Planta
Planta 138, 199-203 (1978)
by Springer-Verlag 1978
Genetic Control of Chalcone-Flavanone Isomerase Activity in Cailistephus chinensis B. Kuhn, G. F o r k m a n n and W. Seyffert [nstitut far Biologie II, Lehrstuhl ffir Genetik der Universitfit Tfibingen, Auf der Morgenstelle 28, D-7400 Tfibingen, Federal Republic of Germany
Abstract. A mutant blocked in anthocyanin synthesis leads to an accumulation of 4,2',4',6'-tetrahydroxychalcone-2'-glucoside (isosalipurposide) in blossoms of Callistephus chinensis (L.) Nees, whereas in genotypes with the wild-type allele, higher oxidized flavonoids and anthocyanins are synthesized. Measurements of chalcone-flavanone isomerase activity of 18 lines of Callistephus chinensis showed a clear correlation between accumulation of chalcone in the recessive genotypes (ch ch) and deficiency of this enzyme activity. Both the chemogenetic and the enzymologic evidence lead to the following conclusions: 1. The first product of the synthesis of the flavonoid skeleton should be tetrahydroxychalcone.-2. The chalconeflavanone isomerase catalyzes the formation of flavanone from chalcone in a stereospecific way and therewith furnishes the substrate for the further reactions in the flavonoid biosynthesis.
Key words: Callistephus -
Chalcone-flavanone isomerase - Genetic block - Flavonoid biosynthesis - 4,2',4',6'_Tetrahydroxychalcone_2'_glucoside.
substrate for further reactions (Grisebach, 1975). An experimental decision is difficult especially since there exists an enzymic equilibrium between chalcone and flavanone and because the respective enzyme, the chalcone-flavanone isomerase, occurs in all plants so far examined (Moustafa and Wong, 1967; Hahlbrock et al., 1970). Likewise the significance of the chalconeflavanone isomerase for the adjustment of the balance between chalcone and flavanone is uncertain. On the basis of tracer experiments, Grisebach (1962) postulated tetrahydroxychalcone as the first product in the synthesis of the flavonoid skeleton. Consequently, the chalcone-flavanone isomerase should catalyze the cyclization of the chalcone to the flavanone in a stereospecific way. However, in experiments with the key enzyme of flavonoid biosynthesis, the flavanone-synthase, naringenin was found as the first reaction product (Kreuzaler and Hahlbrock, 1975). Therefore, it must be assumed that the cyclization to the flavanone takes place already at the synthase molecule. Thus, the possibility that the chalcone-flavanone isomerase does not catalyze the isomerization to the flavanone, but opens the pyrone ring to yield the chalcone (Grisebach, 1975) became evident.
Introduction Suitably substituted chalcones can be considered as central intermediates in flavonoid biosynthesis (Grisebach and Barz, 1969). Up to now, the question remains whether the biosynthesis of the higher oxidized flavonoids and anthocyanins arises from the chalcone or from the flavanone stage. Results of competition tests and enzymic assays suggest on the one side the chalcone and on the other side the flavanone as the Abbreviations." EGME=ethylene glycol monomethyl ether; HOAc= acetic acid~ MeOH= methanol ; PVP = polyvinylpyrrolidone; TBA=tert. butanol-acetic acid-water, 3 : 1: 1; TLC = thinlayer chromatography
Materials and Methods The investigations included several acyanic and anthocyanin-conraining lines of Callistephus chinensis (Table 1). The phenolic compounds were analyzed by paper- and TLC (SeytTert, unpublished; Forkmann, 1977). The multiple alleles at the R locus (R, r', and r) in the anthocyanin-containinglines are responsibie for the degree of substitution of the ring B. The gene M concerns the glycosylation in the 5-position of the anthocyanins (Wit, 1937; Forkmann, 1977). Extensive cross experiments indicated that the anthocyanin synthesis is blocked in the acyanic lines by at least two complementary acting factors, a and f (L6wer: unpublished). Additionally another gene responsible for anthocyanin synthesis (ch) was detected. It exists in the three yellow lines and in the line called "lavendel" in a recessive state and there leads to an accumulation of tetrahydro-
0032-0935/78/0138/0199/$01.00
B. Kuhn et al. : Chalcone-Flavanone Isomerase Activity
200 Table 1. Genotype and phenotype of 18 lines of Callistephus ehinensis Line
Genotype
01 02 03 04 05 06 07 08 09 10a 10b 10c 10d 10e I0f 10g 10h Lavendel
ChCh AA FF ChCh AA FF ChCh AA FF ChCh AA FF ChCh AA FF ChCh AA FF chch aa FF C h C h a a FF ChCh aa FF ChCh AA ff ChCh AA ff ChCh aa FF ChCh aa FF chch A A f f ChCh aa FF C h C h A A ff chch AA ff chch AA FF
RR r'r' rr RR r'r' rr rr rr rr RR RR RR RR rr RR rr r'r'
MM MM MM mm mm mm MM MM MM MM MM MM MM MM MM MM MM
Color
Tetrahydroxychalcone-2'-glucoside (isosalipurposide)
Naringenin glycosides
blue lilac pink violet purple red yeliow ivory white ivory ivory ivory ivory yellow ivory pale ivory yellow yellow with traces of anthocyanins
+++ + ++ + + + + + +
n.i." n.J. n.i. n.i. n.i. n.J. + * * * * * * + * * + +
Apigenin and Ap glycosides
n.i. n.i. n.J. n.i. ~n.i. n.i. + * * * * * * + * * + +
Flavonol glycosides
Anthocyanin glycosides
* * * * * * tr b * tr + + + + + + * * tr * * tr n.i.
