r assays w i t h a c t i v e e n z y m e , t h e s p e c t r a l a n d chromatographic analysis of the reaction mixture ( K u h n et al., 1978) s h o w e d a c o m p l e t e c o n v e r s i o n o f n a r i n g e n i n - c h a l c o n e to n a r i n g e n i n a f t e r 3 m i n incub a t i o n . I n t h e case o f e n z y m e p r e p a r a t i o n s f r o m anthers with yellow or green pollen, however, a part o f t h e a p p l i e d n a r i n g e n i n - c h a l c o n e was still d e t e c t e d , in a d d i t i o n t o s o m e n a r i n g e n i n f o r m e d m a i n l y by non-enzymic isomerisation.
G. Forkmann and B. Kuhn: Chalcone Isomerase Activity in Anthers
03~ 0.6-
< 0.50.40.30203-'
0}1
~
a 013
0!4
015
46
0.~/
O:B
ol 2'5
5;
7'5
1;0
1;0~
b) +75
+ 50
+25
+100
+ 50/d
0[9
1:0mgprotein
al., 1978). The shortage of enzyme solution which could be prepared from anthers precluded more detailed investigations of the substrate specificity and of further enzyme activators and inhibitors. Independent of the situation found in enzyme preparations from anthers of Petunia hybrida, a clear CI activity could be demonstrated in corrolla extracts of each of the four lines. But these measurements also revealed significant differences in CI activity between the two lines V 10 and V 31 with dominant Po alleles and the two lines V 28 and V 33 with recessive po alleles (Table 1). Discussion
Fig. 1. Dependence of the enzymic isomerisation of naringeni.nchalcone on protein concentration, x - - x line V 10; :~--D line V28; o Mixtures containing enzyme extract of line V10 (a) and line V28 (b) in ~tl per assay. The values were plotted against protein contents of two different V 10 enzyme extracts (a). Lines V31 and V33 behaved similarly
Table 2. The effect of some substrates and several additions upon
the enzymic conversion of chalcone to flavanone Additions
191
Substrate
Activity of the enzyme i n %
None
naringeniu-chalcone
100
None
naringenin-chalcone-2"glucoside (isosalipurposide)
0
None
isoliquiritigenin
0
1 mM KCN
naringenin-chalcone
100
10 m M K C N
naringenin-chalcone
100
0.02 M MgClz
naringenin-chalcone
104
0.1 mM p-hydro- naringenin-chalcone xymercuribenzoate
22.2
0.5mM p-hydro- naringenin-chalcone xymercuribenzoate
12,5
The enzyme from anthers of Petunia hybrida has a pH optimum of about pH 8.6. This value corresponds to the values found for chalcone isomerases from other plants (Halbrock et al., 1970; Kuhn et al., 1978). In accordance with the data given by Moustafa and Wong (1967) for CI, the enzymic conversion of naringenin-chalcone to naringenin is not affected by chelating reagents but is strongly inhibited by low concentrations of p-hydroxymercuribenzoate (Table 2). Moreover, the enzyme from anthers of Petunia hybrida showed the same high substrate specificity (Table 2) as the chalcone isomerases from other plants (Hahlbrock et al., 1970; Wiermann, 1972; Kuhn et
The results obtained from chemogenetical investigations of a yellow pigment in pollen of some lines of Petunia hybrida (de Vlaming and Kho, 1976) correspond largely with the results obtained from similar investigations of the flavonoid compounds in yellow flowering lines of Callistephus chinensis (Kuhn et al., 1978). In both plants, a definite gene (Po in Petunia and Ch in Callistephus) controls a discrete step in flavonoid biosynthesis localized between chalcones and higher oxidized flavonoids. An accumulation of chalcones only takes place in recessive genotypes while in genotypes with wild-type alleles flavonols and anthocyanins are synthesized. Contrary to the temporary accumulation of chalcones in anthers of Tulipa reported by Quast and Wiermann (1973), the accumulation of naringenin-chalcone in pollen of Petunia hybrida is not restricted to a specific developmental stage. The recessive alleles of the genes Po and Ch also agree with regard to their performance. Both suppress the synthesis of other flavonoids and anthocyanins but apparently do not interrupt it completely. The close agreement between the genes Po and Ch on chemogenetical basis suggested that, both in the blossoms of Callistephus chinensis and in the pollen of Petunia hybrida, a deficiency of an enzyme catalysing the conversion of chalcones to other classes of flavonoids should be responsible for the chalcone accumulation. In Callistephus chinensis, the enzyme concerned was found to be the chalcone isomerase (Kuhn et al., 1978). The results of the present paper demonstrate that the accumulation of chalcone in pollen of Petunia hybrida is also caused by a nearly complete deficiency of this enzyme. Because of the extremely small amounts of material, the enzymic tests were performed with crude extracts. Nevertheless, CI could be demonstrated in anthers with blue or white pollen (Po dominant) in relatively high activity. In this connection it should be mentioned that compared to the high values of the specific activity of CI found in the corrollas, the
