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1.  Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli 
Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.
doi:10.1128/AEM.71.7.3617-3623.2005
PMCID: PMC1169036  PMID: 16000769
2.  Flower colour and cytochromes P450† 
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.
doi:10.1098/rstb.2012.0432
PMCID: PMC3538422  PMID: 23297355
anthocyanin; anthocyanidin; flavonoid; flavonoid 3′-hydroxylase; flavonoid 3′,5′-hydroxylase; flavone synthase
3.  Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics 
Metabolomics  2007;3:399-412.
Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.
doi:10.1007/s11306-007-0074-2
PMCID: PMC4309898
GC/MS; LC/MS; Metabolic engineering; Metabolomics; Tomato
4.  Genome-wide transcriptome analysis of genes involved in flavonoid biosynthesis between red and white strains of Magnolia sprengeri pamp 
BMC Genomics  2014;15(1):706.
Background
Magnolia sprengeri Pamp is one of the most highly valuable medicinal and ornamental plants of the Magnolia Family. The natural color of M. sprengeri is variable. The complete genome sequence of M. sprengeri is not available; therefore we sequenced the transcriptome of white and red petals of M. sprengeri using Illumina technology. We focused on the identity of structural and regulatory genes encoding the enzymes involved in the determination of flower color.
Results
We sequenced and annotated a reference transcriptome for M. sprengeri, and aimed to capture the transcriptional determinanats of flower color. We sequenced a normalized cDNA library of white and red petals using Illumina technology. The resulting reads were assembled into 77,048 unique sequences, of which 28,243 could be annotated by Gene Ontology (GO) analysis, while 48,805 transcripts lacked GO annotation. The main enzymes involved in the flavonoid biosynthesis, such as phenylalanine ammonia-Lyase, cinnamat-4-Hydroxylase, dihydroflavonol-4-reductase, flavanone 3-hydroxylase, flavonoid-3′-hydroxylase, flavonol synthase, chalcone synthase and anthocyanidin synthase, were identified in the transcriptome. A total of 270 transcription factors were sorted into three families, including MYB, bHLH and WD40 types. Among these transcription factors, eight showed 4-fold or greater changes in transcript abundance in red petals compared with white petals. High-performance liquid chromatography analysis of anthocyanin compositions showed that the main anthocyanin in the petals of M. sprengeri is cyanidin-3-O-glucoside chloride and its content in red petals was 26-fold higher than that in white petals.
Conclusion
This study presents the first next-generation sequencing effort and transcriptome analysis of a non-model plant from the Family Magnoliaceae. Genes encoding key enzymes were identified and the metabolic pathways involved in biosynthesis and catabolism of M. sprengeri flavonoids were reconstructed. Identification of these genes and pathways adds to the current knowledge of the molecular biology and biochemistry of their production in plant. Such insights into the mechanisms supporting metabolic processes could be used to genetically to enhance flower color among members of the Magnoliaceae.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-706) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-706
PMCID: PMC4156625  PMID: 25150046
Transcriptome; Flavonoid biosynthesis; Magnolia sprengeri; Flower color
5.  Glycosyltransferase efficiently controls phenylpropanoid pathway 
BMC Biotechnology  2008;8:25.
Background
In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR).
Results
Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins.
Conclusion
In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants.
doi:10.1186/1472-6750-8-25
PMCID: PMC2294120  PMID: 18321380
6.  Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products 
BMC Plant Biology  2014;14:84.
Background
The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana.
Results
This study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3′-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase, secondary wall-associated NAC domain protein1, MYB58 and MYB63 resulting in higher accumulation of lignin content compared to the control.
Conclusions
These results indicate that the additions of microbially based soil additives have a perceptible influence on phenylpropanoid pathway gene regulation and its production of secondary metabolites. These findings open an avenue of research to investigate the mode of action of microbially-based soil additives which may assist in the sustainable production of food, feed, fuel and fiber.
doi:10.1186/1471-2229-14-84
PMCID: PMC4021374  PMID: 24690446
Arabidopsis; Metabolites; Microbes; Transcriptional profiling; Plant Growth Promoting Rhizobacteria; Soil Builder
7.  Light-induced vegetative anthocyanin pigmentation in Petunia 
Journal of Experimental Botany  2009;60(7):2191-2202.