+ + + + + + +
+ + + + + +
+ + + + + +
+ + + = high concentration; + = l o w concentration; - = not detectable; * = p r e s e n t but concentration not measured a b
n.i. = n o t investigated tr = traces
xychalcone-T-glucoside (isosalipurposide). In these lines also small amounts of apigenin, apigenin glucosides, and naringenin glucosides are found (Seyffert, unpublished). The so-called lavendel type occurs as a new combination in the Fz after cross experiments with yellow and anthocyanin-containing lines. Here anthocyanin synthesis obviously is affected only by the recessive cb gene, whereas in the three yellow lines, one of the complementary factors, a or f, is additionally present in a recessive state (L6wer, unpublished). This leads to complete inhibition of the anthocyanin synthesis. The enzymic investigations were carried out with all lines listed in Table 1. The flowers were picked the day before utilization, the stems placed in water, and kept overnight at 4 ~ C to equilibrate the water content.
buffer and enzyme. All values were corrected for the rate of the spontaneous isomerization.
Identification of the Reaction Product An assay mixture (5 ml) as given above and another containing additionally 0.4 m M p-hydroxymercuribenzoate were extracted after 3 min with 2 x 5 ml ethyl acetate. The combined ethyl acetate fractions were evaporated to dryness in a vacuum and the solid residue was redissolved in 0.1 ml MeOH. The reaction product was identified by spectral analysis and TLC on 0.1 mm cellulose plates with naringenin and tetrahydroxychalcone using both 30% HOAc and TBA.
Preparation of Chalcones Enzyme Preparation From each line 10 g of flowers were homogenized in a Waring Blender at 4~ with 80 ml 0.05 M Soerensen buffer, pH 6.8, containing 1.4raM mercaptoethanol and 5g insoluble PVP (Serva) equilibrated in the Soerensen buffer. Filtration and centrifugation led to total removal of PVP and debris. The supernatant was fi'actionated with solid (NH4)2SO4. The precipitation between 35% and 75% saturation, dissolved in 4 ml 0.05 M sodium phosphate buffer, pH 7.5, was used as enzyme source.
Enzyme Assay The reaction mixture (1.7 ml) contained 0.05 M sodium phosphate, pH 7,5, 20 gl enzyme solution (0.08 mg protein) and 0.17 m M chalcone in EGME. The reaction was started by addition of the chalcone. The decrease in absorptivity at the absorption m a x i m u m of the chalcone (385 nm) was plotted against time in a registrating spectral photometer (Zeiss RPQ 20) at 30 ~ C. The blank contained
4,2',4',6'-tetrahydroxychalcone was prepared from naringenin (Sigma) according to Moustafa and Wong (1967). Isoliquiritigenin was synthesized according to Geissmann and Clinton (1946). Isosalipurposide (4,2',4'06'-tetrahydroxychalcone-2'-glucoside) was isolated from yellow commercial strains of Dianthus caryophyllus (Harborne, 1966).
Determination of pH Optimum The " B r i t t o n - R o b i n s o n I " buffer was used (Rauen, 1964). The reaction rates were measured at the absorption maxima of tetrahydroxychalcone at the respective pH values. Since the heights of the absorption maxima were slightly different in response to pH all measured values were related to the m a x i m u m measured at pH 7.5.
Determination of Protein The method of Bradford (1976) was used.
B. K u h n et al. : Chalcone-Flavanone Isomerase Activity
201
Results
1.0"
The assays of the chalcone-free lines (cf. Table 1) exhibited in all cases a very rapid decrease of the applied tetrahydroxychalcone. At the conditions chosen the reaction remained linear for about 90 s. The part due to spontaneous isomerization of tetrahydroxychalcone (in vitro) amounts to approximately 28% (Fig, 1). On the contrary, in the strains t h a t accumulate isosalipurposide (07, 10e, 10h and "lavendel") no decrease in absorptivity exceeding spontaneous isomerization of tetrahydroxychalcone could be observed (Fig. 1). These results are characteristic not only for flowers of the lines investigated but also for buds and even for leaves. For the chalcone-free lines a linear dependence exists between protein concentration and the reaction rate (Fig. 2). In the chalcone-rich lines, even at high protein concentration, no decrease of absorption exceeding spontaneous isomerization could be found. Mixtures of enzyme extracts with high activity and no activity behaved additively. This proved to be the case even if the enzyme extract was prepared from a mixture (1:1) of flowers from a chalcone-rich and a chalconefree strain (Fig. 2). The spectral and chromatographic analyses of the assay mixture showed complete conversion of tetrahydroxychalcone to naringenin in the chalcone-free lines. In the assays from the chalconerich Iines and in all tests in the presence of p-hydroxymercuribenzoate a portion of the applied tetrahydroA
0g0.6~9 04"
P,
.ca
0.2054 2~
0dB 5p
121~ 7~5
0.2 mg protein o/oadiveenzyme
Fig. 2. Dependenceof the enzymic isomerization of tetrahydroxychalcone from protein concentration, x x Lines with isomerase activity (representative measurement); 9 lines 07, 10e, 10h, and "lavendel" (representative measurement); e--o mixtures from enzyme extracts with high activity and with those of no activity; z~ activity in mixtures from flowers of a chaleone-free and a chalcone-containingline (1 : 1, w/w)
Table 2. Identification of the reaction product
R f ( x 100) in
2~
TBA
30% H O A c
Reaction mixture
MeOH
4,2',4',6'-Tetrahydroxychalcone
85
17
385
364
Naringenin
91
70
322
288
Reaction product
91
69
322
288
in (rim)
Table 3. Effect of several enzyme activators and inhibitors upon the enzymic conversion of tetrahydroxychalcone to naringenin
Additions
o.sJ
0.1'6 1(}0
Activity of the enzyme
(%) None 10 m M N a N 3 1 mM KCN 0.01 M E D T A 0.1 m M p hydroxymercuribenzoate 0.5 m M p-hydroxymercuribenzoate 0.02 M MgC12 6 m M dithiothreitol
0.z,
100 100 100 I00 I3.5 0 106 i26.7
02-
1
2
3
4 rain
Fig. 1. Enzymatic and spontaneous isomerization of tetrahydroxychalcone in the 18 investigated lines, x - - x Disappearance of chalcone (measured by the decrease of absorptivity at 385 nm) in the presence of enzyme from chalcone-free lines (representative measurement); 9 9 disappearance of chalcone in the presence of an enzyme from the chalcone-rich lines (07, 10e, I0h, and "lavendel'3 (representative measurement); 9 - 9 spontaneous isomerization
xychalcone was still detected in addition to naringenin (Table 2). The influence of several activators and inhibitors upon the activity of the enzyme from Callistephus chinensis is in good accordance with the data given by Moustafa and Wong (1967) for the chalcone-flavanone isomerase (Table 3). The enzyme has a pH optimum of about pH 8.6 (Fig. 3). Similar values were also found for the chalcone-flavanone isomerase from Petroselinum hortense, Cicer arietinum, and Phaseolus aureus (Hahlbrock et aL, 1970).
