Regulation Of Flavonoid Biosynthesis Research Articles

Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis

TT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYB-bHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated. Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches. Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA- and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized. Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.

Levels of constitutive flavonoid biosynthetic enzymes in carnation (Dianthus caryophyllus L.) cultivars with differential response to Fusarium oxysporum f. sp. dianthi

We have assessed the constitutive total phenolic and flavonoid contents and the antioxidant activity levels in the stems and roots of nine commercial cultivars of carnation grown in Colombia showing differential response to Fusarium oxysporum f. sp. dianthi. In stems, these parameters were not correlated with resistance, but in roots, resistant cultivars exhibited higher levels of flavonoids and antioxidant activity than those found in susceptible cultivars. The activities of enzymes involved in the biosynthesis of these compounds were evaluated in the root level. Differences in the phenylalanine ammonia-lyase enzyme (PAL) activity were not correlated with resistance, but the activities of chalcone synthase (CHS) and chalcone isomerase (CHI) were higher in the roots of disease-resistant cultivars. Likewise, the transcript levels of CHS and CHI were significantly higher in resistant cultivars, as evaluated by real-time RT-PCR. These results suggested that the constitutive levels of flavonoids in the root level are most likely associated with resistance to F. oxysporum f. sp. dianthi. Such differences may be attributed to increased expression of genes in the phenylpropanoid pathway, specifically CHS and CHI, which are the enzymes that have been previously associated with the regulation of flavonoid biosynthesis in other species.

Identification of TT2 gene from floral transcriptome in Fagopyrum tataricum

In plants, the MYB family transcription factor has selectively expanded, particularly through the large family of R2R3-MYB. MYB proteins are key factors in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses. AtMYB123/TT2 encodes a R2R3 type MYB transcription factor, and plays a key role in regulating flavonoid late biosynthetic genes in Arabidopsis. Here, we firstly identified a TT2/AtMYB123 homologous protein FtMYB123L through bioinformatics analysis from the floral transcriptome data from Fagopyrum tataricum. We speculate that FtMYB123L also involves the regulation of flavonoid biosynthesis in F. tataricum.

Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape

Biosynthesis of phenolic compounds is known to be sensitive to light environments, which reflects the possible role of these compounds for photoprotection in plants. Herein, the effects of UV and visible light on biosynthesis of flavonoids was investigated, i.e., proanthocyanidins (PAs) and flavonols, in young berry skins of a red-wine grape, Vitis vinifera cv. Cabernet Sauvignon. Shading with light-proof boxes from the flowering stage until 49days after treatment (DAT) partially decreased PA concentrations, and completely decreased flavonol concentrations in the berry skins. Shading decreased the transcript abundance of a flavonol-related gene more remarkably than those of PA-related genes. In addition, light exclusion influenced the composition of PAs, such as the decrease in the proportion of trihydroxylated subunits and the mean degree of polymerization (mDP) within PAs. However, solar UV exclusion did not affect the concentration and composition of PAs, whereas this exclusion remarkably decreased the flavonol concentration. Consistently, UV exclusion did not influence the transcript levels of PA-related genes, whereas it dramatically decreased that of flavonol-related genes. These findings indicated a different light regulation of the biosynthesis of these flavonoids in young berry skins of wine grape. Visible light primarily induces biosynthesis of PAs and affects their composition, whereas UV light specifically induces biosynthesis of flavonols. Distinct roles of members of a MYB transcription factor family for light regulation of flavonoid biosynthesis were proposed.

R2R3 MYB transcription factors: key regulators of the flavonoid biosynthetic pathway in grapevine

