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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02103-5.

Stem Cells as a Source of Pancreatic Cells for Production of 3D Bioprinted Bionic Pancreas in the Treatment of Type 1 Diabetes.

Type 1 diabetes (T1D) is the third most common autoimmune disease which develops due to genetic and environmental risk factors. Often, intensive insulin therapy is insufficient, and patients require a pancreas or pancreatic islets transplant. However, both solutions are associated with many possible complications, including graft rejection. The best approach seems to be a donor-independent T1D treatment strategy based on human stem cells cultured in vitro and differentiated into insulin and glucagon-producing cells (ß and α cells, respectively). Both types of cells can then be incorporated into the bio-ink used for 3D printing of the bionic pancreas, which can be transplanted into T1D patients to restore glucose homeostasis. The aim of this review is to summarize current knowledge about stem cells sources and their transformation into key pancreatic cells. Last, but not least, we comment on possible solutions of post-transplant immune response triggered stem cell-derived pancreatic cells and their potential control mechanisms.

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.

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia.

In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the T-DNA integration sites in different GM petunia cultivars and breeding lines. By means of genome walking, we isolated different T-DNA sequences, which are located at the junctions between the T-DNA(s) and the petunia DNA. Based on the results obtained we conclude that there are at least two T-DNA copies of different lengths. This is supported by Southern blot analysis. For T-DNA1, the 3'-junction sequence was isolated, whereas the 5'-junction remained unclear. In contrast, for T-DNA2, the 5'-junction sequence was isolated, whereas the sequence isolated from the 3'-region consists only of T-DNA, but did not include the junction from the T-DNA to the petunia DNA. We developed primers for event-specific PCRs and screened a set of three orange GM petunia cultivars and 126 GM offspring from a commercial breeding program. We show that both T-DNA copies are present in all our tested GM petunia samples, which underpins the assumption of a single transgenic origin of the undeclared GM petunia. Most likely, the two T-DNAs are integrated in close proximity into the petunia genome.

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.