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Iron plays a crucial role in plant chlorophyll synthesis, respiration, and plant growth. However, excessive iron content can contribute to ginseng poisoning. We previously discovered that the application of silicon (Si) and potassium (K) can mitigate the iron toxicity on ginseng. To elucidate the molecular mechanism of how Si and K alleviate iron toxicity stress in ginseng. We investigated the physiological and transcriptional effects of exogenous Si and K on Panax ginseng. The results suggested that the leaves of ginseng with Si and K addition under iron stress increased antioxidant enzyme activity or secondary metabolite content, such as phenylalanine amino-lyase, polyphenol oxidase, ascorbate peroxidase, total phenols and lignin, by 6.21%-25.94%, 30.12%-309.19%, 32.26%-38.82%, 7.81%-23.66%, and 4.68%-48.42%, respectively. Moreover, Si and K increased the expression of differentially expressed genes (DEGs) associated with resistance to both biotic and abiotic stress, including WRKY (WRKY1, WRKY5, and WRKY65), bHLH (bHLH35, bHLH66, bHLH128, and bHLH149), EREBP, ERF10 and ZIP. Additionally, the amount of DEGs of ginseng by Si and K addition was enriched in metabolic processes, single-organism process pathways, signal transduction, metabolism, synthesis and disease resistance. In conclusion, the utilization of Si and K can potentially reduce the accumulation of iron in ginseng, regulate the expression of iron tolerance genes, and enhance the antioxidant enzyme activity and secondary metabolite production in both leaves and roots, thus alleviating the iron toxicity stress in ginseng., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Background: The ginseng endophyte Paenibacillus polymyxa Pp-7250 (Pp-7250) has multifaceted roles such as preventing ginseng diseases, promoting growth, increasing ginsenoside accumulation, and degrading pesticide residues, however, these effects still have room for improvements. Composite fungicides are an effective means to improve the biocontrol effect of fungicides, but the effect of Pp-7250 in combination with its symbiotic bacteria on ginseng needs to be further investigated, and its mechanism of action has not been elucidated. In this study, a series of experiments was conducted to elucidate the effect of Paenibacillus polymyxa and Bacillus cereus co-bacterial agent on the yield and quality of understory ginseng, and to investigate their mechanism of action., Results: The results indicated that P. polymyxa and B. cereus co-bacterial agent (PB) treatment improved ginseng yield, ginsenoside accumulation, disease prevention, and pesticide degradation. The mechanism is that PB treatment increased the abundance of beneficial microorganisms, including Rhodanobacter, Pseudolabrys, Gemmatimonas, Bacillus, Paenibacillus, Cortinarius, Russula, Paecilomyces, and Trechispora, and decreased the abundance of pathogenic microorganisms, including Ellin6067, Acidibacter, Fusarium, Tetracladium, Alternaria, and Ilyonectria in ginseng rhizosphere soil. PB co-bacterial agents enhanced the function of microbial metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, biosynthesis of amino acids, carbon fixation pathways in prokaryotes, DNA replication, and terpenoid backbone biosynthesis, and decreased the function of microbial plant pathogens and animal pathogens., Conclusion: The combination of P. polymyxa and B. cereus may be a potential biocontrol agent to promote the resistance of ginseng to disease and improve the yield, quality, and pesticide degradation., (© 2024. The Author(s).)

Ginseng (Panax ginseng C. A. Mey.) is an important and valuable medicinal plant species used in traditional Chinese medicine, and its metabolite ginsenoside is the primary active ingredient. The FAR1/FHY3 gene family members play critical roles in plant growth and development as well as participate in a variety of physiological processes, including plant development and signaling of hormones. Studies have indicated that methyl jasmonate treatment of ginseng adventitious roots resulted in a significant increase in the content of protopanaxadiol ginsenosides. Therefore, it is highly significant to screen the FAR1/FHY3 gene family members in ginseng and preliminarily investigate their expression patterns in response to methyl jasmonic acid signaling. In this study, we screened and identified the FAR1/FHY3 family genes in the ginseng transcriptome databases. And then, we analyzed their gene structure and phylogeny, chromosomal localization and expression patterns, and promoter cis-acting elements, and made GO functional annotations on the members of this family. After that, we treated the ginseng adventitious roots with 200 mM methyl jasmonate and investigated the trend of the expression of four genes containing the largest number of methyl jasmonate cis-acting elements at different treatment times. All four genes were able to respond to methyl jasmonate, the most significant change was in the PgFAR40 gene. This study provides data support for subsequent studies of this family member in ginseng and provides experimental reference for subsequent validation of the function of this family member under methyl jasmonic acid signaling., (© 2024. The Author(s).)

The allelopathic autotoxicity of ginsenosides is an important cause of continuous cropping obstacles in ginseng planting. There is no report on the potential molecular mechanism of the correlation between polarity of ginsenoside components and their allelopathic autotoxicity. This study applied a combination of metabolomics and transcriptomics analysis techniques, combined with apparent morphology, physiological indexes, and cell vitality detection of the ginseng hairy roots, through which the molecular mechanism of correlation between polarity and allelopathic autotoxicity of ginsenosides were comprehensively studied. The hairy roots of ginseng presented more severe cell apoptosis under the stress of low-polarity ginsenoside components (ZG70). ZG70 exerted allelopathic autotoxicity by regulating the key enzyme genes of cis-zeatin (cZ) synthesis pathway, indole-3-acetic acid (IAA) synthesis pathway, and jasmonates (JAs) signaling transduction pathway. The common pathway for high-polarity ginsenoside components (ZG50) and ZG70 to induce the development of allelopathic autotoxicity was through the expression of key enzymes in the gibberellin (GA) signal transduction pathway, thereby inhibiting the growth of ginseng hairy roots. cZ, indole-3-acetamid (IAM), gibberellin A1 (GA1), and jasmonoyl-L-isoleucine (JA-ILE) were the key response factors in this process. It could be concluded that the polarity of ginsenoside components were negatively correlated with their allelopathic autotoxicity.

