Multiple F3′5′H evolution from F3′H has occurred in dicotyledonous plants. Efficient pollinator attraction is probably the driving force behind, as this allowed for the synthesis of delphinidin-based blue anthocyanins.

Products containing the epiphytic yeast Aureobasidium pullulans are commercially available and applied by fruit growers to prevent several fungal and bacterial diseases of fruit trees. The proposed beneficial mechanisms relate to limitations of space and nutrients for the pathogens in presence of the rapidly proliferating yeast cells. These explanations ignore the potential of yeasts to elicit the plant's defense. Our experiments aim at clarifying if an autoclaved and centrifuged suspension of A. pullulans may induce defense mechanisms. As a model system, the biosynthesis and accumulation of stilbene phytoalexins in callus and shoots of grapevine Vitis vinifera grown in vitro was used. Yeast application to the plant tissue stimulated stilbene biosynthesis, sometimes at the cost of flavonoids. The expression of the gene encoding stilbene synthase was enhanced and the enzyme showed higher activity while chalcone synthase activity and expression was reduced in some cases. An accumulation of stilbenes was also found in transgenic apple trees (Malus domestica cv. Holsteiner Cox) harboring the stilbene synthase-gene under control of its own promoter. These results clearly show that the application of A. pullulans may induce defense mechanisms of the treated plants.

Extracts from flowers of different strains of Verbena hybrida were found to contain flavones, flavonols, and anthocyanins. Furthermore, the presence of flavanones and dihydroflavonols was demonstrated. With regard to the B-ring substitution pattern, flavonoids with hydroxy groups at the 4'-, the 3',4'-, and the 3',4',5'-positions were found to occur. Red flowering strains contain 4'- and 3',4'-hydroxylated flavonoids in varying amounts. Enzymic studies revealed that the compounds with the 3',4'-hydroxylation pattern can be established by two different reactions. A small part of them is formed by incorporation of caffeic acid beside p-coumaric acid into the flavonoid skeleton during the condensation reaction catalysed by chalcone synthase. This basic level can be considerably enhanced by hydroxylation of 4'-hydroxylated flavonoids in the 3'-position. The reaction is catalysed by the well-known enzyme flavonoid 3'-hydroxylase. Enzyme activity was found to be correlated with the presence of enhanced amounts of 3',4'-hydroxylated flavonoid compounds in the flowers. Beside chalcone synthase and 3'-hydroxylase a further enzyme activity could be demonstrated in enzyme preparations from flowers of Verbena hybrida which catalyses the oxidation of naringenin to apigenin and of eriodictyol to luteolin. The reaction required NADPH as cofactor and had a pH optimum of about 6.5. Enzyme activity was found to be localized in the microsomal fraction.

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants including pathogen defence. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Modifying flavonoid biosynthesis in fruit crops, such as apple, offers the opportunity to increase plant resistance against pathogens and the health benefit potential of the fruit. Both overexpression and RNAi-based suppression strategies were used to modify flavonoid biosynthesis in apple. Introducing the maize Lc transcription factor gene, responsible for controlling the expression of structural genes of the flavonoid biosynthetic pathway in maize, into Malus domestica Borkh. cv. 'Holsteiner Cox' resulted in increased mRNA levels for most of the structural genes of the flavonoid pathway. Lc transgenic plants accumulated higher levels of the anthocyanin idaein, the flavan-3-ols epicatechin, catechin, and some distinct dimeric proanthocyanidins. In a second approach, the consequences of RNAi silencing of the genes for anthocyanidin synthase (ANS) and UDPgalactose: flavonoid 3-O-galactosyltransferase (FGT) gene to induce a shift towards flavan-3-ols were examined. Preliminary results obtained from transgenic plants of the cv. 'Holsteiner Cox' and of the red-leaved type TNR 31-35 (a descendent from Malus sieversii var. sieversii f. niedzwetzkyana (Dieck)) revealed that suppression of ANS or FGT resulted in increased amounts of flavan-3-ols and flavonols. In the red leaved type TNR 31-35, anthocyanin accumulation decreased after ANS silencing.

