The (Bio)chemical Base of Flower Colour in Bidens ferulifolia.

Bidens ferulifolia is a yellow flowering plant, originating from Mexico, which is increasingly popular as an ornamental plant. In the past few years, new colour combinations ranging from pure yellow over yellow-red, white-red, pure white and purple have emerged on the market. We analysed 16 Bidens ferulifolia genotypes to provide insight into the (bio)chemical base underlying the colour formation, which involves flavonoids, anthochlors and carotenoids. In all but purple and white genotypes, anthochlors were the prevalent pigments, primarily derivatives of okanin, a 6'-deoxychalcone carrying an unusual 2'3'4'-hydroxylation pattern in ring A. The presence of a cytochrome-P450-dependent monooxygenase introducing the additional hydroxyl group in position 3' of both isoliquiritigenin and butein was demonstrated for the first time. All genotypes accumulate considerable amounts of the flavone luteolin. Red and purple genotypes additionally accumulate cyanidin-type anthocyanins. Acyanic genotypes lack flavanone 3-hydroxylase and/or dihydroflavonol 4-reductase activity, which creates a bottleneck in the anthocyanin pathway. The carotenoid spectrum was analysed in two Bidens genotypes and showed strong variation between the two cultivars. In comparison to anthochlors, carotenoids were present in much lower concentrations. Carotenoid monoesters, as well as diesters, were determined for the first time in B. ferulifolia flower extracts.

Molecular and Enzymatic Characterization of Flavonoid 3'-Hydroxylase of Malus × domestica.

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2'-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3'-hydroxylase (F3'H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3'H cDNA clones from apple leaves and tested recombinant Malus F3'Hs for their substrate specificity. From the two isolated cDNA clones, only F3'HII encoded a functionally active enzyme. In the F3'HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3'HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely.

Dahlia variabilis cultivar 'Seattle' as a model plant for anthochlor biosynthesis.

We investigated the bi-colored dahlia cultivar 'Seattle', which exhibits bright yellow petals with white tips, for its potential use as a model system for studies of the anthochlor biosynthesis. The yellow base contained high amounts of the 6'-deoxychalcones and the structurally related 4-deoxyaurones, as well as flavones. In contrast, only traces of anthochlors and flavones were detected in the white tips. No anthocyanins, flavonols, flavanones or dihydroflavonols were found in the petals. Gene expression studies indicated that the absence of anthocyanins in the petals is caused by a lack of flavanone 3-hydroxylase (FHT) expression, which is accompanied by a lack of expression of the bHLH transcription factor IVS. Expression of other genes involved in anthocyanidin biosynthesis such as dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) was not affected. The yellow and white petal parts showed significant differences in the expression of chalcone synthase 2 (CHS2), which is sufficient to explain the absence of yellow pigments in the white tips. Transcriptomes of both petal parts were de novo assembled and three candidate genes for chalcone reductase (CHR) were identified. None of them showed a significantly higher expression in the yellow base compared to the white tips. In summary, it was shown that the bicolouration is most likely caused by a bottleneck in chalcone formation in the white tip. The relative prevalence of flavones compared to the anthochlors in the white tips could be an indication for the presence of a so far unknown differentially expressed CHR.

The rare orange-red colored Euphorbia pulcherrima cultivar 'Harvest Orange' shows a nonsense mutation in a flavonoid 3'-hydroxylase allele expressed in the bracts.

