Induction of chalcone synthase in cell suspension cultures of carrot (Daucus carota L. spp. sativus) by ultraviolet light: evidence for two different forms of chalcone synthase

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).Abbreviations BSA bovine serum albumin - CHS chalcone synthase - IEF isoelectric focusing - kDa kilodaltons - KP_i potassium phosphate buffer - PAL phenylalanine ammonialyase - pI isoelectric point - UV ultraviolet     

In situ localization of chalcone synthase inLarix needles by indirect immunofluorescence

Summary Chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis, is localized by indirect immunofluorescence in needles ofLarix decidua. In young stages of needle development CHS is present in epidermal cells and individual cells in the region of the vascular bundles which possibly contain tannins. A later developmental stage exhibits immunofluorescence predominantly in the mesophyll of the needle. The epidermis and the cells in the vicinity of the vascular bundles show a considerably weaker and only sporadically detectable fluorescence. CHS is no longer observable at the latest analyzed stages of needle development.     

A chalcone synthase/stilbene synthase DNA probe for conifers

A probe for chalcone synthase (CHS) was generated by PCR using chalcone synthase conserved sequences. The cloned PCR product has high similarity to both chalcone synthase and stilbene synthase sequences. The probe was used to examine the organization of chalcone synthase and stilbene synthase genes in Abies procera, Pinus lambertiana, P. monticola, Picea glauca, P. sitchensis, Pseudostuga menziesii, Taxus brevifolia, and Thuja plicata. A large number of hybridizing bands were found in all species except T. plicata which did not cross hybridize. The hybridization patterns are highly polymorphic between the species and are also polymorphic within several of them.     

Co-suppression in transgenicPetunia hybrida expressing chalcone synthase A (chsA)

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression ofchsA gene in transgenicPetunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal ofchsA gene, and transferred the fusion gene intoPetunia hybrida viaAgrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNAin situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C₂-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7–4.2% of naringenin) and naringenin production by STS (1.4–2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS¹ type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Molecular modeling of the effects of mutant alleles on chalcone synthase protein structure

Chalcone synthase (CHS) catalyzes the first committed step in flavonoid biosynthesis, a major pathway of plant secondary metabolism. An allelic series for the Arabidopsis CHS locus, tt4, was previously characterized at the gene, protein, and end-product levels. In an effort to deduce the molecular basis for the observed phenotypes, homology models were generated for five of the tt4 proteins based on the crystal structure of CHS2 from Medicago. Molecular dynamics simulations provided insights into how even those substitutions that are not in close spatial proximity to key functional residues may still alter the architecture and dynamic movement of the enzyme, with dramatic effects on enzyme function. Simulations carried out at different temperatures pointed to optimized positioning of key residues in the active site or dimerization domain, rather than enhancement of overall structure, as underlying the higher activity of two temperature-sensitive variants at lower temperatures. Extending this type of analysis to account for protein–protein interactions may offer additional insights into the mechanisms by which single amino-acid substitutions can affect diverse aspects of protein function.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

p-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii.

Chalcone synthase and stilbene synthase are plant-specific polyketide synthases. They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions. They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase. Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var. thunbergii. They were expressed in Escherichia coli in order to determine their enzyme functions. Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene. However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein. This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization.     

Signal transduction in the photocontrol of chalcone synthase gene expression*

The photocontrol of chalcone synthase gene expression was studied by means of promoter analyses, in vitro systems, photoreceptor mutants and microinjections, and pharmacological approaches. A 52 bp element of the promoter is necessary and sufficient to transfer light regulation. Chalcone synthase expression is primarily under the control of phyA and blue/UV photo receptors; the latter are functional even in the absence of phyA and phyB. Phytochromes seem to be soluble proteins and, within seconds of irradiation, light-dependent phosphorrylations were observed in membrane-depleted cytosol preparations, indicating very early processes of signal transduction. Microinjection and pharmacological experiments reveal that, in the phyA pathway, heterotrimeric G-proteins, cGMP and a genistein-sensitive protein kinase are involved, whereas the UV pathway includes several trimeric G-proteins and Ca/calmodulin-dependent steps.     

Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.)

Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3′ region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product.     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Phenylalanine ammonia-lyase and chalcone synthase in glands ofPrimula kewensis (W. Wats): immunofluorescence and immunogold localization

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were localized by indirect immunofluorescence and immunogold labeling in glands ofPrimula kewensis. Both enzymes were exclusively present in the head cells of the glands. Phenylalanine ammonialyase was located in the regions of the dense tubular endoplasmic reticulum and occasionally found in more or less spherical organelles that have not yet been identified. Furthermore, an appreciable proportion of the enzyme protein was associated with the plasmalemma and the cell wall of the head cell. In contrast, the occurrence of CHS was restricted to the spherical, unidentified cell compartments. Our findings indicate that the gland cells have the potential for flavonoid biosynthesis. When a mutant ofP. kewensis forming structurally intact glands but incapable of farina excretion was studied, neither PAL nor CHS were found in the head cells.Abbreviations CHS chalcone synthase - IgG immunoglobulin G - PAL phenylalanine ammonia-lyase Financial support from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged. We are grateful to Mrs. Karin Schlattmann and Mrs. Susanne Otter for preparing the ultrathin sections and to Mrs. Marianne Opalka for taking the photographs.     

Differential patterns of phytoalexin accumulation and enzyme induction in wounded and elicitor-treated tissues ofPhaseolus vulgaris

In wounded cotyledons ofPhaseolus vulgaris L. the accumulation of the 5-hydroxy isoflavonoids kievitone and 2-hydroxygenistein precedes the major increases in the levels of the 5-deoxy compounds phaseollin and coumestrol. Increased phytoalexin levels are preceded by transient increases in the extractable activities of L-phenylalanine ammonia-lyase (EC 4.3.1.5.), chalcone synthase and chalcone isomerase (EC 5.5.1.6.). Accumulation of phytoalexins, above wounded control levels, is observed following treatment of excised cotyledons or hypocotyls with crude or fractionated elicitor preparations heat-released from the cell walls ofColletotrichum lindemuthianum. Chalcone synthase levels are also induced in cotyledons, although crude elicitor and all fractions suppress L-phenylalanine ammonia-lyase activity in both tissues. Kievitone is the major phytoalexin induced in cotyledons, whereas in hypocotyls phaseollin predominates. Patterns of phytoalexin accumulation have been studied in response to varying concentrations of the crude and fractionated elicitor; 5-hydroxy isoflavonoid accumulation is highly dependent upon elicitor concentration, the dose-response curves for kievitone accumulation showing maxima at around 1 g glucose equivalents per cotyledon, minima at 2–3 g equivalents and increasing induction at higher concentrations. Similar patterns are observed for L-phenylalanine ammonia-lyase and chalcone synthase levels, although the overall extent of these changes is masked by the high wound response. Accumulation of 5-deoxy isoflavonoids above control levels requires high elicitor concentrations; no experimental conditions were found under which phaseollin accumulated to higher levels than kievitone in cotyledons during the first 48 h after elicitation.Abbreviations CHS chalcone synthase - PAL L-phenylalanine ammonia-lyase     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida: differential effect on the expression of family members

Suppression of gene expression using antisense technology has been successful in various applications. In this paper we report differential inhibition of gene expression of the chalcone synthase (chs) gene superfamily members in transgenic Gerbera hybrida (Asteraceae) plants. We have transformed two different cDNAs of the chs gene family, gchs 1 [4] and gchs2, in antisense orientation under control of the CaMV 35S promoter into gerbera. Gchs1 codes for an enzyme with chalcone synthase activity while gchs2 is a more diverged member of the gene family having distinct structure and expression pattern. Furthermore, gchs2 is evidently not involved in anthocyanin synthesis and encodes an enzyme with novel catalytic properties. In both cases effective blocking of the resident sense gene expression was detected. In addition, the transformation affected differentially the expression of other members of the chs gene family. The degree of inhibition appeared to depend on the sequence homology between the antisense and the target genes. In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.     

Isolation and characterization of buckwheat (Fagopyrum esculentum M.) chalcone synthase and its polyclonal antibodies

Chalcone synthase was isolated from illuminated buckwheat (Fagopyrum esculentum M.) hypocotyls and purified to electrophoretic homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using (NH)4SO4 fractionation, gel filtration on AcA 44, ion exchange chromatography on DEAE-Bio-Gel, and HPLC on hydroxylapatite. The properties of the enzyme were pH optimum, 8.0; Mr approximately 83,000 +/- 1000; Mr subunit, approximately 41,500 +/- 500; isoelectric point, pH 5.2; Km, 1 X 10(-6)M for malonyl-CoA, and 0.6 X 10(-6) M for p-coumaryl-CoA. Buckwheat chalcone synthase used p-coumaryl-CoA as substrate and also utilized caffeyl-CoA and ferulyl-CoA at 20 and 80%, respectively, of the rate of p-coumaryl-CoA in the chalcone synthase reaction. Antibodies against the buckwheat chalcone synthase were developed in a New Zealand white rabbit and characterized for specificity by enzyme-linked immunosorbent assay, Ouchterlony double immunodiffusion, and Western blotting.