Background: Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols. Results: An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLS_Y) and DFR with N133 (DFR_N). In contrast, dicots generally possess FLS_H and DFR_N, DFR_D, and DFR_A. DFR_A, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time. Conclusion: This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies.

Flavonoids and carotenoids are pigments involved in stress mitigation and numerous other processes. Both pigment classes can contribute to flower and fruit coloration. Flavonoid aglycones and carotenoids are produced by a pathway that is largely conserved across land plants. Glycosylations, acylations, and methylations of the flavonoid aglycones can be species-specific and lead to a plethora of biochemically diverse flavonoids. We previously developed KIPEs for the automatic annotation of biosynthesis pathways and presented an application on the flavonoid aglycone biosynthesis. KIPEs3 is an improved version with additional features and the potential to identify not just the core biosynthesis players, but also candidates involved in the decoration steps and in the transport of flavonoids. Functionality of KIPEs3 is demonstrated through the analysis of the flavonoid biosynthesis in Arabidopsis thaliana Nd-1, Capsella grandiflora, and Dioscorea dumetorum. We demonstrate the applicability of KIPEs to other pathways by adding the carotenoid biosynthesis to the repertoire. As a technical proof of concept, the carotenoid biosynthesis was analyzed in the same species and Daucus carota. KIPEs3 is available as an online service to enable access without prior bioinformatics experience. KIPEs3 facilitates the automatic annotation and analysis of biosynthesis pathways with a consistent and high quality in a large number of plant species. Numerous genome sequencing projects are generating a huge amount of data sets that can be analyzed to identify evolutionary patterns and promising candidate genes for biotechnological and breeding applications.

Withanolides are steroidal lactones from nightshade (Solanaceae) plants with untapped drug potential due to limited availability of minor representatives caused by lack of biosynthetic pathway knowledge. Here, we combine phylogenomics with metabolic engineering to overcome this limitation. By sequencing the genome of the medicinal plant ashwagandha (Withania somnifera) and comparing it with nine Solanaceae species, we discover a conserved withanolide biosynthesis gene cluster, consisting of two sub gene clusters with differing expression patterns. We establish metabolic engineering platforms in yeast (Saccharomyces cerevisiae) and the model plant Nicotiana benthamiana to reconstitute the first five oxidations of withanolide biosynthesis, catalysed by the cytochrome P450 monooxygenases CYP87G1, CYP88C7, and CYP749B2 and a short-chain dehydrogenase/reductase, producing the aglycone of withanoside V. Enzyme functions are conserved within both sub gene clusters in W. somnifera and between W. somnifera and Physalis pruinosa. Our work sets the basis for biotechnological withanolide production to unlock their pharmaceutical potential.

Poaceae is a species-rich family of plants in the monocots harboring many important crop species. As grasses, these plants lack striking flowers that are typically pigmented by anthocyanins. Here, we investigated the anthocyanin biosynthesis genes in Poaceae and characterized several striking patterns. (1) An independently evolved MYB lineage (ncaMYB) is responsible for the regulation of the anthocyanin biosynthesis in Poaceae. (2) The independently evolved BZ2 lineage replaced the otherwise widespread TT19/An9 lineage as anthocyanin-related glutathione S-transferase in Poaceae. (3) The anthocyanin biosynthesis genes ANS, arGST (BZ2) , and the anthocyanin biosynthesis activating MYB (ncaMYB) appear to be absent from the genus Brachypodium . These observations could be explained by a loss of the anthocyanin biosynthesis at the basis of the Poaceae , followed by an evolutionarily independent regain and again a loss of the anthocyanin biosynthesis in Brachypodium .

Summary: Plant pigmentation secrets are among the oldest interests of plant scientists, with pigments such as chlorophyll, carotenoids, flavonoids, and betalains contributing to the diversity of hues in higher plants. Anthocyanins, a class of flavonoids responsible for vibrant shades of pink, red, and blue pigmentation, are almost ubiquitous in angiosperms but are replaced by betalains in some families in the order Caryophyllales. We investigated anthocyanin pigmentation in Cucurbitaceae, one of the largest fruit and vegetable families, characterised by white and yellow flowers and red, orange, and green fruits predominantly pigmented by carotenoids. Using a comprehensive collection of 258 datasets representing 183 unique species across all 15 tribes of Cucurbitaceae, along with a phylogenomics approach, we observed a systematic absence of genes involved in anthocyanin and proanthocyanidin biosynthesis. Absence of the structural genes DFR , ANS , arGST , LAR , and ANR , along with the anthocyanin-related regulatory MYB genes, was consistently confirmed by synteny and phylogenetic analysis. These results suggest that anthocyanin loss in angiosperms is more common than previously assumed. In light of this new discovery, we propose a stepwise loss of anthocyanin pigmentation in Cucurbitaceae, likely accompanied by a partial functional replacement with carotenoid pigmentation.

SUMMARY: Whereas genome sequencing and assembly technologies are improving, cost can still be prohibitive for plant species with large, complex genomes. As a consequence, genomics work on some taxa in evolutionarily pivotal positions in the vascular plant tree of life has been hampered. The species‐rich genus Ranunculus (Ranunculaceae) is an important angiosperm group for the study of polyploidy, apomixis, and reticulate evolution. However, neither mitochondrial nor high‐quality nuclear genome sequences are available. This limits phylogenomic, functional, and taxonomic analyses thus far. Here, we tested Illumina short‐read, Oxford Nanopore Technology (ONT) and PacBio (HiFi) long‐read, and hybrid‐read assembly strategies. We sequenced the diploid progenitor species R. cassubicifolius ( R. auricomus species complex) and selected the best assemblies in terms of completeness, contiguity, and quality scores. We first assembled the plastome (156 kbp, 85 genes) and mitogenome (1.18 Mbp, 40 genes) sequences using Illumina and Illumina‐PacBio‐hybrid strategies, respectively. We also present an updated plastome and the first mitogenome phylogeny of Ranunculaceae, including studies of gene loss (e.g., infA , ycf15 , or rps ) with evolutionary implications. For the nuclear genome sequence, we favored a PacBio‐based assembly polished three times with filtered short reads and subsequently scaffolded into eight pseudochromosomes by chromatin conformation data (Hi‐C). We obtained a haploid genome sequence of 2.69 Gbp, with 94.1% complete BUSCO genes found and 35 482 annotated genes, and inferred ancient gene duplications compared to existing Ranunculales genomes. The genomic information presented here will enable advanced evolutionary‐functional analyses for the species complex, but also for the genus and beyond Ranunculaceae.