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Biochemical and Immunological Characterization of Chalcone Synthase from Rye Leaves
Abstract
As part of investigations on the role and the location of the key enzyme of flavonoid biosynthesis, chalcone synthase (CHS) in leaf tissues of Gramineae, CHS from rye (Secale cereale L.) was purified and characterized biochemically and immunologically. Km-values for the substrates p-coumaroyl-CoA and caffeoyl-CoA were 0.6 and 1.45 μM, respectively, the corresponding Km-values for malonyl-CoA were 1.4 and 1.5 μM. The pH-optimum for the formation of 2′,4,4′,6′-tetrahydroxychalcone was 8 and for the formation of 2′,3,4,4′,6′-pentahydroxychalcone, 6.5. A 50% reduction in enzyme activity was caused by 9 μM apigenin, 13 μM luteolin, 36 μM CoA, 45 μM naringenin, 45 μM eriodictyol, and 62 μM isovitexin 2″-O-arabinoside. SDS-PAGE analysis of the purified enzyme revealed two peptides with apparent molecular weights of 43 and 44 kd, respectively. On western blots run with the purified enzyme as well as with crude enzyme extracts, both peptides were immunostained by polyclonal antibodies raised against rye or parsley CHS. The recognition of these peptides by single monoclonal antibodies demonstrated that both peptides were similar and derived from CHS. Applied to western blots run with CHS-containing extracts of parsley, spinach, pea, maize, and oat, the same antibodies labelled only a single peptide, or in some cases probably even more than two peptides.
... Fractions of 500 mL were collected and combined according to the TLC characteristics to afford twelve major fractions (A-L). Combined fraction "H" [53][54][55][56][57], eluted with hexane-EtOAc (2:3), was purified by repeated flash chromatography, Sephadex fitration chromatography, preparative TLC and crystallization to furnish an isomorphic crystalline equilibrium mixture of 8 and 9. ...
... "Apeldoorn" (Liliaceae) 45 and Sorghum bicolor (Poaceae) 46 , however, it has been detected by GC/MS in the peel of tomato fruits Lycopersicum sculentum (Solanaceae) 47 , and in some medicinal plants: Artemisia argyi (Asteraceae) 48 , Populus sieboldii (Salicaceae) 49 and Pouteria lucuma (Sapotaceae) 50 . An interesting aspect to highlight is the frequency with which its biosynthesis from caffeoyl-CoA (6) has been induced in some plants by genetic engineering, using the enzyme chalcone synthase (CHS); in general, plants of nutritional interest have been used: Tomato (Lycopersicum sculentum) 51 , barley (Hordeum vulgare) 52 , rice (Oryza sativa) 53 , carrot (Daucus carota) 54 , apple (Malus x domestica) 55 and pear (Pyrus communis) 55 , which support the possible interest of this chalcone as a phytochemical additive in the diet to be ingested in human consumption 56 . ...
From the leaves and stems of Stevia lucida Lagasca (Asteraceae), an equilibrium chalcone-flavanone isomeric mixture, composed of 2',3,4,4',6'-pentahydroxychalcone (8) (eriodictyol-chalcone) and eriodictyol (9) [ratio 8/9 5:3], was isolated as an isomorphic crystal. The mixture was structurally characterized by spectroscopic methods, including 1D- and 2D-NMR experiments. The presence of chalcones in the genus Stevia is reported here for the second time.
... Fractions of 500 mL were collected and combined according to the TLC characteristics to afford twelve major fractions (A-L). Combined fraction "H" [53][54][55][56][57], eluted with hexane-EtOAc (2:3), was purified by repeated flash chromatography, Sephadex fitration chromatography, preparative TLC and crystallization to furnish an isomorphic crystalline equilibrium mixture of 8 and 9. ...
... "Apeldoorn" (Liliaceae) 45 and Sorghum bicolor (Poaceae) 46 , however, it has been detected by GC/MS in the peel of tomato fruits Lycopersicum sculentum (Solanaceae) 47 , and in some medicinal plants: Artemisia argyi (Asteraceae) 48 , Populus sieboldii (Salicaceae) 49 and Pouteria lucuma (Sapotaceae) 50 . An interesting aspect to highlight is the frequency with which its biosynthesis from caffeoyl-CoA (6) has been induced in some plants by genetic engineering, using the enzyme chalcone synthase (CHS); in general, plants of nutritional interest have been used: Tomato (Lycopersicum sculentum) 51 , barley (Hordeum vulgare) 52 , rice (Oryza sativa) 53 , carrot (Daucus carota) 54 , apple (Malus x domestica) 55 and pear (Pyrus communis) 55 , which support the possible interest of this chalcone as a phytochemical additive in the diet to be ingested in human consumption 56 . ...
From the leaves and stems of Stevia lucida Lagasca (Asteraceae), an equilibrium chalcone-flavanone isomeric mixture, composed of 2',3,4,4',6'-pentahydroxychalcone (8) (eriodictyol-chalcone) and eriodictyol (9) [ratio 8/9 5:3], was isolated as an isomorphic crystal. The mixture was structurally characterized by spectroscopic methods, including 1D- and 2D-NMR experiments. The presence of chalcones in the genus Stevia is reported here for the second time.
