Phylogenetic Analysis of R2R3-MYB Family Genes in Tetrastigma hemsleyanum Diels et Gilg and Roles of ThMYB4 and ThMYB7 in Flavonoid Biosynthesis.

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is an extensively used Chinese folk herb with multiple bioactivities. Among these bioactivities, flavonoids are recognized as the representative active ingredients. We previously found an elevated accumulation of flavonoids in T. hemsleyanum under water stress; however, the mechanism remains unclear. R2R3-MYB transcription factors play vital roles in the plant response to environmental stress and the regulation of secondary metabolites. Herein, a systematic transcriptome identification of R2R3-MYB family genes under water stress in T. hemsleyanum was performed to explore their potential function in the biosynthesis of flavonoids. A total of 26 R2R3-MYB genes were identified, most of which were clustered into functional branches of abiotic stress. ThMYB4 and ThMYB7 were then screened out to be associated with the biosynthesis of flavonoids through a protein-protein interaction prediction. An expression correlation analysis based on RNA-seq further confirmed that ThMYB4 and ThMYB7 were positively related to the flavonoid biosynthetic pathway genes of T. hemsleyanum. In ThMYB4- and ThMYB7-overexpression hairy roots, it was found that the expression of ThCHS and ThCHI was significantly increased, suggesting that ThMYB4 and ThMYB7 may act as regulators in flavonoid biosynthesis. This will shed new light on the promotion of flavonoid production and the medicinal value of T. hemsleyanum by manipulating transcription factors.

Induction, Proliferation, Regeneration and Kinsenoside and Flavonoid Content Analysis of the Anoectochilus roxburghii (Wall.) Lindl Protocorm-like Body.

Anoectochilus roxburghii (Wall.) Lindl has been used in Chinese herbal medicine for treating various ailments. However, its wild resources are endangered, and artificial cultivation of the plant is limited by the low regeneration rate of conventional propagation methods. The lack of A. roxburghii resources is detrimental to the commercial production of the plant and kinsenoside, which is unique to Anoectochilus species. To develop highly efficient methods for A. roxburghii micropropagation and find alternative resources for kinsenoside production, we created an induction, proliferation, and regeneration of PLBs (IPR-PLB) protocol for A. roxburghii. We also analyzed the kinsenoside and flavonoid contents during the induction and proliferation of PLBs. The best media of IPR-PLB for PLB induction and proliferation (secondary PLB induction and proliferation), shoot formation, and rooting medium were Murashige and Skoog (MS) + 3 mg/L 6-benzylaminopurine (6-BA) + 0.5 mg/L naphthaleneacetic acid (NAA) + 0.8 mg/L zeatin (ZT) + 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D), MS + 3 mg/L 6-BA + 0.5 mg/L NAA, and MS + 0.5 mg/L NAA, respectively. On these optimized media, the PLB induction rate was 89 ± 2.08%, secondary PLB induction rate was 120 ± 5%, secondary PLB proliferation rate was 400 ± 10% and 350 ± 10 % in terms of the quantity and biomass at approximately 1 month, shoot induction rate was 10.5 shoots/PLB mass, and root induction rate was 98%. All plantlets survived after acclimation. Darkness or weak light were essential for PLB proliferation, and light was crucial for PLB differentiation on these optimized media. The kinsenoside contents of PLBs and secondary PLBs were 10.38 ± 0.08 and 12.30 ± 0.08 mg/g fresh weight (FW), respectively. Moreover, the peak kinsenoside content during the proliferation of secondary PLBs was 34.27 ± 0.79 mg/g FW, which was slightly lower than that of the whole plant (38.68 ± 3.12 mg/g FW). Two flavonoids exhibited tissue- or temporal-specific accumulation patterns, and astragalin accumulated exclusively during the first 2 weeks of cultivation. The IPR-PLB protocol for A. roxburghii may facilitate the efficient micropropagation of A. roxburghii plants. Furthermore, the PLBs are a good alternative resource for kinsenoside production.

