Widely Targeted Metabolomic Analysis Reveals the Improvement in Panax notoginseng Triterpenoids Triggered by Arbuscular Mycorrhizal Fungi via UPLC-ESI-MS/MS.

Panax notoginseng is a highly valued perennial medicinal herb in China and is widely used in clinical treatments. The main purpose of this study was to elucidate the changes in the composition of P. notoginseng saponins (PNSs), which are the main bioactive substances, triggered by arbuscular mycorrhizal fungi (AMF) via ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). A total of 202 putative terpenoid metabolites were detected, of which 150 triterpene glycosides were identified, accounting for 74.26% of the total. Correlation analysis, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) of the metabolites revealed that the samples treated with AMF (group Ce) could be clearly separated from the CK samples. In total, 49 differential terpene metabolites were identified between the Ce and CK groups, of which 38 and 11 metabolites were upregulated and downregulated, respectively, and most of the upregulated differentially abundant metabolites were mainly triterpene glycosides. The relative abundances of the two major notoginsenosides (MNs), ginsenosides Rd and Re, and 13 rare notoginsenosides (RNs), significantly increased. The differential saponins, especially RNs, were more easily clustered into one branch and had a high positive correlation. It could be concluded that the biosynthesis and accumulation of some RNs share the same pathways as those triggered by AMF. This study provides a new way to obtain more notoginsenoside resources, particularly RNs, and sheds new light on the scientization and rationalization of the use of AMF agents in the ecological planting of medicinal plants.

Comprehensive Analysis of 34 Edible Flowers by the Determination of Nutritional Composition and Antioxidant Capacity Planted in Yunnan Province China.

The main purpose of this study was to reveal the nutritional value and antioxidant activity of 34 edible flowers that grew in Yunnan Province, China, through a comprehensive assessment of their nutritional composition and antioxidant indices. The results showed that sample A3 of Asteraceae flowers had the highest total flavonoid content, with a value of 8.53%, and the maximum contents of vitamin C and reducing sugars were from Rosaceae sample R1 and Gentianaceae sample G3, with values of 143.80 mg/100 g and 7.82%, respectively. Samples R2 and R3 of Rosaceae were the top two flowers in terms of comprehensive nutritional quality. In addition, the antioxidant capacity of Rosaceae samples was evidently better than that of three others, in which Sample R1 had the maximum values in hydroxyl radical (·OH) scavenging and superoxide anion radical (·O2-) scavenging rates, and samples R2 and R3 showed a high total antioxidant capacity and 2,2-diphenyl-1-pyridylhydrazine (DPPH) scavenging rate, respectively. Taken together, there were significant differences in the nutrient contents and antioxidant properties of these 34 flowers, and the comprehensive quality of Rosaceae samples was generally better than the other three families. This study provides references for 34 edible flowers to be used as dietary supplements and important sources of natural antioxidants.

Optimization of the Ultrasonic-Assisted Extraction Technology of Steroidal Saponins from Polygonatum kingianum Collett & Hemsl and Evaluating Its Quality Planted in Different Areas.

Polygonatum kingianum Collett & Hemsl is one of the famous traditional Chinese herbs with satisfactory therapeutic effects on invigorating Qi, nourishing Yin and moistening lungs, in which steroidal saponins are one class of important active substances. The main purpose is to determine the optimal extraction technology of steroidal saponins and evaluate the quality of P. kingianum planted in five different areas. The optimal ultrasonic-assisted extraction (UAE) technology was established by using single-factor experiments and the response surface methodology (RSM), and the determination method of high-performance liquid chromatography (HPLC) for dioscin and diosgenin, two primary types of acid-hydrolyzed steroidal saponins, was constructed with good linear range and precision. The results showed that UAE was an efficient extraction method for steroidal saponins, and the extraction yield was significantly affected by the liquid-solid ratio. The optimal extraction technology was generated following a liquid-solid ratio of 10:1 (mL/g), an ethanol concentration of 85% (v/v), an extraction time of 75 min, an extraction temperature of 50 °C and three extractions, of which these parameters were in line with the predicted values calculated by RSM. Considering only dioscin and diosgenin, the quality of P. kingianum planted at five sample plots presented non-significant difference. However, the content of diosgenin in Pingbian Prefecture (PB) was higher than that of the other four areas with a value of 0.46 mg/g. Taken together, the optimal UAE technology for P. kingianum steroidal saponins was determined via RSM. The quality evaluation revealed that there was a non-significant difference among P. kingianum planted in different areas based on the contents of the sum of dioscin and diosgenin. This work has important reference value for the exploitation and utilization of P. kingianum.