Hawthorn (Crataegus sp.) is a traditional medicinal plant, which shows a broad spectrum of health-related effects. Drug preparation is based on material collected from wild growing plants, which usually show variability in their secondary metabolite composition and concentration. Plantations of selected hawthorn genotypes could form the basis of a consistent homogeneous source of top-quality hawthorn drugs. However, as hawthorn is a member of the Rosaceae family, such plantations would be threatened by the bacterial disease fire blight, which Currently causes dramatic economic losses in apple and pear orchards. We show that prohexadione-Ca induces the formation in Crataegus monogyna leaves of the rare class of 3-deoxyflavonoids, which are responsible for better fire blight resistance and that the quality and quantity of the health related ingredients are not negatively influenced by prohexadione-Ca treatment.

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Here, we report on metabolic engineering of the flavonoid biosynthetic pathways in apple ( Malus domestica Borkh.) by overexpression of the maize ( Zea mays L.) leaf colour ( Lc ) regulatory gene. The Lc gene was transferred into the M. domestica cultivar Holsteiner Cox via Agrobacterium tumefaciens -mediated transformation which resulted in enhanced anthocyanin accumulation in regenerated shoots. Five independent Lc lines were investigated for integration of Lc into the plant genome by Southern blot and PCR analyses. The Lc -transgenic lines contained one or two Lc gene copies and showed increased mRNA levels for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3 beta-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductases (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). HPLC-DAD and LC-MS analyses revealed higher levels of the anthocyanin idaein (12-fold), the flavan 3-ol epicatechin (14-fold), and especially the isomeric catechin (41-fold), and some distinct dimeric proanthocyanidins (7 to 134-fold) in leaf tissues of Lc -transgenic lines. The levels of phenylpropanoids and their derivatives were only slightly increased. Thus, Lc overexpression in Malus domestica resulted in enhanced biosynthesis of specific flavonoid classes, which play important roles in both phytopathology and human health.

Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3β-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.

Modern biotechnology has developed powerful tools for genetic engineering and flower colours are an excellent object to study possibilities and limitations of engineering strategies. Osteospermum hybrida became a popular ornamental plant within the last 20 years. Many cultivars display rose to lilac flower colours mainly based on delphinidin-derived anthocyanins. The predominant synthesis of delphinidin derivatives is referred to a strong endogenous flavonoid 3′,5′-hydroxylase (F3′5′H) activity. Furthermore, since dihydroflavonol 4-reductase (DFR) of Osteospermum does not convert dihydrokaempferol (DHK) to leucopelargonidin, synthesis of pelargonidin-based anthocyanins is naturally not realised. In order to redirect anthocyanin biosynthesis in Osteospermum towards pelargonidin derivatives, we introduced cDNAs coding for DFRs which efficiently convert DHK to LPg. But neither the expression of Gerbera hybrida DFR nor of Fragaria × ananassa DFR – the latter is characterised by an unusual high substrate preference for DHK – altered anthocyanin composition in flowers of transgenic plants. However, chemical inhibition of F3′5′H activity in ray florets of dfr transgenic plants resulted in the accumulation of pelargonidin derivatives. Accordingly, retransformation of a transgenic plant expressing Gerbera DFR with a construct for RNAi-mediated suppression of F3′5′H activity resulted in double transgenic plants accumulating predominantly pelargonidin derivatives in flowers.