The Lc petunia system, which displays enhanced, light-induced vegetative pigmentation, was used to investigate how high light affects anthocyanin biosynthesis, and to assess the effects of anthocyanin pigmentation upon photosynthesis. Lc petunia plants displayed intense purple anthocyanin pigmentation throughout the leaves and stems when grown under high-light conditions, yet remain acyanic when grown under shade conditions. The coloured phenotypes matched with an accumulation of anthocyanins and flavonols, as well as the activation of the early and late flavonoid biosynthetic genes required for flavonol and anthocyanin production. Pigmentation in Lc petunia only occurred under conditions which normally induce a modest amount of anthocyanin to accumulate in wild-type Mitchell petunia [Petunia axillaris×(Petunia axillaris×Petunia hybrida cv. ‘Rose of Heaven’)]. Anthocyanin pigmentation in Lc petunia leaves appears to screen underlying photosynthetic tissues, increasing light saturation and light compensation points, without reducing the maximal photosynthetic assimilation rate (Amax). In the Lc petunia system, where the bHLH factor Leaf colour is constitutively expressed, expression of the bHLH (Lc) and WD40 (An11) components of the anthocyanin regulatory system were not limited, suggesting that the high-light-induced anthocyanin pigmentation is regulated by endogenous MYB transcription factors.
doi:10.1093/jxb/erp097
PMCID: PMC2682507  PMID: 19380423
Anthocyanin; bHLH; flavonol; Lc; Leaf colour; light; MYB; photosynthesis; vegetative pigmentation
8.  Functional analysis of Flavonoid 3′,5′-hydroxylase from Tea plant (Camellia sinensis): critical role in the accumulation of catechins 
BMC Plant Biology  2014;14(1):347.
Background
Flavonoid 3′,5′-hydroxylase (F3′5′H), an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the CYP75A subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3′,4′,5′-hydroxylated derivatives. However, whether B-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown.
Results
The Camellia sinensis F3′5′H (CsF3′5′H) gene was isolated from tea cDNA library. Expression pattern analysis revealed that CsF3′5′H expression was tissue specific, very high in the buds and extremely low in the roots. CsF3′5′H expression was enhanced by light and sucrose. Over-expression of CsF3′5′H produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. Heterologous expressions of CsF3′5′H in yeast were carried out to demonstrate the function of CsF3′5′H enzyme in vitro. Heterologous expression of the modified CsF3′5′H (CsF3′5′H gene fused with Vitis vinifera signal peptide, FSI) revealed that 4′-hydroxylated flavanone (naringenin, N) is the optimum substrate for CsF3′5′H, and was efficiently converted into both 3′4′- and 3′4′5′-forms. The ratio of 3′4′5′- to 3′4′-hydroxylated products in FSI transgenic cells was significantly higher than VvF3′5′H cells.
Conclusions
CsF3′5′H is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4′-hydroxylated flavanone into 3′4′5′- and/or 3′4′-hydroxylated products. These findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0347-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0347-7
PMCID: PMC4275960  PMID: 25490984
Camellia sinensis; Flavonoid 3′5′-hydroxylase; Functional analysis; Heterologous expression; Catechins
9.  Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers 
Journal of Experimental Botany  2012;63(18):6505-6517.
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.
doi:10.1093/jxb/ers306
PMCID: PMC3504500  PMID: 23125348
Early flavonoid biosynthesis; flavone; flower colour; Japanese gentian; R2R3-MYB; transcription factor
10.  Stress-responsive hydroxycinnamate glycosyltransferase modulates phenylpropanoid metabolism in Populus  
Journal of Experimental Botany  2014;65(15):4191-4200.
Summary
UGT84A produces a dynamic pool of hydroxycinnamoyl-glucose esters in vegetative tissues and can modulate phenylpropanoid metabolism in response to developmental and environmental cues, such as nitrogen limitation, in Populus.
The diversity of phenylpropanoids offers a rich inventory of bioactive chemicals that can be exploited for plant improvement and human health. Recent evidence suggests that glycosylation may play a role in the partitioning of phenylpropanoid precursors for a variety of downstream uses. This work reports the functional characterization of a stress-responsive glycosyltransferase, GT1-316 in Populus. GT1-316 belongs to the UGT84A subfamily of plant glycosyltransferase family 1 and is designated UGT84A17. Recombinant protein analysis showed that UGT84A17 is a hydroxycinnamate glycosyltransferase and able to accept a range of unsubstituted and substituted cinnamic and benzoic acids as substrates in vitro. Overexpression of GT1-316 in transgenic Populus led to plant-wide increases of hydroxycinnamoyl-glucose esters, which were further elevated under N-limiting conditions. Levels of the two most abundant flavonoid glycosides, rutin and kaempferol-3-O-rutinoside, decreased, while levels of other less abundant flavonoid and phenylpropanoid conjugates increased in leaves of the GT1-316-overexpressing plants. Transcript levels of representative phenylpropanoid pathway genes were unchanged in transgenic plants, supporting a glycosylation-mediated redirection of phenylpropanoid carbon flow as opposed to enhanced phenylpropanoid pathway flux. The metabolic response of N-replete transgenic plants overlapped with that of N-stressed wild types, as the majority of phenylpropanoid derivatives significantly affected by GT1-316 overexpression were also significantly changed by N stress in the wild types. These results suggest that UGT84A17 plays an important role in phenylpropanoid metabolism by modulating biosynthesis of hydroxycinnamoyl-glucose esters and their derivatives in response to developmental and environmental cues.
