Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

Flavonoid metabolons (weakly‐bound multi‐enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein–protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4‐reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3′‐hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage‐dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co‐suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long‐suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.     

Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco.

Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene (SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a pI of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.     

Localization of flavonoid enzymes in Arabidopsis roots

Immunofluorescence and immuno-electron microscopy have been used to test the hypothesis that flavonoid metabolism is organized as a membrane-associated enzyme complex. The cellular and subcellular locations of chalcone synthase (CHS) and chalcone isomerase (CHI), the first two enzymes of this pathway, were examined in Arabidopsis roots. High levels of both enzymes were found in the epidermal and cortex cells of the elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. Co-localization of CHS and CHI was observed at the endoplasmic reticulum and tonoplast in these cells, and also in electron-dense regions that are, as yet, unidentified. In addition, a striking asymmetric distribution was observed for these enzymes in cortex cells of the elongation zone, which may provide clues about the physiological function of flavonoids in roots. The accumulation of CHS and CHI was also examined in tt7(88), a mutant in the gene for flavonoid 3'-hydroxylase (F3'H), which has been postulated to serve as a membrane anchor for the flavonoid enzyme complex. CHS and CHI accumulated to lower levels in cortex cells and higher levels in epidermal cells in the roots of this mutant as compared with wild-type plants. Moreover, the electron-dense regions containing these two enzymes were not observed. However, localization of CHS and CHI to the ER and tonoplast did not appear to be affected, suggesting that other proteins may function in recruiting the "soluble" flavonoid enzymes to membranes. Staining of flavonoid endproducts with DPBA was consistent with expression of CHS and CHI in these seedlings.     

Regulation and manipulation of flavonoid gene expression in anthers of petunia: the molecular basis of the Po mutation.

Molecular mechanisms governing development of the male reproductive organs of flowers, the anthers, are largely unknown. In this article, we report on the investigation of the molecular basis of a mutation involving the expression of a gene encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase (CHI) in anthers of petunia. In petunia, the gene Po regulates the expression of CHI in anthers: PoPo petunia lines contain CHI enzyme activity in petals and anthers, whereas popo lines contain the CHI enzyme only in petals but not in anthers. As a result of the Po mutation, the substrate of CHI accumulates and therefore the pollen of a popo line are yellow or greenish. The genome of petunia contains two chi genes, chiA and chiB. In a restriction fragment length polymorphism analysis, a 100% linkage was observed between Po and chiA. This result suggested that Po is identical to chiA and that Po is not a regulatory gene of chiA. Introduction of a chiA gene isolated from a PoPo line into a popo line resulted in a complementation of the mutation that was directly visible because the pollen color shifted from yellow to white. This proved that chiA and Po are identical. Because chiA encodes a functional CHI enzyme in flower petals of a popo line, we propose that the Po mutation is a mutation in the regulatory region of chiA abolishing chiA promoter activity in anthers but not in corollas. This change in anther color is a fine illustration of how floral pigmentation can be manipulated in a predictable way and suggests the use of CHI as a visible marker.     

Gold color in onions (<Emphasis Type="Italic"> Allium cepa</Emphasis>): a natural mutation of the chalcone isomerase gene resulting in a premature stop codon

Unusual gold-colored onions were selected from a F₃ family originating from a cross between US-type yellow and Brazilian yellow onions. HPLC analysis showed that the gold onions contained a significantly reduced amount of quercetin, the most abundant flavonoid in onions. This result indicated that an early step in the flavonoid biosynthesis pathway might be abnormal in these onions. The expression of flavonoid synthesis genes isolated from onions was examined in gold onions and compared to that in onions of other colors by RT-PCR. The results showed that all genes were transcribed in gold onions as in red onions. In order to identify any critical mutations in flavonoid synthesis genes encoding enzymes involved in early steps of the pathway, the genomic sequence of chalcone isomerase (CHI) was obtained. A premature stop codon and a subsequent single base-pair addition causing a frameshift were identified in the coding region of the CHI gene in the gold onions. Co-segregation of the mutant allele of the CHI gene and the gold phenotype was investigated in the original F₂ segregating population. Genotyping of three color groups (red, yellow and gold) of F₂ onions revealed perfect co-segregation of the mutant CHI allele with the gold phenotype. All tested gold F₂ onions were homozygous for the mutant CHI allele. This perfect co-segregation implies that the presence of a premature stop codon in the gold CHI gene results in an inactive CHI. Inactivation of CHI results in a block in the flavonoid biosynthesis pathway and the accumulation of chalcone derivatives, including a yellow pigment which might be responsible for the gold color in onions.Communicated by R. Hagemann     

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

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.     