Traditional selection markers for the production of transgenic plants such as genes for antibiotic resistance and herbicide tolerance are not accepted by the public opinion. A promising alternative system consists in the use of mannose as selective agent. Mannose can not be metabolized by many plant species and accumulates as mannose-6-phosphate resulting in growth inhibition. By the use of a gene encoding phosphomannose isomerase (PMI) as a selection marker, transgenic cells are enabled to use mannose-6-phophate as carbon source, since PMI catalyses its conversion to fructose-6-phosph ate. A mannose-based selection system has already been successfully established for the production of transgenic crops such as sugar beet, rice, maize or potato. In this paper we used this "positive" selection system for the transformation of Torenia hybrids. The integration and expression of pmi gene was confirmed by PCR analysis. Furthermore, a spectrophotometrical enzyme assay led to a clear detection of PMI activity in enzyme extracts of the transgenic material. In contrast, despite the high mannose-tolerance of Torenia, no endogenous PMI activity was found in non-transgenic Torenia tissues. Taken together, the mannosebased selection system can be successfully applied for the generation of transgenic Torenia plants. Compared to selection by kanamycin, the mannose-based selection decreases regeneration time up to 25%, the transgenic shoots show favourable rooting even on mannose-media and the transformation efficiency is generally higher.

Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3'- or the 3'- and 5'-position, respectively. Sequence identity between F3'H and F3'5'H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3'5'H from an F3'H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3'H and F3'5'H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5'-hydroxylation activity to recombinant Gerbera hybrida F3'H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3'5'H into an F3'H with low remaining 5'-hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3'- to 3',5'-hydroxylation activity.

2-Oxoglutarate-dependent dioxygenases (2-ODDs) catalyze numerous steps in biosynthetic pathways of plants. Prohexadione-Ca is a known inhibitor of such reactions, due to its structural similarity to 2-oxoglutarate. In apple (Malus domestica) and pear (Pyrus communis) leaves, the transient inhibition of 2-ODDs flavanone 3β-hydroxylase (FHT) and flavonol synthase (FLS) by prohexadione-Ca results in distinct changes in the flavonoid spectrum, which are responsible for an enhanced resistance against two major pome fruit diseases, fire blight (caused by Erwinia amylovora) and apple scab (caused by Venturia inaequalis). We used recombinant apple and pear FHT and apple FLS for screening 23 structural analogues of 2-oxoglutarate, mostly cyclohexanediones, pyridine dicarboxylic acids and N-heterocycles with carbonyl functions for other dioxygenase inhibitors. Activations, which were also observed for some compounds, are interpreted as in vitro effects due to Fe2+-chelating ability. Apart from structural similarity to 2-oxoglutarate, close structural similarity of cyclohexanediones and some pyridine dicarboxylic acids to flavonoid substrates was identified. Beyond the competitive inhibition for the co-substrate 2-oxoglutarate, flavonoid converting 2-ODDs may also be inhibited at the substrate binding site by these inhibitors. All compounds found to be active as inhibitors may prove useful for studying the reaction mechanisms and substrate specificities of various 2-ODDs.

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.

Catechin and epicatechin biosyntheses were studied of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crop leaves, since these monomers and the derived proanthocyanidins are important disease resistance factors. Grape and apple leucoanthocyanidin 4-reductase (LAR; EC 1.17.1.3) enzymes were characterized on basis of plant and recombinant enzymes. In case of grape, two LAR cDNAs were cloned by assembling available EST sequences. Grape and apple leaf anthocyanidin reductase (ANR; EC 1.3.1.77) cDNAs were also obtained and the respective plant and recombinant enzymes were characterized. Despite general low substrate specificity, within the respective flavonoid biosyntheses of grape and apple leaves, both enzyme types deliver differently hydroxylated catechins and epicatechins, due to substrate availability in vivo. Furthermore, for LAR enzymes conversion of 3-deoxyleucocyanidin was shown. Beside relevance for plant protection, this restricts the number of possible reaction mechanisms of LAR. ANR enzyme activity was demonstrated for a number of other crop plants and its correlation with (–)-epicatechin and obvious competition with UDP-glycosyl:flavonoid-3-O-glycosyltransferases was considered.