202
B. Kuhn et al. : Chalcone-Flavanone Isomerase Activity
020 c_
~ 015 010
7'0
2'5
8~3 8'5
90
1(]0
110pH
Fig. 3. pH-Dependence of enzyme activity from Callistephus chil~lensis
Since the chalcone-flavanone isomerase can exhibit a high substrate specificity (Hahlbrock et al., 1970; Wiermann, 1972), isoliquritigenin (4,2',4'-trihydroxychalcone) and isosalipurposide (4,2',4',6'-tetrahydroxychalcone-T-glucoside) were tested as substrates in the assay for the enzyme from Callistephus chinensis. However, only 4,2',4',6'-tetrahydroxychalcone was converted to the flavanone.
Discussion
The present work should contribute toward an answer to the questions mentioned in the Introduction, by means of measurements of the chalcone-flavanone isomerase activity in chemogenetically defined lines of Callistephus chinensis. Analysis of the phenolic compounds already suggests that in the genotypes which accumulate isosalipurposide (lines 07, 10e, 10h, and "lavendel") a genetic block suppresses the synthesis of higher oxidized flavonoids but apparently does not interrupt it completely. The cross experiments proved that the genetic block is identical in these lines and the feature "chalcone accumulation" segregates monogenically. Only in recessive genotypes does this lead to an accumulation of isosalipurposide. The most obvious interpretation is a deficiency of an enzyme that catalyzes the conversion of chalcone to other classes of flavonoids. A comparable situation was found recently in the pollen of defined lines of Petunia (de Vlaming and Kho, 1976): Here a gene W leads in the recessive state to an accumulation of chalcone whereas under the influence of dominant alleles higher oxidized flavonoids are synthesized. Since in appropriate genotypes, in addition to chalcones, anthocyanins also can be detected, the deficiency likewise seems to be incomplete. On the basis of previous knowledge about flavonoid biosynthesis, the concerned enzyme could be in all probability the chalcone-flavanone isomerase.
Yet it must be taken into consideration that the accumulation of isosalipurposide might possibly be attributed to a deficiency of a chalcone peroxidase. Such an enzyme was detected in Phaseolus vulgaris and Cicer arietinurn (Rathmell and Bendall, 1972; Wong and Wilson, 1972). It catalyzes the conversion of isoliquiritigenin to auron and via dihydroflavonol to garbanzol. It is completely inhibited by KCN. We could demonstrate the same reaction in all examined lines of Callistephus chinensis, but only with isoliquiritigenin as substrate. 4,2',4',6'-Tetrahydroxychalcone, which really should function as the natural substrate, was never converted to auron or flavonol. Furthermore the concerned enzyme of the genetic block in Callistephus is neither inhibited by KCN nor affected in its activity by other chelating agents. These results prove the improbability of chalcone accumulation being caused by an absence of a peroxidase reaction. On the contrary, that chalcone-flavanone isomerase is the concerned enzyme, is supported by the evidence that naringenin is the sole reaction product, and also that there is a broad agreement of all enzymatic results with the data of the chalconeflavanone isomerases from other plants (Moustafa and Wong, 1967; Hahlbrock et al., 1970; Wiermann, 1972). The substrate specificity of the enzyme corresponds completely to expectations, since in Callistephus chinensis only flavonoids with a phloroglucinol type of the ring A occur. Due to the additive behavior of the mixed assays with extracts of high activity and no activity and of the enzyme extracts from mixtures of flowers from the corresponding lines, it seems unlikely that a macromolecular inhibitor or regulation mechanisms are responsible for the enzyme activity deficiency in the lines which accumulate isosalipurposide. The Ch gene should rather be concerned with the synthesis of the chalcone-flavanone isomerase. It should be emphasized that in chalcone-free lines enzyme activity could be observed in flowers, buds, and leaves. In chalcone-rich lines, however, chalconeftavanone isomerase activity was neither detectable in flowers or leaves nor during the time of high chalcone synthesis (buds). On the basis of these results, for the first time a correlation could be established between a genetic block and an enzyme deficiency concerning a step in the actual flavonoid biosynthesis. The results of this work allow, at least for the examined material, a clear statement about the role of the chatcone-flavanone isomerase and consequently also about the substrate for the further reactions. The only interpretation for the accumulation of isosalipurposide in lines without chalcone-flavanone
B. Kuhn et al. : Chalcone-Flavanone Isomerase Activity
