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.

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.

De novo and comparative transcriptomic analysis explain morphological differences in Panax notoginseng taproots.

BACKGROUND: Panax notoginseng (Burk.) F. H. Chen (PN) belonging to the genus Panax of family Araliaceae is widely used in traditional Chinese medicine to treat various diseases. PN taproot, as the most vital organ for the accumulation of bioactive components, presents a variable morphology (oval or long), even within the same environment. However, no related studies have yet explained the molecular mechanism of phenotypic differences. To investigate the cause of differences in the taproot phenotype, de novo and comparative transcriptomic analysis on PN taproot was performed. RESULTS: A total of 133,730,886 and 114,761,595 paired-end clean reads were obtained based on high-throughput sequencing from oval and long taproot samples, respectively. 121,955 unigenes with contig N50 = 1,774 bp were generated by using the de novo assembly transcriptome, 63,133 annotations were obtained with the BLAST. And then, 42 genes belong to class III peroxidase (PRX) gene family, 8 genes belong to L-Ascorbate peroxidase (APX) gene family, and 55 genes belong to a series of mitogen-activated protein kinase (MAPK) gene family were identified based on integrated annotation results. Differentially expressed genes analysis indicated substantial up-regulation of PnAPX3 and PnPRX45, which are related to reactive oxygen species metabolism, and the PnMPK3 gene, which is related to cell proliferation and plant root development, in long taproots compared with that in oval taproots. Furthermore, the determination results of real-time quantitative PCR, enzyme activity, and H2O2 content verified transcriptomic analysis results. CONCLUSION: These results collectively demonstrate that reactive oxygen species (ROS) metabolism and the PnMPK3 gene may play vital roles in regulating the taproot phenotype of PN. This study provides further insights into the genetic mechanisms of phenotypic differences in other species of the genus Panax.

Overexpression of PnMYB2 from Panax notoginseng induces cellulose and lignin biosynthesis during cell wall formation.

MAIN CONCLUSION: Panax notoginseng PnMYB2 is a transcriptional activator of primary and secondary cell wall formation by promoting the PCW-specific gene CesA3 and key lignin biosynthetic gene CCoAOMT1, respectively. R2R3-MYB transcription factors play important roles in regulation secondary cell wall (SCW) formation. However, there are few reports on the functions of MYB transcription factors which involved in both primary cell wall (PCW) and SCW formation. Here, we isolated an R2R3-MYB transcription factor, PnMYB2, from Panax notoginseng roots which are widely used in Chinese traditional medicines and contain abundant cellulose and lignin. The expression pattern of PnMYB2 was similar to the accumulation pattern of cellulose and lignin contents in different organs. PnMYB2 localized in the nucleus and may function as a transcriptional activator. Overexpression of PnMYB2 in Arabidopsis thaliana enhanced cellulose and lignin biosynthesis, and remarkably increased thickness of PCW and SCW in the stem of transgenic plants compared with wild-type plants. The expression levels of genes associated with PCW-specific cellulose synthase (CesA) genes and key SCW-specific lignin biosynthetic genes were significantly increased in PnMYB2-overexpressing plants compared to the wild type plants. Furthermore, yeast one-hybrid, dual-luciferase reporter assays and electrophoretic mobility shift assays (EMSA) results verified that PnMYB2 could bind and activate the promoters of AtCesA3 and PnCesA3, which are the PCW-specific cellulose biosynthetic genes, and AtCCoAOMT1 and PnCCoAOMT1, which are the key lignin biosynthetic genes. These results demonstrated the central role of PnMYB2 in PCW-specific cellulose formation and SCW-specific lignin biosynthesis.

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.

An MYB Transcription Factor Modulates Panax notoginseng Resistance Against the Root Rot Pathogen Fusarium solani by Regulating the Jasmonate Acid Signaling Pathway and Photosynthesis.

Root rot of Panax notoginseng, a precious Chinese medicinal plant, seriously impacts its sustainable production. However, the molecular regulatory mechanisms employed by P. notoginseng against root rot pathogens, including Fusarium solani, are still unclear. In this study, the PnMYB2 gene was isolated, and its expression was affected by independent treatments with four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) as assessed by quantitative real-time PCR. Moreover, the PnMYB2 expression level was induced by F. solani infection. The PnMYB2 protein localized to the nucleus and may function as a transcription factor. When overexpressed in transgenic tobacco, the PnMYB2 gene conferred resistance to F. solani. Jasmonic acid (JA) metabolism and disease resistance-related genes were induced in the transgenic tobacco, and the JA content significantly increased compared with in the wild type. Additionally, transcriptome sequencing, Kyoto Encyclopedia of Genes and Genomes annotation enrichment, and metabolic pathway analyses of the differentially expressed genes in the transgenic tobacco revealed that JA metabolic, photosynthetic, and defense response-related pathways were activated. In summary, PnMYB2 is an important transcription factor in the defense responses of P. notoginseng against root rot pathogens that acts by regulating JA signaling, photosynthesis, and disease-resistance genes.

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.

Genome and transcriptome analysis to understand the role diversification of cytochrome P450 gene under excess nitrogen treatment.

