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.     

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.     

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...     

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.     

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".     

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.     

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.     

Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings

Polyclonal antibodies were developed against the flavonoid biosynthetic enzymes, CHS, CHI, F3H, FLS, and LDOX from Arabidopsis thaliana. These antibodies were used to perform the first detailed analysis of coordinate expression of flavonoid metabolism at the protein level. The pattern of flavonoid enzyme expression over the course of seedling development was consistent with previous studies indicating that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) are encoded by early genes while leucoanthocyanidin dioxygenase (LDOX) is encoded by a late gene. This sequential expression may underlie the variations in flavonoid end-products produced during this developmental stage, as determined by HPLC analysis, which includes a shift in the ratio of the flavonols, quercetin and kaempferol. Moreover, immunoblot and HPLC analyses revealed that several transparent testa lines blocked at intermediate steps of the flavonoid pathway actually accumulated higher levels of specific flavonoid enzymes and end-products. These results suggest that specific intermediates may act as inducers of flavonoid metabolism.     

Organ- and Tissue-specific Biosynthesis of Flavonoids in Seedlings of Oenothera odorata (Onagraceae)

In developing Oenothera odorata seedlings, phytochrome-mediated accumulation of various flavonoids (mainly glycosides of cyanidin and quercetin) is detectable, subsequent to a transient induction of the key enzymes of the general phenylpropanoid metabolism, L-phenylalanine ammonia lyase (PAL) and of flavonoid biosynthesis, chalcone synthase (CHS). Organ- and tissue-specific distribution of these enzymes and of the flavonoid end products was investigated in seedlings, irradiated with continuous far-red light. Anthocyanins and quercetin glycosides are mainly localized in both the upper and lower epidermis of the cotyledons and to a lesser extent also in the epidermal cell layer of the hypocotyl. An obvious organ-specific distribution was observed for the anthocyanins: cyanidin-3,5-O-diglucoside accumulates in the epidermal cells of the cotyledons, whereas cyanidin-3-O-glucoside is restricted to the epidermis of the hypocotyl. By contrast the pattern of quercetin glycosides is the same in the cotyledons and in the hypocotyl. The methylated flavonol aglycone 3-0-methylquercetin was found to be localized in the seed coat. According to this organ- and tissue-specific pattern of flavonoids, immunochemical and immunohistochemical detection of PAL and CHS revealed a predominant localization of theenzymes in the epidermal layers of the cotyledons and the hypocotyl but also in the cells surrounding the vascular bundles. The role of compartmentation in regulation of flavonoid biosynthesis and putative functions of flavonoid compounds are discussed.     

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 gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

柚皮素合成关键基因表达水平对目标产物积累水平的量化影响

柚皮素是一种天然黄酮类化合物,具有抗炎、抗氧化、抗病毒、预防动脉粥样硬化等多种药理活性,也是其他黄酮类化合物合成的重要前体,具有重要的应用价值。目前,微生物法生产柚皮素等黄酮类化合物由于代谢通路不平衡等原因导致产量较低,在很大程度上限制了其工业应用。文中以一株产柚皮素的酿酒酵母菌株Y-01为研究对象,利用启动子和拷贝数控制柚皮素合成代谢途径关键酶4-香豆酸:CoA连接酶(4CL)、查尔酮合成酶(CHS)和查尔酮异构酶(CHI)编码基因的表达水平,考察这些基因的表达水平对目标产物积累水平的量化影响。结果表明,柚皮素产量与4CL或CHI编码基因的表达量之间关联性较低,而与chs基因的表达量存在显著的正相关性。通过调控chs基因的表达水平,获得一株高产柚皮素的酿酒酵母工程菌株Y-04,产量较出发菌株Y-01提高了4.1倍。研究结果表明,CHS是柚皮素合成过程的关键调控靶点,合理调控CHS表达可以显著促进酿酒酵母积累柚皮素。相关结果为采用代谢工程强化微生物合成柚皮素等重要黄酮类化合物提供了重要的理论参考。     

Chalcone synthase and flavonoid products in primary-leaf tissues of rye and maize

During cell and tissue differentiation of developing rye (Secale cereale L.) and maize (Zea mays L.) primary leaves, various flavonoids are synthesized and accumulate in both epidermal and mesophyll tissues. In order to prove either the biosynthetic autonomy of each tissue type and- or intercellular transport of flavonoids, the tissue distributions of chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the pathway, and of flavonoids have been comparatively investigated. Monoclonal antibodies raised against CHS from rye were used to relate enzyme activity in a particular tissue extract to the corresponding amount of CHS protein. A close correlation was found between CHS activities and amounts of CHS protein during leaf development and in the various tissues. The simultaneous occurrence of CHS in both epidermal layers as well as in the mesophyll correlated with the accumulation of flavonoid products in these tissues, indicating tissue autonomy of flavonoid biosynthesis. These data are in contrast to previous reports (Knogge and Weissenböck, 1986, Planta 167, 196–205) on primary leaves of oat (Avena sativa) where CHS and several subsequent enzymes were located mainly in the mesophyll whereas major flavonoid products accumulated predominantly in both epidermal cell layers, indicating that intertissue transport of flavonoids might occur.     

Tissue-distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L.

Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - 4CL 4-coumarate: CoA ligase - CMT S-adenosyl-L-methionine:caffeate 3-O-methyltransferase - FMT S-adenosyl-L-methionine:vitexin 2-O-rhamnoside 7-O-methyltransferase - HPLC high-performance liquid chromatography - IAT uridine 5-diphosphate L-arabinose:isovitexin 2-O-arabinosyltransferase - PAL L-phenylalanine ammonia-lyase     

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.