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.

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.

Cucumber mosaic virus impairs the physiological homeostasis of Panax notoginseng and induces saponin-mediated resistance.

As an important medicinal plant, Panax notoginseng often suffers from various abiotic and biotic stresses during its growth, such as drought, heavy metals, fungi, bacteria and viruses. In this study, the symptom and physiological parameters of cucumber mosaic virus (CMV)-infected P. notoginseng were analyzed and the RNA-seq was performed. The results showed that CMV infection affected the photosynthesis of P. notoginseng, caused serious oxidative damage to P. notoginseng and increased the activity of several antioxidant enzymes. Results of transcriptome analysis and corresponding verification showed that CMV infection changed the expression of genes related to plant defense and promoted the synthesis of P. notoginseng saponins to a certain extent, which may be defensive ways of P. notoginseng against CMV infection. Furthermore, pretreatment plants with saponins reduced the accumulation of CMV. Thus, our results provide new insights into the role of saponins in P. notoginseng response to virus infection.

Integrated metabolome and transcriptome analysis reveals the regulatory mechanism of low nitrogen-driven biosynthesis of saponins and flavonoids in Panax notoginseng.

BACKGROUND: Nitrogen (N) is an important macronutrient involved in the biosynthesis of primary and secondary metabolites in plants. However, the metabolic regulatory mechanism of low-N-induced triterpenoid saponin and flavonoid accumulation in rhizomatous medicinal Panax notoginseng (Burk.) F. H. Chen remains unclear. METHODS: To explore the potential regulatory mechanism and metabolic basis controlling the response of P. notoginseng to N deficiency, the transcriptome and metabolome were analysed in the roots. RESULTS: The N content was significantly reduced in roots of N0-treated P. notoginseng (0 kg·N·667 m-2). The C/N ratio was enhanced in the N-deficient P. notoginseng. N deficiency promotes the accumulation of amino acids (L-proline, L-leucine, L-isoleucine, L-norleucine, L-arginine, and L-citrulline) and sugar (arabinose, xylose, glucose, fructose, and mannose), thus providing precursor metabolites for the biosynthesis of flavonoids and triterpenoid saponins. Downregulation of key structural genes (PAL, PAL3, ACC1, CHS2, PPO, CHI3, F3H, DFR, and FGT), in particular with the key genes of F3H, involved in the flavonoid biosynthesis pathway possibly induced the decrease in flavonoid content with increased N supply. Notoginsenoside R1, ginsenoside Re, Rg1, Rd, F1, R1 + Rg1 + Rb1 and total triterpenoid saponins were enhanced in the N0 groups than in the N15 (15 kg·N·667 m-2) plants. Higher phosphoenolpyruvate (an intermediate of glycolyticwith pathway metabolism) and serine (an intermediate of photorespiration) levels induced by N deficiency possibly promote saponin biosynthesis through mevalonic acid (MVA) and methylerythritol (MEP) pathways. Genes (MVD2, HMGS, HMGR1, HMGR2, DXR, and HMGR1) encoding the primary enzymes HMGS, HMGR, DXR, and MVD in the MVA and MEP pathways were significantly upregulated in the N0-treated P. notoginseng. The saponin biosynthesis genes DDS, DDS, CYP716A52, CYP716A47, UGT74AE2, and FPS were upregulated in the N-deficient plants. Upregulation of genes involved in saponin biosynthesis promotes the accumulation of triterpenoid saponins in the N0-grown P. notoginseng. CONCLUSIONS: N deficiency enhances primary metabolisms, such as amino acids and sugar accumulation, laying the foundation for the synthesis of flavonoids and triterpenoid saponins in P. notoginseng. F3H, DDS, FPS, HMGR, HMGS and UGT74AE2 can be considered as candidates for functional characterisation of the N-regulated accumulation of triterpenoid saponins and flavonoids in future.

The mitochondrial genomes of Panax notoginseng reveal recombination mediated by repeats associated with DNA replication.

