Flower colour and cytochromes P450

Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3′-hydroxylase (F3′H, CYP75B) and flavonoid 3′,5′-hydroxylase (F3′5′H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue flower colour due to deficiency of F3′5′H. Successful expression of heterologous F3′5′H genes in roses and carnations results in delphinidin production, causing a novel blue/violet flower colour. Down-regulation of F3′H and F3′5′H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of flavones that contribute to the bluing of flower colour, and modulation of FNSII gene expression in petunia and tobacco changes their flower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of flower colours.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Functional characterization of key structural genes in rice flavonoid biosynthesis

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl ^w locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T⁶⁴ M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3′H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3′H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering of flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway

Flower color was modified in forsythia (Forsythia x intermedia cv Spring Glory) by inducing anthocyanin synthesis in petals through sequential Agrobacterium-mediated transformation with dihydroflavonol 4-reductase from Antirrhinum majus (AmDFR) and anthocyanidin synthase from Matthiola incana (MiANS) genes. This is the second report of flower color modification of an ornamental shrub after rose, and the first time an ANS gene is used for this purpose. Double transformants (AmDFR+MiANS) displayed a novel bronze-orange petal color, caused by the de novo accumulation of cyanidin-derived anthocyanins over the carotenoid yellow background of wild type (wt), and intense pigmentation of vegetative organs. Transformation with single genes (either AmDFR or MiANS) produced no change in flower color, showing a multistep control of late anthocyanin pathway in petals of forsythia. Analysis of relevant late flavonoid pathway genes – an endogenous flavonoid glycosyltransferase (FiFGT) and transformed DFR and ANS genes – showed appropriate expression in flower organs. Functional characterization of FiFGT expressed in E. coli revealed its ability to metabolize both flavonols and anthocyanidin substrates, a prerequisite for effective anthocyanin accumulation in petals of plants transformed with constructs leading to anthocyanidin synthesis. Biochemical analyses of flavonoid compounds in petals and leaves showed that, besides anthocyanin induction in petals of double transformants, the accumulation pattern of flavan-3-ols was quantitatively and qualitatively modified in petals and leaves of transformants, in agreement with the most recent model proposed for flavan-3-ol synthesis. On the other hand, phenylpropanoid, flavone and flavonol pools were not quantitatively affected, indicating a tight regulation of early flavonoid pathway.     

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3′4′5′-hydroxylated forms through the differential regulation of F3′H and F3′5′H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.     

Functional analysis of <Emphasis Type="Italic">Antirrhinum kelloggii</Emphasis> flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes; critical role in flower color and evolution in the genus <Emphasis Type="Italic">Antirrhinum</Emphasis>

The enzymes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3′5′H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3′H and two F3′5′H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3′Hs and F3′5′Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3′H and F3′5′H, respectively, and that the ancestor of A. majus lost F3′5′H activity after its speciation from the ancestor of A. kelloggii.     

Co-expression of<Emphasis Type="Italic">flavonoid 3′ 5′-hydroxylase</Emphasis> and<Emphasis Type="Italic">flavonoid 3′-hydroxylase</Emphasis> Accelerates Decolorization in Transgenic Chrysanthemum Petals

Theflavonoid 3′,5′-hydroxylase (F3′,5′H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation. Cotyledon expiants were co-cultured withA. tumefaciens LBA 4404 harboring the vector pMBP that carriesF3′,5′H under the control of the CaMV 35S promoter andnptll as a selectable marker gene. After 72 h of co-cultivation, the expiants were placed on an MS medium supplemented with 4 mg L^-1 BA, 0.1 mg L^-1 NAA, 400 mg L^-1 carbenicillin, and 100 mg L^-1; kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were then rooted and acclimatized in potting soil. Integration ofF3′,5′H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed thatF3′,5′H andflavonoid 3′hydroxylase (F3′H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color results from 1) competition between levels of transgenicF3′,5′H and endogenousF3′H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of chrysanthemumDFR (dihydroflavonol 4-reductase).     

Cytochrome P450s in flavonoid metabolism

In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed.     

Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene

Anthocyanins are the major pigments contributing to carnation flowercoloration. Most carnation varieties are sterile and hence molecular breedingis an attractive approach to creating novel colors in this commercially importantcrop. Characterization of anthocyanins in the flowers of the modern carnationcv. Eilat revealed that only the orange pelargonidin accumulates, due to a lackof both flavonoid 3,5-hydroxylase and flavonoid3-hydroxylase activities. To modify flower color in cv. Eilat, we usedantisense suppression to block the expression of a gene encoding flavanone3-hydroxylase, a key step in the anthocyanin pathway. The transgenic plantsexhibited flower color modifications ranging from attenuation to complete lossof their original orange/reddish color. In the latter, only traces ofpelargonidin were detected. Dramatic suppression of flavanone 3-hydroxylaselevel/activity in these transgenes was confirmed by northern blot, RT-PCR andenzymatic assays. The new phenotype has been stable for over 4 years ofvegetative propagation. Moreover, transgenic plants with severe colormodification were more fragrant than control plants. GC-MS headspace analysesrevealed that transgenic anti-f3h flowers emit higherlevels of methyl benzoate. The possible interrelation between pathways leadingto anthocyanin and fragrance production is discussed.     

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3′end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (−60) minimal promoter [AG-I-35S (−60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (−60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (−60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (−60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.     

Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene

The plant-tumorigenic 6b (AK-6b) gene of Agrobacterium tumefaciens strain AKE10 induces morphological alterations to tobacco plants, Nicotiana tabacum. To investigate the molecular mechanisms underlying these processes, we generated transgenic tobacco harboring the AK-6b gene under the control of a dexamethazone-inducible promoter. Upon induction, transgenic tobacco seedlings exhibited distinct classes of aberrant morphologies, most notably adventitious outgrowths and stunted epicotyls. Histological analysis revealed massive proliferation and altered venation in the newly established outgrowths. Prominent vascular development suggested that auxin metabolism or signaling had been altered. Indeed, basipetal auxin transport in the hypocotyls of the transgenic seedlings was reduced by 50–80%, whereas intracellular auxin contents were only slightly reduced. Analysis of cell extracts by HPLC revealed a large accumulation of phenolic compounds, including the flavonoid kaempferol-3-rutinoside, in transgenic plants compared with wild-type seedlings. As some naturally occurring flavonoids have been shown to affect auxin transport, we suggest that the AK-6b gene expression impairs auxin transport via modulation of phenylpropanoid metabolism, and ultimately results in the observed morphological alterations. Electronic Supplementary Material Supplementary material is available for this article at