A chalcone isomerase‐like protein enhances flavonoid production and flower pigmentation

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best‐studied metabolic pathways. Here we have identified three mutations within a gene that result in pale‐colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)‐related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio‐temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3‐MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale‐colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.     

A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicus

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6'-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6'-deoxychalcone and 6'-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1-cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1-3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.     

Characterization and expression analysis of chalcone synthase and chalcone isomerase genes in Phyllanthus emblica (L.)

Journal of Plant Biochemistry and Biotechnology - The two decisive enzymes in flavonoid biosynthetic pathway are chalcone synthase (CHS) and chalcone isomerase (CHI), wherein the former carries the...     

大花美人蕉查尔酮异构酶基因的cDNA克隆和序列分析

以高等植物查尔酮异构酶（CHI）基因的保守区域CKFVKFT、KFTAIGV、AVKWKGK及GPFEKFT、IIGKI-IGV设计简并引物和采用逆转录．聚合酶链式反应（RT-PCR）以及温度非对称交互PCR（TAIL．PCR）方法,从大花美人蕉花瓣组织中扩增查尔酮异构酶基因（倒）的全长cDNA（678bp）,编码226个氨基酸,其氨基酸组成与其它已知的高等植物CHI基因具有很高的同源性,与葡萄、草莓、丁香、柑桔、矮牵牛、洋葱及玉米的同源性分别为82％、79％、80％、80％、79％、81％和76％。     

Decreasing unpalatable flavonoid components in Citrus: the effect of transformation construct

Citrus species accumulate large quantities of flavanone glycosides in their leaves and fruit. The physiological role(s) of these compounds in citrus plants are unknown, but they have been documented to benefit human health upon consumption. Flavanone rutinosides are tasteless, whereas flavanone neohesperidosides, such as naringin, give a bitter taste to fruit and fruit juice products, reducing their palatability. In an effort to alter the types and levels of flavanone neohesperidosides in citrus, an Agrobacterium -mediated genetic transformation approach was employed. Citrus paradisi Macf. (grapefruit) epicotyl stem segments were transformed with sense (S) and antisense (AS) constructs of the target genes chalcone synthase (CHS) and chalcone isomerase (CHI), whose products catalyze the first two steps in the flavonoid biosynthetic pathway. Transformation with each of the individual constructs led to a different and unpredictable combination of viability, phenotypic change, transgene steady-state expression and alteration in flavonoid content in the resulting transgenic plants. These qualities were consistent within the transgenic plants obtained using any particular construct. Transgenic plants with decreased leaf naringin levels were obtained, particularly when the CHS-AS constructs were employed.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Production of yellow colour in flowers: redirection of flavonoid biosynthesis in Petunia

Chalcones are intermediates in the biosynthesis of all flavonoids. In addition, in some species they constitute the major yellow flower pigments. There are two types of chalcones, distinguished by the presence (6′-hydroxychalcones) or absence (6′-deoxychalcones) of a hydroxyl group at the 6′ position of the A-ring. The 6′-deoxychalcones are formed when the enzyme chalcone reductase (CHR) is active in conjunction with chalcone synthase (CHS). In Petunia, only 6′-hydroxychalcones are synthesized, and except in the pollen of some genotypes, they are ephemeral intermediates in flavonoid metabolism. By introducing a CHR cDNA from Medicago sativa under the control of the 35S CaMV promoter into acyanic- or cyanic-flowered lines of Petunia, flower colour was changed from either white to pale yellow or deep purple to pale purple, respectively. Lines were generated that accumulated up to 60% of their petal flavonoids as 6′-deoxychalcones. Several different 6′-deoxychalcones accumulated in the petals of the CHR transgenics. The structures of three of these were determined: one, butein 4-O-glucoside, is a novel plant chalcone. Another chalcone compound was identified in the pollen of the transgenics. The results show that the Petunia chalcone isomerase is unable to use 6′-deoxychalcones as substrates so that 6′-deoxychalcones are stable in Petunia petals, leaves and pollen, but some Petunia flavonoid enzymes can use 6′-deoxychalcones as substrates to modify their structures. The introduction of CHR provides a method to redirect the flavonoid pathway into chalcone production, in order to modify flower colour or to reduce the biosynthesis of other flavonoid types.     