Panax notoginseng is one of the most valuable medicinal species. However, its mitochondrial genome has not been reported yet. We aimed to determine the mitogenome sequence of P. notoginseng. We de novo assembled the mitogenome with Illumina short reads and Nanopore long reads. The mitochondrial genome of P. notoginseng has a multipartite structure consisting of interconversion between a "master circle" and numerous "subgenomic circles" through recombinations mediated by 64 pairs of repetitive sequences. Among the multipartite structure, seven subgenomic circles were best supported. Six of the seven subgenomic circles shared an 852 bp conserved fragment. The complete mitogenome of P. notoginseng was 662,479 bp long including 34 mitochondrial protein-coding genes (PCGs), three rRNA, and 19 tRNA genes. We identified 166 microsatellite repeats and 26 long-tandem repeats. Phylogenetic analysis resolved a tree that was mostly congruent with the phylogeny of Apiales species described in the APG IV system and the tree built with the chloroplast genome sequences. A total of 12 mitochondrial plastid DNA fragments were identified. Lastly, we predicted 591C-to-U RNA editing sites in the coding regions of mitochondrial PCGs. The mitochondrial genome will lay the foundation for understanding the evolution of Panax species.

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.

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.

Genome-wide identification and expression analysis of MYB gene family under nitrogen stress in Panax notoginseng.

The myeloblastosis (MYB) gene family, involved in regulating many important physiological and biochemical processes, is one of the largest transcript factor superfamilies in plants. Since the identification of genome sequencing of Panax notoginseng has been completed, there was little known about the whole genome of its specific MYB gene family and the response to abiotic stresses, in consideration of the excessive application of nitrogen fertilizers in P. notoginseng. In this study, 123 PnMYB genes (MYB genes of P. notoginseng) have been identified and divided into 3 subfamilies by the phylogenetic analysis. These PnMYB genes were unevenly located on 12 chromosomes. Meanwhile, the gene structure and protein conserved domain were established by MEME Suite. The analysis of collinear relationships reflected that there were 121 homologous genes between P. notoginseng and Arabidopsis and 30 between P. notoginseng and rice. Moreover, cis-acting elements of PnMYB gene promoters were predicted which indicated that PnMYBs are involved in biotic, abiotic stress, and hormone induction. The expressions of PnMYB transcription factors in its roots, flowers, and leaves were detected by qRT-PCR and they had tissue-specific expressions and related to the growth of different tissues. Under nitrogen stress, MYB transcription factors had great feedback. Ten R2R3-MYB subfamily genes were significantly induced and indicated the possible function of protecting P. notoginseng from excess nitrogen. With further knowledge on identification of PnMYB gene related to tissue selectivity and abiotic stresses, this study laid the foundation for the functional development of PnMYB gene family and improved the cultivation of P. notoginseng.

Multi-algorithm cooperation research of WRKY genes under nitrogen stress in Panax notoginseng.

WRKY transcription factors play an important role in the immune system and the innate defense response of plants. WRKY transcription factors have great feedback on nitrogen stress. In this study, bioinformatics was used to detect the WRKYs of Panax notoginseng (PnWRKYs). The response of PnWRKYs under nitrogen stress was also well studied. PnWRKYs were distributed on 11 chromosomes. According to PnWRKY and Arabidopsis thaliana WRKY (AtWRKY) domains, these PnWRKY proteins were divided into three groups by phylogenetic analysis. MEME analysis showed that almost every member contained motif 1 and motif 2. PlantCARE online predicted the cis-acting elements of the promoter. PnWRKY gene family members obtained 22 pairs of repeat fragments by collinearity analysis. The expression levels of PnWRKYs in different parts (roots, flowers, and leafs) were analyzed by the gene expression pattern. They reflected tissue-specific expressions. The qRT-PCR experiments were used to detect 74 PnWRKYs under nitrogen stress. The results showed that the expression levels of 8 PnWRKYs were significantly induced. The PnWRKY gene family may be involved in biotic/abiotic stresses and hormone induction. This study will not only lay the foundation to explore the functions of PnWRKYs but also provide candidate genes for the future improvement of P. notoginseng.

Biogas slurry application alters soil properties, reshapes the soil microbial community, and alleviates root rot of Panax notoginseng.