... To investigate the potential inhibitory role of flavonoid molecules [18,58], naringenin was added to the crystallization buffer. In addition to a bound CoA molecule, one naringenin molecule was visible in the concave pocket established at the interface between two PvCHS dimers in a stacked orientation of the A and C rings from each naringenin molecule. ...
Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI’s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.
... Furthermore, CHS is also known to be non-competitively inhibited by the products of flavonoid pathway including naringenin chalcone, naringenin and the derailment products. For instance, 100 μM concentration of naringenin is inhibitory to Parsely (50% inhibition) and Carrot CHSs, whereas the flavonoids luteolin and apigenin have been shown to inhibit Rye CHS in vitro (Hinderer and Seitz 1985;Kreuzaler and Hahlbrock 1975;Peters et al. 1988). ...
Main conclusion
Present review provides a thorough insight on some significant aspects of CHSs over a period of about past three decades with a better outlook for future studies toward comprehending the structural and mechanistic intricacy of this symbolic enzyme.
Abstract
Polyketide synthases (PKSs) form a large family of iteratively acting multifunctional proteins that are involved in the biosynthesis of spectrum of natural products. They exhibit remarkable versatility in the structural configuration and functional organization with an incredible ability to generate different classes of compounds other than the characteristic secondary metabolite constituents. Architecturally, chalcone synthase (CHS) is considered to be the simplest representative of Type III PKSs. The enzyme is pivotal for phenylpropanoid biosynthesis and is also well known for catalyzing the initial step of the flavonoid/isoflavonoid pathway. Being the first Type III enzyme to be discovered, CHS has been subjected to ample investigations which, to a greater extent, have tried to understand its structural complexity and promiscuous functional behavior. In this context, we vehemently tried to collect the fragmented information entirely focussed on this symbolic enzyme from about past three–four decades. The aim of this review is to selectively summarize data on some of the fundamental aspects of CHSs viz, its history and distribution, localization, structure and analogs in non-plant hosts, promoter analyses, and role in defense, with an emphasis on mechanistic studies in different species and vis-à-vis mutation-led changes, and evolutionary significance which has been discussed in detail. The present review gives an insight with a better perspective for the scientific community for future studies devoted towards delimiting the mechanistic and structural basis of polyketide biosynthetic machinery vis-à-vis CHS.
... Chalcone synthase assay was performed as previously described (Schr6der et al. 1979; Peters et al. 1988). The following incubation medium was used: 0.1 M K-phosphate buffer, pH 8.0; 18 mM L-cysteine; 31 mM Na-ascorbate; 0.5% bovine serum albumin (BSA); 11 pM 4-coumaroyl-CoA; 10 !aM [214C]malonyl-CoA; and up to 15 111 enzyme solution in a total volume of 110 I~l. ...
Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.
... There are many studies showing that CHS is inhibited noncompetitively by flavonoid pathway products like naringenin, chalcone naringenin and the other end products of CoA esters. For example, the parsley CHS is 50% inhibited by 100 lM naringenin and 10 lM CoA esters (Hinderer and Seitz 1985; Kreuzaler and Hahlbrock 1975), the flavonoids luteolin and apigenin are inhibitory to rye CHS in vitro (Peters et al. 1988), whereas in carrot, among the range of flavonoids tested, only naringenin and chalcone narigenin can inhibit CHS at 100 lM (Hinderer and Seitz 1985). It seems that flavonoids accumulate in the cytosol to a level that blocks CHS activity to avoid toxic levels for the plant (Whitehead and Dixon 1983), though there is no direct evidence that this inhibition happens in vivo. ...
Chalcone synthase (CHS, EC 2.3.1.74) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway. Besides being part of the plant developmental program the CHS gene expression is induced in plants under stress conditions such as UV light, bacterial or fungal infection. CHS expression causes accumulation of flavonoid and isoflavonoid phytoalexins and is involved in the salicylic acid defense pathway. This review will discuss CHS and its function in plant resistance.
... There are many studies showing that CHS is inhibited noncompetitively by flavonoid pathway products like naringenin, chalcone naringenin and the other end products of CoA esters. For example, the parsley CHS is 50% inhibited by 100 µM naringenin and 10 µM CoA esters [Hinderer and Seitz, 1985;Kreuzaler and Hahlbrock, 1975], the flavonoids luteolin and apigenin are inhibitory to rye CHS in vitro [Peters et al., 1988], whereas in carrot, among the range of flavonoids tested, only naringenin and chalcone narigenin can inhibit CHS at 100 µM [Hinderer and Seitz, 1985]. It seems that flavonoids accumulate in the cytosol to a level that blocks CHS activity to avoid toxic levels for the plant [Whitehead and Dixon, 1983], though there is no direct evidence that this inhibition happens in vivo. ...
Benzothiadiazole (BTH) is a functional analog of the plant endogenous hormone-like compound, salicylic acid (SA), which is required for the induction of plant defense genes leading to systemic acquired resistance (SAR). Previous molecular and genetic studies have suggested that BTH itself might potentiate SAR resulting in the induction of several pathogenesis-related (PR) genes. However, the changes in the metabolome, which occur as a result of BTH-treatment, remain unclear. In this study, metabolic alterations in BTH-treated Arabidopsis thaliana were investigated using nuclear magnetic resonance (NMR) spectroscopy followed by multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). Both PCA and PLS-DA show that increase of glucose, glutamine, inositol, malic acid, sucrose, and threonine as well as BTH and its degraded metabolites contribute to the clear discrimination of the metabolome of BTH-treated Arabidopsis from control plants. However, the levels of phenolic metabolites, which have generally been observed to be induced by other signaling molecules were significantly reduced in BTH-treated Arabidopsis. In addition to these changes due to BTH-treatment, it was also found that the EtOH used as a solvent in this treatment may per se act as an inducer of the accumulation of a flavonoid.
