Flavone Accumulation Research Articles

A multi-omics approach reveals low temperature inhibition of flavones and flavonols accumulation in postharvest bananas via downregulation of MabHLH363

Flavonoids are widely acknowledged for their antioxidative properties. To explore their biosynthetic mechanisms in response to low temperature (7 °C) in bananas, we conducted phenotypic, transcriptomic, metabolomic, and molecular biology analyses. Phenotypic evaluations revealed significant chilling injury in bananas exposed to cold stress. Transcriptomic and metabolomic analyses showed a decrease in flavones and flavonols accumulation at low temperature. Specifically, nicotiflorin, rutin, narcissin, and astragalin were identified as the main metabolites affected by the cold. Importantly, the downregulation of MabHLH363, a positive regulator of MaUGT016, MaUGT028, and MaUGT041 involved in flavones and flavonols glycosylation, was detected at low temperature. In summary, our findings elucidate that low temperature attenuates the expression of MabHLH363, thereby suppressing the accumulation of flavones and flavonols in postharvest bananas.

Multiomics analyses reveal the mechanisms of the responses of subalpine treeline trees to phenology and winter low-temperature stress.

Withstanding extreme cold stress is a prerequisite for alpine treeline trees to persist and survive. However, the underlying mechanism by which treeline trees sense phenological changes and survive hard winters has not been fully elucidated. Here, we investigated the physiology, transcriptome, and metabolome of the subalpine treeline species Larix chinensis to identify the molecular mechanism of phenological and cold resistance. Calcium and antioxidant enzyme activities (e.g., superoxide dismutase and glutathione peroxidase) are essential for coping with winter cold stress in L. chinensis. Transcriptome analysis revealed that circadian rhythm and phytohormone signalling transduction played important roles in regulating L. chinensis phenological changes and cold stress responses. The variations in the transcriptome identified were accompanied by the specific accumulation of flavones, flavonols, and monosaccharides. The flavonoid biosynthesis and phenylpropanoid biosynthesis pathways played important roles in the adaptation of L. chinensis to the extreme winter environment, and flavone and flavonol biosynthesis was an important pathway involved in bud burst. In addition, temperature and photoperiod had synergistic influences on the formation and release of bud dormancy. Thus, our findings provided new insights into the mechanism of subalpine treeline formation.

Biotechnological strategies for controlled accumulation of flavones in hairy root culture of Scutellaria lateriflora L.

Accumulation of medicinally important flavones and acteoside was evaluated in Scutellaria lateriflora hairy root cultures subjected to different experimental strategies – feeding with precursors of phenolics biosynthesis (phenylalanine, cinnamic acid, and sodium cinnamate), addition of elicitors (chitosan, jasmonic acid) and Amberlite XAD-4 and XAD-7 resins and permeabilization with dimethyl sulfoxide (DMSO) and methanol. The production profile of S. lateriflora cultures changed under the influence of the applied strategies. Hairy roots of S. lateriflora were found to be a rich source of wogonoside or wogonin, depending on the treatment used. The addition of sodium cinnamate (1.0 mg/L) was the most effective approach to provide high production of flavonoids, especially wogonoside (4.41% dry weight /DW/; 566.78 mg/L). Permeabilization with DMSO (2 µg/ml for 12 h) or methanol (30% for 12 h) resulted in high biosynthesis of wogonin (299.77 mg/L and 274.03 mg/L, respectively). The obtained results provide new insight into the selection of the optimal growth conditions for the production of in vitro biomass with a significant level of flavone accumulation. The data may be valuable for designing large-scale cultivation systems of hairy roots of S. lateriflora with high productivity of bioactive compounds – wogonin or wogonoside.

Combined metabolomics and transcriptomics analysis reveals the mechanism underlying blue light-mediated promotion of flavones and flavonols accumulation in Ligusticum chuanxiong Hort. microgreens.