Among rare earth elements, cerium has the unique ability of regulating the growth of plant cells and the biosynthesis of metabolites at different stages of plant development. The signal pathways of Ce 3+ -mediated ginsenosides biosynthesis in ginseng hairy roots were investigated. At a low concentration, Ce 3+ improved the elongation and biomass of hairy roots. The Ce 3+ -induced accumulation of ginsenosides showed a high correlation with the reactive oxygen species (ROS), as well as the biosynthesis of endogenous methyl jasmonate (MeJA) and ginsenoside key enzyme genes ( PgSS , PgSE and PgDDS ). At a Ce 3+ concentration of 20 mg L -1 , the total ginsenoside content was 1.7-fold, and the total ginsenosides yield was 2.7-fold that of the control. Malondialdehyde (MDA) content and the ROS production rate were significantly higher than those of the control. The activity of superoxide dismutase (SOD) was significantly activated within the Ce 3+ concentration range of 10 to 30 mg L -1 . The activity of catalase (CAT) and peroxidase (POD) strengthened with the increasing concentration of Ce 3+ in the range of 20-40 mg L -1 . The Ce 3+ exposure induced transient production of superoxide anion (O 2 • - ) and hydrogen peroxide (H 2 O 2 ). Together with the increase in the intracellular MeJA level and enzyme activity for lipoxygenase (LOX), there was an increase in the gene expression level of MeJA biosynthesis including PgLOX , PgAOS and PgJMT . Our results also revealed that Ce 3+ did not directly influence PgSS , PgSE and PgDDS activity. We speculated that Ce 3+ -induced ROS production could enhance the accumulation of ginsenosides in ginseng hairy roots via the direct stimulation of enzyme genes for MeJA biosynthesis. This study demonstrates a potential approach for understanding and improving ginsenoside biosynthesis that is regulated by Ce 3+ -mediated signal transduction.

Brassinosteroids (BRs) play crucial roles in the physiology and development of plants. In the model plant Arabidopsis , BR signaling is initiated at the level of membrane receptors, BRASSINOSTEROIDS INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) complex, thus activating the transcription factors (TFs) BRASSINAZOLE RESISTANT 1/BRI1-EMS-SUPPRESSOR 1 (BZR1/BES1) to coordinate BR responsive genes. BRASSINOSTEROIDS INSENSITIVE 2 (BIN2), glycogen synthase kinase 3 (GSK3) like-kinase, negatively regulates BZR1/BES1 transcriptional activity through phosphorylation-dependent cytosolic retention and shuttling. However, it is still unknown whether this mechanism is conserved in Panax ginseng C. A. Mayer, a member of the Araliaceae family, which is a shade-tolerant perennial root crop. Despite its pharmacological and agricultural importance, the role of BR signaling in the development of P. ginseng and characterization of BR signaling components are still elusive. In this study, by utilizing the Arabidopsis bri1 mutant, we found that ectopic expression of the gain of function form of PgBZR1 ( Pgbzr1-1D ) restores BR deficiency. In detail, ectopic expression of Pgbzr1-1D rescues dwarfism, defects of floral organ development, and hypocotyl elongation of bri1-5 , implying the functional conservation of PgBZR1 in P. ginseng . Interestingly, brassinolide (BL) and BRs biosynthesis inhibitor treatment in two-year-old P. ginseng storage root interferes with and promotes, respectively, secondary growth in terms of xylem formation. Altogether, our results provide new insight into the functional conservation and potential diversification of BR signaling and response in P. ginseng .

Basic helix-loop-helix (bHLH) gene family is a gene family of transcription factors that plays essential roles in plant growth and development, secondary metabolism and response to biotic and abiotic stresses. Therefore, a comprehensive knowledge of the bHLH gene family is paramount to understand the molecular mechanisms underlying these processes and develop advanced technologies to manipulate the processes efficiently. Ginseng, Panax ginseng C.A. Meyer, is a well-known medicinal herb; however, little is known  about the bHLH genes (PgbHLH) in the species. Here, we identified 137 PgbHLH genes from Jilin ginseng cultivar, Damaya, widely cultivated in Jilin, China, of which 50 are newly identified by pan-genome analysis. These 137 PgbHLH genes were phylogenetically classified into 26 subfamilies, suggesting their sequence diversification. They are alternatively spliced into 366 transcripts in a 4-year-old plant and involved in 11 functional subcategories of the gene ontology, indicating their functional differentiation in ginseng. The expressions of the PgbHLH genes dramatically vary spatio-temporally and across 42 genotypes, but they are still somehow functionally correlated. Moreover, the PgbHLH gene family, at least some of its genes, is shown to have roles in plant response to the abiotic stress of saline. These results provide a new insight into the evolution and functional differentiation of the bHLH gene family in plants, new bHLH genes to the PgbHLH gene family, and saline stress-responsive genes for genetic improvement in ginseng and other plant species.

Ginseng is a valuable herb of traditional Chinese medicine and ginsenosides, the main bioactive components of ginseng, have been proven to have multiple functions in human therapies and health. Methyl jasmonate (MeJA) is an elicitor that has been demonstrated to have a vital influence on ginsenoside biosynthesis. Quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in quantification of gene expressions. Here, we report the selection and validation of reference genes desirable for normalization of gene expressions quantified by qRT-PCR in ginseng hairy roots treated with MeJA. Twelve reference genes were selected as candidate genes, and their expressions were quantified by qRT-PCR, and analyzed by geNorm, NormFinder and BestKeeper. CYP and EF-1α were shown to be the most stable reference genes in geNorm, CYP was the most stable reference gene in NormFinder, and 18S was the most stable reference gene in BestKeeper. On this basis, we further quantified the relative expression levels of four genes encoding key enzymes that are involved in ginsenoside biosynthesis using CYP and 18S as the reference genes, respectively. Moreover, correlation analysis was performed between the quantified expressions of four genes and the ginsenoside content in MeJA-treated ginseng hairy roots. The results of relative expressions of the four genes quantified using CYP as the reference gene and their significant correlations with the ginsenoside content were better than those using 18S as the reference gene. The CYP gene, hence, was concluded as the most desirable reference gene for quantification of the expressions of genes in MeJA-treated ginseng hairy roots. This finding, therefore, provides information useful for gene research in ginseng, particularly in MeJA-treated ginseng hairy roots, which includes identification and characterization of genes involved in ginsenoside biosynthesis., Competing Interests: The authors have declared that no competing interests exist.

Root rot caused by Ilyonectria mors-panacis is a devastating fungal disease leading to defect in root quality and causes reduced yield during the perennial life cycle of Panax ginseng Meyer. This indicates the imperative need to understand the molecular basis of disease development and also to enhance tolerance against the fungus. With this idea, the protective effect of silicon (supplied as silica nanoparticles) in P. ginseng root rot pathosystem and its molecular mechanism was investigated in the current study. We have tested different concentrations of silicon (Si) to disease-infected ginseng and found that long term analysis (30 dpi) displayed a striking 50% reduction in disease severity index upon the treatment of Si. Expectedly, Si had no direct degradative effect against the pathogen. Instead, in infected roots it resulted in reduced expression of PgSWEET leading to regulated sugar efflux into apoplast and enhanced tolerance against I. mors-panacis. In addition, under diseased condition, both protopanaxadiol (PPD) and protopanaxatriol (PPT) type ginsenoside profile in roots were higher in Si treated plants. This is the first report indicating the protective role of Si in ginseng-root rot pathosystem, thereby uncovering novel features of ginseng mineral physiology and at the same time, enabling its usage to overcome root rot.

