Comprehensive genomic analysis of Burkholderia arboris PN-1 reveals its biocontrol potential against Fusarium solani-induced root rot in Panax notoginseng.

Panax notoginseng (Burkill) F.H. Chen, a valuable traditional Chinese medicine, faces significant yield and quality challenges stemming from root rot primarily caused by Fusarium solani. Burkholderia arboris PN-1, isolated from the rhizosphere soil of P. notoginseng, demonstrated a remarkable ability to inhibit the growth of F. solani. This study integrates phenotypic, phylogenetic, and genomic analyses to enhance our understanding of the biocontrol mechanisms employed by B. arboris PN-1. Phenotype analysis reveals that B. arboris PN-1 effectively suppresses P. notoginseng root rot both in vitro and in vivo. The genome of B. arboris PN-1 comprises three circular chromosomes (contig 1: 3,651,544 bp, contig 2: 1,355,460 bp, and contig 3: 3,471,056 bp), with a 66.81% GC content, housing 7,550 protein-coding genes. Notably, no plasmids were detected. Phylogenetic analysis places PN-1 in close relation to B. arboris AU14372, B. arboris LMG24066, and B. arboris MEC_B345. Average nucleotide identity (ANI) values confirm the PN-1 classification as B. arboris. Comparative analysis with seven other B. arboris strains identified 4,628 core genes in B. arboris PN-1. The pan-genome of B. arboris appears open but may approach closure. Whole-genome sequencing revealed 265 carbohydrate-active enzymes and identified 9 gene clusters encoding secondary metabolites. This comprehensive investigation enhances our understanding of B. arboris genomes, paving the way for their potential as effective biocontrol agents against fungal plant pathogens in the future.

Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown. RESULTS: We carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis. CONCLUSIONS: These results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

Identification of candidate genes and residues for improving nitrogen use efficiency in the N-sensitive medicinal plant Panax notoginseng.

BACKGROUND: Nitrogen (N) metabolism-related key genes and conserved amino acid sites in key enzymes play a crucial role in improving N use efficiency (NUE) under N stress. However, it is not clearly known about the molecular mechanism of N deficiency-induced improvement of NUE in the N-sensitive rhizomatous medicinal plant Panax notoginseng (Burk.) F. H. Chen. To explore the potential regulatory mechanism, the transcriptome and proteome were analyzed and the three-dimensional (3D) information and molecular docking models of key genes were compared in the roots of P. notoginseng grown under N regimes. RESULTS: Total N uptake and the proportion of N distribution to roots were significantly reduced, but the NUE, N use efficiency in biomass production (NUEb), the recovery of N fertilizer (RNF) and the proportion of N distribution to shoot were increased in the N0-treated (without N addition) plants. The expression of N uptake- and transport-related genes NPF1.2, NRT2.4, NPF8.1, NPF4.6, AVP, proteins AMT and NRT2 were obviously up-regulated in the N0-grown plants. Meanwhile, the expression of CIPK23, PLC2, NLP6, TCP20, and BT1 related to the nitrate signal-sensing and transduction were up-regulated under the N0 condition. Glutamine synthetase (GS) activity was decreased in the N-deficient plants, while the activity of glutamate dehydrogenase (GDH) increased. The expression of genes GS1-1 and GDH1, and proteins GDH1 and GDH2 were up-regulated in the N0-grown plants, there was a significantly positive correlation between the expression of protein GDH1 and of gene GDH1. Glu192, Glu199 and Glu400 in PnGS1 and PnGDH1were the key amino acid residues that affect the NUE and lead to the differences in GDH enzyme activity. The 3D structure, docking model, and residues of Solanum tuberosum and P. notoginseng was similar. CONCLUSIONS: N deficiency might promote the expression of key genes for N uptake (genes NPF8.1, NPF4.6, AMT, AVP and NRT2), transport (NPF1.2 and NRT2.4), assimilation (proteins GS1 and GDH1), signaling and transduction (genes CIPK23, PLC2, NLP6, TCP20, and BT1) to enhance NUE in the rhizomatous species. N deficiency might induce Glu192, Glu199 and Glu400 to improve the biological activity of GS1 and GDH, this has been hypothesized to be the main reason for the enhanced ability of N assimilation in N-deficient rhizomatous species. The key genes and residues involved in improving NUE provide excellent candidates for the breeding of medicinal plants.