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

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

EpABC Genes in the Adaptive Responses of Exophiala pisciphila to Metal Stress: Functional Importance and Relation to Metal Tolerance.

Exophiala pisciphila is one of the dominant dark septate endophytes (DSEs) colonizing metal-polluted slag heaps in southwest China. It shows numerous super-metal-tolerant characteristics, but the molecular mechanisms involved remain largely unknown. In the present study, the functional roles of a specific set of ATP-binding cassette (ABC) transporters in E. pisciphila were characterized. In total, 26 EpABC genes belonging to 6 subfamilies (ABCA to ABCG) were annotated in previous transcriptome sequencing libraries, and all were regulated by metal ions (Pb, Zn, and Cd), which was dependent on the metal species and/or concentrations tested. The results from the heterologous expression of 3 representative EpABC genes confirmed that the expression of EpABC2.1, EpABC3.1, or EpABC4.1 restored the growth of metal-sensitive mutant Saccharomyces cerevisiae strains and significantly improved the tolerance of Arabidopsis thaliana to Pb, Zn, and Cd. Interestingly, the expression of the 3 EpABC genes further altered metal (Pb, Zn, and Cd) uptake and accumulation and promoted growth by alleviating the inhibitory activity in yeast and thale cress caused by toxic ions. These functions along with their vacuolar location suggest that the 3 EpABC transporters may enhance the detoxification of vacuolar compartmentation via transport activities across their membranes. In conclusion, the 26 annotated EpABC transporters may play a major role in maintaining the homeostasis of various metal ions in different cellular compartments, conferring an extreme adaptative advantage to E. pisciphila in metal-polluted slag heaps.IMPORTANCE Many ABC transporters and their functions have been identified in animals and plants. However, little is known about ABC genes in filamentous fungi, especially DSEs, which tend to dominantly colonize the roots of plants growing in stressed environments. Our results deepen the understanding of the function of the ABC genes of a super-metal-tolerant DSE (E. pisciphila) in enhancing its heavy metal resistance and detoxification. Furthermore, the genetic resources of DSEs, e.g., numerous EpABC genes, especially from super-metal-tolerant strains in heavy metal-polluted environments, can be directly used for transgenic applications to improve tolerance and phytoextraction potential.

Functional and transcript analysis of a novel metal transporter gene EpNramp from a dark septate endophyte (Exophiala pisciphila).

Various metal transporters mediate sub-cellular sequestration of diverse metal ions, contribute to cellular metal tolerance, and control metal partitioning, particularly under conditions of high rates of metal influx into organisms. In the current study, a ubiquitous and evolutionary conserved metal transporter gene, homology to natural resistance associated macrophage protein (Nramp), was cloned from a metal-tolerant isolate of dark septate endophyte (DSE, Exophiala pisciphila), and its functional and transcript characterization were analyzed. The full-length Nramp gene from E. pisciphila (named EpNramp) was 1716 bp and expected to encode a polypeptide of 571 amino acid residues. EpNramp fused to green fluorescent protein suggested that EpNramp was a plasma membrane metal transporter, which was consistent with the results of bioinformatics analysis with 11 transmembrane domains. Yeast functional complementation revealed that EpNramp could complement the growth defect of Fe-uptake yeast mutant (fet3fet4 double mutant) by mediating the transport of Fe(2+). Expression of EpNramp increased Cd(2+) sensitivity and Cd(2+) accumulation in yeast. In addition, qPCR data revealed that E. pisciphila significantly down-regulated EpNramp expression with elevated Cd(2+) exposure. Altogether, EpNramp is a bivalent cation transporter localized in cell membrane, which is necessary for efficient translocation of both Fe and Cd, and its activities partly attributed to the tolerance of DSE to toxic and excessive Cd(2+) supplements.

