Fig. 2: Identification of putative enzymes involved in cepharanthine biosynthesis.

A Phylogenetic trees of known enzyme genes and candidate Stephania genes, including norcoclaurine synthase (NCS), O-methyltransferase (OMT), N-methyltransferase (NMT), and cytochrome P450 (CYP450). B Collinearity among the three Stephania species, namely, Stephania japonica (Sjap), Stephania yunnanensis (Syun), and Stephania cepharantha (Scep). Gray lines represent synteny patterns between the genomic regions of neighboring Stephania genomes. Locations of key enzymes in the cepharanthine biosynthetic pathway, namely, NCS, sterol-methyltransferase (SMT), OMT, NMT, and CYP450, are highlighted with colored points. C Illustration of a putative biosynthetic gene cluster (BGC) located in S. japonica chromosome 3 and S. cepharantha chromosome 4. The topologically associating domains (TADs) present in this cluster are conserved between these two species. The blue band with a star denotes the location of a BGC identified using plantiSMASH. Synteny of four biosynthetic genes is highlighted with dark red links. D Illustration of a putative BGC located in S. japonica chromosome 2 and S. cepharantha chromosome 13. The red arrow denotes the position at which the boundary of the S. cepharantha TAD projected onto its corresponding S. japonica TAD. E Expression of BGC genes. Circled numbers denote their locations in Panels C and D. F Enrichment of candidate biosynthetic genes in the same TAD. Violin plots depict TAD count distributions of 1,000 permutations sampled from orthologs or all genes. Dashed red lines denote TAD counts of candidate genes, that is, 17 in S. japonica and 21 in S. cepharantha (box plot represents median and 25th and 75th percentiles-interquartile range; IQR-and whiskers extend to maximum and minimum values; n = 1000 permutations; statistical analysis: two-sided Wilcox test). G Proposed biosynthetic pathway for BIA in Stephania species. Each cube represents the concentration of its closest compound in different tissues, namely, the root, stem, and leaf, of three Stephania species. H Genomic location of two syntenic regions. NCS genes were denoted as irregular pentagons, whereas other genes were denoted as vertical lines. Ortholog gene pairs were linked with dotted lines. I Product profiles of NCS using 4 and 5 as substrates. CcNCS1, NCS gene of Coptis chinensis whose enzymatic activity is known. The experiment has been repeated three times with similar results. J Phylogenetic relationship and expression of NCS genes. Bootstrap values (>50) were labeled in the corresponding branches. Source data are provided as a Source Data file.