BACKGROUND: Panax notoginseng (Burk.) F. H. Chen (P. notoginseng) is a medicinal plant. Cytochrome P450 (CYP450) monooxygenase superfamily is involved in the synthesis of a variety of plant hormones. Studies have shown that CYP450 is involved in the synthesis of saponins, which are the main medicinal component of P. notoginseng. To date, the P. notoginseng CYP450 family has not been systematically studied, and its gene functions remain unclear. RESULTS: In this study, a total of 188 PnCYP genes were identified, these genes were divided into 41 subfamilies and clustered into 9 clans. Moreover, we identified 40 paralogous pairs, of which only two had Ka/Ks ratio greater than 1, demonstrating that most PnCYPs underwent purification selection during evolution. In chromosome mapping and gene replication analysis, 8 tandem duplication and 11 segmental duplication events demonstrated that PnCYP genes were continuously replicating during their evolution. Gene ontology (GO) analysis annotated the functions of 188 PnCYPs into 21 functional subclasses, suggesting the functional diversity of these gene families. Functional divergence analyzed the members of the three primitive branches of CYP51, CYP74 and CYP97 at the amino acid level, and found some critical amino acid sites. The expression pattern of PnCYP450 related to nitrogen treatment was studied using transcriptome sequencing data, 10 genes were significantly up-regulated and 37 genes were significantly down-regulated. Combined with transcriptome sequencing analysis, five potential functional genes were screened. Quantitative real-time PCR (qRT-PCR) indicated that these five genes were responded to methyl jasmonate (MEJA) and abscisic acid (ABA) treatment. CONCLUSIONS: These results provide a valuable basis for comprehending the classification and biological functions of PnCYPs, and offer clues to study their biological functions in response to nitrogen treatment.

High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae.

The Chinese medicinal plant Panax notoginseng has been traditionally used to activate blood flow and circulation, and to prevent blood stasis. P. notoginseng contains protopanaxatriol (PPT)-type saponins as its main active compounds, thus distinguishing it from the other two famous Panax species, P. ginseng and P. quinquefolius. Ginsenoside Rg1 (Rg1), notoginsenoside R1 (NgR1), and notoginsenoside R2 (NgR2) are three major PPT-type saponins in P. notoginseng and possess potential cardiovascular protection activities. However, their use in medical applications has long been hampered by the lack of sustainable and low-cost industrial-scale preparation methods. In this study, a PPT-producing yeast chassis strain was designed and constructed based on a previously constructed and optimized protopanaxadiol (PPD)-producing Saccharomyces cerevisiae strain, and further optimized by systemically engineering and optimizing the expression level of its key P450 biopart. Rg1-producing yeast strains were constructed by introducing PgUGT71A53 and PgUGT71A54 into the PPT chassis strain. The fermentation titer of Rg1 reached 1.95 g/L. A group of UDP-glycosyltransferases (UGT) from P. notoginseng and P. ginseng were characterized, and were found to generate NgR1 and NgR2 by catalyzing the C6-O-Glc xylosylation of Rg1 and Rh1, respectively. Using one of these UGTs, PgUGT94Q13, and the previously identified PgUGT71A53 and PgUGT71A54, the biosynthetic pathway to produce saponins NgR1 and NgR2 from PPT could be available. The NgR1 cell factory was further developed by introducing PgUGT94Q13 and a heterologous UDP-xylose biosynthetic pathway from Arabidopsis thaliana into the highest Rg1-producing cell factory. The NgR2-producing cell factory was constructed by introducing PgUGT71A54, PgUGT94Q13, and the UDP-xylose biosynthetic pathway into the PPT chassis. De novo production of NgR1 and NgR2 reached 1.62 g/L and 1.25 g/L, respectively. Beyond the realization of artificial production of the three valuable saponins Rg1, NgR1, and NgR2 from glucose, our work provides a green and sustainable platform for the efficient production of other PPT-type saponins in engineered yeast strains, and promotes the industrial application of PPT-type saponins as medicine and functional foods.

Analysis of saponins detoxification genes in Ilyonectria mors-panacis G3B inducing root rot of Panax notoginseng by RNA-Seq.

Saponins are kinds of antifungal compounds produced by Panax notoginseng to resist invasion by pathogens. Ilyonectria mors-panacis G3B was the dominant pathogen inducing root rot of P. notoginseng, and the abilities to detoxify saponins were the key to infect P. notoginseng successfully. To research the molecular mechanisms of detoxifying saponins in I. mors-panacis G3B, we used high-throughput RNA-Seq to identify 557 and 1519 differential expression genes (DEGs) in I. mors-panacis G3B with saponins treatments for 4H (Hours) and 12H (Hours) compared with no saponins treatments, respectively. Among these DEGs, we found 93 genes which were simultaneously highly expressed in I. mors-panacis G3B with saponins treatments for 4H and 12H, they mainly belong to genes encoding transporters, glycoside hydrolases, oxidation-reduction enzymes, transcription factors and so on. In addition, there were 21 putative PHI (Pathogen-Host Interaction) genes out of those 93 up-regulated genes. In this report, we analyzed virulence-associated genes in I. mors-panacis G3B which may be related to detoxifying saponins to infect P. notoginseng successfully. They provided an excellent starting point for in-depth study on pathogenicity of I. mors-panacis G3B and developed appropriate root rot disease management strategies in the future.

