Katharina Wolff

Background: Urtica dioica , also known as stinging nettle, is a widespread plant that can indicate high nitrogen availability in the soil. It is probably best known for the pain caused by touching it. U. dioica is also recognized as a medicinal plant with reports claiming applicability against numerous diseases. Results: A highly continuous genome sequence was constructed based on nanopore long read sequencing data. The total assembly size is 1.1 Gbp with an N50 of 40.7 Mbp. RNA-seq data and hints from other species were integrated to produce a high quality annotation of the protein encoding genes. This genomic resource enabled the identification of genes involved in the flavonoid biosynthesis. A particular focus was on anthocyanin biosynthesis genes as these are crucial for high light and nitrogen deprivation stress response, which is revealed by redding of the leaves. Conclusion: This genomic resource provides the basis for future studies unraveling the biosynthesis pathways underlying various medically important compounds produced by stinging nettles.

Summary: Here, we present a comprehensive multiomics analysis of anthocyanin biosynthesis in Rubus armeniacus , known for its dark fruits. A phased genome sequence of the tetraploid blackberry was generated, achieving an N50 of 34 Mb with an assembly size of 1.2 Gbp based on Oxford Nanopore Technology sequencing (ONT). The BUSCO score for the total assembly shows a high completeness of 99.1%. The assembly was separated into 4 pseudohaplophases, with the pseudohaplophase A representing the R. armeniacus genome in 7 chromosome scale contigs, with an N50 of 46 Mbp and 98.8% conserved BUSCO genes. A total of 118,183 protein coding genes were annotated within the genome assembly and all relevant genes encoding enzymes and transcriptional regulators of the anthocyanin biosynthesis pathway were identified within each pseudohaplophase. To further understand the underlying cause of dark pigmentation, the gene expression was analysed during different stages of berry development revealing a strong induction of anthocyanin biosynthesis genes including the anthocyanin activating subgroup 6 MYB transcriptions during the berry ripening process. Further, a quantification of cyanidin-3-O-glucoside in methanolic berry extract, utilizing a UHPLC-HRAM-MS analysis, revealed an approximately 500-fold increase of cyanidin-3-O-glucoside from green to black fruit, indicating that dark pigmentation in R. armeniacus results from high anthocyanin accumulation. Significance statement: This study provides a multiomics analysis of the dark pigmentation of Rubus armeniacus, including a high quality phased assembly and an in-depth analysis of the anthocyanin biosynthesis pathway. A transcriptional and metabolomic analysis revealed that dark berry pigmentation is caused by a high accumulation of cyanidin-3-O-glucoside during fruit ripening.

Digitalis purpurea (foxglove) is a widely distributed ornamental plant. Here, we present a long read sequencing-based genome sequence of a magenta flowering D. purpurea plant and a corresponding prediction of gene models. The high assembly continuity is indicated by the N50 of 4.3 Mbp and the completeness is supported by discovery of about 96% complete BUSCO genes. This genomic resource paves the way for an in-depth investigation of the flower pigmentation of D. purpurea . Structural genes of the anthocyanin biosynthesis and the corresponding transcriptional regulators were identified. The comparison of magenta and white flowering plants revealed a large insertion in the anthocyanidin synthase gene in white flowering plants that most likely renders this gene non-functional and could explain the loss of anthocyanin pigmentation. Furthermore, we found a large insertion in the DpTFL1/CEN gene to be likely responsible for the development of large terminal flowers.

Protocol optimized for the extraction of RNA out of berries of Rubus armeniacus.

Long-read sequencing has shown a rapid technological development during the last years. It has been established as the standard method for the sequencing of plant genomes and has also gained importance for full plasmid sequencing. As Sanger sequencing has a limited read length of about 1 kb, long read sequencing offers a great advantage, as the full plasmid can be sequenced in one read. Here, we present a cost-effective workflow to sequence full plasmids and compare the results against an expectation. The per plasmid cost of this workflow is determined by the number of plasmids investigated simultaneously, but can be lower than the price of a single Sanger sequencing reaction. We developed a workflow for automatic data processing, which allows us to complete sequencing and data analysis within a day.