Growth-promoting nitrogen nutrition affects flavonoid biosynthesis in young apple (Malus domestica Borkh.) leaves

Enhanced shoot growth and a decrease in flavonoid concentration in apple trees grown under high nitrogen (N) supply was observed in previous studies, along with increasing scab susceptibility of cultivar "Golden Delicious" after high N nutrition. Several hypotheses have suggested that there is a trade-off between primary and secondary metabolism because of competition for common substrates, but nothing is known about regulation at the enzyme level. In this study, a set of experiments was performed to elucidate the effect of N nutrition on the activities of key enzymes involved in flavonoid biosynthesis (phenylalanine ammonia-lyase [PAL], chalcone synthase/chalcone isomerase [CHS/CHI}, flavanone 3-hydroxylase [FHT], flavonol synthase [FLS], dihydroflavonol 4-reductase [DFR]) and the accumulation of different groups of phenylpropanoids. The inhibition of flavonoid accumulation by high N nutrition could be confirmed, but the influence of N supply on the flavonoid enzymes CHS/CHI, FHT, DFR, and FLS was not evident. However, PAL activity seems to be downregulated, thus forming a bottleneck resulting in a generally decreased flavonoid accumulation. Furthermore, the response of the scab-resistant cultivar "Rewena" to high N nutrition was not as strong as that of the susceptible cultivar "Golden Delicious".     

A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings

Throughout the plant kingdom expression of the flavonoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status, and environmental stimuli such as light, heat and pathogen attack. Previously we showed that, in developing Arabidopsis seedlings, flavonoid genes are transiently expressed during germination in a light-dependent manner, with maximal mRNA levels occurring in 3-day-old seedlings. Here we describe the relationship between developmental and environmental regulation of flavonoid biosynthesis by examining phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) mRNA levels in germinating Arabidopsis seedlings as a function of light, developmental stage and temperature. We show that seedlings exhibit a transient potential for induction of these four genes, which is distinct from that observed for chlorophyll a/b-binding protein (CAB). The potential for flavonoid gene induction was similar in seedlings grown in darkness and red light, indicating that induction potential is not linked to cotyledon expansion or the development of photosynthetic capacity. The evidence for metabolic regulation of flavonoid genes during seedling development is discussed.     

Cross-talk interactions of sucrose and Fusarium oxysporum in the phenylpropanoid pathway and the accumulation and localization of flavonoids in embryo axes of yellow lupine

This study investigated the effects of cross-talk interactions of sucrose and infection caused by a pathogenic fungus Fusarium oxysporum f.sp. lupini on the regulation of the phenylpropanoid pathway, i.e. the level of expression of genes encoding enzymes participating in flavonoid biosynthesis, as well as cell location and accumulation of these compounds in embryo axes of Lupinus luteus L. cv. Polo. Embryo axes, both non-inoculated and inoculated, were cultured for 96 h on Heller medium with 60 mM sucrose (+Sn and +Si) or without it (−Sn and −Si). Real-time RT-PCR to assess expression levels of the flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS) were used. Sucrose alone strongly stimulated the expression of these genes. There was a very high expression level of these genes in +Si embryo axes in the early phase of infection. Signal amplification by sucrose and the infection was most intense in the 48-h +Si axes, resulting in the highest level of expression of flavonoid biosynthetic genes. In −Si tissues, the expression level of these genes increased at 48 and 72 h after inoculation relative to 24 h; however, the relative level of expression was much lower than in +Si axes, except at 72 h for PAL and CHS.Moreover, at 48 h of culture, considerably higher activity of CHI (EC 5.5.1.6) was observed in axes with a high level of sucrose than in those with a sucrose deficit. CHI activity in +Si axes at 48 and 96 h post-inoculation was over 1.5 and 2 times higher than that in +Sn axes, as well as higher than in −Si axes.Observations of yellow lupine embryo axes under a confocal microscope showed an increased post-infection accumulation of flavonoids, particularly in cells of embryo axes infected with F. oxysporum and cultured on a medium containing sucrose (+Si). Up to 48 h post-infection in +Si axes, a very intensive emission of green fluorescence was observed, indicating high accumulation of these compounds in whole cells. Moreover, a nuclear location of flavonoids was recorded in cells. Strong staining of flavonoid end products in +Si embryo axes was consistent with the expression of PAL, CHS, CHI and IFS.These results indicate that, in the early phase of infection, the flavonoid biosynthesis pathway is considerably enhanced in yellow lupine embryo axes as a strong signal amplification effect of sucrose and the pathogenic fungus F. oxysporum.