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.Peer Reviewe

Reinvestigating substrate specificity of Dihydroflavonol 4-reductase of Petunia × hybrid.

Europäische Kommissio

Molecular studies on the chalcone synthase deficient unstablebicolored Dahlia variabilis

Fonds zur Förderung der Wissenschaftlichen ForschungEuropäische Kommissio

Transcriptome studies deliver multiple candidate sequences for chalcone reductase in the Asteraceae species

Österreichische ForschungsförderungsgesellschaftEuropäische Kommissio

Undeclared genetically engineered orange petunias harbour a special variant of the maize dihydroflavonol 4-reductase

Fonds zur Förderung der Wissenschaftlichen ForschungEuropäische Kommissio

Functionally active recombinant flavonoid 3’-hydroxylase from a pelargonidin accumulating poinsettia cultivar

European Union's Horizon 202

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

Alteration of the phenylpropanoid pathway by watercore disorder in apple (Malus x domestica)

Watercore is a physiological disorder of apple expressed as fluid deposition in intercellular spaces. We analysed the watercore prone cultivar \u27Fuji\u27 to establish a link between enzymatic response and fluid accumulation in the fruit. Individual phenolic content, selected enzyme activities of the phenylpropanoid pathway, sugars and organic acids were quantified in watercore affected flesh and peel. In addition, transcriptome sequencing was performed to obtain a first insight into molecular mechanisms underlying the physiological disorder. Sampled material included: peel (HP) and flesh from healthy fruit (H), peel from watercore affected fruit (WP), healthy flesh from fruit with watercore (HW) and watercore flesh (WW) from fruit with watercore. A sorbitol increase in watercore affected apple was observed not only in the flesh but also in the peel. Moreover, two phenolic groups (hydroxycinnamic acids and flavonols) were significantly affected by watercore and their content was higher in WP in comparison to HP. Dihydrochalcone content was the highest in WW flesh. This was supported by RNA-seq data, which indicated a generally increased expression of genes from the early flavonoid pathway, sorbitol metabolism and polyphenol oxidases. Among the phenylpropanoid pathway enzymes analysed, phloretin 2′-O-glycosyltransferase was the only one exhibiting altered activity

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