Effect of Continuous Cropping Soil on the Biosynthesis of Bioactive Compounds and the Antioxidant Activity of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Continuous monoculture can increase plant morbidity and reduce crop yields. However, the Ganoderma lucidum mushroom possesses unique nutrient acquisition methods and displays an altered response to the generation of reactive oxygen species (ROS). The effects of continuous cropping on G. lucidum remain unclear. Here, we investigated the effects of consecutive monoculture soil on the yield, bioactive components, antioxidant properties, nutrient metabolism, and enzyme activity of G. lucidum. The results showed no significant differences in terms of yield between the control check and treatment groups after 2 years. However, the content of polysaccharides and triterpenoids after 2 years of treatment and the content of total phenols and flavonoids after 3 years of treatment significantly increased. Moreover, the antioxidant activity (AOA) of the polysaccharides under consecutive monoculture soil treatments also significantly increased. These differences were caused by changes in nutrient metabolism and the activities of antioxidant and ligninolytic enzymes. Under continuous monoculture soil treatment, the content of reducing sugars, water-soluble sugars, free amino acids, and water-soluble proteins significantly increased. The activities of superoxide dismutase, manganese peroxidase, and lignin peroxidase increased with the time of continuous monoculture, while the activities of peroxidase, catalase, and laccase decreased. The increase in ligninolytic enzymes was postulated to mediate the ROS scavenging ability of G. lucidum. In conclusion, continuous cropping soil could increase the levels of bioactive components and AOAs of G. lucidum.

Anti-inflammatory effects of Ganoderma lucidum sterols via attenuation of the p38 MAPK and NF-κB pathways in LPS-induced RAW 264.7 macrophages.

Ganoderma lucidum exhibits pronounced anti-inflammatory effects, polysaccharides and triterpenoids are regarded as major constituents displaying the anti-inflammatory activities, whether sterols contribute to this activity remains unclear. Herein Ganoderma lucidum sterols (GLS) were innovatively isolated by a single-step procedure, the profile of GLS was characterized by HPLC-ELSD and shown similar to that of sterols separated by a traditional method, but much higher in content. Furthermore, GLS inhibited inflammation in macrophages by significantly attenuating LPS-induced cell polarization as well as releases and mRNA expressions of pro-inflammatory mediators NO, TNF-α, IL-1ß and IL-6. Moreover, the anti-inflammatory activity of GLS was mediated by MAPK and NF-κB pathways, GLS suppressed MAPK pathways by blocking phosphorylation of p38 but not ERK and JNK, which is complementary with inhibitory effects of Ganoderma polysaccharides and triterpenes on JNK and ERK, indicating Ganoderma sterols may exert synergistic anti-inflammatory effect with polysaccharides and triterpenes. GLS also inhibited NF-κB pathways by restraining phosphorylation and degradation of IκB-α and blocking phosphorylation of NF-κB p65. Molecular docking confirmed that sterols of GLS were directly bound to active sites of p38 and p65 to suppress their activation. Therefore, our findings suggest GLS as natural and safe anti-inflammatory agents to prevent and treat inflammatory diseases.

Historical record of nutrients inputs into the Xin'an Reservoir and its potential environmental implication.

The Xin'an Reservoir is an important water supply source and water conservation area for the Qiantang River. However, after the occurrence of the two algae blooms in 1998 and 1999, the safety of water quality has been put into question. In order to study the historical deposition of nutrients, sediment cores were collected in different regions from the Xin'an Reservoir. The stable isotopes δ13C and δ15N, nutrients, total organic carbon (TOC), and inorganic carbon (IC) in the sediment cores were determined. Radiometric methods (210Pb and 137Cs) were used to obtain sediment chronologies. Spatially, it was found that the average total nitrogen (TN) content in the upper 5 cm of sediments increased from 0.21% in the riverine zone, to 0.33%, and then to 0.57% in the lacustrine zone. The average TP content in the upper 5 cm increased from 0.67 g kg-1 in the riverine zone, to 1.03 g kg-1 in the estuary region, and then to 1.65 g kg-1 in the lacustrine zone. In addition, TOC levels showed a distinct increase from 1.42% in the bottom to 5.97% in the surface of the lacustrine zone. These results demonstrated that although primary productivity and the input of nutrients constantly increased in recent years, algae blooms rarely occurred in the Xin'an Reservoir, due to "depth effect" and an aquatic environment protection-oriented fishery policy. However, high TOC flux and high bio-available phosphorus and nitrogen in surface sediment demonstrated that the reservoir is still confronted with the potential risk of algae blooms.

Structural features of a new heteropolysaccharide from the fruit bodies of Melia azedarach and its effect on cytotoxic activity.

Water-soluble polysaccharides were extracted from the fruit pulps of Melia azedarach and some columns were undertaken to isolate the major polysaccharide (MPS-III). Its structural features were elucidated by IR analysis, carbohydrate analysis, periodate oxidation, Smith degradation, methylation analysis and NMR spectroscopy. Then it was evaluated for the cytotoxic activity in vitro against four human cell lines, using the sulphorhodamine B assay. The data obtained indicated that MPS-III contains a alpha(1-->4) main chain backbone composed of arabinose, mannose in a molar ratio of 1.31:1.0 and has alpha(1-->6) branch structure. And MPS-III showed a strong cytotoxic activity in the BGC-823 cell line.