Flavonoids compose one of the most abundant and important subgroups of secondary metabolites with more than 6,000 compounds detected so far in higher plants. They are found in various compositions and concentrations in nearly all plant tissues. Besides the attraction of pollinators and dispersers to fruits and flowers, flavonoids also protect against a plethora of stresses including pathogen attack, wounding and UV irradiation. Flavonoid content and composition of fruits such as grapes, bilberries, strawberries and apples as well as food extracts such as green tea, wine and chocolate have been associated with fruit quality including taste, colour and health-promoting effects. To unravel the beneficial potentials of flavonoids on fruit quality, research has been focused recently on the molecular basis of flavonoid biosynthesis and regulation in economically important fruit-producing plants such as grapevine (Vitis vinifera L.). Transcription factors and genes encoding biosynthetic enzymes have been characterized, studies that set a benchmark for future research on the regulatory networks controlling flavonoid biosynthesis and diversity. This review summarizes recent advances in the knowledge of regulatory cascades involved in flavonoid biosynthesis in grapevine. Transcriptional regulation of flavonoid biosynthesis during berry development is highlighted, with a particular focus on MYB transcription factors as molecular clocks, key regulators and powerful biotechnological tools to identify novel pathway enzymes to optimize flavonoid content and composition in grapes.

Flavonoids: biosynthesis, biological functions, and biotechnological applications

Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds.

MOLECULAR AND METABOLIC ANALYSES IN DEVELOPING OLIVE FRUIT IN RELATION TO DIFFERENT WATER REGIMES

Despite the global economic importance of olive (Olea europaea L.), little is known about the molecular and metabolic changes during fruit development and the modulation of quality-related metabolic pathways during drought stress. In this work, we report the expression pattern of genes involved in important pathways of secondary metabolism (polyphenols, terpenoids) during fruit development in rainfed or fully irrigated olive plants. Phenolic compounds represent a complex mixture in both olive fruits and oil. The interest on these compounds is due to their demonstrated anti-atherogenic and anti-cancerogenic effects providing to the olive oil important nutraceutical properties. Several parameters affect olive fruit phenolic content and these include genotype, pedo-climatic conditions, agronomic techniques (e.g. irrigation) and the develop¬mental stage of the drupe. To gain insight into the transcriptional regulation of phenolic pathways we analysed the pattern of expression of important genes during olive fruit development. Transcript levels of phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and dihydroflavonol reductase (DFR) genes showed different expression patterns during fruit development. CHS and DFR showed stronger expression in the skin than in the pulp. PAL expression was strong before pit hardening and decreased thereafter. CHS expression peaked at pit hardening and at veraison stage, whereas DFR was only strongly expressed at fruit ripening, suggesting a crucial role for these genes in the regulation of flavonoid biosynthesis in the last developmental stages of olive fruit. Water stress enhanced the expressions of PAL and CHS at pit hardening and of DFR in the skin at ripening. Of the terpenoids, amyrin synthase (AS) showed a decreasing expression trend throughout development, whereas lupeol synthase (LUS) was highly expressed in the skin of ripe fruit. Induction of LUS gene expression was observed at the early developmental stages in fruit of water-stressed plants. To gain insight into the metabolic changes in the fruit under different water regimes in the field, we initiated, in a high-throughput approach, analysis of the mesocarp of ripe fruits by GC-TOF mass spectrometry, and differences in metabolite composition were determined.

Flower Color Modification by Engineering of the Flavonoid Biosynthetic Pathway: Practical Perspectives

The status quo of flavonoid biosynthesis as it relates to flower color is reviewed together with a success in modifying flower color by genetic engineering. Flavonoids and their colored class compounds, anthocyanins, are major contributors to flower color. Many plant species synthesize limited kinds of flavonoids, and thus exhibit a limited range of flower color. Since genes regulating flavonoid biosynthesis are available, it is possible to alter flower color by overexpressing heterologous genes and/or down regulating endogenous genes. Transgenic carnations and a transgenic rose that accumulate delphinidin as a result of expressing a flavonoid 3',5'-hydroxylase gene and have novel blue hued flowers have been commercialized. Transgenic Nierembergia accumulating pelargonidin, with novel pink flowers, has also been developed. Although it is possible to generate white, yellow, and pink-flowered torenia plants from blue cultivars by genetic engineering, field trial observations indicate difficulty in obtaining stable phenotypes.