BACKGROUND: Commercially available poinsettia (Euphorbia pulcherrima) varieties prevalently accumulate cyanidin derivatives and show intense red coloration. Orange-red bract color is less common. We investigated four cultivars displaying four different red hues with respect to selected enzymes and genes of the anthocyanin pathway, putatively determining the color hue. RESULTS: Red hues correlated with anthocyanin composition and concentration and showed common dark red coloration in cultivars 'Christmas Beauty' and 'Christmas Feeling' where cyanidin derivatives were prevalent. In contrast, orange-red bract color is based on the prevalent presence of pelargonidin derivatives that comprised 85% of the total anthocyanin content in cv. 'Premium Red' and 96% in cv. 'Harvest Orange' (synonym: 'Orange Spice'). cDNA clones of flavonoid 3'-hydroxylase (F3'H) and dihydroflavonol 4-reductase (DFR) were isolated from the four varieties, and functional activity and substrate specificity of the corresponding recombinant enzymes were studied. Kinetic studies demonstrated that poinsettia DFRs prefer dihydromyricetin and dihydroquercetin over dihydrokaempferol, and thus, favor the formation of cyanidin over pelargonidin. Whereas the F3'H cDNA clones of cultivars 'Christmas Beauty', 'Christmas Feeling', and 'Premium Red' encoded functionally active enzymes, the F3'H cDNA clone of cv. 'Harvest Orange' contained an insertion of 28 bases, which is partly a duplication of 20 bases found close to the insertion site. This causes a frameshift mutation with a premature stop codon after nucleotide 132 and, therefore, a non-functional enzyme. Heterozygosity of the F3'H was demonstrated in this cultivar, but only the mutated allele was expressed in the bracts. No correlation between F3'H-expression and the color hue could be observed in the four species. CONCLUSIONS: Rare orange-red poinsettia hues caused by pelargonidin based anthocyanins can be achieved by different mechanisms. F3'H is a critical step in the establishment of orange red poinsettia color. Although poinsettia DFR shows a low substrate specificity for dihydrokaempferol, sufficient precursor for pelargonidin formation is available in planta, in the absence of F3'H activity.

Great Cause-Small Effect: Undeclared Genetically Engineered Orange Petunias Harbor an Inefficient Dihydroflavonol 4-Reductase.

A recall campaign for commercial, orange flowering petunia varieties in spring 2017 caused economic losses worldwide. The orange varieties were identified as undeclared genetically engineered (GE)-plants, harboring a maize dihydroflavonol 4-reductase (DFR, A1), which was used in former scientific transgenic breeding attempts to enable formation of orange pelargonidin derivatives from the precursor dihydrokaempferol (DHK) in petunia. How and when the A1 cDNA entered the commercial breeding process is unclear. We provide an in-depth analysis of three orange petunia varieties, released by breeders from three countries, with respect to their transgenic construct, transcriptomes, anthocyanin composition, and flavonoid metabolism at the level of selected enzymes and genes. The two possible sources of the A1 cDNA in the undeclared GE-petunia can be discriminated by PCR. A special version of the A1 gene, the A1 type 2 allele, is present, which includes, at the 3'-end, an additional 144 bp segment from the non-viral transposable Cin4-1 sequence, which does not add any functional advantage with respect to DFR activity. This unequivocally points at the first scientific GE-petunia from the 1980s as the A1 source, which is further underpinned e.g., by the presence of specific restriction sites, parts of the untranslated sequences, and the same arrangement of the building blocks of the transformation plasmid used. Surprisingly, however, the GE-petunia cannot be distinguished from native red and blue varieties by their ability to convert DHK in common in vitro enzyme assays, as DHK is an inadequate substrate for both the petunia and maize DFR. Recombinant maize DFR underpins the low DHK acceptance, and, thus, the strikingly limited suitability of the A1 protein for a transgenic approach for breeding pelargonidin-based flower color. The effect of single amino acid mutations on the substrate specificity of DFRs is demonstrated. Expression of the A1 gene is generally lower than the petunia DFR expression despite being under the control of the strong, constitutive p35S promoter. We show that a rare constellation in flavonoid metabolism-absence or strongly reduced activity of both flavonol synthase and B-ring hydroxylating enzymes-allows pelargonidin formation in the presence of DFRs with poor DHK acceptance.

Evaluation of a MdMYB10/GFP43 fusion gene for its suitability to act as reporter gene in promoter studies in Fragaria vesca L. 'Rügen'.