Biotransformation approach to produce rare ginsenosides F1, compound Mc1, and Rd2 from major ginsenosides.

The stems and leaves of Panax notoginseng contain high saponins, but they are often discarded as agricultural waste. In this study, the predominant ginsenosides Rg1, Rc, and Rb2, presented in the stems and leaves of ginseng plants, were biotransformed into value-added rare ginsenosides F1, compound Mc1 (C-Mc1), and Rd2, respectively. A fungal strain YMS6 (Penicillium sp.) was screened from the soil as a biocatalyst with high selectivity for the deglycosylation of major ginsenosides. Under the optimal fermentation conditions, the yields of F1, C-Mc1, and Rd2 were 97.95, 68.64, and 79.58%, respectively. This study provides a new microbial resource for the selective conversion of protopanaxadiol-type and protopanaxatriol-type major saponins into rare ginsenosides via the whole-cell biotransformation and offers a solution for the better utilization of P. notoginseng waste.

Multi-layered effects of Panax notoginseng on immune system.

Recent research has demonstrated the immunomodulatory potential of Panax notoginseng in the treatment of chronic inflammatory diseases and cerebral hemorrhage, suggesting its significance in clinical practice. Nevertheless, the complex immune activity of various components has hindered a comprehensive understanding of the immune-regulating properties of Panax notoginseng, impeding its broader utilization. This review evaluates the effect of Panax notoginseng to various types of white blood cells, elucidates the underlying mechanisms, and compares the immunomodulatory effects of different Panax notoginseng active fractions, aiming to provide the theory basis for future immunomodulatory investigation.

Acetylcholine Analog-Modified Albumin Nanoparticles for the Enhanced and Synchronous Brain Delivery of Saponin Components of Panax Notoginseng.

BACKGROUND: Panax notoginseng saponins (PNS) are commonly used first-line drugs for treating cerebral thrombosis and stroke in China. However, the synchronized and targeted delivery of active ingredients in traditional Chinese medicine (TCM) poses a significant challenge for modern TCM formulations. METHODS: Bovine serum albumin (BSA) was modified using 2-methacryloyloxyethyl phosphorylcholine (MPC), an analog of acetylcholine, and subsequently adsorbed the major PNS onto the modified albumin to produce MPC-BSA@PNS nanoparticles (NPs). This novel delivery system facilitated efficient and synchronized transport of PNS across the blood-brain barrier (BBB) through active transport mediated by nicotinic acetylcholine receptors. RESULTS: In vitro experiments demonstrated that the transport rates of R1, Rg1, Rb1, and Rd across the BBB were relatively synchronous in MPC-BSA@PNS NPs compared to those in the PNS solution. Additionally, animal experiments revealed that the brain-targeting efficiencies of R1 + Rg1 + Rb1 in MPC-BSA@PNS NPs were 2.02 and 7.73 times higher than those in BSA@PNS NPs and the free PNS group, respectively. CONCLUSIONS: This study presents a simple and feasible approach for achieving the targeted delivery of complex active ingredient clusters in TCM.

PnNAC2 promotes the biosynthesis of Panax notoginseng saponins and induces early flowering.

KEY MESSAGE: PnNAC2 positively regulates saponin biosynthesis by binding the promoters of key biosynthetic genes, including PnSS, PnSE, and PnDS. PnNAC2 accelerates flowering through directly associating with the promoters of FT genes. NAC transcription factors play an important regulatory role in both terpenoid biosynthesis and flowering. Saponins with multiple pharmacological activities are recognized as the major active components of Panax notoginseng. The P. notoginseng flower is crucial for growth and used for medicinal and food purposes. However, the precise function of the P. notoginseng NAC transcription factor in the regulation of saponin biosynthesis and flowering remains largely unknown. Here, we conducted a comprehensive characterization of a specific NAC transcription factor, designated as PnNAC2, from P. notoginseng. PnNAC2 was identified as a nuclear-localized protein with transcription activator activity. The expression profile of PnNAC2 across various tissues mirrored the accumulation pattern of total saponins. Knockdown experiments of PnNAC2 in P. notoginseng calli revealed a significant reduction in saponin content and the expression level of pivotal saponin biosynthetic genes, including PnSS, PnSE, and PnDS. Subsequently, Y1H assays, dual-LUC assays, and electrophoretic mobility shift assays (EMSAs) demonstrated that PnNAC2 exhibits binding affinity to the promoters of PnSS, PnSE and PnDS, thereby activating their transcription. Additionally, an overexpression assay of PnNAC2 in Arabidopsis thaliana witnessed the acceleration of flowering and the induction of the FLOWERING LOCUS T (FT) gene expression. Furthermore, PnNAC2 demonstrated the ability to bind to the promoters of AtFT and PnFT genes, further activating their transcription. In summary, these results revealed that PnNAC2 acts as a multifunctional regulator, intricately involved in the modulation of triterpenoid saponin biosynthesis and flowering processes.