Arabidopsis NAC Transcription Factor, ANAC078, Regulates Flavonoid Biosynthesis under High-light

We have isolated a combination of high-light and heat-shock (HL + HS) stress-inducible genes, including a NAC transcription factor designated ANAC078. Here we explored the physiological function of ANAC078 under HL stress. Yeast transcription activity assays showed that ANAC078 functions as a transcriptional activator. A fusion protein composed of green fluorescent protein (GFP) and full-length ANAC078 was detected in the nucleus and cytoplasm, while fusion proteins comprising GFP and ANAC078 deleted of a putative transmembrane motif were found only in the nucleus. In ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078-43), the transcription of 166 genes was up-regulated compared with the levels in wild-type plants under HL (1200 micromol m(-2) s(-1), 30 degrees C). These genes included some for transcription factors regulating the expression of genes related to the biosynthesis of flavonoids. Interestingly, the transcript levels of some genes related to flavonoid biosynthesis and the levels of anthocyanins were significantly increased in Ox-ANAC078 plants and reduced in knockout ANAC078 plants (KO-ANAC078) compared with wild-type plants under HL stress. The present findings suggest that ANAC078 protein is associated with the induction of genes related to flavonoid biosynthesis, leading to the accumulation of anthocyanins, in response to HL stress.

Single MYB-type transcription factor AtCAPRICE: A new efficient tool to engineer the production of anthocyanin in tobacco

The single MYB-type protein, CAPRICE has long been thought to be a positive regulator of root hair development in Arabidopsis thaliana. To further probe the role of AtCPC and how conserved this role is across plant species, the AtCPC gene was constitutively overexpressed in tobacco ( Nicotiana tabacum). Here, we show that flowers of transformants overexpressing AtCPC displayed an unexpected defection in pigmentation. A reduction in the level of anthocyanin was detected and the severity of abnormal flavonoid phenotype correlates well with the increased expression level of AtCPC gene. We also found that transcripts of three late biosynthetic genes were substantially down-regulated. Furthermore, AtCPC could interact with other known anthocyanin regulators. Our data suggest that in a heterologous host, AtCPC acts as a repressor of anthocyanin production, from which a new evidence for better understanding of the transcriptional regulation of flavonoid biosynthesis and, an implication for the development of new varieties of tobacco and other commercially important ornamental plants are provided.

CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis

Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis.

Ectopic Expression of VvMybPA2 Promotes Proanthocyanidin Biosynthesis in Grapevine and Suggests Additional Targets in the Pathway

Grapevine (Vitis vinifera) proanthocyanidins contribute to plant defense mechanisms against biotic stress and also play a critical role in organoleptic properties of wine. In grapevine berry, these compounds are mainly accumulated in exocarps and seeds in the very early stages of development. A previous study has already identified VvMybPA1 as the first transcription factor involved in the regulation of the proanthocyanidin pathway during seed development in grapevine. A novel Myb factor, VvMybPA2, which is described in this study, is in contrast mainly expressed in the exocarp of young berries and in the leaves. This transcription factor shows very high protein sequence homology with other plant Myb factors, which regulate flavonoid biosynthesis. Ectopic expression of either VvMybPA1 or VvMybPA2 in grapevine hairy roots induced qualitative and quantitative changes of the proanthocyanidin profiles. High-throughput transcriptomic analyses of transformed grapevine organs identified a large set of putative targets of the VvMybPA1 and VvMybPA2 transcription factors. Both genes significantly activated enzymes of the flavonoid pathway, including anthocyanidin reductase and leucoanthocyanidin reductase 1, the specific terminal steps in the biosynthesis of epicatechin and catechin, respectively, but not leucoanthocyanidin reductase 2. The functional annotation of the genes whose expression was modified revealed putative new actors of the proanthocyanidin pathway, such as glucosyltransferases and transporters.

MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana

In Arabidopsis thaliana, several MYB and basic helix-loop-helix (BHLH) proteins form ternary complexes with TTG1 (WD-Repeats) and regulate the transcription of genes involved in anthocyanin and proanthocyanidin (PA) biosynthesis. Similar MYB-BHLH-WDR (MBW) complexes control epidermal patterning and cell fates. A family of small MYB proteins (R3-MYB) has been shown to play an important role in the regulation of epidermal cell fates, acting as inhibitors of the MBW complexes. However, so far none of these small MYB proteins have been demonstrated to regulate flavonoid biosynthesis. The genetic and molecular analyses presented here demonstrated that Arabidopsis MYBL2, which encodes a R3-MYB-related protein, is involved in the regulation of flavonoid biosynthesis. The loss of MYBL2 activity in the seedlings of two independent T-DNA insertion mutants led to a dramatic increase in the accumulation of anthocyanin. In addition, overexpression of MYBL2 in seeds inhibited the biosynthesis of PAs. These changes in flavonoid content correlate well with the increased level of mRNA of several structural and regulatory anthocyanin biosynthesis genes. Interestingly, transient expression analyses in A. thaliana cells suggested that MYBL2 interacts with MBW complexes in planta and directly modulates the expression of flavonoid target genes. These results are fully consistent with the molecular interaction of MYBL2 with BHLH proteins observed in yeast. Finally, MYBL2 expression studies, including its inhibition by light-induced stress, allowed us to hypothesise a physiological role for MYBL2. Taken together, these results bring new insights into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation of its developmental and environmental regulation.

AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis

In Arabidopsis, MYB transcription factors regulate flavonoid biosynthesis via the formation of protein complexes with a basic helix-loop-helix (bHLH) transcription factor and a WD40 repeat protein. Several R3-type single-MYB proteins (R3-MYB), such as CPC and TRY, act as negative regulators of the development of epidermal cells. However, such regulators of flavonoid biosynthesis have not yet been reported, to our knowledge. We show here that an R3-MYB protein, AtMYBL2, acts as a transcriptional repressor and negatively regulates the biosynthesis of anthocyanin in Arabidopsis. In an AtMYBL2 knockout line (mybl2), the expression of the DFR and TT8 genes was enhanced and resulted in the ectopic accumulation of anthocyanin, while ectopic expression of AtMYBL2 or of a chimeric repressor that is a dominant negative form of AtMYBL2 suppressed the expression of DFR and TT8, and the biosynthesis of anthocyanin. The expression of AtMYBL2 was detected in various tissues but not in those in which anthocyanin accumulated or TT8 was expressed. The minimal repression domain of AtMYBL2 was found to be the six amino acids (TLLLFR) at the carboxyl terminus, and TLLLFR appears to be a novel repression motif that is different from the ERF-associated amphiphilic repression (EAR) motif. The defective phenotype of mybl2 mutants was complemented by 35S:AtMYBL2 but enhanced by a truncated form of AtMYBL2 from which the repression domain had been deleted. AtMYBL2 bound directly to TT8 protein and this complex suppressed the expression of DFR and TT8. The repression activity of AtMYBL2 appears to play a critical role in the regulation of anthocyanin biosynthesis.

Functional Differentiation of Lotus japonicus TT2s, R2R3-MYB Transcription Factors Comprising a Multigene Family

Leguminous plants have many paralogous genes encoding enzymes involved in the flavonoid biosynthetic pathway. Duplicate genes are predicted to contribute to the production of various flavonoid compounds and to have resulted in a diversity of legume species. We identified gene duplication in the transcription factors regulating flavonoid biosynthesis in the model legume Lotus japonicus. Three copies of a homolog of Arabidopsis thaliana TRANSPARENT TESTA2 (TT2), which is a MYB transcription factor that regulates proanthocyanidin biosynthesis, were present in the L. japonicus genome. The organ specificity and stress responsiveness differed among the three LjTT2s, and correlations between proanthocyanidin accumulation and the expression levels of LjTT2s were observed during seedling development. Moreover, three LjTT2s functionally complemented TT2 in transient expression experiments in A. thaliana leaf cells. The different reporter activity caused by LjTT2a was consistent with the affinity of physical interactions with TT8 and TTG1 in yeast two-hybrid experiments as well as the branching pattern of the phylogenetic tree. These results suggest that LjTT2 factors have diverse functions in the tissues in which they are expressed; in particular, LjTT2a is predicted to have evolved flexibility in interaction with other transcription regulators to resist environmental stresses.

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera "Cabernet Sauvignon", and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3'4'5'-hydroxylated forms through the differential regulation of F3'H and F3'5'H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.

Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato.

Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have been identified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatory elements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1 and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressed in the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such as nontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars but not in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars contained anthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest that IbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stable transformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyanin genes and anthocyanin accumulation in the flesh of tuberous roots, as well as in heterologous tissues or heterologous plant species.