Dark pigmentation can be observed in various parts of the plant, ranging from foliage to petals and berries. Here, we review the available knowledge about dark pigmentation in plants and the potential for biotechnological applications. The molecular basis of black pigmentation appears to vary among species, with anthocyanins playing a significant role, although specific anthocyanin types and their mechanisms differ. These findings suggest that the development of phenotypes is species‐specific or varies between larger taxonomic groups; this is further supported by the polyphyletic nature of dark pigmentation. Additionally, several different regulatory mechanisms have been described for the occurrence of dark pigmentation. First, the repression or knockout of the competing flavone biosynthesis has been shown to lead to darker pigmentation while another mechanism is based on the activation and upregulation of the anthocyanin biosynthesis genes in the presence of MYB transcription factors. Potential ecological functions of dark pigmentation were identified as protection of the photosynthesis apparatus, camouflage against herbivores, and the attraction of pollinators. Promising industrial applications include microbial factories for the production of natural food colourants, induction of novel phenotypes for the ornamental plant industry and, lastly, increase of anthocyanins within agriculturally relevant crops. Understanding the genetic basis of dark pigment accumulation would facilitate biotechnological and agricultural applications.

Aquilegia vulgaris is a widespread ornamental plant. The species is well known for its extensive variation of different flower colors and can even be considered as a model species for the evolution of flower morphology. Anthocyanins are a major pigment group responsible for pigmentation of flowers in A. vulgaris and many plant species. Here, we report a highly continuous genome sequence of an European Aquilegia vulgaris plant displaying purple flowers and a genome sequence of a plant displaying white flowers. The corresponding annotation facilitates research on flower color and morphology evolution. Long-read alignments revealed different structural variants in an anthocyanidin synthase ( ANS ) gene, crucial for pigment biosynthesis, that could explain the white flower phenotype. Candidate genes for all steps in the core anthocyanidin biosynthesis were identified. The identification of a flavonoid 3’,5’ hydroxylase ( F3’5’H ), a gene essential for the biosynthesis of bluish delphinidin derivatives, corroborates previous reports about metabolites and transcripts in A. vulgaris .

Theobroma cacao is well known for its role in producing cacao. Many different cacao varieties are cultivated in equatorial regions, each distinguished by unique phenotypic traits. An instance of this is the pod color variation, which has been linked to differences in anthocyanin content. Anthocyanins are produced by one branch of the flavonoid biosynthesis. The highly continuous genome sequence and comprehensive gene set generated in this study enabled an investigation of the genetic basis of the flavonoid biosynthesis. Expression differences between paralogs in the proanthocyanidin biosynthesis suggest subfunctionalization. In pod husks, one copy of the leucoanthocyanidin reductase gene ( LAR ) displayed substantially higher expression than the other copy.

Victoria cruziana is well known for its huge floating leaves covered with sharp spines and its night blooming. Reports indicate that white flowers open during the first night and turn light pinkish during the following day and the second night. Here, we set out to unravel the molecular basis and ecological function of the flower color change in V. cruziana . A high quality genome sequence with a contig N50 of 44.2 Mbp, a scaffold N50 of 300 Mbp, and a total assembly size of 3.5 Gbp was generated as the genetic basis for this study. Comparative transcriptomics revealed the genes required for anthocyanin biosynthesis genes and their transcriptional regulators as differentially expressed between the white and the light pinkish stage of a flower. Structural genes with expression differences between white and light pinkish flower stages include VcrF3’H , VcrF3’5’H , VcrDFR , VcrANS , and VcrarGST . The expression pattern of the corresponding transcription factors VcrMYB123 , VcrMYB-SG6_a , VcrMYB-SG6_b, VcrTT8 , and VcrTTG1 also showed differences that aligned with the flower color.

Digitalis purpurea (foxglove) is a widely distributed ornamental plant. Here, we present a long read sequencing-based genome sequence of a magenta flowering D. purpurea plant and a corresponding prediction of gene models. The high assembly continuity is indicated by the N50 of 4.3 Mbp and the completeness is supported by discovery of about 96% complete BUSCO genes. This genomic resource paves the way for an in-depth investigation of the flower pigmentation of D. purpurea . Structural genes of the anthocyanin biosynthesis and the corresponding transcriptional regulators were identified. The comparison of magenta and white flowering plants revealed a large insertion in the anthocyanidin synthase gene in white flowering plants that most likely renders this gene non-functional and could explain the loss of anthocyanin pigmentation. Furthermore, we found a large insertion in the DpTFL1/CEN gene to be likely responsible for the development of large terminal flowers.