First Report of Panax notoginseng Leaf spot Disease Caused by Boeremia exigua in China.

Panax notoginseng a perennial herb native to China, is widely grown in the Yunnan Province. (Yang et al. 2022). From July to August 2022, a new leaf spot disease was observed on fully expanded leaves of P. notoginseng from a planting base in the Xundian, Yunnan Province, China. Approximately 250 ha. of P. notoginseng is the cultivated area, and the incidence of leaf spot disease was around 10-15%. Round spots appeared on the infected leaves and as the disease progressed these leaves fell off the plant. A total 21 symptomatic leaves were randomly collected from the planting base to isolate the pathogens and further study in the laboratory. The surface of infected leaves were sanitized with 0.5% sodium hypochlorite for 2 min. and 75% alcohol for 1 min., and then rinsed thrice with sterile water. Once drying, the samples were placed on potato dextrose agar (PDA), plates and incubated at 25 °C for 5 days. The fungus was isolated from the symptomatic tissue, but only three isolates were preserved for further identification. Pure cultures of the representative strain Zhaochanglin 118 were obtained using the singlespore method, and the colonies obtained were dark-green to dark-black in appearance. The pycnidia were dark brown, solitary, or congregated with an inconspicuous neck. The conidia were colorless, ellipsoidal, and measured between 4.5 to 7 × 2 to 3 µm (n = 30). These morphological characteristics were similar to those described for Boeremia exigua (Valenzuela-Lopezi et al. 2018). The genomic DNA of the isolate was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit. The internal transcribed spacer (ITS), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF1) genes were amplified via polymerase chain reaction using the primers ITS1/ITS4 (White et al. 1990), Af/Cf (Matheny et al. 2002), and EF1-983F/EF1-2218R (Chen et al. 2015), respectively. All sequences were deposited in GenBank (OQ996531 for ITS; OR291158 for RPB2 and OR291159 for TEF1). A BLASTN homology search using the ITS nucleotide sequence indicated that this has 99.6% identity with the sequence MH859059, named B. exigua from CBS culture collection (517/519 bp); the RPB2 sequence has 97.5% identity with sequence GU371780, named B. exigua from CBS culture collection (704/722 bp); and the TEF1 sequence has 98.4% identity with sequence GU349080, named B. exigua from CBS culture collection (871/885 bp). To test Koch's postulates, a pathogenicity test was carried out on the leaves of six fully expanded P. notoginseng plants in the Xundian planting base. Conidial suspensions were prepared for one isolates at a concentration of 106 spores per milliliter. Three leaflets on different plants were applied with 20µl spore suspension and the other three leaflets were drop of 20 µl sterile distilled water. The whole experiment was repeated three times. The P. notoginseng plants were incubated under sterile conditions at 25°C for 7 days. Inoculated leaves showed the characteristic brown round spots, while control leaves were asymptomatic so, Koch's postulates were fulfilled by re-isolating the pathogen from symptomatic tissue, which was subsequently confirmed as B. exigua through morphological and molecular analyses. Koch's postulates were fulfilled. To our knowledge, this is the first report of B. exigua causing leaf spot disease in P. notoginseng in China, which lays a foundation for further study and developing disease control methods.

Alleviatory Role of Panax Notoginseng Saponins in Modulating Inflammation and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease: mechanisms and Implications.

