Metabolome and transcriptome analyses provide new insights into the mechanisms underlying the enhancement of medicinal component content in the roots of Acanthopanax senticosus (Rupr. et Maxim.) Harms through foliar application of zinc fertilizer.

Acanthopanax senticosus (Rupr. et Maxim.) Harms is a perennial shrub of the Acanthopanax genus in the Araliaceae family and has a high medicinal value. The application of zinc fertilizer can improve the yield and quality of medicinal materials. However, there are limited reports on approaches to increase the content of medicinal components in A. senticosus, hindering the improvement of its medicinal quality. In this study, A. senticosus was treated with 0.1% (LZn) and 0.4% (HZn) zinc sprayed on the leaf surface. The effects of zinc treatment on the medicinal components in the roots of A. senticosus were analyzed by comprehensive metabolomics and transcriptomics analyses. A total of 316 metabolites were detected, with a prevailing occurrence of terpenoids and phenylpropanoids. We identified metabolites related to the medicinal components that were upregulated after Zn treatment, including 43 terpenoids, 19 phenylpropanoids, eight phenols, and three flavonoids. Combining differential gene expression and K-means analysis, we found 95, 65, and 25 upregulated genes related to phenylpropanoid biosynthesis, terpenoid biosynthesis, and flavonoid biosynthesis, respectively. Under different concentrations of Zn treatment, the upregulated metabolite biosynthesis-related genes and differentially expressed transcription factors varied. Pearson correlation network analysis revealed significant correlations among terpenoids, phenylpropanoids, flavonoids biosynthetic genes, and several transcription factors (ERFs, WRKYs, bHLHs, NACs, and MYBs). This study lays the foundation for understanding the metabolic processes in response to varying levels of zinc foliar spray and provides a theoretical basis for enhancing the efficiency of zinc fertilizer utilization in A. senticosus.

Metabolomic and antioxidant analyses of Salvia miltiorrhiza Bunge and Salvia prattii Hemsl. seeds.

Salvia miltiorrhiza and Salvia prattii seeds are rich in metabolites that are beneficial to human health and can be utilised as nutritional supplements. In this study, UPLC-MS and GC-MS based on extensively focused metabolomics were used to compare the seed metabolomics of the two species. LC-MS detected 118 metabolites, primarily Lipids and phenylpropanoids. GC- MS detected a total of 188 metabolites, mainly organic acids and their derivatives, of which Salvia prattii seeds contain high levels of nutrients. In addition, we experimentally determined antioxidant activity of two Salvia species, and the results showed that the antioxidant activity of Salvia prattii seeds was about twice as high as that of Salvia miltiorrhiza seeds. We used WGCNA to group the metabolites, and found the central metabolites in the focal modules including flavonoids and terpenoids. Our study contributes valuable knowledge for future research on the chemical makeup of Salvia prattii seeds.

Germplasm resources and secondary metabolism regulation in Reishi mushroom (Ganoderma lucidum).

Ganoderma lucidum is a valuable medical macrofungus with a myriad of diverse secondary metabolites, in which triterpenoids are the major constituents. This paper introduced the germplasm resources of genus Ganoderma from textual research, its distribution and identification at the molecular level. Also we overviewed G. lucidum in the components, the biological activities and biosynthetic pathways of ganoderic acid, aiming to provide scientific evidence for the development and utilization of G. lucidum germplasm resources and the biosynthesis of ganoderic acid.

LC-MS metabolomics profiling of Salvia aegyptiaca L. and S. lanigera Poir. with the antimicrobial properties of their extracts.

