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Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two Km values were determined, these were KmH, 2·9 × 10−4 M and KmL, 4·3 × 10−5 M. Strong inhibition of the enzyme was exerted by d -phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d -phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.

The incorporation of shikimate-[ 14 C] and cinnamate-[ 14 C] into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins was studied in isolated petals of Petunia hybrida . According to the dilution values, the incorporation of shikimate-[ 14 C] was about 3–6 times better than that of cinnamate-[ 14 C]. However a comparison of the incorporation of the 2 precursors on a relative basis showed no significant differences in the relative proportions of the specific activities of the 3′,4′-dihydroxysubstituted cyanidin-3-monoglucoside and the 3′,4′,5′-trihydroxysubstituted delphinidin-3-monoglucoside. This result and the [ 14 C]-incorporation behaviour of the 3′-methoxy-4′-hydroxysubstituted peonidin-3-monoglucoside do not support the hypothesis that there are alternative pathways of flavonoid biosynthesis.

In vitro cultured corollas of the white flowering mutant W18 of Petunia hybrida synthesize anthocyanins after administration of a bud extract of the white flowering 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.

Gibberellic acid (GA3), given at low, physiological concentrations, was found to inhibit anthocyanin and flavonoid biosynthesis markedly in both growing and nongrowing cultures of two species of Spirodela. In growing cultures, very low concentrations of GA3 (10−6–10−7M) promoted anthocyanin formation in the first 7–10 days, but the inhibition increased with time, and after 3 weeks' growth under 300 ft.-c white light a concentration as low as 3 × 10−6M sufficed for 50% inhibition. At these levels, gibberellin had no effect on growth. In total darkness, GA3 decreased the synthesis in S. polyrrhiza by 50% at 1 × 10−7M. In nongrowing cultures, the effect of light was found to overcome the inhibiting effect of GA3 on anthocyanin synthesis. Spirodela oligorrhiza was much less sensitive to inhibition than S. polyrrhiza in such conditions. In both types of cultures, the lower the light intensity, the more readily was the synthesis inhibited. The type of inhibition thus differs from those previously studied, which were most effective in full light; it also differs in not being reversed by riboflavin.

Parallel competitive feeding experiments have been carried out in which either ( a ) 14 C-isoliquiritigenin (2′,4′,4-trihydroxychalcone) diluted with an equal amount of (−)-liquiritigenin (4′,7-dihydroxyflavanone), or ( b ) 14 C-(−)-liquiritigenin diluted similarly with isoliquiritigenin were fed to subterranean clover seedlings and to cell-free extracts of garbanzo beans. The radioactive products, 4′,7-dihydroxyflavone, daidzein, formononetin and garbanzol were isolated and their specific activities determined. The results show that the specific activities for the products from ( a ) are higher than those from ( b ). This is interpreted as indicating that the chalcone, and not the flavanone, is the more immediate precursor for other classes of flavonoid. The possible roles of “oxidized chalcone” intermediates in the biosynthesis of flavonoids are discussed.

The incorporation of p -tritio-cinnamic acid [3- 14 C] into the flavonols kaempferol and quercetin in pea seedlings ( Pisum sativum ) and into the isoflavones formononetin and biochanin A in chana seedlings ( Cicer arietinum ) was investigated. The retention of tritium found was: kaempferol, 85 per cent; quercetin, 49 per cent; formononetin, 80 per cent; and biochanin A, 81 per cent. Degradation of the labelled formononetin to 3-nitro-5-tritio-4-methoxybenzoic acid proved that the tritium had migrated to a position ortho to the 4′-hydroxyl group. The results are discussed in terms of the chronological sequence of steps during flavonoid biosynthesis, in particular when hydroxylation in ring B occurs.

Summary Tracer experiments have established, that chalcones are precursors in the biosynthetic pathway of flavonoids. We synthesized 2′,4′6′,3,4-penta-hydroxy-chalcone and worked out a method to isolate chalcones. By using the penta-hydroxy-chalcone as carrier in trapping experiments, we prouved the existence of chalcones/flavanones in petals of Petunia hybrida in the stage of intensive and linear anthocyanin synthesis. The results support the hypothesis, that chalcone are not only precursors, but also natural intermediates in flavonoid biosynthesis.

The incorporation of 14 C-labelled l -phenylalanine and sucrose into caffeic acid in sunflower discs has been determined under a number of conditions which affect the net rate of synthesis of the latter compound. The results indicate that l -phenylalanine may not be an obligate precursor in the biosynthesis of phenylpropanoid compounds, including flavonoids, in higher plants.

The flavonoid compounds are one of the largest classes of naturally occurring phenolic compounds. They are C15 compounds (exclusive of O-alkyl groups and secondary substitution) composed of two phenolic nuclei connected by a three-carbon unit. The A-ring is characteristic of the phloroglucinol or resorcinol hydroxylation pattern and the B-ring usually 4-, 3,4- or 3,4,5-hydroxylated. Depending on the oxidation level of the three-carbon portion of the flavonoid molecules or of the pattern of the ring closure or subsequent modifications, we know the chalcones (V) e.g. isoliquiritigenin (Va), the flavanones, e.g. naringenin (VIa), the flavanonols, e.g. garbanzol (VII), the flavones, e.g. apigenin (VIII), the flavonols, e.g. quercetin (IX), the anthocyanidins, e.g. cyanidin (X), the catechins (XI), the aurones, e.g. hispidol (XII), the isoflavones, e.g. formononetin (XIII) and biochanin-A (XIV), some 3-arylcoumarins like coumestrol (XV) and others.

2′,4,4′-Trihydroxy[14C]chalcone or its 4′-glucoside is transformed by chana seedlings in vivo or in cell-free extracts into 4′,7-dihydroxyflavanone, 4′,7-dihydroxyflavanol and 7-hydroxy-4′-methoxyisoflavone. 4′,7-Dihydroxy[14C]flavanolol is not transformed into 7-hydroxy-4′-methoxyisoflavone when administered in vivo or with cell-free extracts.

1. 1. Concomitant increases in the activities of phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, and p -coumarate:CoA ligase occurred after a lag period of approx. 2.5 h when parsley cells from 10-day-old suspension cultures were transferred to fresh medium. 2. 2. Changes in the activities of these enzymes were measured over a period of 30 h. Activities of phenylalanine ammonia-lyase and p -coumarate:CoA ligase reached maximum values about 15 h after inoculation and decreased subsequently, while linear increases in the activity of the microsomal enzyme, cinnamic acid 4-hydroxylase, were measured from 2.5 to 30 h. 3. 3. Activities of a number of enzymes related to primary cell metabolism or specifically involved in flavonoid biosynthesis did not change during this period of time. 4. 4. Results obtained from these experiments are strikingly similar to those previously described for light-induced increases in the activities of the three enzymes investigated in this study. Thus, further evidence is presented that these enzymes are regulated interdependently.

The synthesis and accumulation of several phenylpropanes in the anther content (pollen+tapetum fraction) during microsporogenesis has been investigated by chromatographic techniques in Narcissus pseudonarcissus, Lilium candidum, and in the Darwin tulip “Apeldoorn”. In these species, the pigmentation process is initiated by the synthesis of several cinnamic acid derivates (mainly derivates of ferulic acid) during meiosis II. In Narcissus, and intense synthesis of kaempferol glycosides takes place during the separation of the tetrad which follows immediately upon its formation. In Tulipa and Lilium, however, chalcones are synthesized in an intermediate phase before flavonols and anthocyanins (in Tulipa) are produced in significant amounts. In Tulipa, the investigations revealed the following sequence in the pigmentation process: cinnamic acid derivatives-chalcone-flavonols-anthocyanins. The sequence is discussed in relation to flavonoid biosynthesis. Because of biogenetic considerations a special emphasis is laid on the “chalcone stage”. Chromatographic and spectroscopic data show that the isomerization product of the chalcone is eriodictyol. Accordingly, this chalcone must be 2′,3,4,4′,6′-pentahydroxychalcone. Other chalcones could not be identified. During anthesis the following aglycones are accumulated in the pollen of Tulipa cv. “Apeldoorn”: ferulic acid, p-coumaric acid, kaempferol, quercetin, isorhamnetin, delphinidin, and small traces of the pentahydroxy-chalcone, which is the main pigment in the intermediate stages of microsporogenesis. On the basis of histochemical findings, it is suggested that at least the final steps of synthesis leading to flavonol and anthocyanidin glycosides take place on the pollen wall in the loculus of the anthers, that is, in the extracellular space.

Isosalipurposide, the 2′-glucoside of chalconaringenin, has been shown to be the principal flavonoid of young bark of Salix purpurea ; in the old bark it is accompanied by isomeric forms of naringenin 5-glucoside, salipurposide. The leaf of S. purpurea shows a very different flavonoid pattern; luteolin 7-glucoside predominates, together with smaller amounts of the 7-glucosides of eriodictyol and naringenin. The distribution of these compounds among Salix species has been surveyed, and their possible significance in flavonoid biosynthesis is discussed.

CHALCONES and flavanones have been shown to be the products from (a) are higher than those from precursors for many classes of flavonoid com- (b). Furthermore, within each experiment, the pounds.l Enzymic and in vitro feeding experi- specific activities of these compounds follow ments, however, show that chalcone and (-)- closely that of the chalcone but not that of the flavanone are interconvertible, and the question ( - )-flavanone. still remains as to whether both are directly These results strongly suggest that, contrary to involved as intermediates in flavonoid biosynthesis. expectations: flavones and isoflavones are formed TABLE Specific activities* of $avonoid compounds from clover seedlings fed [14C]chalcone or jlavanone

The photolysis of 2-hydroxychalcone in alcoholic solvents using 3500 A light yields the corresponding 2-alkoxy-flav-3-ene together with small quantities of flavone.