Background: Panax ginseng seeds have strong dormancy and a prolonged germination period in comparison to other seeds; thus, it is a great challenge to propagate ginseng. Seed longevity is closely associated with germination rate and viability, so we assumed that if a seed loses its viability, specific metabolic alterations regarding plant growth factors might occur. In this study, we divided ginseng seeds into normal and accelerated-aging groups. Both groups were treated with gibberellic acid, which is one of the most important plant-growth regulators. Afterward, gas chromatography-mass spectrometry (GC-MS) was used to analyze the samples, to identify the metabolic alterations between the two groups., Results: Forty-four endogenous metabolites in normal and accelerated aging groups were putatively identified. To determine the differential significance of these metabolites, t-tests and fold-change analysis were conducted followed by principal component analysis and partial least-squares discriminant analysis to determine the metabolites that showed distinct responses between the groups. Among the differentially expressed metabolites (P value < 0.05 and FDR < 0.1), nine metabolites were selected as potential biomarker candidates for the prediction of seed longevity., Conclusion: Nine metabolites related to ginseng seed longevity were identified by comparing metabolomes. Our findings suggest that ginseng propagation can be facilitated by the regulation of these distinctive metabolic features of the seeds. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The MYB transcription factor family members have been reported to play different roles in plant growth regulation, defense response, and secondary metabolism. However, MYB gene expression has not been reported in Panax ginseng . In this study, we isolated a gene from ginseng adventitious root, PgMYB2, which encodes an R2R3-MYB protein. Subcellular localization revealed that PgMYB2 protein was exclusively detected in the nucleus of Allium cepa epidermis. The highest expression level of PgMYB2 was found in ginseng root and it was significantly induced by plant hormones methyl jasmonate (MeJA). Furthermore, the binding interaction between PgMYB2 protein and the promoter of dammarenediol synthase (DDS) was found in the yeast strain Y1H Gold. Moreover, the electrophoretic mobility shift assay (EMSA) identified the binding site of the interaction and the results of transiently overexpressing PgMYB2 in plants also illustrated that it may positively regulate the expression of PgDDS. Based on the key role of PgDDS gene in ginsenoside synthesis, it is reasonable to believe that this report will be helpful for the future studies on the MYB family in P. ginseng and ultimately improving the ginsenoside production through genetic and metabolic engineering.

Panax quinquefolium hairy root cultures synthesize triterpenoid saponins named ginsenosides, that have multidirectional pharmacological activity. The first rate-limiting enzyme in the process of their biosynthesis is 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In this study, a 741 bp fragment of the P. quinquefolium HMGR gene ( PqHMGR ), consisting of a proximal promoter, 5'UTR (5' untranslated region) and 5'CDS (coding DNA sequence) was isolated. In silico analysis of an isolated fragment indicated a lack of tandem repeats, miRNA binding sites, and CpG/CpNpG elements. However, the proximal promoter contained potential cis -elements involved in the response to light, salicylic, and abscisic acid (ABA) that was represented by the motif ABRE (TACGTG). The functional significance of ABA on P. quinquefolium HMGR gene expression was evaluated, carrying out quantitative RT-PCR experiments at different ABA concentrations (0.1, 0.25, 0.5, and 1 mg·L -1 ). Additionally, the effect of abscisic acid and its time exposure on biomass and ginsenoside level in Panax quinquefolium hairy root was examined. The saponin content was determined using HPLC. The 28 day elicitation period with 1 mg·L -1 ABA was the most efficient for Rg2 and Re (17.38 and 1.83 times increase, respectively) accumulation; however, the protopanaxadiol derivative content decreased in these conditions.

Rationale: Obesity has become a worldwide health challenge. In elderly patients, obesity induces a decrease in skeletal muscle strength and mass, which is linked to age-related sarcopenia. These are important predictors of hospitalization and death. Herein, we report on 2 elderly male patients who were treated by pharmacopuncture with wild ginseng complex (WGC)., Patient Concerns: Case 1 was that of a 62-year-old man who was mainly concerned about his appearance as his abdominal fat was steadily increasing, without any changes to his body weight. He also had an aversion to any type of modern Western medicine. In addition, the patient was averse to changing his eating habits or exercise style. Case 2 was that of a 66-year-old man who was concerned about his appearance due to an increase in abdominal fat accumulation. Furthermore, he had gained 1.5 inches in waist circumference over the past 2 years. The patient wanted to lose fat in the abdomen, but not in other parts of the body., Diagnoses: In this study, we examined 2 elderly male patients (aged 60 years and above) with abdominal obesity and excessive visceral fat mass. Abdominal obesity is defined as a waist circumference of ≥ 90 cm for men and ≥ 80 cm for women., Interventions: The patients received pharmacopuncture with WGC 5 times over 3 weeks on their abdomen without controlling diet and exercise., Outcome: After 3 weeks of pharmacopuncture with WGC and at a follow-up appointment 3 weeks later, we detected an increase in muscle mass, protein content, and basal metabolic rate in both cases., Lessons: Although we observed only 2 cases, our results indicated that pharmacopuncture with WGC can be used as a new alternative treatment to prevent and improve abdominal obesity in elderly individuals, as well as age-related sarcopenic obesity.

Panax ginseng is an important cash crop in the Asian countries due to its pharmaceutical effects, however the plant is exposed to various abiotic stresses, lead to reduction of its quality. One of them is the Aluminum (Al) accumulation. Plant growth promoting bacteria which able to tolerate heavy metals has been considered as a new trend for supporting the growth of many crops in heavy metal occupied areas. In this study, twelve bacteria strains were isolated from rhizosphere of diseased Korean ginseng roots located in Gochang province, Republic of Korea and tested for their ability to grow in Al-embedded broth media. Out of them, four strains (Pseudomonas simiae N3, Pseudomonas fragi N8, Chryseobacterium polytrichastri N10, and Burkholderia ginsengiterrae N11-2) were able to grow. The strains could also show other plant growth promoting activities e.g. auxins and siderophores production and phosphate solubilization. P. simiae N3, C. polytrichastri N10, and B. ginsengiterrae N11-2 strains were able to support the growth of Arabidopsis thaliana stressed by Al while P. fragi N8 could not. Plants inoculated with P. simiae N3, C. polytrichastri N10, and B. ginsengiterrae N11-2 showed higher expression level of Al-stress related genes, AtAIP, AtALS3 and AtALMT1, compared to non-bacterized plants. Expression profiles of the genes reveal the induction of external mechanism of Al resistance by P. simiae N3 and B. ginsengiterrae N11-2 and internal mechanism by C. polytrichastri N10. Korean ginseng seedlings treated with these strains showed higher biomass, particularly the foliar part, higher chlorophyll content than non-bacterized Al-stressed seedlings. According to the present results, these strains can be used in the future for the cultivation of ginseng in Al-persisted locations., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