A Malus domestica MdMYB10 transcription factor gene was previously used as visible marker for successful plant transformation. We combined the MdMYB10 transcription factor gene with a GFP gene to test its viability as a non-destructive, visual, double reporter system for functional promoter studies in transgenic strawberry plants. The GFP gene was fused to MdMYB10 to provide evidence for promoter activity in red colored cells of transformed plant tissue and to exclude artefacts resulting from stress response or due to other environmental cues. To test this system in a first approach, we evaluated the MdMYB10-GFP43 construct in transgenic strawberries in combination with two constitutive promoters of varying strength, the strong CaMV 35S promoter and a weak flavonoid 3'-hydroxylase (F3'H) promoter isolated from the ornamental plant Cosmos sulphureus. Agrobacterium tumefaciens mediated transformation of Fragaria vesca with the MdMYB10-GFP43 construct combined with the CaMV 35S or F3'H promoter sequences resulted in the regeneration of 6 and 4 transgenic lines, respectively. A complete red coloration of all plant organs was found in four out of six transgenic lines harboring the 35S-MdMYB10-GFP43 construct. Less red coloration of plant organs was found for lines transformed with the F3'H-MdMYB10-GFP43 construct. The MdMYB10 gene shows only limited suitability as a reporter gene for promoter studies in strawberries because weak promoter activity is difficult to distinguish, particularly in tissues showing a strongly colored background such as green leaves. GFP specific fluorescence signals were detectable neither in tissue strongly expressing MdMYB10 nor in green tissue of any transgenic line. The reason for this remained unclear but it can be excluded that it was due to incorrect splicing.

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

MAIN CONCLUSION: Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3'-hydroxylase (F3'H). Using recombinant F3'H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3'H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.

High concentrations of anthocyanins in genuine cherry-juice of old local Austrian Prunus avium varieties.

Antioxidant activity and polyphenols were quantified in vapour-extracted juice of nine Austrian, partially endemic varieties of sweet cherry (Prunus avium): cv. 'Spätbraune von Purbach', cv. 'Early Rivers', cv. 'Joiser Einsiedekirsche', cv. 'Große Schwarze Knorpelkirsche' and four unidentified local varieties. Additionally the effect of storage was evaluated for six of the varieties. A variety showing the highest antioxidant capacity (9.64 µmol Trolox equivalents per mL), total polyphenols (2747 mg/L) and total cyanidins (1085 mg/L) was suitable for mechanical harvest and its juice did not show any losses of antioxidant capacity and total anthocyanin concentration during storage. The juice of cv. 'Große Schwarze Knorpelkirsche' had also high concentrations of total anthocyanins (873 mg/L), but showed substantial losses through storage. The local Austrian sweet cherry varieties from the Pannonian climate zone are particularly suitable for the production of processed products like cherry juice with high content of anthocyanins and polyphenols.

Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3'-hydroxylase on leucoanthocyanidins.

MAIN CONCLUSION: In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3' position by flavonoid 3'-hydroxylase. The cytochrome P450-dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3' or 3' and 5', respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3'H and F3'5'H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3'H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3'-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3',5'-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3'Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.

Isolation of dihydroflavonol 4-reductase cDNA clones from Angelonia x angustifolia and heterologous expression as GST fusion protein in Escherichia coli.

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at -80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.

4-Deoxyaurone formation in Bidens ferulifolia (Jacq.) DC.

The formation of 4-deoxyaurones, which serve as UV nectar guides in Bidens ferulifolia (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6'-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of B. ferulifolia and from plants, which do not accumulate aurones e.g. Arabidopsis thaliana, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in Antirrhinum majus L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general.

Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria × ananassa and Fragaria vesca.

Flavonoid 3'-hydroxylase (F3'H) was studied for the first time in different Fragaria species. The cDNA clones isolated from unripe and ripe fruits of Fragaria x ananassa (garden strawberry) and Fragaria vesca (wild strawberry) showed high similarity (99% at the amino acid level) to the publically available F. vesca genome sequence and no significant differences could be identified between species and developmental stages of the fruits. In contrast, the genomic F3'H clones showed differences in the non-coding regions and 5'-flanking elements. The recombinant F3'Hs were functionally active and showed high specificity for naringenin, dihydrokaempferol, and kaempferol, whereas apigenin was only a minor substrate. During fruit development, a clear difference in the F3'H expression was observed between F. × ananassa and F. vesca. While a drastic decline of F3'H expression occurred during fruit ripening in F. × ananassa, F3'H in F. vesca was highly expressed in all stages. This was reflected by the anthocyanin composition, which showed a prevalence of pelargonidin in ripe fruits of F. × ananassa, whereas F. vesca had a high content of cyanidin. Screening of 17 berry species for their anthocyanin and flavonol composition showed that the prevalence of monohydroxylated anthocyanins makes garden strawberry unique among all other fruit species indicating that selection of bright red color during strawberry breeding, which consumers typically associate with freshness and ripeness, has selected phenotypes with a special biochemical background.