... Preliminary data suggest K m values of HvCHS2 at 1 ऌM in the conversion of feruloyl-CoA and of 2 ऌM in the conversion of 4-coumaroyl-CoA (data not shown). These values are comparable to the K m values of 0.6-2.5 ऌM found for chalcone synthases from mustard, rye and parsley [46,34,20]. The substrate specificity of the heterologous expressed enzyme establishes HvCHS2 as a novel type of chalcone syn- thase possessing homoeriodictyol/eriodictyol chalcone synthase activity. ...
A cDNA clone was isolated by differential hybridization from a library prepared from barley leaves inoculated with the fungus Blumeria graminis f.sp. hordei (Bgh). The open reading frame of the insert (designated HvCHS2) encoded a polypeptide with 72-79% identity to chalcone synthases (CHS) and 65-68% identity to stilbene synthases. Alignments of the amino acid sequence of HvCHS2 with the consensus sequence of naringenin-CHS (EC 2.3.1.74) reveals significant differences between HvCHS2 and naringenin-CHS. HvCHS2 transcripts accumulate strongly in barley leaves in response to inoculation with Bgh, whereas only insignificant accumulation of barley naringenin-CHS (CHS1) transcripts is seen upon the inoculation. The accumulation of HvCHS2 transcripts is also elicited by UV light. To compare the activity of HvCHS2 with the activity of CHS1, the two enzymes were expressed in Escherichia coli. Both HvCHS2 and CHS1 catalyse the formation of chalcones. However, HvCHS2 and CHS1 differ in their substrate requirements. CHS1 uses cinnamoyl-CoA and 4-coumaroyl-CoA at comparable rates whereas feruloyl-CoA is a poor substrate for this enzyme. In contrast, HvCHS2 converts feruloyl-CoA and caffeoyl-CoA at the highest rate whereas cinnamoyl-CoA is a poor substrate. Thus, HvCHS2 is a novel pathogen and UV light induces homoeriodictyol/eriodictyol CHS involved in the direct production of flavonoids possessing multi-substituted B-rings.
The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis—a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.
Some of the many plant-specific phenylpropanoid branch pathways and the corresponding functional diversity of their products were discussed in the last volume (Schütte 1992). Last year it was mentioned that special topics of the phenylpropanoid metabolism had been discussed by Schütte (1978, 1979,1985) and that recent developments in this area involve major advances in elucidating the structural organization, mode of expression and functional relationships of genes encoding enzymes of phenylpropanoid metabolism in dicotyledonous plants. In this Volume, special interest is given to the quinones, lignans and to capsaicin.
Flavonoid compounds are among the most widely distributed natural plant products. They occur in several conjugated forms, most commonly as glycosylated and methylated derivatives, although other flavonoids may be acylated, prenylated or sulfated.1,2 A variety of flavonoids have been assigned different roles as antimicrobial compounds, stress metabolites or signalling molecules.3-7
As part of an ongoing investigation into the organization and regulation of the flavonoid biosynthetic pathway, two Arabidopsis thaliana expressed sequence tag (EST) clones (153O10T7 and YAY780) with high homology to leucoanthocyanidin dioxygenase (LDOX) or flavonol synthase (FLS) were identified. EST YAY780 was sequenced and found to encode a protein 49 to 78% identical to all LDOX sequences in the database. EST 153O10T7 was used to isolate a genomic clone encoding a protein with 59 to 61% sequence identity to petunia (Petunia hybrida) and potato (Solanum tuberosum) FLS. DNA blot analysis was used to screen the Arabidopsis genome for sequences related to FLS and LDOX and to determine the positions of the two clones on the RI map. The expression patterns of FLS and LDOX in etiolated seedlings moved to white light and in two putative regulatory mutants (ttg and tt8) were determined by RNA blot analysis. These studies indicate that FLS is an "early" flavonoid gene in Arabidopsis seedlings, whereas LDOX is a "late" gene. Furthermore, FLS is the first flavonoid enzyme identified in Arabidopsis that may be encoded by a gene family.
Two cell lines of carrot (Daucus carota L. spp. sativus), grown as cell-suspension cultures in the dark, were irradiated with ultraviolet light (315-420 nm) 10 d after the onset of cultivation. Chalcone synthase (CHS) enzyme activity was induced in both cell lines. Anthocyanin synthesis was only stimulated in the anthocyanin-containing cell line DCb. Parallel to the increase in CHS activity there was an increase with time in the amount of one CHS form with an isoelectric point of 6.5 and a molecular weight of 40 kilodaltons (kDa) per subunit. Whereas the anthocyanin-free cell line DCs failed to accumulate anthocyanin, it did stimulate another CHS form with an isoelectric point at pH 5.5 and a molecular weight of 43 kDa per subunit. Both enzyme activities could be separated by isoelectric focusing and stabilized using sodium hydrosulfite as an oxidation protectant. In carrot plants, CHS was restricted to the dark purple petals of the inflorescence (40 kDa) and to the leaves (43 kDa).