Improvement of Panax notoginseng saponin accumulation triggered by methyl jasmonate under arbuscular mycorrhizal fungi.

Panax notoginseng is a highly valued perennial medicinal herb plant in Yunnan Province, China, and the taproots are the main medicinal parts that are rich in active substances of P. notoginseng saponins. The main purpose of this study is to uncover the physiological and molecular mechanism of Panax notoginseng saponin accumulation triggered by methyl jasmonate (MeJA) under arbuscular mycorrhizal fungi (AMF) by determining physiological indices, high-throughput sequencing and correlation analysis. Physiological results showed that the biomass and saponin contents of P. notoginseng, the concentrations of jasmonic acids (JAs) and the key enzyme activities involved in notoginsenoside biosynthesis significantly increased under AMF or MeJA, but the interactive treatment of AMF and MeJA weakened the effect of AMF, suggesting that a high concentration of endogenous JA have inhibitory effect. Transcriptome sequencing results indicated that differential expressed genes (DEGs) involved in notoginsenoside and JA biosynthesis were significantly enriched in response to AMF induction, e.g., upregulated genes of diphosphocytidyl-2-C-methyl-d-erythritol kinases (ISPEs), cytochrome P450 monooxygenases (CYP450s)_and glycosyltransferases (GTs), while treatments AMF-MeJA and salicylhydroxamic acid (SHAM) decreased the abundance of these DEGs. Interestingly, a high correlation presented between any two of saponin contents, key enzyme activities and expression levels of DEGs. Taken together, the inoculation of AMF can improve the growth and saponin accumulation of P. notoginseng by strengthening the activities of key enzymes and the expression levels of encoding genes, in which the JA regulatory pathway is a key link. This study provides references for implementing ecological planting of P. notoginseng, improving saponin accumulation and illustrating the biosynthesis mechanism.

Physiological response mechanism of heavy metal-resistant endophytic fungi isolated from the roots of Polygonatum kingianum.

This study aims to evaluate the tolerance of endophytic fungi isolated from the fibrous roots of Polygonatum kingianum to arsenic (As) and cadmium (Cd) and their physiological response mechanisms. Five isolated strains were obtained with EC50 values for As(V) ranging from 421 to 1281 mg/L, while the other three strains tolerated Cd(II) with an EC50 range of 407-1112 mg/L. Morphological and molecular identification indicated that these eight strains were Cladosporium spp. belonging to dark septate endophytes (DSEs). The contents of metal ions in mycelium sharply increased, reaching 38.87 mg/kg for strain MZ-11 under As(V) stress and 0.33 mg/kg for fungus PR-2 under Cd(II). The physiological response revealed that the biomass decreased with increasing concentrations of As(V) or Cd(II), and the activity of superoxide dismutase significantly improved under the corresponding EC50 -concentration As/Cd of the strains, as well as the contents of antioxidant substances, including metallothionein, glutathione, malondialdehyde, melanin, and proline. Taken together, the filamentous fungi of Cladosporium spp. accounted for a high proportion of fungi isolated from the fibrous roots of P. kingianum and had a strong capacity to tolerate As(V) or Cd(II) stress by improving antioxidase activities and the content of antioxidant substances, and immobilization of metal ions in hyphae.

A Panax notoginseng phosphate transporter, PnPht1;3, greatly contributes to phosphate and arsenate uptake.