One of the most effective strategies to enhance metabolite biosynthesis and accumulation in biotechnological systems is the use of elicitation processes. This study assesses the influence of different concentrations of yeast extract (YE) on ginsenoside biosynthesis in Panax quinquefolium (American ginseng) hairy roots cultivated in shake flasks and in a nutrient sprinkle bioreactor after 3 and 7 days of elicitation. The saponin content was determined using HPLC. The maximum yield (20 mg g -1 d.w.) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved after application of YE at 50 mg L -1 concentration and 3 day exposure time. The ginsenoside level was 1.57 times higher than that attained in control medium. The same conditions of elicitation (3 day time of exposure and 50 mg L -1 of YE) also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The total ginsenoside content was 32.25 mg g -1 d.w. and was higher than that achieved in control medium and in shake flasks cultures. Obtained results indicated that yeast extract can be used to increase ginsenoside production in hairy root cultures of P. quinquefolium ., Competing Interests: The authors declare no conflict of interest.

Panax ginseng C.A. Meyer is a valuable herb in China that has also gained popularity in the West because of its pharmacological properties. The constituents isolated and characterized in ginseng stems include ginsenosides, fatty acids, amino acids, volatile oils, and polysaccharides. In this study, the effects of fungicide azoxystrobin applied on antioxidant enzyme activity and ginsenosides content in ginseng stems was studied by using Panax ginseng C. A. Mey. cv. (the cultivar of Ermaya) under natural environmental conditions. The azoxystrobin formulation (25% SC) was sprayed three times on ginseng plants at different doses (150ga.i./ha and 225ga.i./ha), respectively. Two new fatty acids esters (ethyl linoleate and methyl linolenate) were firstly detected in ginseng stems by the application of azoxystrobin as foliar spray. The results indicated that activities of enzymatic antioxidants, the content of ginsenosides and two new fatty acids esters in ginseng stems in azoxystrobin-treated plants were increased. Azoxystrobin treatments to ginseng plants at all growth stages suggest that the azoxystrobin-induced delay of senescence is due to an enhanced antioxidant enzyme activity protecting the plants from harmful active oxygen species (AOS). The activity of superoxide dismutase (SOD) in azoxystrobin-treated plants was about 1-3 times higher than that in untreated plants. And the effects was more significant (P=0.05) when azoxystrobin was applied at dose of 225ga.i./ha. This work suggests that azoxystrobin plays an important role in delaying of senescence by changing physiological and biochemical indicators and increasing ginsenosides content in ginseng stems., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Red ginseng, a steamed and sun-dried ginseng, is a popular health-promoting food in Korea and other Asian countries. We introduced nanofertilizer technology using gold nanoparticles in an effort to develop red ginseng with an elevated level of ginsenosides, the main active compounds of ginseng. Shoots of 6-year-old ginseng plants were fertilized three times with colloidal gold nanoparticle sprays. Red ginseng extract was prepared from the main roots. The concentrations of gold and ginsenosides were measured following gold nanoparticle treatment. To evaluate the anti-inflammatory effects, mouse peritoneal macrophages of male BALB/c mouse were stimulated with lipopolysaccharide plus interferon-γ in the presence of extracts from red ginseng with or without gold nanoparticle treatment. The content of ginsenosides, such as Rg1, Re, Rf, and Rb1, increased in ginseng treated with gold nanofertilizer whereas the steaming process increased only the levels of Rd and Rg3. The levels of nitric oxide, inducible nitric oxide synthase, and interleukin-6, but not tumor necrosis factor-α, were more suppressed in macrophages treated with extract from gold nanoparticle-treated red ginseng. Our results show that the use of a colloidal gold nanoparticle fertilizer improved the synthesis of ginsenosides in ginseng and enhanced the anti-inflammatory effects of red ginseng. Further research is required to elucidate the causal factors for the gold-induced change in ginsenoside synthesis and to determine the in vivo effect of gold nanoparticle-treated ginseng.

WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses., (© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.)

In order to search for a new pathway to improve the yield of ginseng through growing at the full sun shine accompanied by salicylic acid (SA), the net photosynthetic rate (P(n)), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), malondialdehyde (MDA) in Panax ginseng leaves, and the content of ginsenosides in roots were compared under various concentrations of SA and full sun shine with the traditional shade shed. Under the full sun shine, 0.05, 0.2 mmol x L(-1) SA increased net photosynthetic rate to a great extent. Under the cloudy day, the average net photosynthetic rate increased by 127.8% and 155.0% over the traditional shade shed, 13.9% and 27.5% over the treatment without SA respectively; under the clear day, 23.5% and 30.4% over the traditional shade shed, 8.6% and 14.6% over the treatment without SA, particularly obvious in the morning and late afternoon. With such concentration, SA increased activities of SOD, CAT, POD, and decreased the contents of the MDA. This difference resulted from different light intensity, rise of light saturation point, and fall of compensation point. Full sun shine decreased ginsenosides contents, but with SA, the ginsenosides regained, the content of Rg1 and Re, Rb1, total six types of ginsenosides in SA 0.2 mmol x L(-1) group were higher than those in the control group (P < 0.05) and other groups. The application of 0.2 mmol x L(-1) SA under full sun shine during a short time has little threat to the P. ginseng in spring, and could enhance the resistance to the adversity, which would improve the yield of ginseng heavily.

Key Message: Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Ginseng is one of the most important plants in oriental medicine. The pharmacological effects of this medicinal herb are mostly correlated to the major bioactive triterpene saponin, called ginsenoside. Due to the long cultivation period of ginseng and increased ginsenoside level in aged root, we need to develop strategies to increase ginseng productivity in cell and tissue culture in a faster way. Elicitation is already considered to improve the yield of this valuable secondary metabolite; especially, different types, timings, and durations of elicitation could affect the ginsenoside production and heterogeneity. Activation of ginsenoside biosynthetic genes and ginsenoside accumulation mediated by elicitor-induced signaling molecules would be helpful for commercial production of individual ginsenosides. Jasmonic acid is the well-known signaling molecule which mainly involved in ginsenoside accumulation. Ca(2+) spiking and reactive oxygen species, nitric oxide, and ethylene production are other messengers which mediate production of ginsenoside. This review highlights the elicitation strategies for production of the ginsenoside based on the principle of putative signal transduction pathways.