BACKGROUND: Salvia L. (Lamiaceae) found in almost all countries in temperate and tropical regions. Both S. aegyptiaca L. and S. lanigera Poir. have a rather wide distribution in Egypt (Mediterranean region, Gebel Elba and nearly the whole Sinai). Salvia species showed antibacterial and antifungal activities against several groups of food microorganisms and pathogens, so they are considered as a natural foods preservatives. AIM: Investigate the phytochemical profiles of S. aegyptiaca & S. lanigera collected from their natural habitats in Egypt and test the antimicrobial activities of both species against some bacteria and fungi pathogenic strains. METHODOLOGY: In the present study, S. aegyptiaca and S. lanigera were collected from their natural habitat. Total phenolics and flavonoids contents were measured for aerial parts of both Salvia spp.. The separation and identification of the pure active materials of both Salvia sp. by using LC-MS system (UHPLC-TSQ Quantum Mass Spectrometer). The antimicrobial activities of the ethanol, water and benzene extracts of the two species were tested against different pathogenic strains and compared with the standard antimicrobial drug (Gentamycin). Antimicrobial activity was determined by using agar disk diffusion method. RESULTS: The phenolics content in S. lanigera 132.61±6.23 mg/g and S. aegyptiaca 125.19±4.97 mg/g, while the flavonoids content was 35.68±1.84 and 40.63±2.11 mg/g, respectively. Through LC-MS analysis, two compounds were detected in both species; heptadecanoyl coenzyme A, that the highest percentage (13.5%) in S. aegyptiaca and (11.5 %) in S. lanigera. Oenin, in a peak area of 3.1% in S. aegyptiaca and 1.2 % in S. lanigera. Ethanol extract of the two species had the most inhibitory effect against all tested microorganisms that exceeded the effect of the standard, except for Mucor reinelloids which was more sensitive to the water extract. Moreover, S. lanigera ethanol extract showed larger inhibition zone than S. aegyptiaca in all tested microorganisms except for Pseudomonas aeruginosa. CONCLUSION: This study shows the important phytochemicals that improve the antibacterial and antifungal activities of Salvia aegyptiaca and S. lanigera.

The complete chloroplast genome sequence of Salvia chienii E.Peter, 1936 (Lamiaceae).

Salvia chienii E.Peter is a medicinal herb mainly distributed in Huangshan Mountain of Anhui province, China. In this study, the first complete chloroplast genome of S. chienii was sequenced and assembled. The genome length was 151,530 bp and encoded 143 genes (91 protein-coding genes, eight rRNA genes, and 37 tRNA genes). The phylogenomic analysis showed that S. chienii was closely related to S. miltiorrhiza. Further evolutionary studies of the genus Salvia could benefit from the complete chloroplast genome of S. chienii present in this study.

Metabolite Profiles Provide Insights into Underlying Mechanism in Bupleurum (Apiaceae) in Response to Three Levels of Phosphorus Fertilization.

Phosphorus (P) deficiency affects plant yield and quality, yet at the same time, excessive phosphorus application does not necessarily promote the growth of plants. How to maintain a balance between biomass accumulation and phosphorus application is a problem. Therefore, the purpose of this research was to explore the relationship between yield and quality of Bupleurum and phosphorus fertilization, based on three phosphorus fertilization levels (20 kg∙ha-1; 10 kg∙ha-1; and 0 kg∙ha-1). We adopted gas chromatography-mass spectrometry to assess the response of primary metabolites of different plant tissues (flowers, main shoots, lateral shoots and roots) to phosphorus fertilization. At the same time, high-performance liquid chromatography was used to quantify saikosaponin A and saikosaponin D, the main active ingredients of Bupleurum. Our research showed that low phosphorus level application has a positive impact on the yield and quality of Bupleurum, especially the above-ground parts increasing the fresh weight of flowers and lateral shoots and the length of main shoots, and moreover, increasing the saikosaponins content in all above-ground parts while decreasing the content in roots which show no significance increase in fresh weight and length. However, high phosphorus level showed a negative impact as it decreases the saikosaponins content significantly in flowers and roots. Furthermore, phosphorus application changed the proportion of saikosaponins, promoting the content of saikosaponin A and inhibiting the content of saikosaponin D in most organs of Bupleurum. Therefore, we can say that high phosphorus application is not preferable to the yield and quality of Bupleurum. To identify the metabolic pathways and special key metabolites, a total of 73 metabolites were discovered, and four differential metabolites-ether, glycerol, chlorogenic and L-rhamnose-were considered to be the key metabolites of Bupleurum's response to phosphorus fertilization. Furthermore, Bupleurum's response to phosphorus fertilization was mainly related to metabolic pathways, such as starch and sucrose metabolism and galactose metabolism. Under the phosphorus level, the content of sugars, organic acids and their derivatives, polyols and their derivatives and alkyl were upregulated in flowers. Furthermore, the contents of compounds in the main shoot and lateral shoots showed the same upward trend, except glycosides and polyols and their derivatives.

