ABSTRACT: Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.
Project description:Anthocyanins are a group of secondary metabolites that colour fruit and flowers orange, red, purple or blue depending on a number of factors, such as the basic structure, co-pigmentation, metal ion complexation and vacuolar pH. The biosynthesis of anthocyanin is regulated at the transcriptional level by a group of transcription factors, the MYB-bHLH-WD40 (MBW) complex. In this study, the purple colouration in several kiwifruit (Actinidia) species was identified and characterised as red cyanidin-based and blue delphinidin-based anthocyanins. The differential pigmentation in the skin and flesh can be attributed to the differential ratio of cyanidin and delphinidin derivatives accumulated in the total anthocyanin profile. The expression of anthocyanin biosynthetic genes chalcone synthase (CHS), flavonoid 3-O-glucosyltransferase (F3GT), flavonoid 3'-hydroxylase (F3'H) and flavonoid 3'5'-hydroxylase (F3'5'H) is crucial for anthocyanin accumulation. However, the balance of expression of the F3'H and F3'5'H genes appears responsible for the ratio of cyanidin and delphinidin derivatives, while a lack of CHS, F3GT and MYB110 expression is responsible for a lack of total anthocyanins. The transcriptional regulation of the F3'H and F3'5'H promoters by the R2R3 MYB transcription factor MYB110 is markedly different in tobacco transient assays. When kiwifruit MYB10 or MYB110 are over-expressed in Actinidia chinensis both cyanidin-based and delphinidin-based anthocyanins are elevated, but F3'H and F3'5'H genes are not strongly correlated with MYB expression. These results suggest that the core kiwifruit anthocyanin pathway genes are dependent on characterised MYB transcription factors, while other regulatory proteins are more directly responsible for the expression of the F3'H and F3'5'H genes.
Project description:Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)-glucose:anthocyanin 3',5'-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3'5'H. Newly synthesized 3',5'-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed "copigmentation," with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.
Project description:BACKGROUND: Glycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat. RESULTS: B09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants. CONCLUSIONS: B09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be a useful tool for studies of the structural and functional properties of F3'5'H genes as well as investigations on the role of flower color in relation to adaptation of G. soja to natural habitats.
Project description:BACKGROUND:Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants, F3'5'H genes are present in low-copy number, but in grapevine they are highly redundant. RESULTS:The first increase in F3'5'H copy number occurred in the progenitor of the eudicot clade at the time of the ? triplication. Further proliferation of F3'5'Hs has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologous F3'5'H has been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. Duplicate F3'5'Hs have spatially and temporally partitioned expression profiles in grapevine. The orphan F3'5'H copy is highly expressed in vegetative organs. More recent duplicate F3'5'Hs are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences in cis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in the F3'5'H mRNA pool across different cultivars. More F3'5'H copies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewer F3'5'H copies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent. CONCLUSIONS:These results suggest that expansion and subfunctionalisation of F3'5'Hs have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.
Project description:Flavonoids are one of the major plant pigments for flower colour. Not only coloured anthocyanins, but also co-pigment flavones or flavonols, accumulate in flowers. To study the regulation of early flavonoid biosynthesis, two R2R3-MYB transcription factors, GtMYBP3 and GtMYBP4, were identified from the petals of Japanese gentian (Gentiana triflora). Phylogenetic analysis showed that these two proteins belong to the subgroup 7 clade (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. Gt MYBP3 and Gt MYBP4 transcripts were detected specifically in young petals and correlated with the profiles of flavone accumulation. Transient expression assays showed that GtMYBP3 and GtMYBP4 enhanced the promoter activities of early biosynthetic genes, including flavone synthase II (FNSII) and flavonoid 3'-hydroxylase (F3'H), but not the late biosynthetic gene, flavonoid 3',5'-hydroxylase (F3'5'H). GtMYBP3 also enhanced the promoter activity of the chalcone synthase (CHS) gene. In transgenic Arabidopsis, overexpression of Gt MYBP3 and Gt MYBP4 activated the expression of endogenous flavonol biosynthesis genes and led to increased flavonol accumulation in seedlings. In transgenic tobacco petals, overexpression of Gt MYBP3 and Gt MYBP4 caused decreased anthocyanin levels, resulting in pale flower colours. Gt MYBP4-expressing transgenic tobacco flowers also showed increased flavonols. As far as is known, this is the first functional characterization of R2R3-MYB transcription factors regulating early flavonoid biosynthesis in petals.