Photosynthetic performance and photosynthesis-related gene expression coordinated in a shade-tolerant species Panax notoginseng under nitrogen regimes.

BACKGROUND: Nitrogen (N) is an essential component of photosynthetic apparatus. However, the mechanism that photosynthetic capacity is suppressed by N is not completely understood. Photosynthetic capacity and photosynthesis-related genes were comparatively analyzed in a shade-tolerant species Panax notoginseng grown under the levels of low N (LN), moderate N (MN) and high N (HN). RESULTS: Photosynthetic assimilation was significantly suppressed in the LN- and HN-grown plants. Compared with the MN-grown plants, the HN-grown plants showed thicker anatomic structure and larger chloroplast accompanied with decreased ratio of mesophyll conductance (gm) to Rubisco content (gm/Rubisco) and lower Rubisco activity. Meanwhile, LN-grown plants displayed smaller chloroplast and accordingly lower internal conductance (gi). LN- and HN-grown individuals allocated less N to light-harvesting system (NL) and carboxylation system (NC), respectively. N surplus negatively affected the expression of genes in Car biosynthesis (GGPS, DXR, PSY, IPI and DXS). The LN individuals outperformed others with respect to non-photochemical quenching. The expression of genes (FBA, PGK, RAF2, GAPC, CAB, PsbA and PsbH) encoding enzymes of Calvin cycle and structural protein of light reaction were obviously repressed in the LN individuals, accompanying with a reduction in Rubisco content and activity. Correspondingly, the expression of genes encoding RAF2, RPI4, CAB and PetE were repressed in the HN-grown plants. CONCLUSIONS: LN-induced depression of photosynthetic capacity might be caused by the deceleration on Calvin cycle and light reaction of photosynthesis, and HN-induced depression of ones might derive from an increase in the form of inactivated Rubisco.

Phosphate transporters, PnPht1;1 and PnPht1;2 from Panax notoginseng enhance phosphate and arsenate acquisition.

BACKGROUND: Panax notoginseng is a medicinally important Chinese herb with a long history of cultivation and clinical application. The planting area is mainly distributed in Wenshan Prefecture, where the quality and safety of P. notoginseng have been threatened by high concentration of arsenic (As) from the soil. The roles of phosphate (Pi) transporters involved in Pi acquisition and arsenate (AsV) tolerance were still unclear in this species. RESULTS: In this study, two open reading frames (ORFs) of PnPht1;1 and PnPht1;2 separated from P. notoginseng were cloned based on RNA-seq, which encoded 527 and 541 amino acids, respectively. The results of relative expression levels showed that both genes responded to the Pi deficiency or As exposure, and were highly upregulated. Heterologous expression in Saccharomyces cerevisiae MB192 revealed that PnPht1;1 and PnPht1;2 performed optimally in complementing the yeast Pi-transport defect, particularly in PnPht1;2. Cells expressing PnPht1;2 had a stronger AsV tolerance than PnPht1;1-expressing cells, and accumulated less As in cells under a high-Pi concentration. Combining with the result of plasma membrane localization, these data confirmed that transporters PnPht1;1 and PnPht1;2 were putative high-affinity H+/H2PO4- symporters, mediating the uptake of Pi and AsV. CONCLUSION: PnPht1;1 and PnPht1;2 encoded functional plasma membrane-localized transporter proteins that mediated a putative high-affinity Pi/H+ symport activity. Expression of PnPht1;1 or PnPht1;2 in mutant strains could enhance the uptake of Pi and AsV, that is probably responsible for the As accumulation in the roots of P. notoginseng.

Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.

[Development and application of chloroplast molecular markers in Panax notoginseng].

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel; cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.

The transcriptome variations of Panaxnotoginseng roots treated with different forms of nitrogen fertilizers.

BACKGROUND: The sensitivity of plants to ammonia is a worldwide problem that limits crop production. Excessive use of ammonium as the sole nitrogen source results in morphological and physiological disorders, and retarded plant growth. RESULTS: In this study we found that the root growth of Panax notoginseng was inhibited when only adding ammonium nitrogen fertilizer, but the supplement of nitrate fertilizer recovered the integrity, activity and growth of root. Twelve RNA-seq profiles in four sample groups were produced and analyzed to identify deregulated genes in samples with different treatments. In comparisons to NH[Formula: see text] treated samples, ACLA-3 gene is up-regulated in samples treated with NO[Formula: see text] and with both NH[Formula: see text] and NO[Formula: see text], which is further validated by qRT-PCR in another set of samples. Subsequently, we show that the some key metabolites in the TCA cycle are also significantly enhanced when introducing NO[Formula: see text]. These potentially enhance the integrity and recover the growth of Panax notoginseng roots. CONCLUSION: These results suggest that the activated TCA cycle, as demonstrated by up-regulation of ACLA-3 and several key metabolites in this cycle, contributes to the increased Panax notoginseng root yield when applying both ammonium and nitrate fertilizer.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.