Ligusticum chuanxiong Hort. (Chuanxiong) is an important Chinese medicinal herb, whose rhizomes are widely used as raw materials for treating various diseases caused by blood stasis. The fresh tender stems and leaves of Chuanxiong are also consumed and have the potential as microgreens. Here, we investigated the effect of light spectra on yield and total flavonoid content of Chuanxiong microgreens by treatment with LED-based white light (WL), red light (RL), blue light (BL), and continuous darkness (DD). The results showed that WL and BL reduced biomass accumulation but significantly increased total flavonoid content compared to RL or DD treatments. Widely targeted metabolomics analysis confirmed that BL promoted the accumulation of flavones and flavonols in Chuanxiong microgreens. Further integration of transcriptomics and metabolomics analysis revealed the mechanism by which BL induces the up-regulation of transcription factors such as HY5 and MYBs, promotes the expression of key genes targeted for flavonoid biosynthesis, and ultimately leads to the accumulation of flavones and flavonols. This study suggests that blue light is a proper light spectra to improve the quality of Chuanxiong microgreens, and the research results lay a foundation for guiding the de-etiolation of Chuanxiong microgreens to obtain both yield and quality in production practice.

Integrated transcriptome and metabolome provide insight into flavonoid variation in goji berries (Lycium barbarum L.) from different areas in China

Goji berries (Lycium barbarum L.) were rich in flavonoids, showing high nutritional and medicinal value. However, a thorough evaluation and comparison of the flavonoids in goji berries from various regions and the possible biological regulation pathways with differences are scanty. Here, we investigated the flavonoid metabolites and gene expression levels of goji berries from three major production areas in China using transcriptomics sequencing and metabolomics. The total flavonoid content and total polyphenol content of goji berry in Ningxia (57.87 μg/g and 183.41 μg/g, respectively) were higher than in Qinghai (50.77 μg/g and 156.81 μg/g) and Gansu (47.86 μg/g and 111.17 μg/g). We identified the 105 differentially accumulated flavonoids (DAFs) and 1858 differentially expressed genes (DEGs) from the goji berries in three habitats. Interestingly, gossypetin-3-O-rutinoside and isorhamnetin were significantly expressed between Ningxia and Qinghai berries. The chalcone isomerase (CHI), chalcone synthase (CHS), and flavonol synthase (FLS) genes also played key roles in the regulation of flavonoid synthesis. In addition, MYB1 positively regulated the expression of quercetin-3-O-glucoside, quercetin-7-O-glucoside and isohyperoside. As a result, we speculated that CHI, CHS, FLS genes, and related transcription factors jointly controlled the variation of flavone accumulation in goji berries. These findings may provide a new perspective for understanding the accumulation and molecular mechanisms of goji flavonoids.

Metabolomics and chemometrics depict the changes in the chemical profile of white lupine (Lupinus albus L.) bioactive metabolites during seed germination

The current study attempts to illustrate how the chemical and biological profile of white lupine seeds varies throughout the course of various germination days using UHPLC-QqQ-MS combined to chemometrics. Abscisic acid showed maximum level in the un-germinated seeds and started to decline with seed germination accompanied by an increase in the levels of gibberellins which were undetectable in un-germinated seeds. Coumaronochromones were the most prevalent constituents detected in un-germinated seeds while day 2 sprouts showed significant accumulation of flavones. The levels of alkaloids showed significant increase upon germination of the seeds reaching its maximum in day 14 sprouts. The OPLS model coefficients plot indicated that lupinalbin D and F, apigenin hexoside, kaempferol hexoside, albine, and hydoxylupanine showed strong positive correlation to the alpha amylase inhibitory activity of the tested samples while lupinalbin A, lupinisoflavone, lupinic acid and multiflorine were positively correlated to the inhibition of alpha glycosidase activity. The results obtained indicated that seed germination has a profound effect on the chemical profile as well as the in-vitro antidiabetic activity of lupine seeds.

ZmSIZ1a and ZmSIZ1b play an indispensable role in resistance against Fusarium ear rot in maize.