Background: Panax notoginseng is an important herbal medicine in China, where this crop is cultivated by replanting of seedlings. Root rot disease threatens the sustainability of P. notoginseng cultivation. Water flooding (WF) is widely used to control numerous soilborne diseases, and biogas slurry shows positive effects on the soil physiochemical properties and microbial community structure and has the potential to suppress soilborne pathogens. Hence, biogas slurry flooding (BSF) may be an effective approach for alleviating root rot disease of P. notoginseng; however, the underlying mechanism needs to be elucidated. Methods: In this study, we conducted a microcosm experiment to determine if BSF can reduce the abundance of pathogens in soil and, alleviate root rot of P. notoginseng. Microcosms, containing soil collected from a patch of P. notoginseng showing symptoms of root rot disease, were subjected to WF or BSF at two concentrations for two durations (15 and 30 days), after which the changes in their physicochemical properties were investigated. Culturable microorganisms and the root rot ratio were also estimated. We next compared changes in the microbial community structure of soils under BSF with changes in WF and untreated soils through high-throughput sequencing of bacterial 16S rRNA (16S) and fungal internal transcribed spacer (ITS) genes amplicon. Results: WF treatment did not obviously change the soil microbiota. In contrast, BSF treatment significantly altered the physicochemical properties and reshaped the bacterial and fungal communities, reduced the relative abundance of potential fungal pathogens (Fusarium, Cylindrocarpon, Alternaria, and Phoma), and suppressed culturable fungi and Fusarium. The changes in the microbial community structure corresponded to decreased root rot ratios. The mechanisms of fungal pathogen suppression by BSF involved several factors, including inducing anaerobic/conductive conditions, altering the soil physicochemical properties, enriching the anaerobic and culturable bacteria, and increasing the phylogenetic relatedness of the bacterial community. Conclusions: BSF application can reshape the soil microbial community, reduce the abundance of potential pathogens, and alleviate root rot in P. notoginseng. Thus, it is a promising practice for controlling root rot disease in P. notoginseng.

Panaxydol Derived from Panax notoginseng Promotes Nerve Regeneration after Sciatic Nerve Transection in Rats.

BACKGROUND: Peripheral nerve regeneration is a coordinated process of Schwann cell (SC) reprogramming and intrinsic neuronal growth program activation. Panaxydol (PND) is a strong biologically active traditional Chinese medicine monomer extracted from Panax notoginseng rhizomes. In vitro, PND protects neurons and SCs from injury and stimulates the expression and secretion of neurotrophic factors (NTFs) by SCs. We hypothesized that PND may also promote peripheral nerve regeneration in adult animals. METHODS: PND (10 mg/kg body weight) was injected intraperitoneally into the Sprague-Dawley (SD) rats for two consecutive weeks after sciatic nerve transection. The morphology of the repaired sciatic nerve was evaluated after 16 weeks, and sensory and motor function recovery was evaluated using functional and behavioral techniques. RESULTS: PND was biologically safe at an injection dose of 10 mg/kg/day. After 14 days, it significantly increased the myelination of regenerated nerve fibers, and promoted sensory and motor function recovery. In the early stage of injury, PND significantly upregulated the mRNA expression of brain-derived neurotrophic factor (BDNF) and its receptors in distal injured nerves, which may represent a possible mechanism by which PND promotes nerve regeneration in vivo. CONCLUSIONS: Our study demonstrated that PND leads to sensory and motor recovery in a sciatic nerve transection model rat. Furthermore, we showed that BDNF mRNA level was significantly increased in the injured distal nerve, potentially contributing to the functional recovery. Further research is warrantied to examine whether direct injection is a more efficient method to increase BDNF expression compared to an exogenous BDNF administration.

Multi-algorithm cooperation comprehensive research of bZIP genes under nitrogen stress in Panax notoginseng.