Comparative metabolomics of two saline-alkali tolerant plants Suaeda glauca and Puccinellia tenuiflora based on GC-MS platform.

Suaeda glauca and Puccinellia tenuiflora are two important saline-alkali tolerant plants that can improve the soil properties. For exploring the different tolerance mechanisms between them, GC-MS-based metabolomics was used to comprehensively evaluate the primary metabolites differences, a total of 51 different metabolites were present in different quantities. The identified compounds were mainly 11 sugars, 7 amino acids, 5 alcohols and 18 organic acids; they play an important role in responding to the saline-alkali stress and distinguish between S. glauca and P. tenuiflora. All identified metabolites classes showed similar trend to largely accumulate in P. tenuiflora roots and S. glauca shoots, this reveals that the two plants used different physiological strategies to cope with saline-alkali stress.

UV-B Radiation Largely Promoted the Transformation of Primary Metabolites to Phenols in Astragalus mongholicus Seedlings.

: Ultraviolet-B (UV-B) radiation (280-320 nm) may induce photobiological stress in plants, activate the plant defense system, and induce changes of metabolites. In our previous work, we found that between the two Astragalus varieties prescribed by the Chinese Pharmacopoeia, Astragalus mongholicus has better tolerance to UV-B. Thus, it is necessary to study the metabolic strategy of Astragalus under UV-B radiation further. In the present study, we used untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-mass spectrometry (LC-MS techniques) to investigate the profiles of primary and secondary metabolic. The profiles revealed the metabolic response of Astragalus to UV-B radiation. We then used real-time polymerase chain reaction (RT-PCR) to obtain the transcription level of relevant genes under UV-B radiation (UV-B supplemented in the field, λmax = 313 nm, 30 W, lamp-leaf distance = 60 cm, 40 min·day-1), which annotated the responsive mechanism of phenolic metabolism in roots. Our results indicated that supplemental UV-B radiation induced a stronger shift from carbon assimilation to carbon accumulation. The flux through the phenylpropanoids pathway increased due to the mobilization of carbon reserves. The response of metabolism was observed to be significantly tissue-specific upon the UV-B radiation treatment. Among phenolic compounds, C6C1 carbon compounds (phenolic acids in leaves) and C6C3C6 carbon compounds (flavones in leaves and isoflavones in roots) increased at the expense of C6C3 carbon compounds. Verification experiments show that the response of phenolics in roots to UV-B is activated by upregulation of relevant genes rather than phenylalanine. Overall, this study reveals the tissues-specific alteration and mechanism of primary and secondary metabolic strategy in response to UV-B radiation.

Metabolomics Analysis Reveals that Ethylene and Methyl Jasmonate Regulate Different Branch Pathways to Promote the Accumulation of Terpenoid Indole Alkaloids in Catharanthus roseus.

The medicinal plant Catharanthus roseus accumulates large numbers of terpenoid indole alkaloids (TIAs), including the pharmaceutically important vinblastine, vincristine, ajmalicine, and serpentine. The phytohormone ethylene or methyl jasmonate (MeJA) can markedly enhance alkaloid accumulation. The interaction between ethylene or MeJA in the regulation of TIA biosynthesis in C. roseus is unknown. Here, a metabolomics platform is reported that is based on liquid chromatography (LC) coupled with time-of-flight mass spectrometry to study candidate components for TIA biosynthesis, which is controlled by ethylene or MeJA in C. roseus. Multivariate analysis identified 16 potential metabolites mostly associated with TIA metabolic pathways and seven targeted metabolites, outlining the TIA biosynthesis metabolic networks controlled by ethylene or MeJA. Interestingly, ethylene and MeJA regulate the 2-C-methyl-d-erythritol 4-phosphate (MEP) and acetate-mevalonate (MVA) pathways through AACT and HMGS and through DXS, respectively, to induce TIA biosynthesis in C. roseus. Overall, both nontargeted and targeted metabolomics, as well as transcript analysis, were used to reveal that MeJA and ethylene control different metabolic networks to induce TIA biosynthesis.