COPD is an inflammatory lung disease that limits airflow and remodels the pulmonary vascular system. This study delves into the therapeutic potential and mechanistic underpinnings of Panax notoginseng Saponins (PNS) in alleviating inflammation and pulmonary vascular remodeling in a COPD rat model. Symmap and ETCM databases provided Panax notoginseng-related target genes, and the CTD and DisGeNET databases provided COPD-related genes. Intersection genes were subjected to protein-protein interaction analysis and pathway enrichment to identify downstream pathways. A COPD rat model was established, with groups receiving varying doses of PNS and a Roxithromycin control. The pathological changes in lung tissue and vasculature were examined using histological staining, while molecular alterations were explored through ELISA, RT-PCR, and Western blot. Network pharmacology research suggested PNS may affect the TLR4/NF-κB pathway linked to COPD development. The study revealed that, in contrast to the control group, the COPD model exhibited a significant increase in inflammatory markers and pathway components such as TLR4, NF-κB, HIF-1α, VEGF, ICAM-1, SELE mRNA, and serum TNF-α, IL-8, and IL-1ß. Treatment with PNS notably decreased these markers and mitigated inflammation around the bronchi and vessels. Taken together, the study underscores the potential of PNS in reducing lung inflammation and vascular remodeling in COPD rats, primarily via modulation of the TLR4/NF-κB/HIF-1α/VEGF pathway. This research offers valuable insights for developing new therapeutic strategies for managing and preventing COPD.

Changes in chemical components and hepatoprotective effect of red Panax notoginseng processed by aspartic acid impregnation treatment.

BACKGROUND: Red Panax notoginseng (RPN) is one of the major processed products of P. notoginseng (PN), with more effective biological activities. However, the traditional processing method of RPN has some disadvantages, such as low conversion rate of ginsenosides and long processing time. RESULTS: In this work, we developed a green, safe, and efficient approach for RPN processing by aspartic acid impregnation pretreatment. Our results showed that the optimized temperature, steaming time, and concentration of aspartic acid were 120 °C, 1 h, and 3% respectively. The original ginsenosides in PN treated by aspartic acid (Asp-PN) were completely converted to rare saponins at 120 °C within just 1 h. The concentration of the rare ginsenosides in Asp-PN was two times higher than that in untreated RPN. In addition, we examined the protective effect of RPN and Asp-PN on acetaminophen-induced liver injury in a mouse model. The results showed that Asp-PN has significantly more potent hepatoprotective action than the RPN. The hepatoprotection of Asp-PN in acetaminophen-induced hepatotoxicity may be due to its anti-oxidative stress, anti-apoptotic, and anti-inflammatory activities. CONCLUSION: These results indicated that aspartic acid impregnation pretreatment may provide an effective method to shorten the steaming time, improve the conversion rate of ginsenosides, and enhance hepatoprotective activity of RPN. © 2024 Society of Chemical Industry.

[Identification of cardioprotective substances in Panax ginseng/P. notoginseng based on mitochondrial morphological characteristics and UPLC-Triple-TOF-MS].

Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength, and P. notoginseng can resolve blood stasis and alleviate pain. P. ginseng and P. notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels. Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology, and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases. In this study, primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R). MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei, respectively. Fluorescence images were then acquired using ImageXpress Micro Confocal. Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus. Subsequently, representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function. The active compounds of P. ginseng and P. notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters. The findings demonstrated that RS-2, RS-4, SQ-1, and SQ-4 significantly increased the values of three key morphometric parameters, including mitochondrial length, branching, and area, which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury. Among the active components of the two medicinal herbs, 20(R)-ginsenoside Rg_3, ginsenoside Re, and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes. Specifically, 20(R)-ginsenoside Rg_3 might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2), and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression. Collectively, they promoted mitochondrial membrane fusion and mitigated mitochondrial damage, thereby exerting protective effects on cardiomyocytes. This study provides experimental support for the discovery of novel therapeutic agents for myocardial ischemia-reperfusion injury from P. ginseng and P. notoginseng and offers a novel approach for large-scale screening of bioactive compounds with cardioprotective effects from traditional Chinese medicines.

[Mechanism of astragaloside â £ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification].

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 µmol·L~(-1)) + PNS(30 µmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P<0.05) and the cell damage rate in the brain tissue(P<0.05), promoted the expression of vWF and VEGFA(P<0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P<0.05, P<0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.05, P<0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P<0.05,P<0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

Genome-wide identification and characterization of members of the LEA gene family in Panax notoginseng and their transcriptional responses to dehydration of recalcitrant seeds.

