First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

The CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia

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

Züchtung von Weihnachtssternen mit neuer Blütenfarbe mittels Genome editing

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersKumulative Dissertation aus fünf ArtikelnPoinsettia (Euphorbia pulcherrima) commonly shows red bract colouration caused by theaccumulation of cyanidin-type anthocyanins. Orange-red cultivars, which accumulatepelargonidin-type anthocyanins occur rarely, whereas blue poinsettias are not naturally presentdue to the lack of delphinidin formation. Breeding is an arduous process, and a lot of effort isnecessary to obtain plants with desired traits. Some phenotypes are not possible to achieve byclassical breeding approaches due to limitations in the genetic background of the plants.Molecular breeding techniques like genome editing can help to overcome those obstacles andobtain novel poinsettia varieties with orange or blue colour in a fast and efficient way. Thecolour of anthocyanin pigments depends on the B-ring hydroxylation pattern and ranges fromorange pelargonidin (one hydroxyl group), dark red to pink cyanidin (two hydroxyl groups)and blue to violet delphinidin (three hydroxyl groups). Two factors are crucial for theestablishment of the B-ring hydroxylation pattern. First, the presence or absence of flavonoid3’-hydroxylase (F3’H) and flavonoid 3’5’-hydroxylase (F3’5’H) activity, and second, thesubstrate specificity of dihydroflavonol 4-reductase (DFR), which provides importantintermediates in the formation of anthocyanins. In this thesis, the physiological background ofcolour formation was studied in petunia as a model plant and in red, and orange poinsettiacultivars. Based on this, a strategy for molecular breeding of orange and blue poinsettia wasestablished. The investigations on the molecular background of pigment formation in orange16

Acyanic white-tipped yellow dahlia unexpectedly expresses a full set of anthocyanin pathway genes in the white tips

Europäische KommissionFonds zur Förderung der Wissenschaftlichen Forschun

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

Europäische Kommissio

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

Fonds zur Förderung der Wissenschaftlichen ForschungEuropäische Kommissio

Metabolite investigations of poinsettia cultivars

Europäische Kommissio

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

European Union's Horizon 202

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