isomerase activity can be that the first product of the flavanone synthase reaction in Callistephus is not naringenin but rather 4,2',4',61-tetrahydroxychalcone. In lines without isomerase activity the synthesis of higher oxidized flavonoids is largely interrupted. In this case tetrahydroxychalcone can function as substrate for a glucosyltransferase and can be converted to isosalipurposide. Whereas in the lines with high isomerase activity no chalcone can be detected, increased amounts of flavones, flavonols, and anthocyanins appear. The role of the chalcone-flavanone isomerase can therefore only be to catalyze the isomerization of chalcone to flavanone in a stereospecific way and so to yield the substrate for the further reactions. Thus, the chalcone-flavanone isomerase plays an important part in flavonoid biosynthesis. These assertions are confirmed by investigations of the chalcone-flavanone isomerase in different stages of development in anthers from Tulipa and Lilium. At the maximal activity of the enzyme a rapid decrease of the previously accumulated chalcone and a corresponding increase of flavonols and anthocyanins occurred (Wiermann, 1972). These statements about the role of the chalconeflavanone isomerase are further confirmed by the evidence that in Petroselinum flavanone and not chalcone is the substrate for the oxidation to the flavone (Sutter et al., 1975). The question as to why small amounts of naringenin glycosides and higher oxidized flavonoids are found in the lines without any detectable isomerase activity must be left open at present. Possibly a low activity of the isomerase is responsible for it. However, even after application of a 5-fold increase in the usual enzyme quantity to the assay mixture no decrease of absorption exceeding spontaneous isomerization could be demonstrated. Perhaps the formation of these flavonoids could be attributed to a spontaneous isomerization of tetrahydroxychalcone to naringenin in vivo. Supposing that, there exists a spontaneous isomerisation in vivo, only the (2S) enantiomer of the formed naringenin could serve as substrate for further reactions. The results of this paper are consistent with those of earlier tracer experiments (Grisebach, 1962), but are contradictory to those from investigations of flavanone synthase, in which naringenin was obtained as the first reaction product (Kreuzaler and Hahlbrock, 1975). In this case it must be considered that
203
the experiments with the flavanone synthase were carried out with cell cultures of Petroselinum hortense, i.e., with a different species and on undifferentiated tissue. Therefore, these results might not be comparable with the conditions in Callistephus chinensis. These investigations were supported by a grant from the Deutsche Forschungsgemeinschaft. The authors are grateful to Prof. H.Grisebach for his critical reading of the manuscript and valuable suggestions.
References Bradford, M.M. : A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry 72, 248-254 (1976) Forkmann, G.: Anthocyanin pigments in Callistephus chinensis. Phytochemistry 16, 299 301 (1977) Geissman, T.A., Clinton, R.Q.: Flavanones and related compounds. I. Preparation of polyhydroxychalcones and -flavanones. J. Am. Chem. Soc. 68, 697-699 (1946) Grisebach, H.: Die Biosynthese der Flavonoide. Planta Medica 10, 385-397 (1962) Grisebach, H.: Enzymologie der Flavonoidbiosynthese. Ber. Deutsch. ,Bot. Ges. 88, 61-69 (1975) Grisebach, H., Barz, W. : Biochemie der Flavonoide. Naturwissenschaften 56, 538 544 (1969) Hahlbrock, K., Wong, E., Schili, L.', Grisebach, H.: Comparison of chalcone-flavanone isomerase heteroenzymes and isoenzymes. Phytochemistry 9, 949 958 (1970) Harborne, H.B.: Comparative biochemistry of flavonoids I. Distribution of chalcone and auroue pigments in plants. Phytochemistry N, 111-115 (1966) Kreuzaler, F., Halbrock, K.: Enzymic synthesis of an aromatic ring from acetate units. Eur. J. Biochem. 56, 205 213 (1975) Moustafa, E., Wong, E.: Purification and properties of chalconeflavanone isomerase from Soya bean seed. Phytochemistry 6, 625-632 (I967) Rathmell, W.G., Bendall, D.S. : The peroxidase-catalysed oxidation of a chalcone and its possible physiological significance. Biochem. J. 127, 125 132 (1972) Rauen, H.M.: Biochemisches Taschenbuch Teil 2. Berlin-G6ttingen-Heidelberg-New York: Springer 1964 Sutter, A., Poulton, J., Grisebach, H.: Oxidation of flavanone to flavone with cell-free extracts from young Parsley leaves. Arch. Biochem. Biophys. 170, 547 556 (1975) Vlaming, P. de, Kho, F.F.K.: 4,2',4',6'-Tetrahydroxychalcone in pollen of Petunia hybrida. Phytochemistry 15, 348 349 (1976) Wiermann, R.: Aktivit/it der Chalkon-Flavanon Isomerase und Akkumulation yon phenylpropanoiden Verbindungen in Antheren. PIanta 102, 55-60 (1972) Wit, F. : Contributions to the genetics of the China Aster. Genetica 19, 1-104 (1937) Wong, E., Wilson, J.M, : The oxidation of chalcone catalyzed by peroxidase. Phytochemistry 11, 875 (1972) Received 7 July; accepted 15 November 1977
Planta 138, 199-203 (1978)
by Springer-Verlag 1978
Genetic Control of Chalcone-Flavanone Isomerase Activity in Cailistephus chinensis B. Kuhn, G. F o r k m a n n and W. Seyffert [nstitut far Biologie II, Lehrstuhl ffir Genetik der Universitfit Tfibingen, Auf der Morgenstelle 28, D-7400 Tfibingen, Federal Republic of Germany
Abstract. A mutant blocked in anthocyanin synthesis leads to an accumulation of 4,2',4',6'-tetrahydroxychalcone-2'-glucoside (isosalipurposide) in blossoms of Callistephus chinensis (L.) Nees, whereas in genotypes with the wild-type allele, higher oxidized flavonoids and anthocyanins are synthesized. Measurements of chalcone-flavanone isomerase activity of 18 lines of Callistephus chinensis showed a clear correlation between accumulation of chalcone in the recessive genotypes (ch ch) and deficiency of this enzyme activity. Both the chemogenetic and the enzymologic evidence lead to the following conclusions: 1. The first product of the synthesis of the flavonoid skeleton should be tetrahydroxychalcone.-2. The chalconeflavanone isomerase catalyzes the formation of flavanone from chalcone in a stereospecific way and therewith furnishes the substrate for the further reactions in the flavonoid biosynthesis.