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

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.

Potent and specific bactericidal effect of juglone (5-hydroxy-1,4-naphthoquinone) on the fire blight pathogen Erwinia amylovora.

A screening of plant quinones for inhibiting effects on the bacterial fire blight pathogen Erwinia amylovora was performed. The most active compound, juglone from walnuts, has a potent and specific bactericidal effect on E. amylovora and minimal inhibitory concentrations of only 2.5-10 µM, with stronger effects at lower, but still physiological, pH values. In vitro tests with juglone and inoculated flowers of apple (Malus domestica) showed an efficacy of 67% in preventing infection. In two years of field tests juglone had variable degrees of efficacy ranging from 40 to 82%, seemingly due to environmental conditions. A phytotoxic reaction to juglone, which is known for its allelopathic effect on plants, was restricted to browning of petals; later fruit russeting was not observed. Juglone is a promising candidate for the development of a new environmentally friendly plant protectant to replace the antibiotic streptomycin currently used in fire blight control.

Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin).

Onobrychis viciifolia (sainfoin) is a traditional fodder legume showing multiple benefits for the environment, animal health and productivity but weaker agronomic performance in comparison to other legumes. Benefits can be mainly ascribed to the presence of polyphenols. The polyphenol metabolism in O. viciifolia was studied at the level of gene expression, enzyme activity, polyphenol accumulation and antioxidant activity. A screening of 37 accessions regarding each of these characters showed a huge variability between individual samples. Principal component analysis revealed that flavonols and flavan 3-ols are the most relevant variables for discrimination of the accessions. The determination of the activities of dihydroflavonol 4-reductase and flavonol synthase provides a suitable screening tool for the estimation of the ratio of flavonols to flavan 3-ols and can be used for the selection of samples from those varieties that have a specific optimal ratio of these compounds for further breeding.

Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility.

Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.

Phloridzin: biosynthesis, distribution and physiological relevance in plants.

The phenolic compound phloridzin (phloretin 2'-O-glucoside, phlorizin, phlorrhizin, phlorhizin or phlorizoside) is a prominent member of the chemical class of dihydrochalcones, which are phenylpropanoids. The apple tree (Malus sp.) accumulates high amounts of phloridzin, whereas few other species contain this compound only in low amounts. Additionally, Malus sp. show a species- and tissue-specific distribution of phloridzin and its derivatives. Whereas the physiological role of phloridzin in planta is not fully understood, the effect on human health - especially diabetes - and membrane permeability is well documented. The biosynthesis of phloridzin was investigated only recently with recombinant enzymes and plant protein extracts and involved a NADPH-dependent dehydrogenase, chalcone synthase and UDP-glucose:phloretin 2'-O-glycosyltransferase.

Measuring flavonoid enzyme activities in tissues of fruit species.

Flavonoids are important secondary metabolites, which are ubiquitously present in plant-derived food. Since flavonoids may show beneficial effects on human health, there is increasing interest in the availability of plants with a tailor-made flavonoid spectrum. Determination of flavonoid enzyme activities and investigations into their substrate specificity are an important precondition for both classical and molecular approaches. We tested two different protocols for enzyme preparation from eight fruit species. In many cases, a protocol adapted for polyphenol-rich tissues was superior. Using a suitable protocol for investigations of kiwi fruits, we show that flavanone 3-hydroxylase is absent in the green-fleshed cultivar Hayward. As flavonoid enzyme activities could be detected in harvested kiwi fruits over a storage period of five months, postharvest modification of the flavonoid spectrum has to be expected.