Fusarium ear rot (FER) is a destructive fungal disease of maize caused by Fusarium verticillioides. FER resistance is a typical complex quantitative trait controlled by micro-effect genes, leading to difficulty in identifying the host resistance genes. SIZ1 encodes a SUMO E3 ligase regulating a wide range of plant developmental processes and stress responses. However, the function of ZmSIZ1 remains poorly understood. In this study, we demonstrate that ZmSIZ1a and ZmSIZ1b possess SUMO E3 ligase activity, and that the Zmsiz1a/1b double mutant, but not the Zmsiz1a or Zmsiz1b single mutants, exhibits severely impaired resistance to FER. Transcriptome analysis showed that differentially expressed genes were significantly enriched in plant disease resistance-related pathways, especially in plant-pathogen interaction, MAPK signalling, and plant hormone signal transduction. Thirty-five candidate genes were identified in these pathways. Furthermore, the integration of the transcriptome and metabolome data revealed that the flavonoid biosynthesis pathway was induced by F.verticillioides infection, and that accumulation of flavone and flavonol was significantly reduced in the Zmsiz1a/1b double mutant. Collectively, our findings demonstrate that ZmSIZ1a and ZmSIZ1b play a redundant, but indispensable role against FER, and provide potential new gene resources for molecular breeding of FER-resistant maize cultivars.

Impact of Blue Light on Plant Growth, Flowering and Accumulation of Medicinal Flavones in Scutellaria baicalensis and S. lateriflora

Scutellaria baicalensis Georgi (Baikal skullcap) and S. lateriflora (American skullcap) are two ethnobotanical medicinal plants used to treat gastrointestinal, respiratory, and inflammatory disorders, in addition to demonstrated anti-cancer properties. The predominant bioactive compounds produced in these species are unique 4′-deoxyflavones, in roots of S. baicalensis and leaves of S. lateriflora, making these two species suitable to study the effects of light quality on flavone accumulation in aerial and underground tissues. Light emitting diodes were used to study the impact of blue-dominated spectrum on the accumulation of bioactive flavones. Eight major flavones, including 4′-deoxyflavones baicalein, wogonin, baicalin, wogonoside and chrysin, along with 4′-hydroxyflavones scutellarein, scutellarin and apigenin, were quantified using HPLC in high flavone accumulating tissues. Aerial tissues directly exposed to blue light in S. lateriflora showed an increase in the concentrations of scutellarein by 18.7%, scutellarin by 296%, and baicalin by 31.6%. While the roots in S. baicalensis also had significant increases in baicalein by 154% and wogonin by 76% in response to blue light, there was a slight reduction in their respective glycosides baicalin and wogonoside as well as a decrease in total flavone content. Blue light resulted in compact skullcap plants with early flowering and modified flavone profiles.

Selenium Application Enhances the Accumulation of Flavones and Anthocyanins in Bread Wheat (Triticum aestivum L.) Grains.

Selenium (Se) biofortification in wheat reduces the risk of Se deficiency in humans. Se biofortification increases the concentration of Se and anthocyanins in wheat grains. However, it is unknown whether Se biofortification can enhance flavonoids other than anthocyanins and the mechanism underlying flavonoid accumulation in wheat grains. Here, foliar application of selenite solution in wheat was conducted 10 days after flowering. Metabolite profiling and transcriptome sequencing were performed in Se-treated grains. A significant increase in the total contents of Se, anthocyanins, and flavonoids was observed in Se-treated mature grains. Twenty-seven significantly increased flavonoids were identified in Se-treated immature grains. The significant accumulation of flavones (tricin, tricin derivatives, and chrysoeriol derivatives) was detected, and six anthocyanins, dihydroquercetin (the precursor for anthocyanin biosynthesis) and catechins were also increased. Integrated analysis of metabolites and transcriptome revealed that Se application enhanced the biosynthesis of flavones, dihydroquercetin, anthocyanins, and catechins by increasing the expression levels of seven key structural genes in flavonoid biosynthesis (two TaF3Hs, two TaDFRs, one TaF3'5'H, one TaOMT, and one TaANR). Our findings shed new light on the molecular mechanism underlying the enhancement in flavonoid accumulation by Se supplementation and pave the way for further enhancing the nutritional value of wheat grains.

