Flavanone Synthase Research Articles

Cloning and functional analysis of flavanone synthase Ⅱ from Dendrobium huoshanense

Flavonoid C-glycosides are a class of natural products that are widely involved in plant defense responses and have diverse pharmacological activities. They are also important active ingredients of Dendrobium huoshanense. Flavanone synthase Ⅱ has been proven to be a key enzyme in the synthesis pathway of flavonoid C-glycosides in plants, and their catalytic product 2-hydroxyflavanone is the precursor compound for the synthesis of various reported flavonoid C-glycosides. In this study, based on the reported amino acid sequence of flavanone synthase Ⅱ, a flavanone synthase Ⅱ gene(DhuFNSⅡ) was screened and verified from the constructed D. huoshanense genome localization database. Functional validation of the enzyme showed that it could in vitro catalyze naringenin and pinocembrin to produce apigenin and chrysin, respectively. The open reading frame(ORF) of DhuFNSⅡ was 1 644 bp in length, encoding 547 amino acids. Subcellular localization showed that the protein was localized on the endoplasmic reticulum. RT-qPCR results showed that DhuFNSⅡ had the highest expression in stems, followed by leaves and roots. The expression levels of DhuFNSⅡ and other target genes in various tissues of D. huoshanense were significantly up-regulated after four kinds of abiotic stresses commonly encountered in the growth process, but the extent of up-regulation varied among treatment groups, with drought and cold stress having more significant effects on gene expression levels. Through the identification and functional analysis of DhuFNSⅡ, this study is expected to contribute to the elucidation of the molecular mechanism of the formation of quality metabolites of D. huoshanense, flavonoid C-glycosides, and provide a reference for its quality formation and scientific cultivation.

The Transcriptome Profiling of Flavonoids and Bibenzyls Reveals Medicinal Importance of Rare Orchid Arundina graminifolia.

Orchids are very important flowering plants that spend long juvenile phases before flowering. Along with aesthetic importance, they are rich sources of medicinal components. However, their long reproductive cycle is the major hurdle to study the medicinal efficacy. Arundina graminifolia is a rare orchid that grows fast, unlike other orchids, and this characteristic makes it an ideal plant to study the medicinal enrichment of orchids. Therefore, this study presents the identification of important medicinal components in various parts of A. graminifolia. Transcriptome analysis was performed for five stages (FD1–FD5) of flower development and four tissue types (mature flower, silique, root, and leaf) to ascertain genetic regulators of flavonoids and bibenzyls. Most of the genes showed the highest expression in roots as compared with other tissues. Weighted gene coexpression network analysis (WGCNA) was performed to identify the coexpression modules and the candidate genes involving biosynthesis pathways of these chemicals. MEyellow module contained the highly coexpressed genes. Moreover, the concentrations of phenylpropanoid, bibenzyls, and flavone were ascertained through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Phenylpropanoid and bibenzyl were comparatively high in the leaf, while flavone showed a high concentration in the stem. The selected candidate genes [bibenzyl biosynthesis (BIBSY212), CYP84A1, CYP73A4, 4CLL7, UGT88B1, UGT73C3, anthocyanin synthase (ANS), phenylalanine ammonia-lyase (PAL), flavanone synthase FLS, and CHS8] were validated through quantitative real-time PCR (qRT-PCR). Most of these genes showed high expression in leaf and root as compared with other tissue. Therefore, the presence of bibenzyls and flavonoids in different parts of A. graminifolia and their molecular regulators can provide a quick source to decipher the medicinal efficacy of orchids.

