Jana Thill

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4‐deoxyaurone formation in Asteraceae was so far unknown. Three novel full‐length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80 kb (cgAUS1) and 1.85 kb (cgAUS2a, 2b), encoding for proteins of 68–69 kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9 kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS–PAGE and shot‐gun type nanoUHPLC–ESI‐MS/MS.

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.

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.

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 (K cat/K m values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa.

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.

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

BackgroundMore 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.ResultsBlack 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.ConclusionsAn 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.

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

Main conclusionIn contrast to current knowledge, theB-ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3′ position by flavonoid 3’-hydroxylase.AbstractThe 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.