Chalcone synthase (CHS) activity was measured in gametophytic tissue of Marchantia polymorpha. The main product of the enzyme reaction was identified as naringenin which results from cyclization of the chalcone. Cross-reactivity under native and denaturing conditions was obtained using antibodies raised against CHS from three different higher plants, indicating the presence of CHS protein in M. polymorpha.
The present study was undertaken in order to investigate the suitability of certain markers for UV plant response. In addition, we attempted to link the internal tissue distribution of specific UV-absorbing compounds to profiles of radiation gradients within intact primary rye leaves (Secale cereale L. cv. Kustro). Etiolated rye seedlings irradiated with low visible light (LL) and/or UV radiation were used to study enzyme activities of the two key enzymes, phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), together with the tissue-specific accumulation of soluble phenylpropanoid products. Plants grown under relatively high visible light (HL) with or without supplementary UV-B radiation were used for further characterization. Apparent quantum yield and fluorescence quenching parameters were monitored to assess potential physiological changes due to UV-B exposure in HL-grown seedlings. A quartz fibreoptic microprobe was used to characterize the internal UV-B gradient of the leaf. The response of the phenylpropanoid metabolism to UV radiation was similar in primary leaves of both etiolated and HL-treated green plants. The epidermis-specific flavonoids together with CHS activity turned out to be suitable markers for assessing the effect of UV on the phenolic metabolism. The functional role of phenylpropanoid compounds was strongly implicated in protecting rye from UV-B radiation.
Chalcone synthase in leaves of Kalanchoe daigremontiana and Acorus calamus, and in phyllodes of Kalanchoe tubiflora was detected both enzymatically and immunochemically. In situ localization by indirect immunofluorescence revealed that, in all three systems, chalcone synthase is highly expressed in tannin-containing idioblasts. In A. calamus the enzyme is additionally present in oil cells. The spatial distribution of chalcone synthase did not change during organ development.
Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.
During cell and tissue differentiation of developing rye (Secale cereale L.) and maize (Zea mays L.) primary leaves, various flavonoids are synthesized and accumulate in both epidermal and mesophyll tissues. In order to prove either the biosynthetic autonomy of each tissue type and- or intercellular transport of flavonoids, the tissue distributions of chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the pathway, and of flavonoids have been comparatively investigated. Monoclonal antibodies raised against CHS from rye were used to relate enzyme activity in a particular tissue extract to the corresponding amount of CHS protein. A close correlation was found between CHS activities and amounts of CHS protein during leaf development and in the various tissues. The simultaneous occurrence of CHS in both epidermal layers as well as in the mesophyll correlated with the accumulation of flavonoid products in these tissues, indicating tissue autonomy of flavonoid biosynthesis. These data are in contrast to previous reports (Knogge and Weissenbck, 1986, Planta 167, 196–205) on primary leaves of oat (Avena sativa) where CHS and several subsequent enzymes were located mainly in the mesophyll whereas major flavonoid products accumulated predominantly in both epidermal cell layers, indicating that intertissue transport of flavonoids might occur.
A new hypothesis concerning the formation of aromatic intermediates in the biosynthesis of hop bitter acids was tested. Two phloroglucinol derivatives, 2-(3-methylbutanoyl)-1,3,5-benzenetriol (phlorisovalerophenone = PIVP) and 2-(2-methylpropanoyl)-1,3,5-benzenetriol (phlorisobutyrophenone = PIBP), were detected in hop extracts. The formation of these compounds was shown to occur in vitro in hop protein extracts using the precursors malonyl-CoA plus isovaleryl-CoA or isobutyryl-CoA, respectively. We conclude that PIVP and PIBP are probably the first aromatic intermediates formed in the biosynthesis of humulone/lupulone and cohumulone/colupulone, respectively. We suggest that the reactions are catalysed by a chalcone synthase-like enzyme. The enzyme chalcone synthase and its activity were found to be present in hop protein extracts. The formation of naringenin, PIVP and PIBP in various hop protein extracts obtained during the development from flower bud to ripe cone was compared.
As part of an ongoing investigation into the organization and regulation of the flavonoid biosynthetic pathway, two Arabidopsis thaliana expressed sequence tag (EST) clones (153O10T7 and YAY780) with high homology to leucoanthocyanidin dioxygenase (LDOX) or flavonol synthase (FLS) were identified. EST YAY780 was sequenced and found to encode a protein 49 to 78% identical to all LDOX sequences in the database. EST 153O10T7 was used to isolate a genomic clone encoding a protein with 59 to 61% sequence identity to petunia (Petunia hybrida) and potato (Solanum tuberosum) FLS. DNA blot analysis was used to screen the Arabidopsis genome for sequences related to FLS and LDOX and to determine the positions of the two clones on the RI map. The expression patterns of FLS and LDOX in etiolated seedlings moved to white light and in two putative regulatory mutants (ttg and tt8) were determined by RNA blot analysis. These studies indicate that FLS is an "early" flavonoid gene in Arabidopsis seedlings, whereas LDOX is a "late" gene. Furthermore, FLS is the first flavonoid enzyme identified in Arabidopsis that may be encoded by a gene family.