Ginsenosides are the abundant secondary metabolites in American ginseng (Panax quinquefolium), it could be released into soil through root exudation and decomposition during plant growth. This study determined ginsenoside contents in American ginseng cultivated soil by HPLC. Three ginsenosides, Rb1, Rb2 and Rd, were detected in the rhizosphere soil of 3-4 years old American ginseng cultivated in Huairou District, Beijing, and their contents were 0.80-3.19 mg x kg(-1). Correspondingly, the contents of the three ginsenosides in soil solution were 4-16 mg x L(-1) at field water-holding capacity of 20%. According to the field soil test data, we designed the concentration of ginsenosides for bioassays (0.2-125 mg x L(-1) in solution or 0.2-125 mg x kg(-1) in soil). The results showed that radicle lengths of American ginseng were reduced by 6%-23% in solution containing 0.2-125 mg x L(-1) ginsenoside extract, and a significant difference was observed at concentration of 125 mg x L(-1) (P < 0.05). The shoot lengths of American ginseng were not significantly inhibited by 0.2-125 mg x L(-1) ginsenosides extractions. After 20 days of growth in nutrient solution amended with 25 mg x L(-1) ginsenosides extraction, plant height of 3-year-old American ginseng seedling was decreased by 28% compared to the control, and the biomass of aerial parts was also reduced by 50% (P < 0.05). However, the growth of newly-grown fibrous root was not significantly inhibited. Comparatively, when American ginseng embryos were cultivated into sterile or non-sterile soil, neither radicle lengths nor shoot lengths were significantly affected by 0.2-125 mg x kg(-1) ginsenoside extracts. In conclusion, ginsenosides showed autotoxic effect on growth of American ginseng radicle and adult seedling, however, this effect was weakened in field soil.

We evaluated the effect of Tween 80 permeabilization on ginsenoside secretion in Panax ginseng hairy roots. Tween 80 (1.2%, w/v) had no significant effect on hairy root vitality. After a 25-day treatment with Tween 80, approximately 76% of the total ginsenosides was released into the surrounding medium. In the case of control, the ginsenosides secreted into the medium were negligible. Furthermore, when compared with control, the level of total ginsenosides was enhanced by approximately threefold under Tween treatment. Additionally, secretion of the typical ginsenoside monomers including Rb1 , Rg1 , and Re was analyzed, indicating that the most of them were released into the medium. Moreover, it was observed that dammarenediol synthase, a key enzyme involved in ginsenoside biosynthesis, was upregulated at both gene expression and enzyme activity levels. The expression of genes CYP716A47 and CYP716A53v2 encoding Cyt P450 enzymes catalyzing the formation of protopanaxadiol from dammarenediol and protopanaxatriol from protopanaxadiol, respectively, was slightly upregulated. These results clearly demonstrated that Tween 80 could act not only as an efficient permeabilizer to enhance ginsenoside secretion from the hairy roots, but also as an elicitor to promote the biosynthesis of ginsenoside., (© 2014 International Union of Biochemistry and Molecular Biology, Inc.)

Panax ginseng Meyer is one of the major medicinal plants in oriental countries belonging to the Araliaceae family which are the primary source for ginsenosides. However, very few genes were characterized for ginsenoside pathway, due to the limited genome information. Through this study, we obtained a comprehensive transcriptome from adventitious roots, which were treated with methyl jasmonic acids for different time points (control, 2h, 6h, 12h, and 24h) and sequenced by RNA 454 pyrosequencing technology. Reference transcriptome 39,304,529 (0.04GB) was obtained from 5,724,987,880 bases (5.7GB) of 22 libraries by de novo assembly and 35,266 (58.5%) transcripts were annotated with biological schemas (GO and KEGG). The digital gene expression patterns were obtained from in vitro grown adventitious root sequences which mapped to reference, from that, 3813 (6.3%) unique transcripts were involved in ≥2 fold up and downregulations. Finally, candidates for ginsenoside pathway genes were predicted from observed expression patterns. Among them, 30 transcription factors, 20 cytochromes, and 11 glycosyl transferases were predicted as ginsenoside candidates. These data can remarkably expand the existing transcriptome resources of Panax, especially to predict existence of gene networks in P. ginseng. The entity of the data provides a valuable platform to reveal more on secondary metabolism and abiotic stresses from P. ginseng in vitro grown adventitious roots., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The paper aimed to evaluate the effects of lead stress on photosynthetic performance and ginsenoside content in ginseng (Panax ginseng). To accomplish this, three years old ginseng were cultivated in pot and in phytotron with different concentrations of lead, ranging from 0 to 1000 mg x kg(-1) soil for a whole growth period (about 150 days). The photosynthetic parameters in leaves and ginsenoside content in roots of ginseng were determined in green fruit stage and before withering stage, respectively. In comparison with the control, net photosynthetic rate and SPAD value in ginseng leaves cultivated with 100 and 250 mg x kg(-1) of lead changed insignificantly, however, ginseng supplied with 500 and 1 000 mg x kg(-1) of lead showed a noticeably decline in the net rate of photosynthesis and SPAD value (P < 0.05), the lowest net photosynthetic rate and SPAD value showed in the treatment supplied with 1 000 mg x kg(-1) of lead, with decline of 57.8%,11.0%, respectively. Total content of ginsenoside in ginseng roots cultivated with 100 mg x kg(-1) of lead showed insignificantly change compared to the control, but the content increased remarkably in treatments supplied with 250, 500, 1 000 mg x kg(-1) of lead (P < 0.05), and highest content appeared in these ginsengs exposed to 1000 mg x kg(-1) of lead. The net photosynthetic rate and SPAD value in leaves of ginseng both showed significantly negative linear correlations with lead stress level (P < 0.01), and significant positive linear correlations between total content of ginsenoside and lead concentration was also observed (P < 0.05). These results strongly indicate that exposing to high level of lead negatively affects photosynthetic performance in ginseng leaves, but benefits for accumulation of secondary metabolism (total content of ginsenoside) in ginseng root.