Pleiotropic effect of flavonoid biosynthesis manipulation in transgenic potato plants

Three approaches were successfully used to manipulate content of flavonoids in transgenic plants. Overexpressing either the adaptor 14-3-3 protein or genes coding the key enzymes of the flavonoid biosynthesis pathway resulted in a significant increase in the compound content in potato tuber epidermis. The opposite effect was observed in transgenic plants in which these proteins were repressed; this strongly supports the view that the gene construct determines transgenic plant features. The most effective construct was, however, the one containing single dihydroflavonol reductase (DFR) gene in sense orientation. In all cases the increase in flavonoid content resulted in the expected enhancement of the antioxidant capacity of tuber extract. At the biochemical level a decrease in the starch content in transgenic plant overexpressing proteins regulating flavonoid biosynthesis was detected. In the case of glucosyl transferase (GT) gene overexpression, the content of phenolic compounds remained at the control level, however, the antioxidant capacity of tuber extracts significantly decreased. The GT plants grew faster and were more resistant to pathogen attacks, the tuber yield was significantly higher than that of nontransformants. Thus it is speculated that it is the chemical structure and degree of glucosylation of flavonoids rather than their quantity which determines transgenic plant features.

Two Cysteines in Plant R2R3 MYB Domains Participate in REDOX-dependent DNA Binding

Plant R2R3 MYB domain proteins comprise one of the largest known families of transcription factors. Discrete evolutionary steps have shaped the plant-specific R2R3 MYB family from the broadly distributed R1R2R3 MYB proteins. R1R2R3 MYB domains have a single Cys residue (Cys-130) that needs to be reduced for DNA binding and transcriptional activity. In contrast, most R2R3 MYB domains contain two cysteines, Cys-49 and Cys-53, with Cys-53 at the equivalent position as Cys-130 in R1R2R3 MYB. Using the maize P1 regulator of flavonoid biosynthesis as a typical R2R3 MYB-domain protein, we investigated here the in vitro REDOX requirement for DNA binding by P1. We show that the C53S mutation requires reducing conditions for DNA-binding, whereas C53A binds DNA under oxidizing and reducing conditions. Neither mutation impairs the in vivo regulatory activity of P1. The C49S and C49A mutants bind DNA in vitro irrespective of the REDOX conditions. A C49I mutant, which simulates the MYB domain of c-MYB, binds DNA only under reducing conditions, and its binding is significantly affected by the C53S replacement. It is interesting that under non-reducing conditions, Cys-49 and Cys-53 form a disulfide bond that prevents the R2R3 MYB domain from binding DNA. Together, our results suggest that the evolutionary origin of Cys-49 within the plants has provided R2R3 MYB domains with a regulatory feature not present in animal MYB domains, highlighting fundamental structural and functional differences between similar DNA-binding domains from plants and animals.

Metabolism of administered (2RS)-naringenin in flavonoid-producing cultured cells of Sophora flavescens

Cultured cells of Sophora flavescens produce (2S)-naringenin-derived prenylated flavanone sophoraflavanone G and liquiritigenin-derived trifolirhizin 6� -O-malonate. The regulation of flavonoid biosynthesis was examined by analyzing the metabolites produced in the cultured cells fed (2RS)-naringenin. The amount of sophoraflavanone G in cells fed 0.1 or 0.3 mM (2RS)-naringenin was two-fold that in control cells, although the conversion ratio was only 5 to 10% of the administered (2S)-naringenin. On the other hand, (2R)-naringenin, which does not occur naturally, was efficiently converted into its 4� ,7-di-O-b-D-glucoside. (2S)-Naringenin prenylation activity was higher at the logarithmic growth stage. The cells fed (2RS)-naringenin at a lower concentration (below 0.1 mM), accumulated sophoraflavanone G as the main prenylated flavanone. In contrast, cells fed 0.3 mM (2RS)-naringenin accumulated 8-prenylnaringenin and leachianone G, intermediates of sophoraflavanone G in large amounts. Accumulation of trifolirhizin 6� -O-malonate was suppressed by the addition of naringenin.