BACKGROUND: Late embryogenesis abundant (LEA) proteins play an important role in dehydration process of seed maturation. The seeds of Panax notoginseng (Burkill) F. H. Chen are typically characterized with the recalcitrance and are highly sensitive to dehydration. However, it is not very well known about the role of LEA proteins in response to dehydration stress in P. notoginseng seeds. We will perform a genome-wide analysis of the LEA gene family and their transcriptional responses to dehydration stress in recalcitrant P. notoginseng seeds. RESULTS: In this study, 61 LEA genes were identified from the P. notoginseng genome, and they were renamed as PnoLEA. The PnoLEA genes were classified into seven subfamilies based on the phylogenetic relationships, gene structure and conserved domains. The PnoLEA genes family showed relatively few introns and was highly conserved. Unexpectedly, the LEA_6 subfamily was not found, and the LEA_2 subfamily contained 46 (75.4%) members. Within 19 pairs of fragment duplication events, among them 17 pairs were LEA_2 subfamily. In addition, the expression of the PnoLEA genes was obviously induced under dehydration stress, but the germination rate of P. notoginseng seeds decreased as the dehydration time prolonged. CONCLUSIONS: We found that the lack of the LEA_6 subfamily, the expansion of the LEA_2 subfamily and low transcriptional levels of most PnoLEA genes might be implicated in the recalcitrant formation of P. notoginseng seeds. LEA proteins are essential in the response to dehydration stress in recalcitrant seeds, but the protective effect of LEA protein is not efficient. These results could improve our understanding of the function of LEA proteins in the response of dehydration stress and their contributions to the formation of seed recalcitrance.

Panax notoginseng transcription factor WRKY15 modulates resistance to Fusarium solani by up-regulating osmotin-like protein expression and inducing JA/SA signaling pathways.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is a valuable traditional Chinese medicinal plant, but its commercial production is seriously affected by root rot caused by some pathogenic fungi, including Fusarium solani. Nevertheless, the genetic breeding for disease resistance of P. notoginseng remains limited. The WRKY transcription factors have been revealed to play important roles in plant defense responses, which might provide an inspiration for resistance improvement in P. notoginseng. RESULTS: In this study, the regulatory mechanism of transcription factor PnWRKY15 on P. notoginseng resistance to F. solani infection was revealed. The suppressed expression of PnWRKY15 via RNA interference increased the sensitivity of P. notoginseng to F. solani and decreased the expression levels of some defense-related genes, including PnOLP1, which encodes an osmotin-like protein that confers resistance to F. solani. Ectopic expression of PnWRKY15 in the model plant tobacco significantly enhanced the resistance to F. solani. Moreover, the transcriptome sequencing analysis discovered that some pathogenesis-related genes were expressed at higher levels in the PnWRKY15-overexpressing tobacco than that in the wild-type tobacco. In addition, the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were evidently induced by PnWRKY15-overexpression, that was evidenced by that the JA and SA contents were significantly higher in the PnWRKY15-overexpressing tobacco than that in the wild-type. Furthermore, PnWRKY15, which was localized in the nucleus, can trans-activate and up-regulate PnOLP1 expression according to the EMSA, yeast one-hybrid and co-expression assays. CONCLUSIONS: PnWRKY15 contributes to P. notoginseng resistance to F. solani by up-regulating the expression of resistance-related gene PnOLP1 and activating JA/SA signaling pathways. These findings will help to further elucidate the transcriptional regulatory mechanism associated with the P. notoginseng defense response to F. solani.