Basic leucine zipper (bZIP) transcription factors play an irreplaceable position in the regulation of plant secondary metabolism, growth and development, and resistance to abiotic stress. Panax notoginseng is a traditional medicinal plant in China, but the systematic identification and the resistance of Panax notoginseng bZIP (PnbZIP) family under nitrogen stress have not been reported before, considering the excessive application of N fertilizers. In this study, we conducted a genome-wide identification of the PnbZIP family and analyzed its phylogeny, tissue selectivity, and abiotic resistence. 74 PnbZIPs were distributed on 12 chromosomes and 8 were not successfully located. Through phylogenetic analysis of Arabidopsis and Panax notoginseng, we divided them into 14 subgroups. In the same subgroup, bZIPs had similiar intron/exon structure and conserved motifs. In the analysis of chromosome structure, two PnbZIP genes were duplicated in tandem on chromosome 3. Intraspecific collinearity analysis showed that 28 PnbZIPs participated in segmental replication. Each PnbZIP promoter contained at least one stress response element or stress-related hormone response element. RNA-seq and qRT-PCR methods were used to analyze the expression patterns of the PnbZIP gene in different tissues (roots, flowers, and leaves) and under different nitrogen stresses. The results showed that the PnbZIP gene had the highest expression level in flowers and reflected tissue-specific expressions. Meanwhile, under the stress of ammonium nitrogen fertilizer and nitrate nitrogen fertilizer, PnbZIPs in roots were differently expressed. 10 PnbZIP stress-responsive genes were screened for significant expression, among which PnbZIP46 was significantly up-regulated, which could be a candidate gene for resistance to Nitrogen stress. This study laid the foundation for functional identification of PnbZIPs and improved the cultivation of Panax notoginseng.

Determination of the Authenticity and Origin of Panax Notoginseng: A Review.

Panax notoginseng, a traditional medicinal and edible plant, is widely used in medicine, health care, cosmetics, and other industries. Affected by the discrepancy between market supply and demand and price, the adulteration of P. notoginseng products with other plant-derived ingredients occurs at times. With the continuous development of technologies such as spectroscopy, chromatography, and DNA barcoding, the detection techniques for rapid and sensitive determination of the authenticity identification and origin of P. notoginseng have become more diversified to meet the needs of different regulatory goals and could effectively control practices that mislead consumers and promote false labeling. This review analyzes and summarizes the existing technologies for determining the authenticity and origin of P. notoginseng from these three aspects: morphological, chemical, and molecular biology methods from the literature since 2001; on this basis, the current problems and future research directions are discussed to provide a reference for the establishment of rapid and accurate methods to verify authenticity and origin to promote the further development and improvement of quality control technology systems for P. notoginseng.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Structural and interactions analysis of a transcription factor PnMYB2 in Panax notoginseng.

The main active ingredients of the traditional Chinese medicinal plant, Panax notoginseng, are the Panax notoginseng saponins (PNS). They can be synthesized via the mevalonate pathway; PnSS and PnSE1 are the key rate-limiting enzymes in this pathway. In this study, an interaction between PnMYB2 and the key enzymes was identified and characterized from the P. notoginseng cDNA library using the Y1H technique. Subsequently, X-α-gal color reaction confirmed the interaction between PnMYB2 and the upstream sequences of PnSS and PnSE1 promoters. Full-length cDNA sequence of PnMYB2 was isolated and characterized. PnMYB2 has an open reading frame of 864 bp, encoding 287 amino acids. 3D structural analysis of PnMYB2 indicated that its structure was similar to that of the template. Phylogenetic analysis revealed that PnMYB2 and PgMYB2 are highly homologous and belong to the R2R3 MYB transcription factor (TF). Subcellular localization analysis showed that PnMYB2 was localized in the nucleus. The recombinant protein PnMYB2 was successfully obtained through prokaryotic expression and was confirmed to be an inclusion body protein. Furthermore, electrophoretic mobility shift assay (EMSA) experiments demonstrated that PnMYB2 specifically binds to MYB core and AC-rich elements. This study provides a theoretical basis for transcriptional regulation of saponin biosynthesis in P. notoginseng.

Characteristics and diversity of endophytic bacteria in Panax notoginseng under high temperature analysed using full-length 16S rRNA sequencing.