Key words: Callistephus -
Chalcone-flavanone isomerase - Genetic block - Flavonoid biosynthesis - 4,2',4',6'_Tetrahydroxychalcone_2'_glucoside.
substrate for further reactions (Grisebach, 1975). An experimental decision is difficult especially since there exists an enzymic equilibrium between chalcone and flavanone and because the respective enzyme, the chalcone-flavanone isomerase, occurs in all plants so far examined (Moustafa and Wong, 1967; Hahlbrock et al., 1970). Likewise the significance of the chalconeflavanone isomerase for the adjustment of the balance between chalcone and flavanone is uncertain. On the basis of tracer experiments, Grisebach (1962) postulated tetrahydroxychalcone as the first product in the synthesis of the flavonoid skeleton. Consequently, the chalcone-flavanone isomerase should catalyze the cyclization of the chalcone to the flavanone in a stereospecific way. However, in experiments with the key enzyme of flavonoid biosynthesis, the flavanone-synthase, naringenin was found as the first reaction product (Kreuzaler and Hahlbrock, 1975). Therefore, it must be assumed that the cyclization to the flavanone takes place already at the synthase molecule. Thus, the possibility that the chalcone-flavanone isomerase does not catalyze the isomerization to the flavanone, but opens the pyrone ring to yield the chalcone (Grisebach, 1975) became evident.
Introduction Suitably substituted chalcones can be considered as central intermediates in flavonoid biosynthesis (Grisebach and Barz, 1969). Up to now, the question remains whether the biosynthesis of the higher oxidized flavonoids and anthocyanins arises from the chalcone or from the flavanone stage. Results of competition tests and enzymic assays suggest on the one side the chalcone and on the other side the flavanone as the Abbreviations." EGME=ethylene glycol monomethyl ether; HOAc= acetic acid~ MeOH= methanol ; PVP = polyvinylpyrrolidone; TBA=tert. butanol-acetic acid-water, 3 : 1: 1; TLC = thinlayer chromatography
Materials and Methods The investigations included several acyanic and anthocyanin-conraining lines of Callistephus chinensis (Table 1). The phenolic compounds were analyzed by paper- and TLC (SeytTert, unpublished; Forkmann, 1977). The multiple alleles at the R locus (R, r', and r) in the anthocyanin-containinglines are responsibie for the degree of substitution of the ring B. The gene M concerns the glycosylation in the 5-position of the anthocyanins (Wit, 1937; Forkmann, 1977). Extensive cross experiments indicated that the anthocyanin synthesis is blocked in the acyanic lines by at least two complementary acting factors, a and f (L6wer: unpublished). Additionally another gene responsible for anthocyanin synthesis (ch) was detected. It exists in the three yellow lines and in the line called "lavendel" in a recessive state and there leads to an accumulation of tetrahydro-
0032-0935/78/0138/0199/$01.00
B. Kuhn et al. : Chalcone-Flavanone Isomerase Activity
200 Table 1. Genotype and phenotype of 18 lines of Callistephus ehinensis Line
Genotype
01 02 03 04 05 06 07 08 09 10a 10b 10c 10d 10e I0f 10g 10h Lavendel
ChCh AA FF ChCh AA FF ChCh AA FF ChCh AA FF ChCh AA FF ChCh AA FF chch aa FF C h C h a a FF ChCh aa FF ChCh AA ff ChCh AA ff ChCh aa FF ChCh aa FF chch A A f f ChCh aa FF C h C h A A ff chch AA ff chch AA FF
RR r'r' rr RR r'r' rr rr rr rr RR RR RR RR rr RR rr r'r'
MM MM MM mm mm mm MM MM MM MM MM MM MM MM MM MM MM
Color
Tetrahydroxychalcone-2'-glucoside (isosalipurposide)
Naringenin glycosides
blue lilac pink violet purple red yeliow ivory white ivory ivory ivory ivory yellow ivory pale ivory yellow yellow with traces of anthocyanins
+++ + ++ + + + + + +
n.i." n.J. n.i. n.i. n.i. n.J. + * * * * * * + * * + +
Apigenin and Ap glycosides
n.i. n.i. n.J. n.i. ~n.i. n.i. + * * * * * * + * * + +
Flavonol glycosides
Anthocyanin glycosides
* * * * * * tr b * tr + + + + + + * * tr * * tr n.i.