Is Photosynthesis Suppressed at Higher Elevations Due to Low CO2 Pressure?

plant cell cultures. Proceedings of the National Academy of Sciences (USA) 89:2389-2393. Haukioja, E., J. Suomela, and S. Neuvonen. 1985. Longterm inducible resistance in birch foliage: triggering cues and efficacy on a defoliator. Oecologia 65:363-369. Karban, R., and J. Meyers. 1989. Induced plant responses to herbivory. Annual Review of Ecology and Systematics 20:331-348. Parsons, T., H. Bradshaw, and M. Gordon. 1989. Systemic accumulation of specific mRNAs in response to wounding in poplar trees. Proceedings of the National Academy of Sciences (USA) 86:7895-7899. Penia-Cortes, H., T. Albrecht, S. Prat, E. Weiler, and L. Willmitzer. 1993. Aspirin prevents wound-induced gene-expression in tomato leaves by blocking jasmonic acid biosynthesis. Planta 191:123-128. Rhoades, D. 1983. Responses of alder and willow to attack by tent caterpillars and webworms: Evidence for pheromonal sensitivity of willows. American Chemical Society Symposium Series 208:55-68. Schr6der, J., F Kreuzaler, E. Schafer, and K. Hahlbrock. 1979. Concomitant induction of phenylalanine ammonialyase and flavanone synthase mRNAs in irradiated plant cells. Journal of Biological Chemistry 254:57-65. Staswick, P. 1992. Jasmonate, genes, and fragrant signals. Plant Physiology 99:804-807. Zeringue, H. 1987. Changes in cotton leaf chemistry induced by volatile elicitors. Phytochemistry 26:1357-1360.

Distribution of Secondary Plant Metabolites and Their Biosynthetic Enzymes in Pea (Pisum sativum L.) Leaves

Leaves of a novel strain of peas (Pisum sativum L.) were used to determine the distribution of secondary metabolites and their biosynthetic enzymes. Leaf epidermal layers in this strain are easily separated from the parenchyma. Anthocyanins and flavonol glycosides were localized in epidermal vacuoles only. Among the biosynthetic enzymes studied, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), S-adenosyl-1-methionine (SAM):caffeic acid and SAM:quercetin methyltransferases (o-dihydric phenol methyltransferase, EC 2.1.1.42) and a flavonoid 7-O-glucosyltransferase (EC 2.4.1.91) were chiefly localized in the parenchyma, whereas trans-cinnamate 4-monooxygenase (EC 1.14.13.11), hydroxycinnamate:CoA ligases (EC 6.2.1.12) and a flavonoid 3-O-glucosyltransferase (EC 2.4.1.91) were found mainly in the epidermis. Flavanone (chalcone) synthase activity was found only in the epidermis, whereas chalcone isomerase (EC 5.5.1.6) was evenly distributed in epidermal and parenchyma tissues.

Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 μg elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 μg ml(-1). The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; α-methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.

"Flavanone Synthase" in Oat Primary Leaves Time Course and Distribution at the Tissue and Subcellular Level

Abstract “Flavanone Synthase” (FS) isolated from oat primary leaves shows maximal activity in the differentiating zone of five day old leaves. Activity of FS is distributed in both the upper and lower epidermis, but dominant in the mesophyll. After fractionation of mesophyll protoplasts of Avena by differential centrifugation, FS activity could only be detected in the “cytosol” and not in intact chloroplasts or other particulate fractions.

Highly purified “flavanone synthase” from parsley catalyzes the formation of naringenin chalcone

The key reaction of flavonoid biosynthesis, the condensation of the acyl residues from one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, has previously been assumed to be catalyzed by a “flavanone synthase.” Results are presented here which indicate that not the flavanone but the isomeric chalcone is the immediate product of the synthase reaction. The new term “chalcone synthase” is therefore suggested for the enzyme.

Chalcone synthesis with enzyme extracts from tulip anther tapetum using a biphasic enzyme assay

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 C 15 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 C 15 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.