Exploring native Scutellaria species provides insight into differential accumulation of flavones with medicinal properties

Scutellaria baicalensis is a well-studied medicinal plant belonging to the Lamiaceae family, prized for the unique 4′-deoxyflavones produced in its roots. In this study, three native species to the Americas, S. lateriflora, S. arenicola, and S. integrifolia were identified by DNA barcoding, and phylogenetic relationships were established with other economically important Lamiaceae members. Furthermore, flavone profiles of native species were explored. 4′-deoxyflavones including baicalein, baicalin, wogonin, wogonoside, chrysin and 4′-hydroxyflavones, scutellarein, scutellarin, and apigenin, were quantified from leaves, stems, and roots. Qualitative, and quantitative differences were identified in their flavone profiles along with characteristic tissue-specific accumulation. 4′-deoxyflavones accumulated in relatively high concentrations in root tissues compared to aerial tissues in all species except S. lateriflora. Baicalin, the most abundant 4′-deoxyflavone detected, was localized in the roots of S. baicalensis and leaves of S. lateriflora, indicating differential accumulation patterns between the species. S. arenicola and S. integrifolia are phylogenetically closely related with similar flavone profiles and distribution patterns. Additionally, the S. arenicola leaf flavone profile was dominated by two major unknown peaks, identified using LC–MS/MS to most likely be luteolin-7-O-glucuronide and 5,7,2′-trihydroxy-6-methoxyflavone 7-O-glucuronide. Collectively, results presented in this study suggest an evolutionary divergence of flavonoid metabolic pathway in the Scutellaria genus of Lamiaceae.

Comprehensive Analysis of Transcriptome and Metabolome Reveals the Flavonoid Metabolic Pathway Is Associated with Fruit Peel Coloration of Melon.

The peel color is an important external quality of melon fruit. To explore the mechanisms of melon peel color formation, we performed an integrated analysis of transcriptome and metabolome with three different fruit peel samples (grey-green ‘W’, dark-green ‘B’, and yellow ‘H’). A total of 40 differentially expressed flavonoids were identified. Integrated transcriptomic and metabolomic analyses revealed that flavonoid biosynthesis was associated with the fruit peel coloration of melon. Twelve differentially expressed genes regulated flavonoids synthesis. Among them, nine (two 4CL, F3H, three F3′H, IFS, FNS, and FLS) up-regulated genes were involved in the accumulation of flavones, flavanones, flavonols, and isoflavones, and three (2 ANS and UFGT) down-regulated genes were involved in the accumulation of anthocyanins. This study laid a foundation to understand the molecular mechanisms of melon peel coloration by exploring valuable genes and metabolites.

Comparative Metabolomic and Transcriptome Analysis Reveal Distinct Flavonoid Biosynthesis Regulation Between Petals of White and Purple Phalaenopsis amabilis

Orchids (Phalaenopsis amabilis), renowned for their extraordinary floral diversity which implies the complex flower color development, are one of the most valuable research materials for molecular biology studies. However, the genetic mechanisms underlying flower color formation in this non-model plant remain unclear. Here, the biochemical and molecular basis of the flavonoid/anthocyanin biosynthesis between purple petal cultivar (PP) and white petal cultivar (WP) was investigated. The results revealed that 142 differential flavonoid-related metabolites were identified using LC–MS/MS method, and the primary anthocyanins were identified as cyanidin derivatives. Moreover, RNA-seq revealed a total of 9523 upregulated and 6941 downregulated genes were detected. GO and KEGG analysis of DEGs indicated metabolic pathways were preferentially upregulated in PP. The expression of most known genes encoding the enzymes in anthocyanin and related derivative biosynthesis was significantly higher in PP than in WP, especially the downstream ones. We hypothesized that altered flavanone and flavone accumulation may lead to pigment elimination in WP, and the limited flux in cyanidin biosynthesis pathway seems to be the most likely reason for the colorless petal. Besides, 115 differentially expressed regulatory genes relating to anthocyanin biosynthesis were also detected, including MYB, bHLH, WD40, WRKY, MADS and bZIP. Subsequently, high consistency was observed between the results of qRT-PCR and those of RNA-seq. collectively, this study provides a comprehensive metabolic and transcriptional dataset for Phalaenopsis, the informative list of candidate structural and regulatory genes associated with purple trait offer a valuable opportunity to unravel the genetic mechanism underlying flower color formation in Phalaenopsis.

Temporal biosynthesis of flavone constituents in flax growth stages

Previous studies showed that chalcone synthase (chs) silencing in flax (Linum usitatisimum) induces a signal transduction cascade that leads to extensive modification of plant metabolism. Result presented in the current study, performed on field grown flax plants – (across the whole vegetation period) demonstrates that, in addition to its role in tannin and lignin biosynthesis, the chs gene also participates in the regulation of flavone biosynthesis during plant growth. Apigenin and luteolin glycosides constitute the flavones, the major group of flavonoids in flax. Alterations in their levels correlate with plant growth, peaking at the flower initiation stage. Suppression of chs gene expression causes significant changes in the ratio of flavone constituents at the early stage of flax growth. A significant correlation between flavonoid 3′-hydroxylase (F3′H) gene expression and accumulation of luteolin glycosides has been found, indicating that flavone biosynthesis during flax growth and development is regulated by temporal expression of this gene. The lack of such a correlation between the flavone synthase (FNS) gene and flavone accumulation in the course of plant growth suggests that the main route of flavone biosynthesis is mediated by eriodictyol. This is the first report indicating the ratio of flavone constituents as a potent marker of flax growth stages and temporal expression of F3′H, the key gene of their biosynthesis.

Effect of Post-Harvest LED and UV Light Irradiation on the Accumulation of Flavonoids and Limonoids in the Segments of Newhall Navel Oranges (Citrus sinensis Osbeck).

To investigate the effect of post-harvest light irradiation on the accumulation of flavonoids and limonoids, harvested Newhall navel oranges were continuously exposed to light-emitting diode (LED) and ultraviolet (UV) light irradiation for 6 days, and the composition and content of flavonoids and limonoids in the segments were determined using UPLC-qTOF-MS at 0, 6, and 15 days after harvest. In total, six polymethoxylated flavonoids (PMFs), five flavone-O/C-glycosides, seven flavanone-O-glycosides, and three limonoids were identified in the segments. The accumulation of these components was altered by light irradiation. Red and blue light resulted in higher levels of PMFs during exposure periods. The accumulation of PMFs was also significantly induced after white light, UVB and UVC irradiation were removed. Red and UVC irradiation induced the accumulation of flavone and flavanone glycosides throughout the entire experimental period. Single light induced limonoid accumulation during exposure periods, but limonoid levels decreased significantly when irradiation was removed. Principal component analysis showed a clear correlation between PMFs and white light, between flavonoid glycosides and red light and UVC, and between limonoids and UVC. These results suggest that the accumulation of flavonoids and limonoids in citrus is regulated by light irradiation. White light, red light and UVC irradiation might be a good potential method for improving the nutrition and flavor quality of post-harvest citrus.

Differentially evolved glucosyltransferases determine natural variation of rice flavone accumulation and UV-tolerance

Decoration of phytochemicals contributes to the majority of metabolic diversity in nature, whereas how this process alters the biological functions of their precursor molecules remains to be investigated. Flavones, an important yet overlooked subclass of flavonoids, are most commonly conjugated with sugar moieties by UDP-dependent glycosyltransferases (UGTs). Here, we report that the natural variation of rice flavones is mainly determined by OsUGT706D1 (flavone 7-O-glucosyltransferase) and OsUGT707A2 (flavone 5-O-glucosyltransferase). UV-B exposure and transgenic evaluation demonstrate that their allelic variation contributes to UV-B tolerance in nature. Biochemical characterization of over 40 flavonoid UGTs reveals their differential evolution in angiosperms. These combined data provide biochemical insight and genetic regulation into flavone biosynthesis and additionally suggest that adoption of the positive alleles of these genes into breeding programs will likely represent a potential strategy aimed at producing stress-tolerant plants.