+ + + + + + +
+ + + + + +
+ + + + + +
+ + + = high concentration; + = l o w concentration; - = not detectable; * = p r e s e n t but concentration not measured a b
n.i. = n o t investigated tr = traces
xychalcone-T-glucoside (isosalipurposide). In these lines also small amounts of apigenin, apigenin glucosides, and naringenin glucosides are found (Seyffert, unpublished). The so-called lavendel type occurs as a new combination in the Fz after cross experiments with yellow and anthocyanin-containing lines. Here anthocyanin synthesis obviously is affected only by the recessive cb gene, whereas in the three yellow lines, one of the complementary factors, a or f, is additionally present in a recessive state (L6wer, unpublished). This leads to complete inhibition of the anthocyanin synthesis. The enzymic investigations were carried out with all lines listed in Table 1. The flowers were picked the day before utilization, the stems placed in water, and kept overnight at 4 ~ C to equilibrate the water content.
buffer and enzyme. All values were corrected for the rate of the spontaneous isomerization.
Identification of the Reaction Product An assay mixture (5 ml) as given above and another containing additionally 0.4 m M p-hydroxymercuribenzoate were extracted after 3 min with 2 x 5 ml ethyl acetate. The combined ethyl acetate fractions were evaporated to dryness in a vacuum and the solid residue was redissolved in 0.1 ml MeOH. The reaction product was identified by spectral analysis and TLC on 0.1 mm cellulose plates with naringenin and tetrahydroxychalcone using both 30% HOAc and TBA.
Preparation of Chalcones Enzyme Preparation From each line 10 g of flowers were homogenized in a Waring Blender at 4~ with 80 ml 0.05 M Soerensen buffer, pH 6.8, containing 1.4raM mercaptoethanol and 5g insoluble PVP (Serva) equilibrated in the Soerensen buffer. Filtration and centrifugation led to total removal of PVP and debris. The supernatant was fi'actionated with solid (NH4)2SO4. The precipitation between 35% and 75% saturation, dissolved in 4 ml 0.05 M sodium phosphate buffer, pH 7.5, was used as enzyme source.
Enzyme Assay The reaction mixture (1.7 ml) contained 0.05 M sodium phosphate, pH 7,5, 20 gl enzyme solution (0.08 mg protein) and 0.17 m M chalcone in EGME. The reaction was started by addition of the chalcone. The decrease in absorptivity at the absorption m a x i m u m of the chalcone (385 nm) was plotted against time in a registrating spectral photometer (Zeiss RPQ 20) at 30 ~ C. The blank contained
4,2',4',6'-tetrahydroxychalcone was prepared from naringenin (Sigma) according to Moustafa and Wong (1967). Isoliquiritigenin was synthesized according to Geissmann and Clinton (1946). Isosalipurposide (4,2',4'06'-tetrahydroxychalcone-2'-glucoside) was isolated from yellow commercial strains of Dianthus caryophyllus (Harborne, 1966).
Determination of pH Optimum The " B r i t t o n - R o b i n s o n I " buffer was used (Rauen, 1964). The reaction rates were measured at the absorption maxima of tetrahydroxychalcone at the respective pH values. Since the heights of the absorption maxima were slightly different in response to pH all measured values were related to the m a x i m u m measured at pH 7.5.
Determination of Protein The method of Bradford (1976) was used.
B. K u h n et al. : Chalcone-Flavanone Isomerase Activity
201
Results
1.0"
The assays of the chalcone-free lines (cf. Table 1) exhibited in all cases a very rapid decrease of the applied tetrahydroxychalcone. At the conditions chosen the reaction remained linear for about 90 s. The part due to spontaneous isomerization of tetrahydroxychalcone (in vitro) amounts to approximately 28% (Fig, 1). On the contrary, in the strains t h a t accumulate isosalipurposide (07, 10e, 10h and "lavendel") no decrease in absorptivity exceeding spontaneous isomerization of tetrahydroxychalcone could be observed (Fig. 1). These results are characteristic not only for flowers of the lines investigated but also for buds and even for leaves. For the chalcone-free lines a linear dependence exists between protein concentration and the reaction rate (Fig. 2). In the chalcone-rich lines, even at high protein concentration, no decrease of absorption exceeding spontaneous isomerization could be found. Mixtures of enzyme extracts with high activity and no activity behaved additively. This proved to be the case even if the enzyme extract was prepared from a mixture (1:1) of flowers from a chalcone-rich and a chalconefree strain (Fig. 2). The spectral and chromatographic analyses of the assay mixture showed complete conversion of tetrahydroxychalcone to naringenin in the chalcone-free lines. In the assays from the chalconerich Iines and in all tests in the presence of p-hydroxymercuribenzoate a portion of the applied tetrahydroA
0g0.6~9 04"
P,
.ca
0.2054 2~
0dB 5p
121~ 7~5
0.2 mg protein o/oadiveenzyme
Fig. 2. Dependenceof the enzymic isomerization of tetrahydroxychalcone from protein concentration, x x Lines with isomerase activity (representative measurement); 9 lines 07, 10e, 10h, and "lavendel" (representative measurement); e--o mixtures from enzyme extracts with high activity and with those of no activity; z~ activity in mixtures from flowers of a chaleone-free and a chalcone-containingline (1 : 1, w/w)
Table 2. Identification of the reaction product
R f ( x 100) in
2~
TBA
30% H O A c
Reaction mixture
MeOH
4,2',4',6'-Tetrahydroxychalcone
85
17
385
364
Naringenin
91
70
322
288
Reaction product
91
69
322
288
in (rim)
Table 3. Effect of several enzyme activators and inhibitors upon the enzymic conversion of tetrahydroxychalcone to naringenin
Additions
o.sJ
0.1'6 1(}0
Activity of the enzyme
(%) None 10 m M N a N 3 1 mM KCN 0.01 M E D T A 0.1 m M p hydroxymercuribenzoate 0.5 m M p-hydroxymercuribenzoate 0.02 M MgC12 6 m M dithiothreitol
0.z,
100 100 100 I00 I3.5 0 106 i26.7
02-
1
2
3
4 rain
Fig. 1. Enzymatic and spontaneous isomerization of tetrahydroxychalcone in the 18 investigated lines, x - - x Disappearance of chalcone (measured by the decrease of absorptivity at 385 nm) in the presence of enzyme from chalcone-free lines (representative measurement); 9 9 disappearance of chalcone in the presence of an enzyme from the chalcone-rich lines (07, 10e, I0h, and "lavendel'3 (representative measurement); 9 - 9 spontaneous isomerization
xychalcone was still detected in addition to naringenin (Table 2). The influence of several activators and inhibitors upon the activity of the enzyme from Callistephus chinensis is in good accordance with the data given by Moustafa and Wong (1967) for the chalcone-flavanone isomerase (Table 3). The enzyme has a pH optimum of about pH 8.6 (Fig. 3). Similar values were also found for the chalcone-flavanone isomerase from Petroselinum hortense, Cicer arietinum, and Phaseolus aureus (Hahlbrock et aL, 1970).
