Chemical profiling of Simiao pill and quantification of main effective constituents in it by ultra-high-performance liquid chromatography coupled with Q Exactive Orbitrap and triple quadrupole mass spectrometry.

Simiao pill is one of the most commonly used prescriptions in traditional Chinese medicine for the treatment of hyperuricemia and gout. However, methods based on more accurate and comprehensive qualitative and quantitative analyses of the active ingredients are not yet perfect due to limited methodology. This not only hinders the elucidation of the pharmacological mechanism of Simiao pill, but also its comprehensive clinical development and utilization. In this study, we employed ultra-high-performance liquid chromatography-Q Exactive Orbitrap-mass spectrometry technology to perform rapid analysis and identification of the chemical constituents in Simiao pill. A total of 101 chemical components were identified, including 26 alkaloids, 15 terpenoids, 11 flavonoids, eight steroids, six fatty acids, five limonoids, four saponins, five phenylpropanoids, and 21 other compounds. In addition, we established a new method by high-throughput ultra-high-performance liquid chromatography-Q Exactive Orbitrap-mass spectrometry combined with ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry technology for quantification of 14 main active ingredients, such as adenosine (1), phellodendrine (2), mangnoflorine (3), ß-ecdysterone (4), 25R-inokosterone (5), 25S-inokosterone (6), jatrorrhizine (7), palmatine (8), chikusetsu saponin IVa (9), limonin (10), atractylenolide III (11), atractylenolide I (12), obacunone (13), and atractylenolide II (14) in Simiao pill. This work laid a foundation for further analysis and quality control of effective components in Simiao pill.

Endophytic fungus Falciphora oryzae enhances salt tolerance by modulating ion homeostasis and antioxidant defense systems in pepper.

Endophytic fungi play an important role in the induction of plant tolerance to abiotic and biotic stresses. However, the role of endophytic fungi in the response of horticultural plants to plant stress remains largely unknown. Here, we addressed the role of the endophytic fungus Falciphora oryzae in enhancing salt tolerance in pepper (Capsicum annuum L.) by inoculation with the endophyte in the rhizosphere. F. oryzae could indeed colonize the roots of pepper and significantly improved the tolerance of pepper to salt stress. This resulted in increased plant growth and photosynthetic performance compared with control plants, which was accompanied by increases in indole acetic acid and abscisic acid biosynthesis and signaling. Furthermore, inoculation with F. oryzae significantly upregulated a subset of transcripts involved in Na+ homeostasis (NHX3, NHX6, NHX8, HKT2-1, and SOS1) and the high-affinity K+ transporter protein-related gene HAK1 in the leaves to maintain Na+ /K+ homeostasis. Moreover, the activity of antioxidant enzymes (catalase, peroxidase, glutathione, and ascorbate peroxidase), the content of glutathione, the transcript level of genes related to antioxidants (catalase, ascorbate peroxidase, glutathione reductase, peroxidase, glutamate-cysteine ligase, and glutamine synthetase) in the leaves were significantly upregulated after inoculation with F. oryzae, which led to decreased levels of lipid peroxidation (malondialdehyde) and reactive oxygen species. These results indicate that inoculation with F. oryzae can enhance the salt tolerance of pepper by promoting ion homeostasis and upregulating antioxidant defense systems.

Two different strategies of Diversispora spurca-inoculated walnut seedlings to improve leaf P acquisition at low and moderate P levels.

Walnut (Juglans regia) is an important nut tree species in the world, whereas walnut trees often face inadequate phosphorus (P) levels of soil, negatively limiting its growth and yield. Arbuscular mycorrhizal fungi (AMF) can colonize walnut roots, but whether and how AMF promotes walnut growth, physiological activities, and P acquisition is unclear. The present study aimed to evaluate the effects of Diversispora spurca on plant growth, chlorophyll component concentrations, leaf gas exchange, sugar and P concentrations, and expression of purple acid phosphatase (PAP) and phosphate transporter (PT) genes in leaves of J. regia var. Liaohe 1 seedling under moderate (100 µmol/L P) and low P (1 µmol/L P) levels conditions. Three months after inoculation, the root mycorrhizal colonization rate and soil hyphal length were 45.6-53.2% and 18.7-39.9 cm/g soil, respectively, and low P treatment significantly increased both root mycorrhizal colonization rate and soil hyphal length. Low P levels inhibited plant growth (height, stem diameter, and total biomass) and leaf gas exchange (photosynthetic rate, transpiration rate and stomatal conductance), while AMF colonization significantly increased these variables at moderate and low P levels. Low P treatment limited the level of chlorophyll a, but AMF colonization did not significantly affect the level of chlorophyll components, independent on soil P levels. AMF colonization also increased leaf glucose at appropriate P levels and leaf fructose at low P levels than non-AMF treatment. AMF colonization significantly increased leaf P concentration by 21.0-26.2% than non-AMF colonization at low and moderate P levels. Low P treatment reduced the expression of leaf JrPAP10, JrPAP12, and JrPT3;2 in the inoculated plants, whereas AMF colonization up-regulated the expression of leaf JrPAP10, JrPAP12, and JrPT3;2 at moderate P levels, although AMF did not significantly alter the expression of JrPAPs and JrPTs at low P levels. It is concluded that AMF improved plant growth, leaf gas exchange, and P acquisition of walnut seedlings at different P levels, where mycorrhizal promotion of P acquisition was dominated by direct mycorrhizal involvement in P uptake at low P levels, while up-regulation of host PAPs and PTs expressions at moderate P levels.

Symbiotic Fungi Alter the Acquisition of Phosphorus in Camellia oleifera through Regulating Root Architecture, Plant Phosphate Transporter Gene Expressions and Soil Phosphatase Activities.