The effect of exogenously applied hydrogen peroxide on salt stress tolerance was investigated in Panax ginseng. Pretreatment of ginseng seedlings with 100 μM H2O2 increased the physiological salt tolerance of the ginseng plant and was used as the optimum concentration to induce salt tolerance capacity. Treatment with exogenous H2O2 for 2 days significantly enhanced salt stress tolerance in ginseng seedlings by increasing the activities of ascorbate peroxidase, catalase and guaiacol peroxidase and by decreasing the concentrations of malondialdehyde (MDA) and endogenous H2O2 as well as the production rate of superoxide radical (O2(-)). There was a positive physiological effect on the growth and development of salt-stressed seedlings by exogenous H2O2 as measured by ginseng dry weight and both chlorophyll and carotenoid contents. Exogenous H2O2 induced changes in MDA, O2(-), antioxidant enzymes and antioxidant compounds, which are responsible for increases in salt stress tolerance. Salt treatment caused drastic declines in ginseng growth and antioxidants levels; whereas, acclimation treatment with H2O2 allowed the ginseng seedlings to recover from salt stress by up-regulation of defense-related proteins such as antioxidant enzymes and antioxidant compounds.

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H2O2) and superoxide (O2(-)) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Key Message: When one of them was inhibited, the two pathways could compensate with each other to guarantee normal growth. Moreover, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside level., Abstract: Ginsenosides, a kind of triterpenoid saponins derived from isopentenyl pyrophosphate (IPP), represent the main pharmacologically active constituents of ginseng. In plants, two pathways contribute to IPP biosynthesis, namely, the mevalonate pathway in cytosol and the non-mevalonate pathway in plastids. This motivates biologists to clarify the roles of the two pathways in biosynthesis of IPP-derived compounds. Here, we demonstrated that both pathways are involved in ginsenoside biosynthesis, based on the analysis of the effects from suppressing either or both of the pathways on ginsenoside accumulation in Panax ginseng hairy roots with mevinolin and fosmidomycin as specific inhibitors for the mevalonate and the non-mevalonate pathways, respectively. Furthermore, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside levels in the hairy roots. These results shed some light on the way toward better understanding of ginsenoside biosynthesis.

In order to screen novel genes involved in the biosynthesis and accumulation of ginsenosides in ginseng for investigating the molecular mechanism of ginsenosides accumulation, the differential expression analysis was performed by cDNA-amplified fragment length polymorphism (cDNA-AFLP) in methyl jasmonate (MeJA) treated ginseng suspension cells and untreated controls. Twenty-five transcript-derived-fragments (TDFs) were obtained and 14 of them was involved in secondary metabolite biosynthesis, transportation, signal transduction and stress response. PgTDF3 (JK974248) was detected in MeJA-treated ginseng cells and its full-length cDNA was subsequently isolated. The gene designated PgPDR3 (GenBank accession number KC013238) has 4515 bp in length containing a 4137 bp open reading frame (ORF). The deduced amino acid sequence of PgPDR3 shares high similarity to other plant pleiotropic drug resistance (PDR) transporters and have the characteristic domain of plant PDR transporter Walker A, Walker B and ABC signature both at the N- and C-terminal ends respectively. RT-PCR and quantitative real-time PCR (qRT-PCR) analysis indicated that PgPDR3 was expressed at a high level in the roots and adventitious roots compared to leaves, seeds and buds, and the expression was induced strongly by MeJA in a time-dependent manner, as it is well known MeJA is a signaling molecule that mediates the biosynthesis and accumulation of secondary metabolites. These results demonstrate a potential role of the PgPDR3 gene in the accumulation of secondary metabolites in MeJA-induced ginseng.

The plant pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters is potentially involved in diverse biological processes. Currently, little is known about their actual physiological functions. A Panax ginseng PDR transporter gene (PgPDR1) was cloned and the cDNA has an open reading frame of 4344 bp. The deduced amino acid sequence contained the characteristic domains of PDR transporters: Walker A, Walker B, and ABC signature. Genomic DNA hybridization analysis indicated that one copy of PgPDR1 gene was present in P. ginseng. Subcellular localization showed that PgPDR1-GFP fusion protein was specifically localized in the cell membrane. Promoter region analysis revealed the presence of cis-acting elements, some of which are putatively involved in response to hormone, light and stress. To understand the functional roles of PgPDR1, we investigated the expression patterns of PgPDR1 in different tissues and under various conditions. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis showed that PgPDR1 was expressed at a high level in the roots and leaves compared to seeds and stems. The expression of PgPDR1 was up-regulated by salicylic acid (SA) or chilling, down-regulated by ABA, and regulated differently at transcript and protein levels by MeJA. These results suggest that PgPDR1 might be involved in responding to environmental stresses and hormones., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The differential transcript patterns of five antioxidant genes, four genes related to the ginsenoside pathway and five P450 genes related to defense mechanism were investigated in in vitro adventitious roots of Panax ginseng after exposure to two different concentrations of heavy metals for 7 days. PgSOD-1 and PgCAT transcription increased in a dose-dependent manner during the exposure to CuCl(2), NiCl(2), and CdCl(2), while all other tested scavenging enzymes didn't show significant increase during heavy metal exposure. Conversely, the mRNA transcripts of PgSQE, PgDDS were highly responsive to CuCl(2) compared to NiCl(2) exposure. However, the transcript profile of Pgβ-AS was highly induced upon NiCl(2) treatment compared to CuCl(2) and CdCl(2) exposure. The expressions of PgCYP716A42, PgCYP71A50U, and PgCYP82C22 were regulated in similar manners, and all showed the highest transcript profile at 100 μM of CuCl(2), CdCl(2), and NiCl(2) except PgCYP71D184, which showed the highest transcript level when subjected to 10 μM CuCl(2) and NiCl(2). Thus it may suggest that in P. ginseng heavy metal interaction on cell membrane induced expression of various defense related genes via jasmonic acid pathway and also possesses cross talk networks with other defense related pathways.

Objective: To investigate the effect of artermisinic acid on the secondary metabolites production of Panax quinquefolium crown galls., Methods: Artemisinic acid was added into the suspended cells of Panax quinquefolium crown galls and co-culture for two days. Products were isolated with chromatographic method., Results: Three hydroxyl octadecenoic acids [9,12,13-trihydroxy-10-octadecenoic acid (1), 11,12,13-trihydroxy-9-octadecenoic acid (2) and 11-hydroxy-12,13-epoxy-9-octadecenoic acid (3)] were isolated from crown galls of Panax quinquefolium., Conclusion: Artermisinic acid as one of the new type of phytohormones that might induce the production of 13-lipoxygenases in crown galls of Panax quinquefolium.