The crisis of arsenic (As) accumulation in rhizomes threatens the quality and safety of Panax notoginseng (Burk.) F.H. Chen, which is a well-known traditional Chinese herb with a long clinical history. The uptake of arsenate (AsV) could be suppressed by supplying phosphate (Pi), in which Pi transporters play important roles in the uptake of Pi and AsV. Herein, the P . notoginseng Pi transporter-encoding gene PnPht1;3 was identified and characterised under Pi deficiency and AsV exposure. In this study, the open reading frame (ORF) of PnPht1;3 was cloned according to RNA-seq and encoded 545 amino acids. The relative expression levels revealed that PnPht1;3 was significantly upregulated under phosphate deficiency and AsV exposure. Heterologous expression in Saccharomyces cerevisiae MB192 demonstrated that PnPht1;3 performed optimally in complementing the yeast Pi-transport defect and accumulated more As in the cells. Combined with the subcellular localisation prediction, it was concluded that PnPht1;3 encodes a functional plasma membrane-localised transporter protein that mediates putative high-affinity Pi/H+ symport activity and enhances the uptake of Pi and AsV. Therefore, a better understanding of the roles of the P . notoginseng Pi transporter could provide new insight for solving As accumulation in medicinal plants.

Enterovirus A71 utilizes host cell lipid ß-oxidation to promote its replication.

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD), which is an infectious disease that endangers children's health. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Metabolism and stress are known to play critical roles in multiple stages of the replication of viruses. Lipid metabolism and ER stress is an important characterization post viral infection. EV-A71 infection alters the perturbations of intracellular lipid homeostasis and induces ER stress. The characterizations induced by viral infections are essential for optimal virus replication and may be potential antiviral targets. In this study, we found that the addition of the chemical drug of ER stress, PKR IN, an inhibitor, or Tunicamycin, an activator, could significantly reduce viral replication with the decrease of lipid. The replication of viruses was reduced by Chemical reagent TOFA, an inhibitor of acetyl-CoA carboxylase (ACC) or C75, an inhibitor of fatty acid synthase (FASN), while enhanced by oleic acid (OA), which is a kind of exogenous supplement of triacylglycerol. The pharmacochemical reagent of carnitine palmitoyltransferase 1 (CPT1) called Etomoxir could knock down CPT1 to induce EV-A71 replication to decrease. This suggests that lipid, rather than ER stress, is the main factor affecting EV-A71 replication. In conclusion, this study revealed that it is the ß-oxidation of lipid that plays a core role, not ER stress, which is only a concomitant change without restrictive effect, on virus replication.

Identification of glutathione S-transferase (GST) genes from a dark septate endophytic fungus (Exophiala pisciphila) and their expression patterns under varied metals stress.

Glutathione S-transferases (GSTs) compose a family of multifunctional enzymes that play important roles in the detoxification of xenobiotics and the oxidative stress response. In the present study, twenty four GST genes from the transcriptome of a metal-tolerant dark septate endophyte (DSE), Exophiala pisciphila, were identified based on sequence homology, and their responses to various heavy metal exposures were also analyzed. Phylogenetic analysis showed that the 24 GST genes from E. pisciphila (EpGSTs) were divided into eight distinct classes, including seven cytosolic classes and one mitochondrial metaxin 1-like class. Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50). Lead (Pb) exposure caused the up-regulation of all EpGSTs, while cadmium (Cd), copper (Cu) and zinc (Zn) treatments led to the significant up-regulation of most of the EpGSTs (p < 0.05 to p < 0.001). Furthermore, although heavy metal-specific differences in performance were observed under various heavy metals in Escherichia coli BL21 (DE3) transformed with EpGSTN-31, the over-expression of this gene was able to enhance the heavy metal tolerance of the host cells. These results indicate that E. Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila. The study represents the first investigation of the GST family in E. pisciphila and provides a primary interpretation of heavy metal detoxification for E. pisciphila.