Exogenous gibberellic acid shortening after-ripening process and promoting seed germination in a medicinal plant Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F.H. Chen is an essential plant in the family of Araliaceae. Its seeds are classified as a type of morphophysiological dormancy (MPD), and are characterized by recalcitrance during the after-ripening process. However, it is not clear about the molecular mechanism on the after-ripening in recalcitrant seeds. RESULTS: In this study, exogenous supply of gibberellic acid (GA3) with different concentrations shortened after-ripening process and promoted the germination of P. notoginseng seeds. Among the identified plant hormone metabolites, exogenous GA3 results in an increased level of endogenous hormone GA3 through permeation. A total of 2971 and 9827 differentially expressed genes (DEGs) were identified in response to 50 mg L-1 GA3 (LG) and 500 mg L-1 GA3 (HG) treatment, respectively, and the plant hormone signal and related metabolic pathways regulated by GA3 was significantly enriched. Weighted gene co-expression network analysis (WGCNA) revealed that GA3 treatment enhances GA biosynthesis and accumulation, while inhibiting the gene expression related to ABA signal transduction. This effect was associated with higher expression of crucial seed embryo development and cell wall loosening genes, Leafy Contyledon1 (LEC1), Late Embryogenesis Abundant (LEA), expansins (EXP) and Pectinesterase (PME). CONCLUSIONS: Exogenous GA3 application promotes germination and shorts the after-ripening process of P. notoginseng seeds by increasing GA3 contents through permeation. Furthermore, the altered ratio of GA and ABA contributes to the development of the embryo, breaks the mechanical constraints of the seed coat and promotes the protrusion of the radicle in recalcitrant P. notoginseng seeds. These findings improve our knowledge of the contribution of GA to regulating the dormancy of MPD seeds during the after-ripening process, and provide new theoretical guidance for the application of recalcitrant seeds in agricultural production and storage.

Panax notoginseng: derived exosome-like nanoparticles attenuate ischemia reperfusion injury via altering microglia polarization.

Cerebral ischemia/reperfusion (CI/R) injury is a clinical conundrum during the treatment of ischemic stroke. Cell-derived exosomes (CDE) were proved to be therapeutically effective for CI/R injury. However, production of CDE is time and effort consuming. Increasing studies reported that plants can also generate exosome-like nanoparticles (ELN) which are therapeutically effective and have higher yield compared with CDE. In this study, a commonly used Chinese herb Panax notoginseng (PN), whose active ingredients were well-documented in the treatment of CI/R injury, was chosen as a source of ELNs. It was found that Panax notoginseng derived exosome like nanoparticles (PDN) could enter the brain without modification and ameliorate cerebral infarct volume, improve behavior outcome and maintained the integrity of BBB. PDNs attenuated CI/R injury by altering the phenotype of microglia from "pro-inflammation" M1 type to "anti-inflammation" M2 type. Also, we found that lipids from PDNs were the major therapeutic effective component. As a mechanism of action, PDN was proved to exert therapeutic effect via activating pI3k/Akt pathway.

Biotransformation of the saponins in Panax notoginseng leaves mediated by gut microbiota from insomniac patients.

Saponins extracted from Panax notoginseng leaves by methanol or water could be orally administrated for insomnia with very low bioavailability, which might be bio-converted by gut microbiota to generate potential bioactive products. Moreover, gut microbiota profiles from insomniac patients are very different from healthy subjects. We aimed to compare the metabolic characteristics and profiles of the two saponins extract by incubation with gut microbiota from insomniac patients. The ginsenosides, notoginsenosides, and metabolites were identified and relatively quantified by high-performance liquid chromatography-tandem mass spectrometry. Gut microbiota was profiled by 16S ribosomal RNA gene sequencing. The results showed that saponins were very different between methanol or water extract groups, which were metabolized by gut microbiota to generate similar yields. The main metabolites included ginsenoside Rd, ginsenoside F2 , ginsenoside C-Mc or ginsenoside C-Y, ginsenoside C-Mx, ginsenoside compound K, and protopanaxadiol in both groups, while gypenoside XVII, notoginsenoside Fe, ginsenoside Rd2 , and notoginsenoside Fd were the intermediates in the methanol group. Moreover, the microbial, Faecalibacterium prausnitzi, could bio-convert the saponins to obtain the corresponding metabolites. Our study implied that saponins extracted from P. notoginseng leaves by methanol or water could be used for insomniac patients due to gut microbiota biotransformation.

The Potential Relevance of PnDREBs to Panax notoginseng Nitrogen Sensitiveness.