The Different Resistance of Two Astragalus Plants to UV-B Stress is Tightly Associated with the Organ-specific Isoflavone Metabolism.

In this work, the changes in isoflavone levels and the expression of genes involved in their biosynthesis were studied in two Astragalus by UPLC-MS and real-time PCR after 10 days of UV-B treatment (λmax  = 313 nm, 804 J m-2 ). Isoflavones were significantly induced by UV-B irradiation. The influence might be activated by the regulation of these target genes. Our results indicate that (1) the resistance of Astragalus membranaceus might not be as good as Astragalus mongholicus in the enhanced UV-B radiation environment; (2) the enhanced accumulation of calycosin and calycosin-7-glucoside with UV-B treatment in roots of A. mongholicus might be derived from formononetin which is synthesized in the leaves; (3) the glycosylation process could be stimulated and activated by the enhanced UV-B radiation in both A. mongholicus and A. membranaceus. In other words, glycosylation of isoflavones might play a crucial role for two Astragalus plants in response to UV-B stress. Overall, this study offered a feasible elicitation strategy to understand the accumulation pattern of isoflavone in A. mongholicus and A. membranaceus, and also provided a reference for the changes in isoflavone levels of Astragalus in UV-B enhanced environment in the future.

Seed metabolite profiling of Vicia species from China via GC-MS.

In this study, we examined Vicia seeds using gas chromatography-mass spectrometry (GC-MS). The metabolic differences of seeds of twelve Vicia species were assessed. 184 metabolites were identified. Vicia species were classified via multivariate data analyses into four clusters. V. unijuga was most enriched in fatty acids and anthraquinones contents while highest levels of amino acids, alcohols and phenolic were in V. costata. Clustering analysis of biochemical profiles matched with the pervious phenotypic observation with all examined species from section Cracca grouped together under one sub-cluster, except for V. costata.

Ethylene Improves Root System Development under Cadmium Stress by Modulating Superoxide Anion Concentration in Arabidopsis thaliana.

This work aims at identifying the effects of ethylene on the response of Arabidopsis thaliana root system to cadmium chloride (CdCl2) stress. Two ethylene-insensitive mutants, ein2-5 and ein3-1eil1-1, were subjected to (25, 50, 75, and 100 µM) CdCl2 concentrations, from which 75 µM concentration decreased root growth by 40% compared with wild type Col-0 as a control. Ethylene biosynthesis increased in response to CdCl2 treatment. The length of primary root and root tip in ein2-5 and ein3-1eil1-1 decreased compared with wild type after CdCl2 treatment, suggesting that ethylene play a role in root system response to Cd stress. The superoxide concentration in roots of ein2-5 and ein3-1eil1-1 was greater than in wild type seedlings under Cd stress. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) (a precursor of ethylene biosynthesis) in different concentrations (0.01, 0.05 and 0.5 µM) decreased superoxide accumulation in Col-0 root tips and increased the activities of superoxide dismutase (SOD) isoenzymes under Cd stress. This result was reversed with 5 µM of aminoisobutyric acid AIB (an inhibitor of ethylene biosynthesis). Moreover, it was accompanied by increase in lateral roots number and root hairs length, indicating the essential role of ethylene in modulating root system development by controlling superoxide accumulation through SOD isoenzymes activities. The suppressed Cd-induced superoxide accumulation in wild type plants decreased the occurrence of cells death while programmed cell death (PCD) was initiated in the root tip zone, altering root morphogenesis (decreased primary root length, more lateral roots and root hairs) to minimize the damage caused by Cd stress, whereas this response was absent in the ein2-5 and ein3-1eil1-1 seedlings. Hence, ethylene has a role in modulating root morphogenesis during CdCl2 stress in A. thaliana by increasing the activity of SOD isoenzymes to control superoxide accumulation.

GC-MS Metabolomic Analysis to Reveal the Metabolites and Biological Pathways Involved in the Developmental Stages and Tissue Response of Panax ginseng.