192 substantially lower values in the anthers are partly caused by the very high protein concentration of anther extracts in comparison to those of the corrolla extracts. The very low activity of CI found in anthers with yellow or green pollen (Po recessive) is obviously not sufficient for a complete conversion of naringeninchalcone to naringenin in vivo. But this low enzyme activity is probably responsible for the presence of appreciable amounts of dihydroflavonol and flavonol in yellow pollen and anthocyanin in green pollen in addition to naringenin-chalcone. Due to the largely additive behaviour of the mixed extracts from anthers with high enzyme activity and those with very low activity, it is unlikely that an inhibitor is responsible for the deficiency of CI activity in pollen which accumulate naringenin-chalcone. Thus, the Po gene is more likely to be concerned with the synthesis of the enzyme. As far as is known, in pollen of Petunia hybrida only flavonoids with a phloroglucinol type of A-ring occur. Therefore, the substrate specificity of the CI in pollen corresponds completely to expectations. Furthermore, chalcone glucosides were also not isomerized by chalcone-isomerases from other plants (Hahlbrock et al., 1970; G r a m b o w and Grisebach, 1971). Application of 10 mM K C N caused no decrease in reaction velocity. This proved that the reaction rate measured is due only to CI and not partly caused by peroxidase (Boland and Wong, 1975). Both the chemogenetic and enzymologic evidence allows a clear statement regarding the role of CI in the biosynthesis of flavonoids in pollen of Petunia hybrida. In blue and white pollen, the CI catalyses the conversion of naringenin-chalcone to naringenin and yields by this reaction the substrate for the formation of flavonols and anthocyanins. In yellow and green pollen, however, the synthesis of flavonols and anthocyanins is largely interrupted by reason of the nearly complete deficiency of CI activity. Since, in this case, naringenin-chalcone is accumulated, the first reaction product of the flavanone synthase in pollen of Petunia hybrida is more likely to be chalcone rather than flavanone, as found by Kreuzaler and Hahlbrock (1975) in Petroselinum. Thus, in pollen of Petunia hybrida, CI plays the same important role as in blossoms of Callistephus chinensis. The situation found in Callistephus chinensis, therefore, seems not to be an exceptional case. This is also supported by a further correlation between chalcone accumulation and deficiency of CI activity demonstrated on chemogenetic and enzymic investigations of yellow flowering lines of Dianthus caryophyllus (Forkmann and Kuhn, in preparation). Moreover, similar conclusions could be drawn from
G. Forkmann and B. Kuhn: Chalcone Isomerase Activity in Anthers the results of investigations on anthers of Tulipa and Lilium (Wiermann, 1972) although in this case the accumulation of chalcone and the activity of CI were not measured in relation to a definite gene but to different developmental stages of the anthers. The clear CI activity demonstrated in the corrollas of each line of Petunia hybrida investigated can not be explained by activation and inactivation of the gene Po in different plant tissues alone; it seems preferable to postulate that another gene is mainly responsible for the enzyme activity measured in the corrollas. Of course, this postulated gene has to be inactivated in pollen. The gene Po, however, could be active in both pollen and corrollas. Possibly, the significantly higher activities found in corrollas of the lines V 10 and V 31 with dominant Po alleles could be caused by two genes, the postulated isomerase gene and the Po gene. These investigations were supported by a grant from the Deutsche Forschungsgemeinschaft. The authors are very grateful to Dr. de Vlaming, Institute of Genetics, University of Amsterdam for her generous gift of seeds of the four genetically defined lines of Petunia hybrida and to Prof. H. Grisebach for his critical reading of the manuscript and valuable suggestions.
References Boland, M.J., Wong, E.: Purification and kinetic properties of chalcone-flavanone-isomerase from Soya bean. Eur. J. Biochem. 50, 383-389 (1975) Grambow, H.J., Grisebach, H.: Further studies on biosynthesis of flavonoids in Datisea cannabina. Phytochemistry 10, 789 (1971) Grisebach, H. : Enzymologieder Flavonoidbiosynthese. Ber. dtsch. Bot. Ges. 88, 61-69 (1975) Hahlbrock, K., Wong, E., Schill, L., Grisebach, H.: Comparison of chalcone-flavanone isomerase heteroenzymes and isoenzymes. Phytochemistry9, 949-958 (1970) Hahlbrock, K., Grisebach, H. : Biosynthesis of flavonoids. In : The flavonoids. Harborne, J.B. Mabry, T.J., Mabry, H., ed., p. 866 915. London: Chapman and Hail I975 Kreuzaler, F., Hahlbrock, K.: Enzymic synthesis of an aromatic ring from acetate units. Eur. J. Biochem. 56, 205-213 (1975) Kuhn, B., Forkmann, G., Seyffert, W.: Genetic control of chalcone-flavanone isomerase activity in Callisthephus chinensis. Planta 138, 199 203 (1978) Moustafa, E., Wong, E.: Purification and properties of chalconeflavanone isomerase from Soya bean seed. Phytochemistry 6, 625 632 (1967) Quast, L., Wiermann, R. : Uber das Vorkommen verschieden substituierter Chalkone wS,hrend der Mikrosporogenese. Experienta 29, 1165 (1973) de u P_, Kho, F.F.K.: 4,T,4',C-Tetrahydroxychalconein pollen of Petunia hybrida. Phytochemistry 15, 348 349 (1976) Wiering, H.: Genetics of flower colour in Petunia hybrida hort. Gen. Phaenen. 17, 117-134 (1974) Wiermann, R.: Aktivitfit der Chalkon-Flavanon Isomerase und Akkumulation yon phenylpropanoiden Verbindungen in Antheren. Planta 102, 55-60 (1972) Received 10 July ; accepted 6 September 1978