Panax notoginseng is a traditional Chinese medicinal herb with diverse properties that is cultivated in a narrow ecological range because of its sensitivity to high temperatures. Endophytic bacteria play a prominent role in plant response to climate warming. However, the endophytic bacterial structures in P. notoginseng at high temperatures are yet unclear. In the present study, the diversity and composition of the endophytic bacterial community, and their relationships with two P. notoginseng plants with different heat tolerance capacities were compared using the full-length 16S rRNA PacBio sequencing system. The results revealed that the diversity and richness of endophytic bacteria were negatively associated with the heat tolerance of P. notoginseng. Beneficial Cyanobacteria, Rhodanobacter and Sphingomonas may be recruited positively by heat-tolerant plants, while higher amounts of adverse Proteobacteria such as Cellvibrio fibrivorans derived from soil destructed the cellular protective barriers of heat-sensitive plants and caused influx of pathogenic bacteria Stenotrophomonas maltophilia. Harmonious and conflicting bacterial community was observed in heat-tolerant and heat-sensitive P. notoginseng, respectively, based on the co-occurrence network. Using functional gene prediction of metabolism, endophytic bacteria have been proposed to be symbiotic with host plants; the bacteria improved primary metabolic pathways and secondary metabolite production of plants, incorporated beneficial endophytes, and combated adverse endophytes to prompt the adaptation of P. notoginseng to a warming environment. These findings provided a new perspective on the function of endophytes in P. notoginseng adaptation to high temperatures, and could pave the way for expanding the cultivable range of P. notoginseng.

Transcriptome analysis reveals putative pathogenesis genes in Alternaria panax during infecting Panax notoginseng leaves.

BACKGROUND: Alternaria panax is the causative agent of black spot disease in Panax notoginseng, which causes significant yield loss. However, the molecular mechanisms underlying its pathogenicity remain mostly unknown. OBJECTIVE: We sequenced the transcriptome of A. panax during infecting P. notoginseng leaves using next-generation RNA-seq to understand the molecular aspects of black spot disease. METHODS: In this study, we sequenced the A. panax transcriptome during infecting P. notoginseng leaves through next-generation sequencing to explore the pathogenesis genes that may be responsible for black spot disease on P. notoginseng. RESULT: The de novo transcriptome assembly of A. panax produced 23,036 unigenes, of which 18,096 genes were functionally annotated by at least one protein database. GO enrichment analysis and KEGG pathways of differentially up-regulated genes suggest that most genes are associated with metabolic processes, catalytic activity, starch, and sucrose metabolism during infection. Many pathogenesis-associated genes, including genes encoding secreted proteins, candidate secreted effectors, cell wall degrading enzymes, transcription factors, and transporters, were up-regulated in A. panax during infection. In addition, the secondary metabolite biosynthesis genes, including cytochrome P450, and nonribosomal peptide synthetases, were also identified in this study. CONCLUSIONS: Differential gene expression analysis has confirmed that A. panax infection was mainly present in the middle and final stages. The findings show that these pathogenesis-associated genes in A. panax may be critical for the P. notoginseng black spots disease.

Binding proteins PnCOX11 and PnDCD strongly respond to GA and ABA in Panax notoginseng.

Panax notoginseng saponins (PNS) are one of the main active ingredients of Panax notoginseng, a representative plant of the genus Panax. However, the detailed regulation mechanism of PNS biosynthesis remains elusive. Therefore, a sequence of upstream promoters of PnSS and PnSE were cloned and analyzed firstly. GUS quantitative results showed that the upstream promoters could specifically and significantly respond to exogenous GA and ABA signals. To further identify the binding proteins that respond to peripheral hormones, PnCOX11 and PnDCD were screened and identified from the P. notoginseng cDNA library. The Y1H experiment verified the interaction between the above two binding proteins and the promoters. Several online software was used to analyze the domains, secondary structures, three-dimensional structures, and phylogenetic trees of the two binding proteins. Subcellular localization analysis exhibited that PnCOX11 was mainly located in the chloroplast, while PnDCD was located in the cytoplasm and nucleus. Prokaryotic expression demonstrated that the recombinant proteins had a high concentration under the induction of IPTG. This study can provide a fundamental date for the subsequent thorough investigation of the transcription regulatory mechanism of PNS biosynthesis.