Butylphthalide is one of the first-line drugs for ischemic stroke therapy, while no biosynthetic enzyme for butylphthalide has been reported. Here, we present a haplotype-resolved genome of Ligusticum chuanxiong , a long-cultivated and phthalide-rich medicinal plant in Apiaceae. On the basis of comprehensive screening, four Fe(II)- and 2-oxoglutarate–dependent dioxygenases and two CYPs were mined and further biochemically verified as phthalide C-4/C-5 desaturases (P4,5Ds) that effectively promoted the forming of ( S )-3- n -butylphthalide and butylidenephthalide. The substrate promiscuity and functional redundancy featured for P4,5Ds may contribute to the high phthalide diversity in L. chuanxiong . Notably, comparative genomic evidence supported L. chuanxiong as a homoploid hybrid with Ligusticum sinense as a potential parent. The two haplotypes demonstrated exceptional structure variance and diverged around 3.42 million years ago. Our study is an icebreaker for the dissection of phthalide biosynthetic pathway and reveals the hybrid origin of L. chuanxiong , which will facilitate the metabolic engineering for ( S )-3- n -butylphthalide production and breeding for L. chuanxiong .

Furochromones are specific bioactive secondary metabolites of many Apiaceae plants. Their biosynthesis remains largely unexplored. In this work, we dissect the complete biosynthetic pathway of major furochromones in the medicinal plant Saposhnikovia divaricata by characterizing prenyltransferase, peucenin cyclase, methyltransferase, hydroxylase, and glycosyltransferases. De novo biosynthesis of prim-O-glucosylcimifugin and 5-O-methylvisamminoside is realized in Nicotiana benthamiana leaves. Through comparative genomic and transcriptomic analyses, we further find that proximal duplication and high expression of a pentaketide chromone synthase gene SdPCS, together with the presence of a lineage-specific peucenin cyclase gene SdPC, lead to the predominant accumulation of furochromones in the roots of S. divaricata among surveyed Apiaceae plants. This study paves the way for metabolic engineering production of furochromones, and sheds light into evolutionary mechanisms of furochromone biosynthesis among Apiaceae plants.

Sauropus androgynus (S. androgynus) (2n = 4x = 52) is one of the most popular functional leafy vegetables in South and Southeast Asia. With its rich nutritional and pharmaceutical values, it has traditionally had widespread use for dietary and herbal purposes. Here, the genome of S. androgynus was sequenced and assembled, revealing a genome size of 1.55 Gb with 26 pseudo-chromosomes. Phylogenetic analysis traced back the divergence of Sauropus from Phyllanthus to approximately 29.67 million years ago (Mya). Genome analysis revealed that S. androgynus polyploidized around 20.51 Mya and shared a γ event about 132.95 Mya. Gene function analysis suggested that the expansion of pathways related to phloem development, lignin biosynthesis, and photosynthesis tended to result in the morphological differences among species within the Phyllanthaceae family, characterized by varying ploidy levels. The high accumulation of ascorbic acid in S. androgynus was attributed to the high expression of genes associated with the L-galactose pathway and recycling pathway. Moreover, the expanded gene families of S. androgynus exhibited multiple biochemical pathways associated with its comprehensive pharmacological activity, geographic adaptation and distinctive pleasurable flavor. Altogether, our findings represent a crucial genomic asset for S. androgynus, casting light on the intricate ploidy within the Phyllanthaceae family.

Oxalis articulata is a widely distributed perennial herb known for its adaptability to diverse environmental conditions. Here, we report the first haplotype-resolved, chromosome-scale genome assembly for O. articulata (Oxalidaceae), generated through PacBio HiFi long reads and Hi-C technology. The resulting assembly comprises two haplotypes with sizes of 377.04 Mb (contig N50 = 31.13 Mb) and 342.70 Mb (contig N50 = 47.46 Mb), respectively. We predicted 36,063 protein-coding genes (94.33% functionally annotated) and 38,292 protein-coding genes (93.80% functionally annotated) in the two haplotypes, respectively. This high-quality genome assembly lays a solid groundwork for future initiatives in ecological and biochemical research for O. articulata.

Butylphthalide, a prescription medicine recognized for its efficacy in treating ischemic strokes approved by the State Food and Drug Administration of China in 2005, is sourced from the traditional botanical remedy Ligusticum chuanxiong. While chemical synthesis offers a viable route, limitations in the production of isomeric variants with compromised bioactivity necessitate alternative strategies. Addressing this issue, biosynthesis offers a promising solution. However, the intricate in vivo pathway for butylphthalide biosynthesis remains elusive. In this study, we examined the distribution of butylphthalide across various tissues of L. chuanxiong and found a significant accumulation in the rhizome. By searching transcriptome data from different tissues of L. chuanxiong, we identified four rhizome-specific genes annotated as 2-oxoglutarate-dependent dioxygenase (2-OGDs) that emerged as promising candidates involved in butylphthalide biosynthesis. Among them, LcSAO1 demonstrates the ability to catalyze the desaturation of senkyunolide A at the C-4 and C-5 positions, yielding the production of butylphthalide. Experimental validation through transient expression assays in Nicotiana benthamiana corroborates this transformative enzymatic activity. Notably, phylogenetic analysis of LcSAO1 revealed that it belongs to the DOXB clade, which typically encompasses genes with hydroxylation activity, rather than desaturation. Further structure modelling and site-directed mutagenesis highlighted the critical roles of three amino acid residues, T98, S176, and T178, in substrate binding and enzyme activity. By unraveling the intricacies of the senkyunolide A desaturase, the penultimate step in the butylphthalide biosynthesis cascade, our findings illuminate novel avenues for advancing synthetic biology research in the realm of medicinal natural products.

Prunus mahaleb is widely utilized as rootstock for cherries in Central Asia due to its exceptional resistance to environmental stressors. In this study, we generated a haplotype-resolved, chromosome-scale genome assembly for P. mahaleb using PacBio HiFi long reads and Hi-C technology. The resulting assembly comprises two haplotypes with sizes of 272.64 Mb (contig N50 = 26.60 Mb) and 271.76 Mb (contig N50 = 30.26 Mb), respectively. We identified 27,965 protein-coding genes (27,627 functionally annotated) and 27,931 protein-coding genes (27,607 functionally annotated) in the two haplotypes, respectively. Additionally, we annotated 145.64 Mb repetitive elements, 2,263 rRNAs, 430 tRNAs, and 780 other non-coding RNAs in haplotype A, and 144.04 Mb repetitive elements, 1,999 rRNAs, 442 tRNAs, and 734 other non-coding RNAs in haplotype B. Further analysis indicated that these haplotypes diverged approximately 2.67 million years ago. This high-quality genome assembly lays a solid groundwork for future initiatives in molecular breeding and functional genomics research for P. mahaleb.