Subcellular localization of flavonoid synthesizing enzymes in Pisum, Phaseolus, Brassica and Spinacia cultivars

Abstract Functionally-intact chloroplasts were obtained from 11-day-old pea ( Pisum sativum cv Midfreezer) seedlings. Enzyme-distribution studies with ribulose bisphosphate carboxylase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase showed that ca 2.1% of the total tissue chloroplasts were present in the chloroplast preparation. The rate of intactness of chloroplast preparations was 34–82%. SAM:caffeic acid methyltransferase, flavanone synthase, UDPG:flavonoid-3- O -glucosyltransferase and SAM:quercetin methyltransferase activities were measured in the homogenate, supernatant and chloroplast lysate fractions. Significant activities of the above four enzymes could only be detected in the homogenate and supernatant fractions. Similar experiments with 11-day-old seedlings of green beans ( Phaseolus vulgaris cv Early Gallatin), red cabbage ( Brassica oleracea cv Red Danish) and 6-week-old plants of spinach ( Spinacia oleracea cv Bloomsdale) showed a similar distribution of the flavonoid synthesizing enzymes. We conclude that under the reported conditions chloroplasts are not involved in flavonoid biosynthesis.

Enzymes of General Phenylpropanoid Metabolism and of Flavonoid Glycoside Biosynthesis in Parsley

Several enzymes of phenylpropanoid metabolism showed large changes in their inducibility by light during the growth cycle of cell suspension cultures from parsley (Petroselinum hortense Hoffm.). Two of the three enzymes of general phenylpropanoid metabolism (group I) and six of the approximately 13 enzymes of the flavone and flavonol glycoside pathways (group II) were investigated. Both enzymes of group I (phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase) were most efficiently induced at two different stages: first, soon after starting a new culture, and second, near the beginning of the stationary phase. In contrast, the enzymes of group II (acetyl-coenzyme A carboxylase, flavanone synthase, chalcone isomerase, UDP-apiose synthase, and at least one of two malonyltransferases) were maximally induced during exponential growth of the culture. This result supports the conclusions drawn from previous data that the two groups are regulated differentially and that the enzymes within each group are regulated in a coordinated manner.

Flavanone synthase from Petroselinum hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue.

Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77 000 for the enzyme and about 42 000 for the subunits were determined respectively by sedimentation-equilibrium measurements and disc-gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrifugation and gel electrophoresis under partially denaturing conditions. Values of about 17 S and Mr = 0.62 X 10(6) were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical. Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, beta-alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3-oxoacyl-(acyl carrier protein) synthase is discussed.

Flavanone synthase: Simple and rapid assay for the key enzyme of flavonoid biosynthesis

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.

Light induced changes in anthocyanin concentration, activity of phenylalanine ammonia-lyase and flavanone synthase and some of their properties in Brassica oleracea

Seedlings of red cabbage, Brassica oleracea cv Red Danish, germinated in the dark, rapidly produced anthocyanins upon illumination. The anthocyanin production increased up to six days of illumination time. The activity of phenylalanine ammonia-lyase increased rapidly in illuminated seedlings to a maximum at 8 hr and declined thereafter to dark levels. During this period the activity of flavanone synthase, the first enzyme responsible for the establishment of C 15 flavonoid skeleton, paralleled that of the anthocyanin concentration. The crude flavanone synthase has a pH optimum at around 8, a molecular weight of ca 120 000, and is able to utilize only p-coumaryl-CoA as co-substrate for the production of flavonoids.

Concomitant induction of phenylalanine ammonia-lyase and flavanone synthase mRNAs in irradiated plant cells.

Irradiation of previously dark-grown cell suspension cultures from parsley (Petroselinum hortense Hoffm.) with ultraviolet light caused large, concomitant increases in mRNA activities for two characteristic enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, and flavanone synthase. The rates of enzyme synthesis both in vitro in a reticulocyte lysate and in vivo were quantitated by direct immunoprecipitation of the labeled enzyme subunits. Following a period of about 2 h, during which possible changes were below the limits of detectability, the two mRNA activities increased rapidly in irradiated cells for serveral hours. Phenylalanine ammonia-lyase mRNA reached a peak in activity a few hours earlier than flavanone synthase mRNA. The apparent half-lives of the enzyme activities were about 7 to 10 h for phenylalanine ammonia-lyase and 5 to 7 h for flavanone synthase. These data were used to calculate the expected, light-induced changes in enzyme activity from the measured changes in mRNA activity. The results for both enzymes were in agreement with experimental data, indicating that the light-induced changes in enzyme activity can be explained by changes in the respective mRNA activity. Some data concerning changes in the degree of polyadenylation of the mRNAs and the inhibitory effects of 7-methylguanosine 5'-phosphate are presented.