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs and contains pharmacologically active components, ginsenosides, in its roots. Ginsenosides, a class of tetracyclic triterpene saponins, are thought to be synthesized from dammarenediol-II after hydroxylation by the Cyt P450 (CYP) enzyme and then glycosylation by glycosyltransferase (GT). However, no genes encoding the hydroxylation and glycosylation in ginsenoside biosynthesis have been identified. Here, we identify protopanaxadiol synthase, which is a CYP enzyme (CYP716A47), to be involved in the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol. Nine putative full CYP sequences were isolated from the expressed sequence tags (ESTs) of methyl jasmonate (MeJA)-treated adventitious ginseng roots. The CYP716A47 gene product was selected as the putative protopanaxadiol synthase because this gene was transcriptionally activated not only by MeJA treatment but also in transgenic ginseng that overexpresses squalene synthase and overproduces ginsenosides. In vitro enzymatic activity assays revealed that CYP716A47 catalyzed the oxidation of dammarenediol-II to produce protopanaxadiol. Ectopic expression of CYP716A47 in recombinant WAT21 yeasts that were fed dammarenediol-II yielded protopanaxadiol. Furthermore, co-expression of the dammarenediol synthase gene (PgDDS) and CYP716A47 in yeast yielded protopanaxadiol without adding dammarenediol-II. The chemical structures of the protopanaxadiol products from dammarenediol-II were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Thus, CYP716A47 is a dammarenediol 12-hydroxylase that produces protopanaxadiol from dammarenediol-II.

Ginseng has a prominent position on the list of best-selling herbal products in the world, and its main active constituents are thought to be ginsenosides. Compared with the long history of use and widespread research on Asian ginseng, studies of American ginseng are relatively limited, especially regarding cancer chemoprevention. In recent studies of American ginseng, steaming or heating altered the ginsenoside profile and thereby increased anticancer effects. Yet the ginsenoside structures and their activities have not been systematically elucidated. In this commentary, we introduce the different ginsenosides in American ginseng, both the naturally occurring compounds and those resulting from steaming or biotransformation. We briefly review American ginseng's reported anticancer effects and their mechanisms of action, and explore the possible structural-function relationship with a focus on sugar molecules, hydroxyl groups and stereoselectivity in ginsenosides. Understanding these relationships may produce insights into chemical and pharmacological approaches for enhancing the chemopreventive effects of ginsenoside and for developing novel anticancer agents., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Objective: To optimize the culture condition of adventitious roots of Panax ginseng., Method: The adventitious roots were obtained through tissue culture by manipulation of inoculum, various sucrose concentrations and salt strength. The contents of ginsenosides Re, Rb1 and Rg1 were determined by HPLC while the contents of polysaccharides were determined by ultraviolet spectrophotometry., Result: The multiplication of adventitious roots reached the peak when the inoculum was 20 g x L(-1). The effects of sucrose concentration and salt strength on adventitious roots were observed. The contents of polysaccharides were higher when the medium contained more sucrose. 40 g x L(-1) sucrose was favorable for roots growth and biosynthesis of Re, while 30 g x L(-1) was favorable for the biosynthesis of Rb1 and Rg1. 3/4MS medium was benefit for the growth of adventitious roots and the biosynthesis of ginsenosides. The contents of polysaccharides were decreased with the increase of salt strength., Conclusion: The results showed that inoculum, various sucrose concentrations and salt strength have significant influences on adventitious roots growth, secondary metabolite and polysaccharide synthesis in P. ginseng.

Ginseng (Panax ginseng C. A. Meyer) is one of the most famous oriental medicinal plants used as crude drugs in Asian countries, and now it is being used worldwide for preventive and therapeutic purposes. Among diverse constituents of ginseng, saponins (ginsenosides) have been found to be major components responsible for their biological and pharmacological actions. On the other hand, difficulties in the supply of pure ginsenosides in quantity prevent the development of ginseng for clinical medicines. Cultivation of ginseng in fields takes a long time, generally 5-7 years, and needs extensive effort regarding quality control since growth is susceptible to many environmental factors including soil, shade, climate, pathogens and pests. To solve the problems, cell and tissue cultures have been widely explored for more rapid and efficient production of ginseng biomass and ginsenosides. Recently, cell and adventitious root cultures of P. ginseng have been established in large scale bioreactors with a view to commercial application. Various physiological and engineering parameters affecting the biomass production and ginsenoside accumulation have been investigated. Advances in adventitious root cultures including factors for process scale-up are reviewed in this chapter. In addition, biosafety analyses of ginseng adventitious roots are also discussed for real application.

The involvement of NO in O(2)(.-) generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O(2)(.-) generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O(2)(.-) anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H(2)O(2)levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O(2) (.-) by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.

The objective of this study was to improve the accumulation of ginsenosides by the adventitious root cultures of ginseng, which are important secondary metabolites with pharmaceutical applications. The adventitious roots were cultured in bioreactors for 50 d using 1.5-strength Murashige and Skoog (MS) medium supplemented with 10 mg/l indole acetic acid and 75 g/l sucrose. Kinetic studies of the nutrient composition of the spent medium revealed the gradual depletion of various inorganic nutrients and sugars. Cultures were supplied with fresh nutrient medium (medium exchange or replenishment with 0.75- and 1.0-strength MS medium) after 10 and 20 d of culture initiation to fulfill the nutritional requirements of adventitious roots. Medium replenishment strategy (with 1.0-strength MS medium after 20 d of culture) significantly improved the growth of adventitious roots and the biosynthesis of ginsenosides by the adventitious roots. This work is useful for the large-scale cultivation of adventitious roots for the production of ginsenosides.

The growth and development of Sanqi in the lack of nutritional elements had been done by the water culture. The results indicated that Sanqi plants had different symptoms in the lack of nutritional elements. According to the symptom the symptomatic list was made in this paper.

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important signalling molecule and antioxidant. Here we investigated the protective effect of NO against the toxicity caused by excess CuSO(4) (50 microM) in the adventitious roots of mountain ginseng. It was found that NO donor, sodium nitroprusside (SNP), was effective in reducing Cu-induced toxicity in the mountain ginseng adventitious roots. Protective effect of SNP, as indicated by extent of lipid peroxidation, was reversed by incorporation of 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (CPTIO), a NO scavenger, in the medium suggesting that the protective effect of SNP is attributable to NO released, which was revealed from in situ confocal laser scanning microscopic localization of NO in the adventitious roots of mountain ginseng. Results obtained in the present study suggest that reduction of excess Cu-induced toxicity by SNP is most likely mediated through the modulation in the activities of antioxidant enzymes involved in H(2)O(2) detoxification (catalase, peroxidase, ascorbate peroxidase) and in the maintenance of cellular redox couples (glutathione reductase), and contents of molecular antioxidants (particularly non-protein thiol, ascorbate and its redox status). Exogenous NO supply also improved the activity of superoxide dismutase, an enzyme responsible for O*(2) (-) dismutation, and NADPH oxidase, an enzyme responsible for O*(2) (-) generation, in excess Cu supplied adventitious roots of mountain ginseng.