doi:10.1093/jxb/eru192
PMCID: PMC4112628  PMID: 24803501
Glycosylation; hydroxycinnamate glucose ester; metabolite profiling; phenylpropanoid; Populus; stress; UGT84A.
11.  'Le Rouge et le Noir': A decline in flavone formation correlates with the rare color of black dahlia (Dahlia variabilis hort.) flowers 
BMC Plant Biology  2012;12:225.
Background
More 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.
Results
Black 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.
Conclusions
An 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.
doi:10.1186/1471-2229-12-225
PMCID: PMC3557166  PMID: 23176321
Dahlia variabilis hort; Asteraceae; Flower colour; Black flower; Flavonoids; Anthocyanins; Flavones; Flavone synthase II; Transcription factors
12.  A R2R3-MYB Transcription Factor from Epimedium sagittatum Regulates the Flavonoid Biosynthetic Pathway 
PLoS ONE  2013;8(8):e70778.
Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.
doi:10.1371/journal.pone.0070778
PMCID: PMC3731294  PMID: 23936468
13.  Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin 
BMC Genomics  2006;7:12.
Background
Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines.
Results
Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals.
Conclusion
We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between transcript profiles and the kinetics of accumulation of red/cyanidin- and blue/delphinidin-based anthocyanins indicated that VvF3'H and VvF3'5'H expression is consistent with the chromatic evolution of ripening bunches. Local physical maps constructed around the VvF3'H and VvF3'5'H loci should help facilitate the identification of the regulatory elements of each isoform and the future manipulation of grapevine and wine colour through agronomical, environmental and biotechnological tools.
doi:10.1186/1471-2164-7-12
PMCID: PMC1403756  PMID: 16433923
14.  Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony (Paeonia lactiflora Pall.) 
BMC Genomics  2014;15(1):689.
Background
Herbaceous peony (Paeonia lactiflora Pall.) is a traditional flower in China and a wedding attractive flower in worldwide. In its flower colour, yellow is the rarest which is ten times the price of the other colours. However, the breeding of new yellow P. lactiflora varieties using genetic engineering is severely limited due to the little-known biochemical and molecular mechanisms underlying its characteristic formation.
Results
In this study, two cDNA libraries generated from P. lactiflora chimaera with red outer-petal and yellow inner-petal were sequenced using an Illumina HiSeq™ 2000 platform. 66,179,398 and 65,481,444 total raw reads from red outer-petal and yellow inner-petal cDNA libraries were generated, which were assembled into 61,431 and 70,359 Unigenes with an average length of 628 and 617 nt, respectively. Moreover, 61,408 non-redundant All-unigenes were obtained, with 37,511 All-unigenes (61.08%) annotated in public databases. In addition, 6,345 All-unigenes were differentially expressed between the red outer-petal and yellow inner-petal, with 3,899 up-regulated and 2,446 down-regulated All-unigenes, and the flavonoid metabolic pathway related to colour development was identified using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of 10 candidate differentially expressed genes (DEGs) involved in the flavonoid metabolic pathway were examined, and flavonoids were qualitatively and quantitatively analysed. Numerous anthoxanthins (flavone and flavonol) and a few anthocyanins were detected in the yellow inner-petal, which were all lower than those in the red outer-petal due to the low expression levels of the phenylalanine ammonialyase gene (PlPAL), flavonol synthase gene (PlFLS), dihydroflavonol 4-reductase gene (PlDFR), anthocyanidin synthase gene (PlANS), anthocyanidin 3-O-glucosyltransferase gene (Pl3GT) and anthocyanidin 5-O-glucosyltransferase gene (Pl5GT).