Flavonoids are closely related to a plant's antioxidative ability. Because chalcone synthase (CHS) is the first enzyme to act as part of the flavonoid biosynthesis pathway, its expression and regulation are important. Here we present the expression of a full-length chs cDNA with 1225bp from grape seedlings as well as the preparation of an antibody against the expressed protein. A full-length chs cDNA was introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and SalI restriction sites. pET-chs was found to be highly expressed in Escherichia coli BL21(DE3) pLysS cells with isopropyl-beta-d-thiogalactoside (IPTG) induction. A fusion protein with the His.tag label was purified by Ni-NTA His. Bind Resin and then used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was then further precipitated by 50% saturated ammonium sulfate and DEAE-Sepharose FF column chromatography to obtain the immunoglobulin G (IgG) fraction. The resulting antibody was found capable of immuno-recognizing the CHS of the crude protein extracts from different grape tissues with a molecular mass of 43kDa.
We have develop ed a technique for the large-scale isolation of epidermal and mesophyll protoplasts, as well as the vascular strands, of rye primary leaf blades.
Separation of the two types of protoplasts has been successful only from leaves harvested at the end of a 13-h light period, when chloroplasts were enriched in starch.
The occurrence of different flavonoid compounds, and amounts, in epidermal and mesophyll protoplasts can be used as criteria for protoplast purity and viability since C-glucosylflavone O-glycosides are characteristic of epidermal protoplasts whereas flavone O-glucuronides and anthocyanins are typical of mesophyll protoplasts. Several non-flavonoid phenolic compounds are found only in the epidermal protoplast. These patterns of secondary product accumulation reflect the high tissue specificity of the rye leaf.
Chalcone synthase activity was found in enzyme preparations from spinach. In homogenates of young leaves two different activities of the enzyme could be separated by DEAE-ion exchange chromatography and chromatofocusing. Both activities formed naringenin with [2-14C] CoA and 4-coum aroyl-CoA as substrates. They exhibited only slight differences in substrate specificity. For both activities 4-coum aroyl-CoA proved to be the most suitable substrate at both pH 6.8 and 8.0. Eriodictyol and hom oeriodictyol formation from caffeoyl-CoA and feruloyl- respectively, only occured at pH 6.8. The formation of naringenin by the two activities was maximal at pH 7.5-8.0 and dependent upon the DTE-concentration intheassay mixture.
Incubation of soybean cell suspension cultures in B-5 medium containing 0.4 ᴍ sucrose caused induction of chalcone synthase (CHS) up to a specific activity of 8 µkat/kg. CHS from these cultures was purified to apparent homogeneity by a 5-step procedure. Isoelectric focussing of pure CHS gave a major band at pH 5.45 and two weaker bands at pH 5.35 and 5.5. At least the bands at pH 5.35 and 5.45 had CHS activity. Analysis of pure CHS by two-dimensional electrophoresis gave a set ot six proteins with a M, of 40 kDa and pIs between 6.0 and 6.6. One-dimensional PAGE of CHS under non-denaturing conditions gave three closely spaced protein bands. A specific antibody was raised against soybean CHS which cross-reacted with parsley CHS.
Attempts to find synthesis of deoxychalconc or of the corresponding 7.4′-dihydroxyflavanonc with CHS of different purification stages and with various cofactors failed.
Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.
Acyl-CoA derivatives of several hydroxylated cinnamic acids have been synthesized in 30 to 50% yield via a. acyl phenyl thiol esters, b. acyl N-hydroxysuccinimide esters, and c. glucocinnamoyl derivatives. Properties of the intermediates have been determined. The cinnamyol-CoA thiol esters were characterized by their chromatographic behaviour and UV spectra. The molar extinction coefficients of these important intermediates in plant phenylpropane metabolism have been unequivocally determined. Recently published values 13 for the molar extinction coefficients of these derivatives are incorrect; the methodological reason for this error has been established.
1. “Flavanone synthase” was isolated from anthers of Tulipa cv. “Apeldoorn” and partially purified by (NH4) 2SO4 fractionation, gel chromatography and isoelectric focussing. The enzyme preparation was free of chalcone-flavanone isomerase activity.
2. p-Coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were found to be efficient substrates of the synthase. The products formed were naringenin (5,7,4′-trihydroxyflavanone), eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) and homoeriodictyol (5,7,4′-trihydroxy-3′-methoxyflavanone), respectively. Addition of thiol reagents at concentrations exceeding 10⁻³ м caused inhibition of the enzyme. “ Release products” , however, were not detectable. Although exclusively chalcones accumulate in the tulip anther, only flavanones but no chalcones were detectable in our in vitro system.
3. The apparent Km values for p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were 1 .7× 10-6 м, 1.6× 10-6 м and 2 .5 ×10-6 м, respectively. Similar data were observed for malonyl- CoA.
4. No cofactors are required for the synthase reaction. The enzyme is strongly inhibited by the reaction products flavanone and coenzyme A . Maximum enzyme activity was found at pH 8.0 and 30 °. The molecular weight was approx. 55,000.
5. Synthase activity develops in early postmeiotic stages of microsporogenesis. Highest specific activities of the enzyme coincide with a maximum in chalcone accumulation within the anthers.
6. The contents of anthers was separated into two fractions, pollen and tapetum. Highest specific activities were observed with tapetum fractions, while pollen fractions exhibited only very low activities. The high enzyme activity in the tapetum fraction points to the important role of the tapetum in the biosynthesis of flavonoids in the loculus of anthers.
Coniferin specific- and isoflavone 7-glucoside specific β-glucosidases have been localized in stem and root sections of chick pea (Cicer arietinum L.) seedlings by the indirect immunofluorometrical method. The coniferin specific β-glucosidase has been found in the cell walls of the tracheary elements and of the endo-, epi-, and exodermis. All these tissues are known to contain either lignin or polymers, like suberin and cutin, which consist partially of phenylpropanoid elements. The localization of this β-glucosidase is therefore in agreement with its postulated relationship to the phenylpropanoid metabolism. The isoflavone 7-glucoside specific β-glucosidase, on the other hand, is predominantly located in the parenchymatic cortex cells, and obviously in the cytoplasm. These cells are known to contain the isoflavone formononetin, which has been shown to undergo turnover in chick pea seedlings. We therefore have good reason to assume that this β-glucosidase is involved in the metabolism of the 7-glucoside of this isoflavone.
Phytochrome was determined in small sections of maize (Zea mays L.) seedlings by means of a highly specific double sandwich enzyme immunoassay which uses a monoclonal anti-phytochrome antibody for binding phytochrome and anti-phytochrome serum to detect the bound phytochrome. The distribution of phytochrome in maize seedlings was followed from germination to the 7th d after soaking the caryopses. Regions of high phytochrome accumulation were found in the coleoptile tip, the root cap and the shoot apex: the values for 5-d-old seedlings were 120, 80 and 70 μg phytochrome per g fresh weight (or 0.91, 0.61 and 0.53 nmol·g(-1)), respectively. The mesocotyl and the leaves contained relatively low amounts of phytochrome (less than 10 μg·g(-1)FW), which were almost uniformly distributed throughout these organs. As might be expected, regions of these organs adjacent to the shoot apex showed higher levels. The root, other than root tip, was almost devoid of phytochrome (0.2 to 0.5 μg·g(-1)). The general distribution of phytochrome in organs did not change during the development of seedlings. The amount of phytochrome, however, did fluctuate: up to the 5th or 6th d after soaking the caryopses, the levels increased in the regions of high phytochrome accumulation but thereafter decreased. After the 6th d the roots were 15 cm or longer and the coleoptiles became prone to penetration by primary leaves. The tips of adventitious roots, emerging after the 6th d, were also found to contain phytochrome. When the root cap was illuminated (4.3 W·m(-1)), phytochrome was degraded as in illuminated shoots. Degradation of phytochrome in coleoptile, mesocotyl and shoot apex started with a lag phase but phytochrome degradation in the root cap and the leaves started without a lag. In contrast to shoot phytochrome, which was almost completely degraded under continuous illumination, about 3% of initial phytochrome was measured in root caps after 24 h continuous illumination. Some of the data, obtained by immunological measurements, may indicate differences between phytochrome, or its synthesis or degradation, in the root cap and shoots. The results are discussed with a view to different red-light-mediated responses of grass seedlings.
Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (λmax 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (λmax 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.
Primary leaves of rye (Secale cereale L.) show a close correlation between tissue differentiation and metabolism of flavones (luteolin derivatives), anthocyanins (cyanidin glycosides) and C-glycosylflavones (6-C-hexosyl-apigenin). The first two groups of flavonoids are located exclusively in the mesophyll, whereas the latter accumulate predominantly in the lower and upper epidermis. Flavonoids accumulate rapidly in the first five days of leaf development. In subsequent developmental stages the amount of the mesophyll flavonoids, luteolin and cyanidin derivatives, drastically drops, reaching at day 8 half the concentration present at day 5. In contrast, the two major epidermal 6-C-hexosyl-apigenins increase slightly up to day 8.
An enzyme catalysing the conversion of chalcones to the corresponding flavanones has been purified about 150-fold from soaked soya bean seed (Soja hispida). This enzyme is stable when stored as a freeze-dried preparation at 2–4°. It is not inhibited by azide, cyanide, diethyldithiocarbamate or EDTA but is strongly inhibited by low concentrations of p-hydroxymercuribenzoate. The pH optimum is about 7–5. The enzymic reaction is not dependent on coenzymes or activators. Substrate specificity of the enzyme was studied using seven chalcones.
Avena sativa leaves, stems and inflorescences contain a range of new C-glycosylflavone 2″-O-glycosides, including vitexin and isoswertisin 2″-rhamnosides, isovitexin and isoorientin 2″-arabinosides. The structure of ‘vitexin 4′-rhamnoside’ from Crataegus oxyacantha is revised in vitexin 2″-rhamnoside.
Preparation and assay of chalcone synthase in presence of sodium ascorbate and exclusion of oxygen during some steps gives improved yield and purity of 2S-naringenin.
Flavanone synthase was isolated and purified ca 62-fold from cell suspension cultures of Haplopappus gracilis. The enzyme preparation catalysed the formation of naringenin from 4-coumaryl-CoA and malonyl-CoA with a pH optimum of ca 8. The same enzyme was also capable of synthesizing eriodictyol from caffeyl-CoA and malonyl-CoA; in this case the pH optimum lay between 6.5 and 7. The homogeneous flavanone synthase from cell suspension cultures of parsley showed the same dependence of the pH optimum on the nature of the cinnamyl-CoA. It can be concluded that both naringenin and eriodictyol are natural products of the synthase reaction.