202
B. Kuhn et al. : Chalcone-Flavanone Isomerase Activity
020 c_
~ 015 010
7'0
2'5
8~3 8'5
90
1(]0
110pH
Fig. 3. pH-Dependence of enzyme activity from Callistephus chil~lensis
Since the chalcone-flavanone isomerase can exhibit a high substrate specificity (Hahlbrock et al., 1970; Wiermann, 1972), isoliquritigenin (4,2',4'-trihydroxychalcone) and isosalipurposide (4,2',4',6'-tetrahydroxychalcone-T-glucoside) were tested as substrates in the assay for the enzyme from Callistephus chinensis. However, only 4,2',4',6'-tetrahydroxychalcone was converted to the flavanone.
Discussion
The present work should contribute toward an answer to the questions mentioned in the Introduction, by means of measurements of the chalcone-flavanone isomerase activity in chemogenetically defined lines of Callistephus chinensis. Analysis of the phenolic compounds already suggests that in the genotypes which accumulate isosalipurposide (lines 07, 10e, 10h, and "lavendel") a genetic block suppresses the synthesis of higher oxidized flavonoids but apparently does not interrupt it completely. The cross experiments proved that the genetic block is identical in these lines and the feature "chalcone accumulation" segregates monogenically. Only in recessive genotypes does this lead to an accumulation of isosalipurposide. The most obvious interpretation is a deficiency of an enzyme that catalyzes the conversion of chalcone to other classes of flavonoids. A comparable situation was found recently in the pollen of defined lines of Petunia (de Vlaming and Kho, 1976): Here a gene W leads in the recessive state to an accumulation of chalcone whereas under the influence of dominant alleles higher oxidized flavonoids are synthesized. Since in appropriate genotypes, in addition to chalcones, anthocyanins also can be detected, the deficiency likewise seems to be incomplete. On the basis of previous knowledge about flavonoid biosynthesis, the concerned enzyme could be in all probability the chalcone-flavanone isomerase.
Yet it must be taken into consideration that the accumulation of isosalipurposide might possibly be attributed to a deficiency of a chalcone peroxidase. Such an enzyme was detected in Phaseolus vulgaris and Cicer arietinurn (Rathmell and Bendall, 1972; Wong and Wilson, 1972). It catalyzes the conversion of isoliquiritigenin to auron and via dihydroflavonol to garbanzol. It is completely inhibited by KCN. We could demonstrate the same reaction in all examined lines of Callistephus chinensis, but only with isoliquiritigenin as substrate. 4,2',4',6'-Tetrahydroxychalcone, which really should function as the natural substrate, was never converted to auron or flavonol. Furthermore the concerned enzyme of the genetic block in Callistephus is neither inhibited by KCN nor affected in its activity by other chelating agents. These results prove the improbability of chalcone accumulation being caused by an absence of a peroxidase reaction. On the contrary, that chalcone-flavanone isomerase is the concerned enzyme, is supported by the evidence that naringenin is the sole reaction product, and also that there is a broad agreement of all enzymatic results with the data of the chalconeflavanone isomerases from other plants (Moustafa and Wong, 1967; Hahlbrock et al., 1970; Wiermann, 1972). The substrate specificity of the enzyme corresponds completely to expectations, since in Callistephus chinensis only flavonoids with a phloroglucinol type of the ring A occur. Due to the additive behavior of the mixed assays with extracts of high activity and no activity and of the enzyme extracts from mixtures of flowers from the corresponding lines, it seems unlikely that a macromolecular inhibitor or regulation mechanisms are responsible for the enzyme activity deficiency in the lines which accumulate isosalipurposide. The Ch gene should rather be concerned with the synthesis of the chalcone-flavanone isomerase. It should be emphasized that in chalcone-free lines enzyme activity could be observed in flowers, buds, and leaves. In chalcone-rich lines, however, chalconeftavanone isomerase activity was neither detectable in flowers or leaves nor during the time of high chalcone synthesis (buds). On the basis of these results, for the first time a correlation could be established between a genetic block and an enzyme deficiency concerning a step in the actual flavonoid biosynthesis. The results of this work allow, at least for the examined material, a clear statement about the role of the chatcone-flavanone isomerase and consequently also about the substrate for the further reactions. The only interpretation for the accumulation of isosalipurposide in lines without chalcone-flavanone
B. Kuhn et al. : Chalcone-Flavanone Isomerase Activity