Plant roots can be colonized by many symbiotic fungi, whereas it is unclear whether and how symbiotic fungi including arbuscular mycorrhizal fungi and endophytic fungi promote phosphorus (P) uptake in Camellia oleifera plants. The objective of the present study was to analyze the effect of inoculation with a culturable endophytic fungus (Piriformospora indica), three arbuscular mycorrhizal fungi (Funneliformis mosseae, Diversispora versiformis, and Rhizophagus intraradices), and mixture of F. mosseae, D. versiformis and R. intraradices on plant growth, root architecture, soil Olsen-P, soil phosphatase activities, leaf and root P concentrations, and phosphate transporter gene expressions, in order to explore the potential and mechanism of these symbiotic fungi on P acquisition. All the symbiotic fungi colonized roots of C. oleifera after 16 weeks, with P. indica showing the best effect on fungal colonization. All the symbiotic fungi significantly increased acid, neutral, and total phosphatase activities in the soil, accompanied with an elevation of soil Olsen-P, of which P. indica presented the best effect. All symbiotic fungal treatments, except D. versiformis, significantly promoted plant growth, coupled with an increase in root total length, area, and volume. Symbiotic fungi almost up-regulated root CoPHO1-3 expressions as well as leaf CoPHO1-1, CoPHO1-3, and CoPHT1;4 expressions. Correlation analysis showed that P concentrations in leaves and roots were significantly positively correlated with root morphological variables (length, volume, and surface area) and soil acid, neutral and total phosphatase activities. It is concluded that symbiotic fungi, especially P. indica, played an important role in P uptake of C. oleifera plants through regulating root architecture, part plant phosphate transporter gene expressions and soil phosphatase activities.

Differential Effects of Exogenous Glomalin-Related Soil Proteins on Plant Growth of Trifoliate Orange Through Regulating Auxin Changes.

Multiple functions of glomalin released by arbuscular mycorrhizal fungi are well-recognized, whereas the role of exogenous glomalins including easily extractable glomalin-related soil protein (EE-GRSP) and difficultly extractable glomalin-related soil protein (DE-GRSP) is unexplored for plant responses. Our study was carried out to assess the effects of exogenous EE-GRSP and DE-GRSP at varying strengths on plant growth and chlorophyll concentration of trifoliate orange (Poncirus trifoliata) seedlings, along with changes in root nutrient acquisition, auxin content, auxin-related enzyme and transporter protein gene expression, and element contents of purified GRSP. Sixteen weeks later, exogenous GRSP displayed differential effects on plant growth (height, stem diameter, leaf number, and biomass production): the increase by EE-GRSP and the decrease by DE-GRSP. The best positive effect on plant growth occurred at exogenous EE-GRSP at ½ strength. Similarly, the GRSP application also differently affected total chlorophyll content, root morphology (total length, surface area, and volume), and root N, P, and K content: positive effect by EE-GRSP and negative effect by DE-GRSP. Exogenous EE-GRSP accumulated more indoleacetic acid (IAA) in roots, which was associated with the upregulated expression of root auxin synthetic enzyme genes (PtTAA1, PtYUC3, and PtYUC4) and auxin influx transporter protein genes (PtLAX1, PtLAX2, and PtLAX3). On the other hand, exogenous DE-GRSP inhibited root IAA and indolebutyric acid (IBA) content, associated with the downregulated expression of root PtTAA1, PtLAX1, and PtLAX3. Root IAA positively correlated with root PtTAA1, PtYUC3, PtYUC4, PtLAX1, and PtLAX3 expression. Purified EE-GRSP and DE-GRSP showed similar element composition but varied in part element (C, O, P, Ca, Cu, Mn, Zn, Fe, and Mo) concentration. It concluded that exogenous GRSP triggered differential effects on growth response, and the effect was associated with the element content of pure GRSP and the change in auxins and root morphology. EE-GRSP displays a promise as a plant growth biostimulant in citriculture.

The Change in Fatty Acids and Sugars Reveals the Association between Trifoliate Orange and Endophytic Fungi.

Endophytes have the ability to improve plant nutrition alongside their agronomic performance, among which arbuscular mycorrhizal fungi provide the most benefits to their host. Previously, we reported for the first time that an arbuscular mycorrhizal-like fungus Piriformospora indica had the ability to colonize roots of trifoliate orange (Poncirus trifoliata) and conferred positive effects on nutrient acquisition. Present study showed the changes in fatty acids and sugars to unravel the physiological and symbiotic association of trifoliate orange with P. indica and an arbuscular mycorrhizal fungus, Funneliformis mosseae singly or in combination. All the endophytic fungi collectively increased fructose, glucose, and sucrose content in leaves and roots, along with a relatively higher increase with P. indica inoculation than with F. mosseae alone or dual inoculation. Treatment with P. indica increased the concentration of part unsaturated fatty acids such as C18:3N6, C20:2, C20:3N6, C20:4N6, C20:3N3, C20:5N3, C22:1N9, and C24:1. Additionally, P. indica induced the increase in the concentration of part saturated fatty acids such as C6:0, C8:0, C13:0, C14:0, and C24:0. F. mosseae hardly changed the content of fatty acids, except for increase in C14:0 and C20:5N3. Double inoculation only reduced the C21:0, C10:0, C12:0, C18:3N3, and C18:1 content and increased the C20:5N3 content. These endophytic fungi up-regulated the root PtFAD2, PtFAD6, PtΔ9, and PtΔ15 gene expression level, coupled with a higher expression of PtFAD2 and PtΔ9 by P. indica than by F. mosseae. It was concluded that P. indica exhibited a stronger response, for sugars and fatty acids, than F. mosseae on trifoliate orange. Such results also reveal the Pi (an in vitro culturable fungus) as a bio-stimulator applying to citriculture.