The dehydration response element-binding (DREB) transcription factor is a subfamily of AP2/ERF. It actively responds to various abiotic stresses in plants. As one of the representative plants, Panax notoginseng is sensitive to Nitrogen (N). Here, bioinformatics analysis, the identification, chromosomal location, phylogeny, structure, cis-acting elements, and collinearity of PnDREBs were analyzed. In addition, the expression levels of PnDREBs were analyzed by quantitative reverse transcription PCR. In this study, 54 PnDREBs were identified and defined as PnDREB1 to PnDREB54. They were divided into 6 subfamilies (A1-A6). And 44 PnDREBs were irregularly distributed on 10 of 12 chromosomes. Each group showed specific motifs and exon-intron structures. By predicting cis-acting elements, the PnDREBs may participate in biotic stress, abiotic stress, and hormone induction. Collinear analysis showed that fragment duplication events were beneficial to the amplification and evolution of PnDREB members. The expression of PnDREBs showed obvious tissue specificity in its roots, flowers, and leaves. In addition, under the action of ammonium nitrogen and nitrate nitrogen at the 15 mM level, the level of PnDREB genes expression in roots varied to different degrees. In this study, we identified and characterized PnDREBs for the first time, and analyzed that PnDREBs may be related to the response of P. Notoginseng to N sensitiveness. The results of this study lay a foundation for further research on the function of PnDREBs in P. Notoginseng.

A new Panax notoginseng Leaf Spot Caused by Boeremia linicola in Yunnan, China.

Panax notoginseng-also known as Tianqi and Sanqi-is one of the most highly valued medicinal perennial herbs in the world (Wang et al. 2016). In August 2021, leaf spot was observed on P. notoginseng leaves in Lincang sanqi base (23º43´10˝N, 100º7´32˝E, 13.33 hm2). Symptoms expanded from water soaked areas on the leaves to form irregular round or oval leaf spots with transparent or grayish-brown centers containing black granular matter, with an incidence of 10 to 20%. To identify the causal agent, ten symptomatic leaves were randomly selected from ten P. notoginseng plants. Symptomatic leaves were cut into small pieces (5 mm2) with asymptomatic tissue margins, disinfected in 75% ethanol for 30s and in 2% sodium hypochlorite for 3 min, and rinsed three times with sterile distilled water. The tissue portions were placed on potato dextrose agar (PDA) plates incubated at 20℃ with a 12 h light/dark photoperiod. Seven pure isolates were obtained with similar colony morphology, dark gray (top view) or taupe (back view) coloration, with flat and villous surfaces. Pycnidia were globose to subglobose, glabrous or with few mycelial outgrowths, dark brown to black, 22.46 to 155.94 (av. 69.57) µm × 18.20 to 130.5 (av. 57.65) µm (n=50) in size. Conidia were ellipsoidal to cylindrical, thinwalled, smooth, hyaline, aseptate, and measured 1.47 to 6.81 (av. 4.29) µm long and 1.01 to 2.97 (av. 1.98) µm thick (n=100). The isolated strains were preliminarily identified as Boeremia sp. based on the morphological characteristics of colonies and conidia. (Aveskamp et al. 2010; Schaffrath et al. 2021). To confirm pathogen identity, the total genomic DNA of two isolates (LYB-2 and LYB-3) was extracted using the T5 Direct PCR kit. The internal transcribed spacer (ITS), 28S large subunit nrRNA gene (LSU), and ß-tubulin (TUB2) gene regions were PCR-amplified using primers ITS1/ITS4, LR0Rf/LR5r, and BT2F/BT4R (Chen et al. 2015), respectively. Sequences have been deposited in GenBank (ON908942-ON908943 for ITS, ON908944-ON908945 for LSU, ON929285-ON929286 for TUB2). BLASTn searches of generated DNA sequences from 2 purified isolates (LYB-2 and LYB-3) against GenBank showed high similarity (>99%) with the sequences of Boeremia linicola. Moreover, a phylogenetic tree was constructed based on the neighbor-joining method in MEGA-X (Kumar et al. 2018) and revealed that the 2 isolates were closest to B. linicola (CBS 116.76). Pathogenicity tests were conducted with the 2 isolates (LYB-2 and LYB-3) as described by Cai et al. (2009) with slight modifications. Each isolate was inoculated with three healthy annual P. notoginseng plants, and each leaf was inoculated with three drops of conidia suspension (106 spores/mL). Three P. notoginseng plants inoculated with sterile water were used as controls. All plants were covered with plastic bags incubated in a greenhouse (20℃, 90%RH, 12 h light/dark photoperiod). Fifteen days post-inoculation, all inoculated leaves showed similar lesions, and the symptoms were identical to those in the field. The pathogen was reisolated from symptomatic leaf spots, and the colony characteristics were identical to the original isolates. Control plants remained healthy, and no fungus was re-isolated. Morphological characteristics, sequence alignment and pathogenicity tests confirmed that B. linicola was the cause of P. notoginseng leaf spot disease. This is the first report of B. linicola causing leaf spot on P. notoginseng in Yunnan, China. The identification of B. linicola as the causal agent of the observed leaf spot on P. notoginseng is critical to the prevention and control of this disease in the future.