Conclusion
Transcriptome sequencing (RNA-Seq) analysis based on the high throughput sequencing technology was an efficient approach to identify critical genes in P. lactiflora and other non-model plants. The flavonoid metabolic pathway and glucide metabolic pathway were identified as relatived yellow formation in P. lactiflora, PlPAL, PlFLS, PlDFR, PlANS, Pl3GT and Pl5GT were selected as potential candidates involved in flavonoid metabolic pathway, which inducing inhibition of anthocyanin biosynthesis mediated yellow formation in P. lactiflora. This study could lay a theoretical foundation for breeding new yellow P. lactiflora varieties.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-689) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-689
PMCID: PMC4159507  PMID: 25134523
Anthocyanins; Flower colour; Flavonoids; Herbaceous peony; Transcriptome; Yellow
15.  Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors 
BMC Plant Biology  2013;13:68.
Background
Flavonoids such as anthocyanins, flavonols and proanthocyanidins, play a central role in fruit colour, flavour and health attributes. In peach and nectarine (Prunus persica) these compounds vary during fruit growth and ripening. Flavonoids are produced by a well studied pathway which is transcriptionally regulated by members of the MYB and bHLH transcription factor families. We have isolated nectarine flavonoid regulating genes and examined their expression patterns, which suggests a critical role in the regulation of flavonoid biosynthesis.
Results
In nectarine, expression of the genes encoding enzymes of the flavonoid pathway correlated with the concentration of proanthocyanidins, which strongly increases at mid-development. In contrast, the only gene which showed a similar pattern to anthocyanin concentration was UDP-glucose-flavonoid-3-O-glucosyltransferase (UFGT), which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. Expression of flavonol synthase (FLS1) correlated with flavonol levels, both temporally and in a tissue specific manner. The pattern of UFGT gene expression may be explained by the involvement of different transcription factors, which up-regulate flavonoid biosynthesis (MYB10, MYB123, and bHLH3), or repress (MYB111 and MYB16) the transcription of the biosynthetic genes. The expression of a potential proanthocyanidin-regulating transcription factor, MYBPA1, corresponded with proanthocyanidin levels. Functional assays of these transcription factors were used to test the specificity for flavonoid regulation.
Conclusions
MYB10 positively regulates the promoters of UFGT and dihydroflavonol 4-reductase (DFR) but not leucoanthocyanidin reductase (LAR). In contrast, MYBPA1 trans-activates the promoters of DFR and LAR, but not UFGT. This suggests exclusive roles of anthocyanin regulation by MYB10 and proanthocyanidin regulation by MYBPA1. Further, these transcription factors appeared to be responsive to both developmental and environmental stimuli.
doi:10.1186/1471-2229-13-68
PMCID: PMC3648406  PMID: 23617716
Anthocyanin; Transcriptional regulation; MYB; Peach; Nectarine; Prunus persica; Light
16.  Production of 7-O-Methyl Aromadendrin, a Medicinally Valuable Flavonoid, in Escherichia coli 
7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate–coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.
doi:10.1128/AEM.06274-11
PMCID: PMC3264098  PMID: 22101053
17.  Coexpression Analysis of Tomato Genes and Experimental Verification of Coordinated Expression of Genes Found in a Functionally Enriched Coexpression Module 
Gene-to-gene coexpression analysis is a powerful approach to infer the function of uncharacterized genes. Here, we report comprehensive identification of coexpression gene modules of tomato (Solanum lycopersicum) and experimental verification of coordinated expression of module member genes. On the basis of the gene-to-gene correlation coefficient calculated from 67 microarray hybridization data points, we performed a network-based analysis. This facilitated the identification of 199 coexpression modules. A gene ontology annotation search revealed that 75 out of the 199 modules are enriched with genes associated with common functional categories. To verify the coexpression relationships between module member genes, we focused on one module enriched with genes associated with the flavonoid biosynthetic pathway. A non-enzyme, non-transcription factor gene encoding a zinc finger protein in this module was overexpressed in S. lycopersicum cultivar Micro-Tom, and expression levels of flavonoid pathway genes were investigated. Flavonoid pathway genes included in the module were up-regulated in the plant overexpressing the zinc finger gene. This result demonstrates that coexpression modules, at least the ones identified in this study, represent actual transcriptional coordination between genes, and can facilitate the inference of tomato gene function.
doi:10.1093/dnares/dsq002
PMCID: PMC2853382  PMID: 20130013
coexpression; flavonoid; Solanum lycopersicum; tomato; zinc finger
18.  The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower 
PLoS ONE  2013;8(8):e72364.
Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.
doi:10.1371/journal.pone.0072364
PMCID: PMC3741151  PMID: 23951318
19.  Functional Characterization of Dihydroflavonol-4-Reductase in Anthocyanin Biosynthesis of Purple Sweet Potato Underlies the Direct Evidence of Anthocyanins Function against Abiotic Stresses 
PLoS ONE  2013;8(11):e78484.
Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.
doi:10.1371/journal.pone.0078484
PMCID: PMC3817210  PMID: 24223813
20.  Berry skin development in Norton grape: Distinct patterns of transcriptional regulation and flavonoid biosynthesis 
BMC Plant Biology  2011;11:7.
Background
The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways.
Results
A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin.
Conclusions
This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.
doi:10.1186/1471-2229-11-7
PMCID: PMC3025947  PMID: 21219654
21.  A flavonoid 3-O-glucoside:2″-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana 
The Plant Journal  2014;79(5):769-782.
Flavonol 3-O-diglucosides with a 1→2 inter-glycosidic linkage are representative pollen-specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild-type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild-type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP-glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen-specific flavonol structure. Kaempferol and quercetin 3-O-glucosyl-(1→2)-glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild-type plants. Recombinant UGT79B6 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucosyl-(1→2)-glucoside. UGT79B6 recognized 3-O-glucosylated/galactosylated anthocyanins/flavonols but not 3,5- or 3,7-diglycosylated flavonoids, and prefers UDP-glucose, indicating that UGT79B6 encodes flavonoid 3-O-glucoside:2″-O-glucosyltransferase. A UGT79B6-GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.
doi:10.1111/tpj.12580
PMCID: PMC4282749  PMID: 24916675
glucosyltransferase; At5g54010; NM_124780; tapetum; pollen; glycosyltransferase; flavonol; flavonoid; Arabidopsis thaliana
22.  Arabidopsis CAPRICE (MYB) and GLABRA3 (bHLH) Control Tomato (Solanum lycopersicum) Anthocyanin Biosynthesis 
PLoS ONE  2014;9(9):e109093.
In Arabidopsis thaliana the MYB transcription factor CAPRICE (CPC) and the bHLH transcription factor GLABRA3 (GL3) are central regulators of root-hair differentiation and trichome initiation. By transforming the orthologous tomato genes SlTRY (CPC) and SlGL3 (GL3) into Arabidopsis, we demonstrated that these genes influence epidermal cell differentiation in Arabidopsis, suggesting that tomato and Arabidopsis partially use similar transcription factors for epidermal cell differentiation. CPC and GL3 are also known to be involved in anthocyanin biosynthesis. After transformation into tomato, 35S::CPC inhibited anthocyanin accumulation, whereas GL3::GL3 enhanced anthocyanin accumulation. Real-time reverse transcription PCR analyses showed that the expression of anthocyanin biosynthetic genes including Phe-ammonia lyase (PAL), the flavonoid pathway genes chalcone synthase (CHS), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS) were repressed in 35S::CPC tomato. In contrast, the expression levels of PAL, CHS, DFR, and ANS were significantly higher in GL3::GL3 tomato compared with control plants. These results suggest that CPC and GL3 also influence anthocyanin pigment synthesis in tomato.
doi:10.1371/journal.pone.0109093
PMCID: PMC4182634  PMID: 25268379
23.  Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen 
BMC Plant Biology  2010;10:107.
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.
doi:10.1186/1471-2229-10-107
PMCID: PMC3095274  PMID: 20540805
24.  Fruit-Surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-Regulated Transcriptional Network 
PLoS Genetics  2009;5(12):e1000777.
The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB–type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.
Author Summary
A major step in the evolution of land plants was the formation of a cuticular layer on their outer surfaces. Despite the cuticle's key role in organ development and in protecting against a variety of stresses, very little is known about the regulation of the metabolic pathways that generate its building blocks. Flavonoids, often embedded in the cuticle, have been suggested to impact the characteristics of this structure and to provide protection against radiation and pathogens. Flavonoids are an integral part of the human diet and are likely responsible for the observed beneficial effects of a fruit-rich diet. Here, we examine in detail the tomato colorless peel y mutant, which lacks the yellow flavonoid pigment naringenin chalcone, a major constituent of the fruit cuticle. Extensive transcript and metabolite profiling of this mutant revealed SlMYB12 as a key regulator in a transcription network that controls flavonoid accumulation in tomato peel. Moreover, the change in cuticle flavonoid composition enabled us to evaluate the importance of these constituents as barriers in the cuticle layer. Finally, because most commercial cultivars in the Far East are based on the y genetic background, discovery of the y gene will contribute also to tomato breeding programs.
doi:10.1371/journal.pgen.1000777
PMCID: PMC2788616  PMID: 20019811
25.  Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines 
BMC Plant Biology  2007;7:46.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2229-7-46
PMCID: PMC2147006  PMID: 17760970

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