Project description:<h4>Background</h4>Fruit coloration of red-skinned grapevines is mainly due to anthocyanin pigments. We analysed a panel of nine cultivars that included extreme phenotypes for berry colour, ranging from green (absence of anthocyanins) to red, purple, violet and blue. Expression of six genes of the anthocyanin pathway coding for flavanone-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT), glutathione-S-transferase (GST), O-methyltransferase (OMT) and four transcription factors (MybA, MybB, MybC, MybD) was analysed by quantitative RT-PCR at four developmental stages from before the onset of ripening until full maturity and compared to anthocyanin metabolites.<h4>Results</h4>Total anthocyanin content at full maturity correlated well with the cumulative expression of F3H, UFGT and GST throughout ripening. Transcripts of the last two genes were absent in the green-skinned cultivar 'Sauvignonasse', also known as 'Tocai friulano', and were at least 10-fold less abundant in pale red cultivars, such as 'Pinot gris' and 'Gewürztraminer', compared to fully coloured cultivars. Predominance of tri-hydroxylated anthocyanins (delphinidin, petunidin and malvidin) in cultivars bearing dark berries with violet and blue hue was associated with higher ratios of F3'5'H/F3'H transcription, compared to red-skinned cultivars. Higher levels of OMT transcripts were observed in berries of cultivars that accumulated methoxylated forms of anthocyanins more abundantly than non-methoxylated forms.<h4>Conclusion</h4>Colour variation of the grape berry conforms to a peculiar pattern of genotype-specific expression of the whole set of anthocyanin genes in a direct transcript-metabolite-phenotype relationship. Cumulative mRNA levels of the structural genes and their relative abundance throughout ripening explained per se the final phenotype for anthocyanin content, anthocyanin composition, colour intensity and colour hue of grapes at berry maturity.
Project description:BACKGROUND: Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. RESULTS: A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. CONCLUSION: To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen.
Project description:The flowers of flax (linseed) are blue-hued, ephemeral and self-pollinating, and the seeds are typically brown. A century-old interest in natural yellow seed variants and a historical model point to recessive alleles in B1, D and G loci being responsible, but the functional aspects had remained unknown. Here, we characterized the "D" locus by quantitative trait loci (QTL) mapping and identified a FLAVONOID 3'5' HYDROXYLASE (F3'5'H) gene therein. It does not belong to the F3'5'H clade, but resembles biochemically characterized F3'Hs (flavonoid 3' hydroxylase) but without F3'H activity. The genome lacks other F3'H or F3'H-like genes. The apparent neo-functionalization from F3'H is associated with a Thr498???Ser498 substitution in a substrate recognition site (SRS). The yellow seed and white flower phenotypes of the classical d mutation was found to be due to one nucleotide deletion that would truncate the deduced product and remove three of the six potential SRS, negatively impacting delphinidin synthesis. Delphinidin is sporadic in angiosperms, and flax has no known pollination syndrome(s) with functional pollinator group(s) that are attracted to blue flowers, raising questions on the acquisition of F3'5'H. The appearance of d allele is suggestive of the beginning of the loss of F3'5'H in this species.
Project description:Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.
Project description:Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium 'LPi' and C × morifolium 'LPu', that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3'H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3'H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3'H using RNAi and overexpressed the Senecio cruentus F3'5'H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3'H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3'5'H only exhibited F3'H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum.