High nitrogen inhibits photosynthetic performance in a shade-tolerant and N-sensitive species Panax notoginseng.

Nitrogen (N) is a primary factor limiting leaf photosynthesis. However, the mechanism of high-N-driven inhibition on photosynthetic efficiency and photoprotection is still unclear in the shade-tolerant and N-sensitive species such as Panax notoginseng. Leaf chlorophyll (Chl) content, Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity and content, N allocation in the photosynthetic apparatus, photosynthetic performance and Chl fluorescence were comparatively analyzed in a shade-tolerant and N-sensitive species P. notoginseng grown under the levels of moderate nitrogen (MN) and high nitrogen (HN). The results showed that Rubisco content, Chl content and specific leaf nitrogen (SLN) were greater in the HN individuals. Rubisco activity, net photosynthetic rate (Anet), photosynthetic N use efficiency (PNUE), maximum carboxylation rate (Vcmax) and maximum electron transport rate (Jmax) were lower when plants were exposed to HN as compared with ones to MN. A large proportion of leaf N was allocated to the carboxylation component under the levels of MN. More N was only served as a form of N storage and not contributed to photosynthesis in HN individuals. Compared with the MN plants, the maximum quantum yield of photosystem II (Fv/Fm), non-photochemical quenching of PSII (NPQ), effective quantum yield and electron transport rate were obviously reduced in the HN plants. Cycle electron flow (CEF) was considerably enhanced in the MN individuals. There was not a significant difference in maximum photo-oxidation P700+ (Pm) between the HN and MN individuals. Most importantly, the HN individuals showed higher K phase in the fast chlorophyll fluorescence induction kinetic curve (OJIP kinetic curve) than the MN ones. The results obtained suggest that photosynthetic capacity might be primarily inhibited by the inactivated Rubisco in the HN individuals, and HN-induced depression of photoprotection might be caused by the photodamage to the donor side of PSII oxygen-evolving complex.

Total Panax notoginseng saponin inhibits balloon injury-induced neointimal hyperplasia in rat carotid artery models by suppressing pERK/p38 MAPK pathways

Total Panax notoginseng saponin (TPNS) is the main bioactivity compound derived from the roots and rhizomes of Panax notoginseng (Burk.) F.H. Chen. The aim of this study was to investigate the effectiveness of TPNS in treating vascular neointimal hyperplasia in rats and its mechanisms. Male Sprague-Dawley rats were randomly divided into five groups, sham (control), injury, and low, medium, and high dose TPNS (5, 10, and 20 mg/kg). An in vivo 2F Fogarty balloon-induced carotid artery injury model was established in rats. TPNS significantly and dose-dependently reduced balloon injury-induced neointimal area (NIA) (P<0.001, for all doses) and NIA/media area (MA) (P<0.030, for all doses) in the carotid artery of rats, and PCNA expression (P<0.001, all). The mRNA expression of smooth muscle (SM) α-actin was significantly increased in all TPNS groups (P<0.005, for all doses) and the protein expression was significantly increased in the medium (P=0.006) and high dose TPNS (P=0.002) groups compared to the injury group. All the TPNS doses significantly decreased the mRNA expression of c-fos (P<0.001). The medium and high dose TPNS groups significantly suppressed the upregulation of pERK1/2 protein in the NIA (P<0.025) and MA (P<0.004). TPNS dose-dependently inhibited balloon injury-induced activation of pERK/p38MAPK signaling in the carotid artery. TPNS could be a promising agent in inhibiting cell proliferation following vascular injuries.

Total Panax notoginseng saponin inhibits balloon injury-induced neointimal hyperplasia in rat carotid artery models by suppressing pERK/p38 MAPK pathways.

Total Panax notoginseng saponin (TPNS) is the main bioactivity compound derived from the roots and rhizomes of Panax notoginseng (Burk.) F.H. Chen. The aim of this study was to investigate the effectiveness of TPNS in treating vascular neointimal hyperplasia in rats and its mechanisms. Male Sprague-Dawley rats were randomly divided into five groups, sham (control), injury, and low, medium, and high dose TPNS (5, 10, and 20 mg/kg). An in vivo 2F Fogarty balloon-induced carotid artery injury model was established in rats. TPNS significantly and dose-dependently reduced balloon injury-induced neointimal area (NIA) (P<0.001, for all doses) and NIA/media area (MA) (P<0.030, for all doses) in the carotid artery of rats, and PCNA expression (P<0.001, all). The mRNA expression of smooth muscle (SM) α-actin was significantly increased in all TPNS groups (P<0.005, for all doses) and the protein expression was significantly increased in the medium (P=0.006) and high dose TPNS (P=0.002) groups compared to the injury group. All the TPNS doses significantly decreased the mRNA expression of c-fos (P<0.001). The medium and high dose TPNS groups significantly suppressed the upregulation of pERK1/2 protein in the NIA (P<0.025) and MA (P<0.004). TPNS dose-dependently inhibited balloon injury-induced activation of pERK/p38MAPK signaling in the carotid artery. TPNS could be a promising agent in inhibiting cell proliferation following vascular injuries.

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.

Fungicidal Activity of Essential Oils from Cinnamomum cassia against the Pathogenic Fungi of Panax notoginseng Diseases.

The frequent disease of Panax notoginseng caused by the pathogenic fungi in field cultivation has become the major threaten to the sustainable development of it. The present study was conducted to find natural agent with potential inhibition against pathogen. Therefore, the inhibitory effects of Cinnamomum cassia (L.) J.Presl essential oils (EOs) against P. notoginseng associated pathogenic fungi were conducted both in vitro and in vivo experiments. The results of the Oxford cup test revealed that C. cassia dry bark EO (50 mg/mL) had significant inhibitory activity on the growth of all tested fungi, and the growth of various pathogens was completely inhibited, except for that of Fusarium solani. Therefore, the constituents of C. cassia EOs were analyzed by GC/MS, and the research demonstrated that the main constituents of C. cassia dry bark EO were trans-cinnamaldehyde (75.65 %), (E)-2-methoxycinnamaldehyde (6.08 %), cinnamaldehyde (3.47 %) and cinnamyl acetate (1.02 %). The MIC results showed that C. cassia dry bark EO and the main compounds had good antifungal effect on the tested strains, and the inhibitory effect was similar to that of hymexazol (chemical pesticide). By analyzing the value of the fraction inhibitory concentration index (FICI), additive effects, irrelevant effects and synergistic effects were observed after the mixture of hymexazol against various pathogens. Moreover, in vivo model showed that C. cassia dry bark EO could reduce the occurrence of anthrax in P. notoginseng. To widen the resources of C. cassia available, the compositions of both C. cassia fresh bark and leaf EOs were also tested and many common compositions existed among them. Taken together, it was concluded that C. cassia EO had the potential use in the field to reduce the pathogenic disease.

A transcriptome analysis uncovers Panax notoginseng resistance to Fusarium solani induced by methyl jasmonate.

BACKGROUND: Panax notoginseng is a famous Chinese herbal medicine, but the root rot disease mainly caused by Fusarium solani severely reduces the yield and quality of its medicinal materials. OBJECTIVE: The defense priming in P. notoginseng through exogenous application of signaling molecule will supply theoretical support for the exogenous regulation of disease resistance in P. notoginseng. METHODS: In this study, the exogenous application of methyl jasmonate (MeJA) increased P. notoginseng's resistance to F. solani. Furthermore, the P. notoginseng transcriptome during F. solani infection was investigated through next-generation sequencing to uncover the resistance mechanism of P. notogingseng induced by MeJA. RESULTS: The de novo assembly of transcriptome sequences produced 80,551 unigenes, and 36,771 of these unigenes were annotated by at least one database. A differentially expressed gene analysis revealed that a large number of genes related to terpenoid backbone biosynthesis, phenylalanine metabolism, and plant-pathogen interactions were predominantly up-regulated by MeJA. Moreover, jasmonic acid (JA) biosynthesis-related genes and the JA signaling pathway genes, such as linoleate 13S-lipoxygenase, allene oxide cyclase, allene oxide synthase, TIFY, defensin, and pathogenesis-related proteins, showed increased transcriptional levels after inoculation with F. solani. Notably, according to the gene expression analysis, JA and ethylene signaling pathways may act synergistically to positively regulate the defense responses of P. notoginseng to F. solani. CONCLUSION: JA signaling appears to play a vital role in P. notoginseng responses to F. solani infection, which will be helpful in improving the disease resistance of P. notoginseng cultivars as well as in developing an environmentally friendly biological control method for root rot disease.

Effects of Essential Oils from Zingiberaceae Plants on Root-Rot Disease of Panax notoginseng.

Replanting obstacles of Panax notoginseng caused by complex factors, including pathogens, have received great attention. In this study, essential oils (EOs) from either Alpinia officinarum Hance or Amomum tsao-ko (Zingiberaceae) were found to inhibit the growth of P. notoginseng-associated pathogenic fungi in vitro. Subsequent GC-MS analysis revealed the chemical profiles of two plant derived EOs. Linalool and eucalyptol were found to be abundant in the EOs and tested for their antifungal activities. In addition, the synergistic effects of A. tsao-ko EOs and hymexazol were also examined. These findings suggested that Zingiberaceae EOs might be a good source for developing new green natural pesticides fighting against root-rot of P. notoginseng.

Impaired terpenoid backbone biosynthesis reduces saponin accumulation in Panax notoginseng under Cd stress.

Panax notoginseng saponins (PNS) are major secondary metabolite of Panax notoginseng (Burk.) F.H. Chen. Previous studies identified that P. notoginseng planting soil usually with high content of Cd. However, the effects of Cd stress on the accumulation of PNS and the corresponding regulation mechanisms have yet to be reported. In the present study, the impact of Cd stress on the PNS accumulation of P. notoginseng was studied in pot culture experiments. The effect of Cd stress on antioxidant enzyme activity was studied using hydroponics. In addition, transcriptase sequencing analysis was used to study the effect of Cd stress on the expression of PNS metabolism transcripts in hydroponic experiments. Cd treatments significantly decreased the accumulation of PNS in the rhizome and main root. The sensitive concentration of antioxidant enzyme activity for both leaf and stem was 2.5µM, whereas the sensitive concentration for the root was 5.0µM. Transcriptome analysis showed that 5132 genes (2930 up- and 2202 downregulated) were regulated by 5.0µM Cd stress in the root of P. notoginseng. Among them, six upregulated differentially expressed genes (DEGs) were related to the methylerythritol 4-phosphate (MEP) pathway, whereas three of the downregulated DEGs were mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and geranylgeranyl diphosphate synthase (type II, GGPS). Of the 15 transcripts selected for real-time quantitative-PCR, 13 were expressed in the same manner as identified using RNA-seq. In conclusion, Cd stress inhibited the accumulation of PNS in the root of P. notoginseng by reducing the expression of MVK, PMK, and GGPS in the terpenoid backbone biosynthesis pathway, and also caused by the removal of reactive oxygen species.

Jasmonic acid and methyl dihydrojasmonate enhance saponin biosynthesis as well as expression of functional genes in adventitious roots of Panax notoginseng F.H. Chen.

Panax notoginseng, an important herbal medicine, has wide uses for its bioactive compounds and health function. In this work, we compared the content of saponin in cultivation and adventitious root. The total content of saponins in adventitious root (8.48 mg⋅g-1 ) was found lower than in the native one (3-year-old) (34.34 mg⋅g-1 ). To enhance the content of bioactive compounds, we applied elicitors jasmonic acid (JA) and methyl dihydrojasmonate (MDJ) to the adventitious root culture. It was observed that the highest total content of saponins (71.94 mg⋅g-1 ) was achieved after treatment with 5 mg⋅L-1 JA, which was 2.09-fold higher than native roots and 8.45-fold higher than the control group. The findings from high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis showed that six new compounds were present after the treatment with the elicitors. Furthermore, we found that JA and MDJ significantly upregulated the expression of the geranyl diphosphate synthase, farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, dammarenediol synthase, and CYP716A47 and CYP716A53v2 (CYP450 enzyme) genes; downregulated the expression of the cycloartenol synthase gene; and increased superoxide dismutase and peroxidase activities.

Nitrate reductase-mediated NO production enhances Cd accumulation in Panax notoginseng roots by affecting root cell wall properties.

Panax notoginseng (Burk) F. H. Chen is a traditional medicinal herb in China. However, the high capacity of its roots to accumulate cadmium (Cd) poses a potential risk to human health. Although there is some evidence for the involvement of nitric oxide (NO) in mediating Cd toxicity, the origin of Cd-induced NO and its function in plant responses to Cd remain unknown. In this study, we examined NO synthesis and its role in Cd accumulation in P. notoginseng roots. Cd-induced NO production was significantly decreased by application of the nitrate reductase inhibitor tungstate but not the nitric oxide synthase inhibitor L-NAME (N(G)-methyl-l-arginine acetate), indicating that nitrate reductase is the major contributor to Cd-induced NO production in P. notoginseng roots. Under conditions of Cd stress, sodium nitroprusside (SNP, an NO donor) increased Cd accumulation in root cell walls but decreased Cd translocation to the shoot. In contrast, the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and tungstate both significantly decreased NO-increased Cd retention in root cell walls. The amounts of hemicellulose 1 and pectin, together with pectin methylesterase activity, were increased with the addition of SNP but were decreased by cPTIO and tungstate. Furthermore, increases or decreases in hemicellulose 1 and pectin contents as well as pectin methylesterase activity fit well with the increased or decreased retention of Cd in the cell walls of P. notoginseng roots. The results suggest that nitrate reductase-mediated NO production enhances Cd retention in P. notoginseng roots by modulating the properties of the cell wall.

[Physiological response and bioaccumulation of Panax notoginseng to cadmium under hydroponic].

The physiological response and bioaccumulation of 2-year-old Panax notoginseng to cadmium stress was investigated under a hydroponic experiment with different cadmium concentrations (0, 2.5, 5, 10 µmol · L(-1)). Result showed that low concentration (2.5 µmol · L(-1)) of cadmium could stimulate the activities of SOD, POD, APX in P. notoginseng, while high concentration (10 µmol · L(-1)) treatment made activities of antioxidant enzyme descended obviously. But, no matter how high the concentration of cadmium was, the activities of CAT were inhibited. The Pn, Tr, Gs in P. notoginseng decreased gradually with the increase of cadmium concentration, however Ci showed a trend from rise to decline. The enrichment coefficients of different parts in P. notoginseng ranked in the order of hair root > root > rhizome > leaf > stem, and all enrichment coefficients decreased with the increase of concentration of cadmium treatments; while the cadmium content in different parts of P. notoginseng and the transport coefficients rose. To sum up, cadmium could affect antioxidant enzyme system and photosynthetic system of P. notoginseng; P. notoginseng had the ability of cadmium enrichment, so we should plant it in suitable place reduce for reducing the absorption of cadmium; and choose medicinal parts properly to lessen cadmium intake.

[Effect of Panax notoginseng seedlings physiological response under simulated drought stress by polyethylene glycol (PEG 6000)].

The physiological effects of Panax notoginseng seedlings under simulated drought stress by PEG 6000 on antioxidant enzymes, osmotic substances and root activities were studied. The results showed that the activity of POD and APX in roots and leaves kept rising with increasing processing concentration and time. However, on the one hand, at the same processing time, SOD in roots and leaves firstly increased and then decreased with the increase of processing concentration. On the other hand, at the same processing concentration, SOD kept rising with the extension of processing time. In addition, the activity of CAT in roots and leaves tended to increase with the increasing concentration at the same processing time, while it increased at first and then decreased with the extension of time at the same concentration. The activity of SOD and APX in stem did not change obviously, whereas CAT activity in stem increased with the increasing processing time and concentration. With the increase of processing concentration and the extension of processing time, the MDA, soluble protein, proline content and root activity in leaves and roots apparently rose. Moreover, fluorescence signal of H2O2 and NO in root tip enhanced as the processing concentration increased after treated for 1 d. In summary, P. notoginseng seedlings could deal with drought stress by means of adjusting the system of antioxidant enzyme, permeating stress substances and impeded stress signal substances. Thus, when the concentration of PEG 6000 was more than 5%, it would have harm on P. notoginseng seedlings.

Autotoxic ginsenosides in the rhizosphere contribute to the replant failure of Panax notoginseng.

BACKGROUND AND AIMS: Sanqi ginseng (Panax notoginseng) growth is often hampered by replant failure. In this study, we aimed to examine the role of autotoxicity in Sanqi replant failures and assess the role of ginsenosides in autotoxicity. METHODS: The autotoxicities were measured using seedling emergence bioassays and root cell vigor staining. The ginsenosides in the roots, soils, and root exudates were identified with HPLC-MS. RESULTS: The seedling emergence and survival rate decreased significantly with the continuous number of planting years from one to three years. The root exudates, root extracts, and extracts from consecutively cultivated soils also showed significant autotoxicity against seedling emergence and growth. Ginsenosides, including R1, Rg1, Re, Rb1, Rb3, Rg2, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg1, Re, Rg2, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R1, Rg1, Re, Rg2, and Rd, caused autotoxicity against seedling emergence and growth and root cell vigor at a concentration of 1.0 µg/mL. CONCLUSION: Our results demonstrated that autotoxicity results in replant failure of Sanqi ginseng. While Sanqi ginseng consecutively cultivated, some ginsenosides can accumulate in rhizosphere soils through root exudates or root decomposition, which impedes seedling emergence and growth.

Arsenic accumulation and resistance mechanism in Panax notoginseng, a traditional rare medicinal herb.

Panax notoginseng, a traditional rare Chinese medicinal herb, was recently found to bring health risk to consumers, mainly because soil in its major plantation area was contaminated by arsenic (As). We investigated the effect of soil As pollution on the growth and As uptake of pot-cultured P. notoginseng, and the associated mechanisms of As stressed response. Results showed that, comparing with P. notoginseng growing in a low-As soil, the root, stem, and leaf biomasses of those growing in a high-As soil significantly reduced by 0.75, 0.09 and 0.21 g seedling(-1), respectively. Arsenic concentrations in roots, stems and leaves of the seedlings growing in high-As soil were 22, 15 and 3 times higher than those growing in low-As soil, respectively. Regardless of the soil As concentration, As existed in plants mainly as As(III), suggesting that the reduction of As(V) is a key step in As metabolism. Arsenic was distributed primarily in cell walls (51.7% for plants growing in the low-As soil, and 51.5% in the high-As soil), followed by cytoplasm supernatant, with cell organelles containing the least As. Compared with plants growing in the low-As soil, those in the high-As soil had increased superoxide dismutase and peroxidase activities in their roots, stems, and leaves, which would be associate with improving the resistance of P. notoginseng to As stress. The results suggest that there exists some special mechanisms of As-tolerance in P. notoginseng and the study is of significance in developing measures to reduce As in the herb.

Manipulation of ginsenoside heterogeneity of Panax notoginseng cells in flask and bioreactor cultivations with addition of phenobarbital.

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg(1) + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg(1) + Re in the ALR was increased from 42.5 +/- 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 +/- 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg(1) + Re in the ALR reached 5.66 +/- 0.38 mg L(-1) d(-1), which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb(1)) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.

Role of jasmonic acid in alteration of ginsenoside heterogeneity in elicited cell cultures of Panax notoginseng.

The efficient manipulation of ginsenoside heterogeneity of Panax notoginseng cells using a recently synthesized elicitor, 2-hydroxyethyl jasmonate (HEJ, at 200 microM), has been reported. In this work, the activities of two enzymes related to ginsenoside heterogeneity (distribution), protopanaxdiol 6-hydroxylase (P6H) and UDPG-ginsenoside Rd glucosyltransferase (UGRdGT), were examined in cell cultures of P. notoginseng elicited by HEJ. P6H and UGRdGT activities were increased by HEJ with corresponding changes in Rb/Rg ratio and Rb1/Rd ratio. Endogenous jasmonic acid (JA) seemed to mediate the induction of UGRdGT activation, but was not involved in P6H activation. The results suggest that JA, as a signal transducer, may play an important role in the alteration of ginsenoside heterogeneity in elicited P. notoginseng cells.

[Adventitious root induction and in vitro culture of Panax notoginseng].

OBJECTIVE: To investigate the induction and culture of adventitious root of Panax notoginseng. METHOD: Three ways, induction from the explants of three-year-old P. notoginseng. The explants of regenerated shoots and calluses, were used to induce adventitious roots. The effects of 2, 4-dichlorophenoxyacetic acid, indole-3-butyric acid and naphthylacetic acid on adventitious root induction were investigated respectively. The effects of four modes of separating adventitious roots from the parent tissues on culture in vitro were compared. RESULT: Adventitious roots were successfully induced by three methods, of which the young flower bud callus was the best material for the induction of adventitious root. Indole-3-butyric acid possessed the strongest potency for induction. The liquid culture system was established by continuous culture of adventitious roots together with their parent tissues before separated. CONCLUSION: The acquisition and culture in vitro in liquid culture system of adventitious roots of P. notoginseng lay a foundation for the next investigation.