Biosynthesis is well elucidated for 5-hydroxyflavonoids (phloroglucinol type), but for 5-deoxyflavonoids (resorcinol type) the knowledge is still limited. We provide detailed and optimized protocols for the synthesis of (14C)-labeled 6′-deoxychalcones, 5-deoxyflavanones, 5-deoxydihydroflavonols and 5-deoxyleucoanthocyanidins. With the exception of the formation of 6′-deoxychalcones, all steps were performed enzymatically using enzymes normally involved in the formation of 5-hydroxyflavonoids. The availability of (14C)-labeled substrates will facilitate future work on the hitherto largely unknown biosynthesis of 5-deoxyflavonoids. In particular, the 5-deoxyleucoanthocyanidins, which are more stable than the corresponding 5-hydroxy compounds, may provide excellent tools for investigating enzymes, which use the unstable 5-hydroxyleucoanthocyanidins as natural substrates. As a first example, the conversion of (14C)-labeled 5-deoxyleucoanthocyanidins to dihydroflavonols in the presence of NADP+ was shown. Studies with defined genotypes of Matthiola incana possessing or lacking dihydroflavonol 4-reductase activity and genetically modified yeast expressing the Matthiola enzyme confirmed that the reaction is catalyzed by the well-known dihydroflavonol 4-reductase, which catalyzes the conversion of dihydroflavonols to leucoanthocyanidins (forward reaction). Thus, the reverse reaction of dihydroflavonol 4-reductase could be demonstrated for the first time. The forward reaction shows an optimum at pH 6.25, the reverse reaction at pH 7.75. The impact of the results on the regulation of flavonoid accumulation is discussed.

A comprehensive study of the complex polyphenol biosynthesis in developing leaves of apple (Malus domestica) was performed comprising gene expression, enzyme activities and polyphenol composition. During leaf development, an early increase in gene expression was observed for phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), flavanone 3‐hydroxylase (FHT, EC 1.14.11.9) and dihydroflavonol 4‐reductase/flavanone 4‐reductase (DFR/FNR, EC 1.1.1.219). Their enzyme activities showed a corresponding trend during the time course. A parallel set of experiments was carried out with leaves treated with prohexadione‐Ca (ProCa), which is an enzyme inhibitor of 2‐oxoglutarate dependent dioxygenases (2‐ODDs). ProCa is known to induce changes in polyphenol biosynthesis, which are accompanied by a reduced incidence of fire blight and scab, the two major pome fruit diseases. The application of ProCa led to an increase in activities of PAL, CHS, FHT and DFR/FNR, which was based on an enhanced gene expression. In contrast, an inhibition of gene expression was detected for anthocyanidin synthase (EC 1.14.11.19). These effects are interpreted as a feedback regulation by changed polyphenol levels. Because of the inhibition of the 2‐ODDs FHT and flavonol synthase (EC 1.14.11.23), some pronounced changes in polyphenol composition were observed. Eriodictyol, the substrate of FHT, accumulated as eriodictyol‐7‐O‐glucoside and 6″‐O‐trans‐p‐coumaroyleriodictyol 3′‐O‐glucoside. In addition, the 3‐deoxycatechin luteoliflavan was formed which is not present in untreated apple leaves. Hence, beyond the redirection of polyphenol biosynthesis by the enzyme inhibitor, changed polyphenol levels obviously cause a distinct induction of gene expression by feedback regulation.

Plant species of the family Apiaceae are known to accumulate flavonoids mainly in the form of flavones and flavonols. Three 2-oxoglutarate-dependent dioxygenases, flavone synthase or flavanone 3 β-hydroxylase and flavonol synthase are involved in the biosynthesis of these secondary metabolites. The corresponding genes were cloned recently from parsley (Petroselinum crispum) leaves. Flavone synthase I appears to be confined to the Apiaceae, and the unique occurrence as well as its high sequence similarity to flavanone 3β-hydroxylase laid the basis for evolutionary studies. In order to examine the relationship of these two enzymes throughout the Apiaceae, RT-PCR based cloning and functional identification of flavone synthases I or flavanone 3β-hydroxylases were accomplished from Ammi majus, Anethum graveolens, Apium graveolens, Pimpinella anisum, Conium maculatum and Daucus carota, yielding three additional synthase and three additional hydroxylase cDNAs. Molecular and phylogenetic analyses of these sequences were compatible with the phylogeny based on morphological characteristics and suggested that flavone synthase I most likely resulted from gene duplication of flavanone 3β-hydroxylase, and functional diversification at some point during the development of the apiaceae subfamilies. Furthermore, the genomic sequences from Petroselinum crispum and Daucus carota revealed two introns in each of the synthases and a lack of introns in the hydroxylases. These results might be explained by intron losses from the hydroxylases occurring at a later stage of evolution.