A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA (Southern blotting) transferred to nitrocellulose for reaction with antibodies or nucleic acid probes is described. The techniques utilize nonfat dry milk as a protein-nucleic acid source for blocking nonspecific reactions, as an incubation medium, and for subsequent washing to remove unreacted reagents. The incubation cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), is superior to bovine serum albumin or gelatin for preventing nonspecific absorption in Western blot analyses and does not require the use of detergents or chaotropic agents to effect efficient reduction of background. BLOTTO, at the proper dilution in NaClNa citrate, is just as efficient in Southern blot analyses as more complicated cocktails typically used in the latter technique. We also found that BLOTTO works well for blocking, incubating, and washing ELISA plate assays relative to the normal BSA carrier, at a considerable savings to the laboratory.
Guard cells of the lower epidermis of leaflets of Vicia faba L. cv. Weißkernige Hangdown contain several kaempferol 3,7-O-glycosides. This was demonstrated for the first time by the use of isolated, highly purified guard cell protoplasts for flavonol estimation and quantitation. From a total of ca 12 kaempferol glycosides, three were identified by comparative thin layer chromatography and high performance liquid chromatography as kaempferol 3-O-glucoside 7-O-rhamnoside (major component), 3-O-rhamnogalactoside 7-O-rhamnoside and 3,7-O-bisglucoside (minor components). On average, the total flavonol content was estimated to be 85 fmol protoplast−1. From comparative investigations including alkaline-induced (green) fluorescence characteristics of flavonols and UV-microscopical studies we suggest that kaempferol glycosides are present in guard cells and epidermal cells in similar quantities, and that these compounds are in the vacuole.
By contrast, mesophyll protoplasts have a low flavonol content (one sixth that of guard cells). In spite of the different total flavonol contents, individual components of each cell-type are the same. However, they show differences in their quantitative distribution.
Guard cells and epidermal cells of the abaxial (lower) and adaxial (upper) epidermis ofPisum sativum L., mutant Argenteum, are the predominant sites of flavonoid accumulation within the leaf. This was demonstrated by the use of a new method of simultaneous isolation and separation of intact, highly-purified guard cell and epidermal cell protoplasts from both epidermal layers and of protoplasts from the mesophyll. Isolated guard and epidermal protoplasts retained flavonoid patterns of the parent epidermal tissue; quercetin 3-triglucoside and its p-coumaric acid ester as major constituents, kaempferol 3-triglucoside and its p-coumaric acid ester as minor compounds. Total flavonoid content in the lower epidermis was estimated to be ca. 80 fmol per guard cell protoplast and 500 fmol per epidermal cell protoplast. Protoplasts isolated from the upper epidermis had about 20–30% as much of these flavonoids. Mesophyll protoplasts retained only about 25 fmol total flavonoid per protoplast.
By fluorescence microscopy, using the alkaline-induced yellow-green fluorescence characteristics of flavonols, we suggest that these flavonol glycosides are present in cell vacuoles. There was no indication for the presence of flavine-like compounds.
It is suggested that flavanone synthase activity should be measured when the key reaction of flavonoid biosynthesis is to be tested. A simple and rapid procedure for the determination of flavanone synthase activity, based on extraction of the 14C -labelled product(s) into ethylacetate, is described. The enzyme can be stored under appropriate conditions for several weeks without significant loss of activity. Results obtained with cell suspension cultures of parsley indicate that the activity of flavanone synthase is regulated differently from the activity of phenylalanine ammonia-lyase, an enzyme frequently referred to as a key enzyme of flavonoid biosynthesis.
A new technique, the quantitative determination of total enzyme concentrations by specific immunoprecipitation with purified, radioiodinated antibodies, were used to investigate the presence and possible roles of inactive enzyme in the regulation of chalcone synthase. Dark-grown cell suspension cultures from parsley (P. hortense) contained neither catalytically active nor detectable amounts of immunoprecipitable chalcone synthase. Irradiation induced large increases and subsequent decreases of both. Significant differences in the peak positions and in the half-lives of active and total chalcone synthase indicated that induced cells contained inactive as well as active enzyme forms. The presence of inactive enzyme could be explained by 2 different modes of regulation, simultaneous de novo synthesis of active and inactive enzyme (simultaneous model), or de novo synthesis of active enzyme only, with sequential steps of inactivation and degradation (sequential model). Both models were compatible with experimental results, as analyzed mathematically by investigating the relations between curves for rate of enzyme synthesis, enzyme activity, total enzyme, and half-lives of active and total enzyme. The simultaneous model postulated that de novo synthesis of inactive enzyme represented always the vast majority of total enzyme synthesis, while the sequential model integrated inactive enzyme with facility in a sequence of irreversible inactivation and degration of active enzyme. Experiments with repeated induction indicated that cells containing large amounts of inactive enzyme increased enzyme activity by de novo synthesis rather than by activation of preexisting inactive enzyme.
Flavanone synthase was isolated and purified about 300‐fold from fermenter‐grown, light‐induced cell suspension cultures of Petroselinum hortense . The enzyme catalyzed the formation of the flavanone naringenin from p ‐coumaroyl‐CoA and malonyl‐CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed.
No cofactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl‐CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30°C, and at an ionic strength of 0.1–0.3 M potassium phosphate. EDTA, Mg ²⁺ , Ca ²⁺ , or Fe ²⁺ at concentrations of about 0.7 μM did not affect the enzyme activity. Apparent molecular weights of approx. 120000, 50000, and 70000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl‐CoA : flavonoid glycoside malonyl transferase.