Ginsenosides, the major compounds present in ginseng, are known to have numerous physiological and pharmacological effects. The physiological processes, enzymes and genes involved in ginsenoside synthesis in P. ginseng have been well characterized. However, relatively little information is known about the dynamic metabolic changes that occur during ginsenoside accumulation in ginseng. To explore this topic, we isolated metabolites from different tissues at different growth stages, and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 30, 16, 20, 36 and 31 metabolites were identified and involved in different developmental stages in leaf, stem, petiole, lateral root and main root, respectively. To investigate the contribution of tissue to the biosynthesis of ginsenosides, we examined the metabolic changes of leaf, stem, petiole, lateral root and main root during five development stages: 1-, 2-, 3-, 4- and 5-years. The score plots of partial least squares-discriminate analysis (PLS-DA) showed clear discrimination between growth stages and tissue samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in the same tissue at different growth stages indicated profound biochemical changes in several pathways, including carbohydrate metabolism and pentose phosphate metabolism, in addition, the tissues displayed significant variations in amino acid metabolism, sugar metabolism and energy metabolism. These results should facilitate further dissection of the metabolic flux regulation of ginsenoside accumulation in different developmental stages or different tissues of ginseng.

The integration of GC-MS and LC-MS to assay the metabolomics profiling in Panax ginseng and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary metabolites and ginsenosides accumulation.

The traditional medicine Ginseng mainly including Panax ginseng and Panax quinquefolius is the most widely consumed herbal product in the world. Despite the extensive investigation of biosynthetic pathway of the active compounds ginsenosides, our current understanding of the metabolic interlink between ginsenosides synthesis and primary metabolism at the whole-plant level. In this study, the tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng were investigated by gas chromatography- and liquid chromatography coupled to mass spectrometry. A complex continuous coordination of primary- and secondary-metabolic network was modulated by tissues and species factors during growth. The results showed that altogether 149 primary compounds and 10 ginsenosides were identified from main roots, lateral roots, stems, petioles and leaves in P. ginseng and P. quinquefolius. The partial least squares-discriminate analysis (PLS-DA) revealed obvious compounds distinction among tissue-specific districts relative to species. To survey the dedication of carbon and nitrogen metabolism in different tissues to the accumulation of ginsenosides, we inspected the tissue-specific metabolic changes. Our study testified that the ginsenosides content was dependent on main roots and lateral roots energy metabolism, whereas independent of leaves and petiole photosynthesis during ginsenosides accumulation. When tow species were compared, the results indicated that high rates of C assimilation to C accumulation are closely associated with ginsenosides accumulation in P. ginseng main roots and P. quinquefolius lateral roots, respectively. Taken together, our results suggest that tissue-specific metabolites profiling dynamically changed in process of ginsenosides biosynthesis, which may offer a new train of thoughts to the mechanisms of the ginsenosides biosynthesis at the metabolite level.

Simultaneous determination of six active metabolites in Astragalus mongholicus (Fisch.) Bge. under salt stress by ultra-pressure liquid chromatography with tandem mass spectrometry.

Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus, family Leguminosae) is one of the most important traditional Chinese herbs because it contains lots of bioactive metabolites, which have beneficial and pharmacological effects on health. Simultaneously, it has been proved to be a salt-tolerant plant-one of the potential species to control the soil salinization. Therefore, a sensitive and specific ultra-pressure liquid chromatography coupled with tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the simultaneous determination of six main bioactive metabolites, astragaloside IV, cycloastragenol, calycosin-7-O-ß-d-glucoside, calycosin, ononin and formononetin in different organs of A. mongholicus. The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization source operating in the positive ionization mode. Calibration curves offered linear ranges of two orders of magnitude with R(2) > 0.99. The method was fully validated for the linearity, intra-day and inter day precisions, accuracy, recovery, matrix effect and stability. Then this method was successfully applied to detect the content of major bioactive metabolites in different plant organs of A. mongholicus under salt stress. Significant variations in the content of six bioactive metabolites were observed after been processed by different levels of salinity in different part of plant. The results support for further exploration of the salt-tolerant mechanisms in A. mongholicus and its possibility as the species that control the soil salinization. Meanwhile, we established a UPLC-MS/MS assay of the trace components in seedling of A. mongholicus in this study.