Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong.

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.

LcSAO1, an Unconventional DOXB Clade 2OGD Enzyme from Ligusticum chuanxiong Catalyzes the Biosynthesis of Plant-Derived Natural Medicine Butylphthalide.

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.

Multiomics comparison among populations of three plant sources of Amomi Fructus.

Amomi Fructus (Sharen, AF) is a traditional Chinese medicine (TCM) from three source species (or varieties), including Wurfbainia villosa var. villosa (WVV), W. villosa var. xanthioides (WVX), or W. longiligularis (WL). Among them, WVV has been transplanted from its top-geoherb region, Guangdong, to its current main production area, Yunnan, for >50 years in China. However, the genetic and transcriptomic differentiation among multiple AF source species (or varieties) and between the origin and transplanted populations of WVV is unknown. In our study, the observed overall higher expression of terpenoid biosynthesis genes in WVV than in WVX provided possible evidence for the better pharmacological effect of WVV. We also screened six candidate borneol dehydrogenases (BDHs) that potentially catalyzed borneol into camphor in WVV and functionally verified them. Highly expressed genes at the P2 stage of WVV, Wv05G1424 and Wv05G1438, were capable of catalyzing the formation of camphor from (+)-borneol, (-)-borneol and DL-isoborneol. Moreover, the BDH genes may experience independent evolution after acquiring the ancestral copies, and the following tandem duplications might account for the abundant camphor content in WVV. Furthermore, four populations of WVV, WVX, and WL are genetically differentiated, and the gene flow from WVX to WVV in Yunnan contributed to the greater genetic diversity in the introduced population (WVV-JH) than in its top-geoherb region (WVV-YC), which showed the lowest genetic diversity and might undergo genetic degradation. In addition, terpene synthesis (TPS) and BDH genes were selected among populations of multiple AF source species (or varieties) and between the top- and non-top-geoherb regions, which might explain the difference in metabolites between these populations. Our findings provide important guidance for the conservation, genetic improvement, and industrial development of the three source species (or varieties) and for identifying top-geoherbalism with molecular markers, and proper clinical application of AF.

The chromosome-level genome of female ginseng (Angelica sinensis) provides insights into molecular mechanisms and evolution of coumarin biosynthesis.

Coumarins are natural products with important medicinal values, and include simple coumarins, furanocoumarins and pyranocoumarins. Female ginseng (Angelica sinensis) is a renowned herb with abundant coumarins, originated in China and known for the treatment of female ailments for thousands of years. The molecular basis of simple coumarin biosynthesis in A. sinensis and the evolutionary history of the genes involved in furanocoumarin biosynthesis are largely unknown. Here, we generated the first chromosome-scale genome of A. sinensis. It has a genome size of 2.37 Gb, which was generated by combining PacBio and Hi-C sequencing technologies. The genome was predicted to contain 43 202 protein-coding genes dispersed mainly on 11 pseudochromosomes. We not only provided evidence for whole-genome duplication (WGD) specifically occurring in the Apioideae subfamily, but also demonstrated the vital role of tandem duplication for phenylpropanoid biosynthesis in A. sinensis. Combined analyses of transcriptomic and metabolomic data revealed key genes and candidate transcription factors regulating simple coumarin biosynthesis. Furthermore, phylogenomic synteny network analyses suggested prenyltransferase genes involved in furanocoumarin biosynthesis evolved independently in the Moraceae, Fabaceae, Rutaceae and Apiaceae after ζ and ε WGD. Our work sheds light on coumarin biosynthesis, and provides a benchmark for accelerating genetic research and molecular breeding in A. sinensis.

Antiproliferative effect of panaxynol on RASMCs via inhibition of ERK1/2 and CREB.

Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.

Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms.

Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.

In vitro anti-coxsackievirus B(3) effect of ethyl acetate extract of Tian-hua-fen.

AIM: To investigation the anti-coxsackievirus B(3) (CVB(3m)) effect of the ethyl acetate extract of Tian-hua-fen on HeLa cells infected with CVB(3m). METHODS: HeLa cells were infected with CVB(3m) and the cytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A(600) was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A(600). RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration. Consistent results were got through these two methods. We also investigated the mechanism of its anti-CVB(3m) effect and the results indicated that the extract represented an inhibitory effect through all the processes of CVB(3m) attachment, entry, biosynthesis and assemble in cells. CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significant protective effect on HeLa cells infected with CVB(3m) in a dose-dependent manner and this effect exists through the process of CVB(3m) attachment, entry, biosynthesis and assemble in cells, suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.