The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl‐CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6‐methyl‐salicylic acid synthesis from “acetate units”, are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.
The role of chalcone synthase in the regulation of flavonoid biosynthesis during organogenesis of oat primary leaves has been investigated at the level of enzyme activity and mRNA translation in vitro. Chalcone synthase was purified about 500-fold. The apparent Km values were 1.5 and 6.3 microM for 4-coumaroyl-CoA and malonyl-CoA, respectively. The end products of oat flavonoid biosynthesis, three C-glucosylflavones, did not inhibit the reaction at concentrations as measured up to 60 microM each. Apigenin (4',5,7-trihydroxyflavone), a stable structural analog of the reaction product, 2',4,4',6'-tetrahydroxychalcone, was found to be a strong competitive inhibitor of 4-coumaroyl-CoA binding and a strong noncompetitive inhibitor of malonyl-CoA binding. Although apigenin is not supposed to be an intermediate of C-glucosylflavone biosynthesis, this compound might be a valuable tool for future kinetic studies. To date, there is no indication of chalcone synthase regulation by feedback or similar mechanisms which modulate enzyme activity. Mathematical correlation of chalcone synthase activity and flavonoid accumulation during leaf development, however, indicates that chalcone synthase is the rate-limiting enzyme of the pathway. By in vitro translation studies using preparations of total RNA from different leaf stages, we could demonstrate for the first time that the translational activity of chalcone synthase mRNA undergoes marked daily changes. The high values found at the end of the dark phase suggest that light does not exert direct influence on flavonoid biosynthesis but probably functions by controlling the basic diurnal rhythm.
Chalcone synthase (CHS) has been partially purified about 35-fold. Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps. In order to determine CHS activity, two procedures [according to Schröder et al. (1979) Plant Sci. Lett. 14, 281-286] were applied. The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC. The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay. Both effects were synergistic, but BSA did not promote "side products" as 2-mercaptoethanol did. BSA (10 mg ml-1) and 2-mercaptoethanol (1.4 mM) were components of the standard assay. Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8). The apparent Km values were 0.6 microM for 4-coumaroyl-CoA (pH 7.9), 7.7 microM for caffeoyl-CoA (pH 6.8), and 3.0 microM for malonyl-CoA (pH 7.9). Substrate inhibition was observed with 4-coumaroyl-CoA (greater than 10 microM) and malonyl-CoA (greater than 50 microM). The inhibitory activity of various flavonoids and related compounds (100 microM) was investigated. Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50%. In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40%. It was shown that the inhibition with naringenin was fully uncompetitive. These in vitro data support the view that the true substrate of CHS in D. carota is 4-coumaroyl-CoA.
This chapter describes an electrophoretic system capable of stacking and fractionating protein and glycoprotein-dodecyl sulfate complexes over a range of 2300–320,000 daltons. The system combines the advantages of electrophoresing protein-SDS complexes pioneered by Shapiro and the advantages of achieving thin starting zones by use of discontinuous buffers discovered by Ornstein and Davis. This SDS discontinuous system was developed to fractionate plasma membrane proteins solubilized in SDS and provides high resolution patterns of membrane protein subunits and reliable estimates of protein subunit molecular weights. The methods described in the chapter have been used in the laboratory for the past two years and give highly reproducible results providing that the essential variables are controlled.
A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots." The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which had been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.
The in vitro synthesis of chalcones has been demonstrated using a special biphasic enzyme assay. The highly viscous lower phase in this assay stems from a tapetum fraction of anthers of Tulipa cv. “Apeldoorn” which has been used an enzyme source. The upper phase of this system consists of a reaction mixture of the normal “flavanone synthase” assay. It is suggested that chalcone synthesis occurs at the boundary layer between the two phases. To prevent spontaneous as well as enzymatic cyclization of the chalcones formed (phloroglucinyl type), the pH of the upper phase must not be allowed to exceed pH 4.0. Under these pH conditions, chalcone formation by a reverse reaction of chalcone-flavanone isomerase can be excluded. The measured substrate specificity of the “chalcone synthase” corresponds to the conditions of chalcone formation in the natural system. Using p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, respectively, as substrates, the enzyme system forms the correspondingly substituted chalcones which are also accumulated in the loculus of tulip anthers. It is suggested that this chalcone synthase is identical to the previously described “flavanone synthase”. The results can be further explained as follows. (i) Not flavanones, but rather chalcones are the first C15 intermediates of flavonoid biosynthesis in tulip anthers. (ii) In this Tulipa system, the substitution pattern of three different hydroxycinnamic acids can be transferred unchanged into the flavonoid C15 stage. (iii) The role of chalcone-flavanone isomerase is to cyclize chalcones to flavanones on the direct biosynthetic pathway to the further accumulated flavonol glycosides. (iv) The sensitivity of the reaction with regard to chalcone production points to the localization of chalcone synthase in a most unstable and, up to now, unknown tapetal compartment. Since purification of the enzyme results in exclusive production of flavanones, it is suggested that certain “chalcone stabilizing factors” must occur in the natural system. (v) The phenomenon of chalcone accumulation in tulip anthers, however, must be caused by a complex system, distinguished by cooperation of certain biochemical and physiological conditions, and, finally, by special compartmentation of the enzymes which are responsible for the biosynthesis of flavonoids.