Indole-3-butyric acid at 25 microM with methyl jasmonate (MJ) at 100 microM in Panax ginseng synergistically stimulated both root growth and ginsenoside accumulation compared with 100 microM MJ alone. Productivity of ginsenoside was 10 mg l(-1) d(-1) compared to 7.3 mg l(-1) d(-1) with MJ elicitation alone.

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5-30 mg l(-1). HS and OS at 30 mg l(-1), fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to approximately 20 g l(-1) from 13 g l(-1) and the total saponin content of roots by more than 1-fold to approximately 3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.

To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 microM methyl jasmonate (MJ) or salicylic acid (SA) in a 5 L bioreactor suspension culture (working volume 4 L). Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2) contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH) and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reducing activity were increased by MJ and SA. Fresh weight (FW) and dry weight (DW) decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%), DPPH activity (40%), flavonoids (88%), ascorbic acid (55%), NPSH (33%), and cysteine (62%) contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase) were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and beta-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.

The suppression subtractive hybridization technique was used to identify differentially expressed genes between hormone-autotrophic and hormone-dependent Panax ginseng callus lines. A tonoplast intrinsic protein cDNA (PgTIP1) was found to be highly and specifically expressed in hormone-autotrophic ginseng cells, which was slightly up-regulated by cytokinin while significantly down-regulated when treated with auxin. PgTIP1 encodes a polypeptide of 250 amino acids which shows sequence and structure similarity with tonoplast aquaporins in plants. The water channel activity of PgTIP1 was demonstrated by its expression in Xenopus laevis oocytes. When over-expressed in Arabidopsis thaliana, PgTIP1 substantially altered the plant's vegetative and reproductive growth and development. Arabidopsis plants over-expressing PgTIP1 showed significantly enhanced seed size and seed mass plus greatly increased growth rate compared with those of the wild type. Moreover, the seeds from PgTIP1 over-expressing Arabidopsis had 1.85-fold higher fatty acid content than the wild-type control. These results demonstrate a significant function of PgTIP1 in the growth and development of plant cells.

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 microM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 microM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 muM but decreased at 50 microM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O2*-) accumulation, and H2O2 content at 5 and 10 microM Cu-treated roots were not increased but strongly increased at 50 microM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 microM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 microM Cu, but both G-POD and CAT activities were inhibited at 50 microM Cu. Glutathione metabolism enzymes such as gamma-glutamylcysteine synthetase (gamma-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 microM Cu but were strongly inhibited at 50 microM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 microM Cu was associated to the strong induction of gamma-glutamyltranspeptidase (gamma-GGT) activity. These results indicate that plant could grow under Cu stress (5-25 microM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O2-), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 microM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested 3, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O2- accumulation. Activity of superoxide dismutase (SOD) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), SOD, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O2- stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.

Chemically synthesized 2-hydroxyethyl jasmonate (HEJA) was for the first time employed to induce the ginsenoside biosynthesis and to manipulate the product heterogeneity in plant cell cultures. The dose response and timing of HEJA elicitation were investigated in cell suspension cultures of Panax notoginseng. The optimal concentration and timing of HEJA addition for both cell growth and ginsenoside accumulation was identified to be 200 microM added on day 4. It was interestingly found that HEJA could stimulate ginsenosides biosynthesis and change their heterogeneity more efficiently than methyl jasmonate (MJA), i.e., the total ginsenoside content and the Rb/Rg ratio increased about 60 and 30% with HEJA elicitation than that by MJA, respectively. The activity of Rb1 biosynthetic enzyme, i.e., UDPG-ginsenoside Rd glucosyltransferase (UGRdGT), was also higher in the former case. A maximal production titer of ginsenoside Rg1, Re, Rb1, and Rd was 47.4+/-4.8, 52.3+/-4.4, 190+/-18, and 12.1+/-2.5 mg/l with HEJA elicitation, which was about 1.3-, 1.3-, 1.7-, and 2.1-fold than that using MJA, respectively. Early signal events in plant defense response, including oxidative burst and jasmonic acid (JA) biosynthesis, were also examined. Levels of H2O2 and NO in medium and L-phenylalanine ammonia lyase activity in cells were not affected by addition of MJA and HEJA. On the other hand, the JA content in cells was increased with external jasmonates elicitation, and it was inhibited with the addition of JA biosynthesis inhibitors. The results suggest that oxidative burst might not be involved in the jasmonates-elicited signal transduction pathway, and MJA and HEJA may induce the ginsenoside biosynthesis via induction of endogenous JA biosynthesis and key enzymes (such as UGRdGT) in the ginsenoside biosynthetic pathway of P. notoginseng cells. The information is useful for hyperproduction of plant-specific heterogeneous products.

Plants generally produce secondary metabolites in nature as a defense mechanism against pathogenic and insect attack. In this study, we applied several abiotic elicitors in order to enhance growth and ginseng saponin biosynthesis in the hairy roots of Panax ginseng. Generally, elicitor treatments were found to inhibit the growth of the hairy roots, although simultaneously enhancing ginseng saponin biosynthesis. Tannic acid profoundly inhibited the hairy root growth during growth period. Also, ginseng saponin content was not significantly different from that of the control. The addition of selenium at inoculum time did not significantly affect ginseng saponin biosynthesis. However, when 0.5 mM selenium was added as an elicitor after 21 d of culture, ginseng saponin content and productivity increased to about 1.31 and 1.33 times control levels, respectively. Also, the addition of 20 microM NiSO4 resulted in an increase in ginseng saponin content and productivity, to about 1.20 and 1.23 times control levels, respectively, and also did not inhibit the growth of the roots. Sodium chloride treatment inhibited hairy root growth, except at a concentration of 0.3% (w/v). Increases in the amounts of synthesized ginseng saponin were observed at all concentrations of added sodium chloride. At 0.1% (w/v) sodium chloride, ginseng saponin content and productivity were increased to approx 1.15 and 1.13 times control values, respectively. These results suggest that processing time for the generation of ginseng saponin in a hairy root culture can be reduced via the application of an elicitor.

Adventitious roots from leafstalks and lateral roots were obtained directly from explants of Panax notoginseng. The lateral root explants were more sensitive to the induction of adventitious roots using indole-3-butyric acid. HPLC analysis of saponins extracted from the adventitious roots indicated that several protopanaxatriol saponins were present but ginsenoside Rd was missing, compared with the saponins extracted from the raw herbs. The dry weight of primary adventitious root culture of Panax notoginseng increased 5.25 times during multiplication in a classical shaking-flask system, suggesting that it is a culture system with great potential for scale-up.