Genetic control of chalcone isomerase activity in anthers of Petunia hybrida

The gene Po in pollen of Petunia hybrida Vilm. controls a discrete step in flavonoid biosynthesis. In recessive genotypes, naringenin-chalcone (4, 2',4',6'-tetrahydroxychalcone) is accumulated, whereas, under the influence of the wild-type allele flavonols and anthocyanins are formed. Enzymic investigations on anthers of four genetically defined lines with different pollen colouration revealed a clear correlation between accumulation of naringenin-chalcone and deficiency of chalcone isomerase (EC 5.5.1.6). The results allow the conclusion that chalcone is the first product of the flavanone synthase reaction in anthers of Petunia hybrida and that chalcone isomerase is essential for the formation of flavonols and anthocyanins. These results were similar to those previously obtained with Callistephus chinensis (L.) Nees.

Characterization, Development and Localization of "Flavanone Synthase" in Tulip Anthers

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.

Changes in the levels of enzymes of phenylpropanoid and flavonoid synthesis during phaseollin production in cell suspension cultures of Phaseolus vulgaris

The extractable activities of phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, p-coumaric acid-coenzyme A ligase, flavanone synthase and chalcone—flavanone isomerase showed significant increases in bean cell cultures in response to treatment with autoclaved ribonuclease, a potent inducer of phaseollin biosynthesis in this system. Increases in the activities of all the enzymes occurred within 6 h after ribonuclease treatment, but the subsequent development of activities was not coordinated. The flavanone synthase catalysed the formation of naringenin (4′,5,7-trihydroxyflavanone) from malonyl coenzyme A and p-coumaroyl coenzyme A. Synthesis of liquiritigenin (4′,7-dihydroxyflavanone) was not detected when soluble cytoplasmic or particulate preparations were incubated with the above substrates in the presence of NADPH or NADH generating systems. The results are discussed in terms of current views on isoflavonoid biosynthesis and the known changes in phenylpropanoid metabolism occurring in the cultures.

Flavanone synthase catalyzes CO 2 exchange and decarboxylation of malonyl-CoA

Flavanone synthase catalyzes CO 2 exchange and decarboxylation of malonyl-CoA

Induction of phytoalexin synthesis in Soybean: Elicitor-induced increase in enzyme activities of flavonoid biosynthesis and incorporation of mevalonate into glyceollin

Soybean (Glycine max) cotyledons removed from 6-to 8-day-old seedlings were treated with mycelial walls of Phytophthora megasperma var. sojae (elicitor) and the activity changes of the enzymes phenylalanine ammonia-lyase and flavanone synthase were determined. Large increases and subsequent decreases in the activities of both enzymes were found in the elicitor-induced cotyledons. No changes in enzyme activities, occurred in control cut cotyledons with elicitor omitted. Concomitant with the elicitor-stimulated changes in enzyme activities there was accumulation of the soybean phytoalexin, glyceollin. [2-14C] Mevalonic acid was incorporated into glyceollin in elicitor-treated but not in untreated cotyledons. The results prove that stimulation of the enzymes of flavonoid biosynthesis and probably glyceollin biosynthesis are caused by the effect of the P. megasperma elicitor and not merely by wounding the plant material. The induced enzyme levels could control glyceollin biosynthesis.

Flavanone synthase from cell suspension cultures of Haplopappus gracilis and comparison with the synthase from parsley

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.