First Report of Panax notoginseng Wilt Disease Caused by Fusarium graminearum in China.

Panax notoginseng is one of the important economic crops under the forest, which is widely planted in Yunnan Province, China. In August of 2022, a survey in Xundian county (25º26' N, 12 103°7' E), was accomplished to verify the occurrence of wilt disease in P. notoginseng and understand its aetiology. The site is an underforest of organic P. notoginseng, covering an area of over 40 ha. Disease symptoms included severe stunting, leaf chlorosis, red or yellow stalks, and rotting roots. The entire plant gradually wilted and died with disease progression (Fig. 1). To identify the causal agent, we collected more than 30 wilted P. notoginseng plants and got the plant tissues from the symptomatic leaves, stalks, and roots. The tissues surface sterilised with 0.5% sodium hypochlorite for 2 min, followed by 75% alcohol for 1 min, and rinsed in sterilised water three times. Upon drying, samples were placed onto potato dextrose agar (PDA) incubated in the dark at 25°C (Bilgi et al. 2011). Isolates were then transferred to carnation leaf agar (CLA) to induce sporulation. Colonies on PDA were yellow, orange to red, with abundant fluffy aerial mycelia with a dark red pigment on the undersides; Colonies on CLA were orange to yellow (Fig. 2). Fusiform macroconidia and bottle-shaped conidiogenous cells were visible under a microscope. Microconidia were not observed. Macroconidia were measured as 18.5-40.5 µm × 3-4.7 µm (n = 60) (Fig. 3), and possessed 2 to 6 septa. These are similar to previously reported morphological characteristics of Fusarium graminearum (Shikur et al. 2018; Martinez et al. 2019). Cetyltrimethylammonium bromide rapid plant genome extraction kit-DN14 was used to obtain genomic DNA from two representative isolate, the ITS, TEF1 and RPB2 gene were amplified by Polymerase Chain Reaction using primers ITS5/ITS4 (White et al, 1990), EF1-983F/EF1-2218R (Rehner et al, 2005), bRPB2-6F/bRPB2-7.1R (Matheny et al, 2002), respectively. BLAST homology search for nucleotide sequences revealed > 99% similarity to F. graminearum ITS (550bp; MG274308, KU847854), TEF1 (1000bp; MH572248, MH572252) and RPB2 (1000bp; KT855203, KT855206) sequences. All sequences from this study were deposited in GenBank (OP617343 and OP617344 for ITS; OP930951 and OP930952 for TEF1; OP930953 and OP930954 for RPB2). In the phylogenetic tree, the isolates (SWFU 0000116, SWFU 0000117) clustered with the representative strains of F. graminearum. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. graminearum. Koch's postulates were used to confirm that the symptoms in wilted P. notoginseng were attributable to F. graminearum. First, healthy leaves were gently wounded with a needle and sprayed with spore suspension (1.0 × 106 spores mL-1) in a hand sprayer (Martinez et al, 2019). All P. notoginseng plants were then replanted in pots with a diameter of 20 cm (1 plants/pot) filled with mixture of sterilised soil, and incubated at 25-27°C. The blank control comprised sterile cotton soaked in sterile water and inactivated mycelia sprayed on the leaves. After 7d of incubation, all inoculated leaves and stalks developed necrosis and developed pale red mycelia, while control plants remained symptomless (Fig. 4-5). The pathogen was successfully isolated from these inoculated plants and identified as F. graminearum. Koch's postulates were implemented. To the best of our knowledge, this is the first report from China with evidence of F. graminearum infecting P. notoginseng.