Enhanced Light Intensity Increases Flavonol and Anthocyanin Concentrations but Reduces Flavone Levels in the Cotyledons of Common Buckwheat Seedlings

The effects of two light intensities on the concentration of several flavonoids were investigated in the cotyledons of common buckwheat seedlings. The study was performed on four days old seedlings of cvs. Hruszowska, Panda, Kora and Red Corolla. One group of seedlings was grown under exposure to 180 ± 20 μmol · m−2 · s−1 photosynthetically active radiation, whereas the other group was exposed to 360 ± 20 μmol · m−2 · s−1. The experiment lasted 5 days. The results revealed that light intensity induces changes in the levels of flavonols and flavones. Increased light intensity contributed to a decrease in the concentrations of all flavone C-glucosides: orientin (luteolin-8-C-glucoside) and iso-orientin (luteolin-6-C-glucoside), and apigenin: vitexin (apigenin-8-C-glucoside) and iso-vitexin (apigenin-6-C-glucoside). Simultaneously, a substantial increase in the content of flavonols, i.e. quercetin O-glycosides, was found. To the best of our knowledge, this is the first evidence to demonstrate the contrary responses of plant flavonols and flavones to light intensity. The content of anthocyanin also increased under exposure to higher light intensity. Our results indicate that quercetin O-glycosides can play a similar role to anthocyanins in the cotyledons of common buckwheat seedlings. Results of correlation analysis indicate that the increase in flavonol and anthocyanin concentrations in response to higher light intensity is maintained through reduced accumulation of flavones and proanthocyanidins.

Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis

Carbohydrate sources play important roles in energy and growth of plants. Therefore, in this study, we investigated the optimal carbohydrate source in hairy root cultures (HRCs) of Scutellaria baicalensis infected with Agrobacterium rhizogenes strain R1000. The hairy roots were cultured in half-strength B5 liquid medium supplemented with seven different carbohydrates sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol and maltose), each at a concentration of 100 mM, in order to identify the best carbon sources for the production of major flavones, such as wogonin, baicalin and baicalein. Sucrose, galactose and fructose markedly influenced the production of major flavones and were therefore chosen for subsequent experiments. HRC growth and flavone accumulation were examined following culture with 30, 100 and 150 mM sucrose, galactose and fructose, respectively. From these data, 150 mM sucrose was found to be the optimal carbon source for the enhancement of baicalein production and growth of S. baicalensis HRCs. Fructose caused the greatest increase in baicalin accumulation. Additionally, galactose was the optimal carbon source for wogonin production. These results provide important insights into the optimal growth conditions, particularly the appropriate carbohydrate source, for S. baicalensis.

Flavone synthases from Lonicera japonica and L. macranthoides reveal differential flavone accumulation.

Flavones are important secondary metabolites found in many plants. In Lonicera species, flavones contribute both physiological and pharmaceutical properties. However, flavone synthase (FNS), the key enzyme responsible for flavone biosynthesis, has not yet been characterized in Lonicera species. In this study, FNSII genes were identified from Lonicera japonica Thunb. and L. macranthoides Hand.-Mazz. In the presence of NADPH, the recombinant cytochrome P450 proteins encoded by LjFNSII-1.1, LjFNSII-2.1, and LmFNSII-1.1 converted eriodictyol, naringenin, and liquiritigenin to the corresponding flavones directly. The different catalytic properties between LjFNSII-2.1 and LjFNSII-1.1 were caused by a single amino acid substitution at position 242 (glutamic acid to lysine). A methionine at position 206 and a leucine at position 381 contributed considerably to the high catalytic activity of LjFNSII-1.1. In addition, LjFNSII-1.1&2.1 and LmFNSII-1.1 also biosynthesize flavones that were further modified by O-glycosylation in transgenic tobacco. The expression levels of the FNSII genes were consistent with flavone accumulation patterns in flower buds. Our findings suggested that the weak catalytic activity of LmFNSII-1.1 and the relatively low expression of LmFNSII-1.1 in flowers might be responsible for the low levels of flavone accumulation in flower buds of L. macranthoides.

Comparison of anthocyanin components, expression of anthocyanin biosynthetic structural genes, and TfF3′H1 sequences between Tulipa fosteriana ‘Albert heijn’ and its reddish sport

A reddish flower tulip sport Tulipa fosteriana ‘SN09’ was bred from a pink flower cultivar T. fosteriana ‘Albert heijn’ (AH). To investigate the molecular basis underlying this color difference, SN09 and AH were compared using biochemical and molecular approaches. Ultra performance liquid chromatography (UPLC) profiles revealed that major cyanidin 3-rutinoside and minor pelargonidin 3-rutinoside were accumulated in the petals of AH, whereas major pelargonidin anthocyanins (pelargonidin 3-acetylrutinoside and pelargonidin 3-rutinoside) and minor cyanidin 3-glucoside were detected in SN09. The total anthocyanin content in petals of AH and SN09 were not significantly different at Stage 4, and correlated with the expression patterns of dihydroflavonol 4-reductase (TfDFR1) and anthocyanidin synthase (TfANS1). In addition, more transcripts of chalcone synthase (TfCHS1), chalcone isomerase (TfCHI2), and flavanone 3-hydroxylase (TfF3H1) contributed to higher accumulation of flavone and flavonol in SN09 than in AH. The transcription of flavonoid 3′-hydroxylase (TfF3′H1) was significantly lower in SN09 than in AH throughout flower development. Accordingly, the nucleotide sequences of TfF3′H1 in AH and SN09 were compared. Results showed that the deduced amino acids of TfF3′H1 in AH and SN09 were not different, but an extra fragment (255bp) was found in the promoter of TfF3′H1 in SN09. Consequently, the promoters of TfF3′H1 in AH and SN09 were isolated, fused with the β-glucuronidase (GUS), and then transformed to tobacco and Arabidopsis thaliana. Results demonstrated that the GUS activity in Arabidopsis with TfF3′H1 promoter from SN09 was only 5.4% of that from AH, suggesting that the insert mutation in the TfF3′H1 promoter hampered the accumulation of cyanidin anthocyanins in SN09 through the reduction of TfF3′H1 transcription.

'Le Rouge et le Noir': a decline in flavone formation correlates with the rare color of black dahlia (Dahlia variabilis hort.) flowers.

BackgroundMore than 20,000 cultivars of garden dahlia (Dahlia variabilis hort.) are available showing flower colour from white, yellow and orange to every imaginable hue of red and purple tones. Thereof, only a handful of cultivars are so-called black dahlias showing distinct black-red tints. Flower colour in dahlia is a result of the accumulation of red anthocyanins, yellow anthochlors (6’-deoxychalcones and 4-deoxyaurones) and colourless flavones and flavonols, which act as copigments. White and yellow coloration occurs only if the pathway leading to anthocyanins is incomplete. Not in all cultivars the same step of the anthocyanin pathway is affected, but the lack of dihydroflavonol 4-reductase activity is frequently observed and this seems to be based on the suppression of the transcription factor DvIVS. The hitherto unknown molecular background for black colour in dahlia is here presented.ResultsBlack cultivars accumulate high amounts of anthocyanins, but show drastically reduced flavone contents. High activities were observed for all enzymes from the anthocyanin pathway whereas FNS II activity could not be detected or only to a low extent in 13 of 14 cultivars. cDNA clones and genomic clones of FNS II were isolated. Independently from the colour type, heterologous expression of the cDNA clones resulted in functionally active enzymes. FNS II possesses one intron of varying length. Quantitative Real-time PCR showed that FNS II expression in black cultivars is low compared to other cultivars. No differences between black and red cultivars were observed in the expression of transcription factors IVS and possible regulatory genes WDR1, WDR2, MYB1, MYB2, 3RMYB and DEL or the structural genes of the flavonoid pathway. Despite the suppression of FHT expression, flavanone 3-hydroxylase (FHT, synonym F3H) enzyme activity was clearly present in the yellow and white cultivars.ConclusionsAn increased accumulation of anthocyanins establishes the black flowering phenotypes. In the majority of black cultivars this is due to decreased flavone accumulation and thus a lack of competition for flavanones as the common precursors of flavone formation and the anthocyanin pathway. The low FNS II activity is reflected by decreased FNS II expression.