3-Deoxyanthocyanins provide bright orange-red colours to flowers of some members of the Gesneriaceae, including sinningia (Sinningia cardinalis). We examined 3-deoxyanthocyanin biosynthesis in sinningia, in particular, the expression of key flavonoid biosynthetic genes and the activities of the encoded proteins. Two abundant 3-deoxyanthocyanins, luteolinidin 5-O-glucoside and apigeninidin 5-O-glucoside, three flavone glycosides, luteolin 7-O-glucoside, luteolin 7-O-glucuronide and apigenin 7-O-glucuronide, and the cinnamic acid verbascoside were identified in sinningia petal tissue. Small amounts of a 3-hydroxyanthocyanin were also detected in a limited region of the petal. cDNA clones for three flavonoid enzymes, flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase/flavanone 4-reductase (DFR/FNR) and anthocyanidin synthase (ANS), were isolated from a sinningia cDNA library made from petal RNA and used to measure transcript abundance during petal development. Only very low levels of F3H transcript were detected, while DFR/FNR transcript was highly abundant. ANS transcript levels were intermediate between these two. The F3H cDNA was shown to encode a functional F3H protein by complementation of the phenotype of an Antirrhinum majus F3H mutant. The recombinant DFR/FNR had activity against both flavanone and dihydroflavonol substrates to a comparable extent. The results suggest a mechanism of 3-deoxyflavonoid biosynthesis in sinningia similar to that reported for Zea mays, in which lack of F3H activity allows action of the DFR/FNR on flavanone substrates and production of flavan-4-ols. These are then likely converted to 3-deoxyanthocyanins through the action of the ANS and subsequent glucosylation.

Petals of Osteospermum exhibit white, yellow and rose to lilac colours. Chemical and biochemical investigations elucidated the basis of flower colour in Osteospermum. Besides carotenoids, which were shown to be responsible for the yellow colour, flavonoids were identified to be the main colour-giving compounds. Different amounts of derivatives of the anthocyanidin delphinidin cause the rose to lilac colour range. In addition to delphinidin, derivatives of flavonols were found to be present in the petals.Enzyme assays elucidated the biosynthetic path leading to the formation of the 3'-, 4'-, and 5'-hydroxylated delphinidin. Because of distinct substrate specifities of the early enzymes of the flavonoid pathway, chalcone synthase, chalcone isomerase, and flavanone 3-hydroxylase, dihydrokaempferol is supposed to be the main branch point. This compound was shown to be hydroxylated in 3'- and 5% position by flavonoid 3', 5'-hydroxylase to dihydromyricetin which is subsequently reduced by dihydroflavonol 4-reductase to leucodelphinidin, the precursor of delphinidin.Molecular biological investigations of two key steps allowed a confirmation of the enzymological findings. The protein of a heterologously expressed full length chalcone synthase cDNA-clone was shown to preferentially use 4-coumaroyl-CoA as a substrate leading to the formation of naringenin after isomerisation by chalcone isomerase. In contrast to this, caffeoyl-CoA as a substrate resulted in the formation of only low amounts of eriodictyol. 3', 5'-hydroxylation, which is indispensable for the formation of delphinidin, was demonstrated with the protein of a heterologously expressed F3'5'H full-length cDNA-clone. Fragments of the other main structural flavonoid genes of Osteospermum are now available and further molecular biological investigation will be performed.