isomerase activity can be that the first product of the flavanone synthase reaction in Callistephus is not naringenin but rather 4,2',4',61-tetrahydroxychalcone. In lines without isomerase activity the synthesis of higher oxidized flavonoids is largely interrupted. In this case tetrahydroxychalcone can function as substrate for a glucosyltransferase and can be converted to isosalipurposide. Whereas in the lines with high isomerase activity no chalcone can be detected, increased amounts of flavones, flavonols, and anthocyanins appear. The role of the chalcone-flavanone isomerase can therefore only be to catalyze the isomerization of chalcone to flavanone in a stereospecific way and so to yield the substrate for the further reactions. Thus, the chalcone-flavanone isomerase plays an important part in flavonoid biosynthesis. These assertions are confirmed by investigations of the chalcone-flavanone isomerase in different stages of development in anthers from Tulipa and Lilium. At the maximal activity of the enzyme a rapid decrease of the previously accumulated chalcone and a corresponding increase of flavonols and anthocyanins occurred (Wiermann, 1972). These statements about the role of the chalconeflavanone isomerase are further confirmed by the evidence that in Petroselinum flavanone and not chalcone is the substrate for the oxidation to the flavone (Sutter et al., 1975). The question as to why small amounts of naringenin glycosides and higher oxidized flavonoids are found in the lines without any detectable isomerase activity must be left open at present. Possibly a low activity of the isomerase is responsible for it. However, even after application of a 5-fold increase in the usual enzyme quantity to the assay mixture no decrease of absorption exceeding spontaneous isomerization could be demonstrated. Perhaps the formation of these flavonoids could be attributed to a spontaneous isomerization of tetrahydroxychalcone to naringenin in vivo. Supposing that, there exists a spontaneous isomerisation in vivo, only the (2S) enantiomer of the formed naringenin could serve as substrate for further reactions. The results of this paper are consistent with those of earlier tracer experiments (Grisebach, 1962), but are contradictory to those from investigations of flavanone synthase, in which naringenin was obtained as the first reaction product (Kreuzaler and Hahlbrock, 1975). In this case it must be considered that
203
the experiments with the flavanone synthase were carried out with cell cultures of Petroselinum hortense, i.e., with a different species and on undifferentiated tissue. Therefore, these results might not be comparable with the conditions in Callistephus chinensis. These investigations were supported by a grant from the Deutsche Forschungsgemeinschaft. The authors are grateful to Prof. H.Grisebach for his critical reading of the manuscript and valuable suggestions.
References Bradford, M.M. : A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry 72, 248-254 (1976) Forkmann, G.: Anthocyanin pigments in Callistephus chinensis. Phytochemistry 16, 299 301 (1977) Geissman, T.A., Clinton, R.Q.: Flavanones and related compounds. I. Preparation of polyhydroxychalcones and -flavanones. J. Am. Chem. Soc. 68, 697-699 (1946) Grisebach, H.: Die Biosynthese der Flavonoide. Planta Medica 10, 385-397 (1962) Grisebach, H.: Enzymologie der Flavonoidbiosynthese. Ber. Deutsch. ,Bot. Ges. 88, 61-69 (1975) Grisebach, H., Barz, W. : Biochemie der Flavonoide. Naturwissenschaften 56, 538 544 (1969) Hahlbrock, K., Wong, E., Schili, L.', Grisebach, H.: Comparison of chalcone-flavanone isomerase heteroenzymes and isoenzymes. Phytochemistry 9, 949 958 (1970) Harborne, H.B.: Comparative biochemistry of flavonoids I. Distribution of chalcone and auroue pigments in plants. Phytochemistry N, 111-115 (1966) Kreuzaler, F., Halbrock, K.: Enzymic synthesis of an aromatic ring from acetate units. Eur. J. Biochem. 56, 205 213 (1975) Moustafa, E., Wong, E.: Purification and properties of chalconeflavanone isomerase from Soya bean seed. Phytochemistry 6, 625-632 (I967) Rathmell, W.G., Bendall, D.S. : The peroxidase-catalysed oxidation of a chalcone and its possible physiological significance. Biochem. J. 127, 125 132 (1972) Rauen, H.M.: Biochemisches Taschenbuch Teil 2. Berlin-G6ttingen-Heidelberg-New York: Springer 1964 Sutter, A., Poulton, J., Grisebach, H.: Oxidation of flavanone to flavone with cell-free extracts from young Parsley leaves. Arch. Biochem. Biophys. 170, 547 556 (1975) Vlaming, P. de, Kho, F.F.K.: 4,2',4',6'-Tetrahydroxychalcone in pollen of Petunia hybrida. Phytochemistry 15, 348 349 (1976) Wiermann, R.: Aktivit/it der Chalkon-Flavanon Isomerase und Akkumulation yon phenylpropanoiden Verbindungen in Antheren. PIanta 102, 55-60 (1972) Wit, F. : Contributions to the genetics of the China Aster. Genetica 19, 1-104 (1937) Wong, E., Wilson, J.M, : The oxidation of chalcone catalyzed by peroxidase. Phytochemistry 11, 875 (1972) Received 7 July; accepted 15 November 1977
