Study on structure and properties of galactomannan and enzyme changes during fenugreek seeds germination.

Fenugreek (Trigonella foenum-graecum L) galactomannan play an important role in the food and pharmaceutical sectors due to its attractive physicochemical properties. In this study, the changes of structure, properties and biological activity of fenugreek galactomannan (FG) during germination are analyzed by the activity and mechanism of endogenous enzymes (α-D-galactosidase and ß-D-mannanase). The enzymes generally increased during germination and synergistically altered the structure of GM by cutting down the main chains and removing partial side residues. The mannose to galactose ratio (M/G) increased from 1.11 to 1.59, which is accompanied by a drastic decrease in molecular weight from 3.606 × 106 to 0.832 × 106 g/mol, and the drop of viscosity from 0.27 to 0.06 Pa·sn. The degraded macromolecules are attributed to the increase in solubility (from 64.55 % to 88.62 %). In terms of antioxidation and antidiabetic ability, germinated fenugreek galactomannan has the ability to scavenge 67.17 % ABTS free radicals and inhibit 86.89 % α-glucosidase. This galactomannan with low molecular weight and excellent biological activity precisely satisfies the current demands of pharmaceutical reagents and food industry. Seeds germination holds promise as a means of industrial scale production of low molecular weight galactomannans.

Tartary Buckwheat (Fagopyrum tataricum) FtTT8 Inhibits Anthocyanin Biosynthesis and Promotes Proanthocyanidin Biosynthesis.

Tartary buckwheat (Fagopyrum tataricum) is an important plant, utilized for both medicine and food. It has become a current research hotspot due to its rich content of flavonoids, which are beneficial for human health. Anthocyanins (ATs) and proanthocyanidins (PAs) are the two main kinds of flavonoid compounds in Tartary buckwheat, which participate in the pigmentation of some tissue as well as rendering resistance to many biotic and abiotic stresses. Additionally, Tartary buckwheat anthocyanins and PAs have many health benefits for humans and the plant itself. However, little is known about the regulation mechanism of the biosynthesis of anthocyanin and PA in Tartary buckwheat. In the present study, a bHLH transcription factor (TF) FtTT8 was characterized to be homologous with AtTT8 and phylogenetically close to bHLH proteins from other plant species. Subcellular location and yeast two-hybrid assays suggested that FtTT8 locates in the nucleus and plays a role as a transcription factor. Complementation analysis in Arabidopsis tt8 mutant showed that FtTT8 could not recover anthocyanin deficiency but could promote PAs accumulation. Overexpression of FtTT8 in red-flowering tobacco showed that FtTT8 inhibits anthocyanin biosynthesis and accelerates proanthocyanidin biosynthesis. QRT-PCR and yeast one-hybrid assay revealed that FtTT8 might bind to the promoter of NtUFGT and suppress its expression, while binding to the promoter of NtLAR and upregulating its expression in K326 tobacco. This displayed the bidirectional regulating function of FtTT8 that negatively regulates anthocyanin biosynthesis and positively regulates proanthocyanidin biosynthesis. The results provide new insights on TT8 in Tartary buckwheat, which is inconsistent with TT8 from other plant species, and FtTT8 might be a high-quality gene resource for Tartary buckwheat breeding.

Comparative proteomic analyses of Tartary buckwheat (Fagopyrum tataricum) seeds at three stages of development.

Tartary buckwheat is among the valuable crops, utilized as both food and Chinese herbal medicine. To uncover the accumulation dynamics of the main nutrients and their regulatory mechanism of Tartary buckwheat seeds, microscopic observations and nutrient analysis were conducted which suggested that starch, proteins as well as flavonoid gradually accumulated among seed development. Comparative proteomic analysis of rice Tartary buckwheat at three different developmental stages was performed. A total of 78 protein spots showed differential expression with 74 of them being successfully identified by MALDI-TOF/TOF MS. Among them, granule bound starch synthase (GBSS1) might be the critical enzyme that determines starch biosynthesis, while 11 S seed storage protein and vicilin seemed to be the main globulin and affect seed storage protein accumulation in Tartary buckwheat seeds. Two enzymes, flavanone 3-hydroxylase (F3H) and anthocyanidin reductase (ANR), involved in the flavonoid biosynthesis pathway were identified. Further analysis on the expression profiles of flavonoid biosynthetic genes revealed that F3H might be the key enzyme that promote flavonoid accumulation. This study provides insights into the mechanism of nutrition accumulation at the protein level in Tartary buckwheat seeds and may facilitate in the breeding and enhancement of Tartary buckwheat germplasm.

Integrated Transcriptomics and Widely Targeted Metabolomics Analyses Provide Insights Into Flavonoid Biosynthesis in the Rhizomes of Golden Buckwheat (Fagopyrum cymosum).

Golden buckwheat (Fagopyrum cymosum) is used in Traditional Chinese Medicine. It has received attention because of the high value of its various medicinal and nutritional metabolites, especially flavonoids (catechin and epicatechin). However, the metabolites and their encoding genes in golden buckwheat have not yet been identified in the global landscape. This study performed transcriptomics and widely targeted metabolomics analyses for the first time on rhizomes of golden buckwheat. As a result, 10,191 differentially expressed genes (DEGs) and 297 differentially regulated metabolites (DRMs) were identified, among which the flavonoid biosynthesis pathway was enriched in both transcriptome and metabolome. The integration analyses of the transcriptome and the metabolome revealed a network related to catechin, in which four metabolites and 14 genes interacted with each other. Subsequently, an SG5 R2R3-MYB transcription factor, named FcMYB1, was identified as a transcriptional activator in catechin biosynthesis, as it was positively correlated to eight flavonoid biosynthesis genes in their expression patterns and was directly bound to the promoters of FcLAR2 and FcF3'H1 by yeast one hybrid analysis. Finally, a flavonoid biosynthesis pathway was proposed in the rhizomes of golden buckwheat, including 13 metabolites, 11 genes encoding 9 enzymes, and 1 MYB transcription factor. The expression of 12 DEGs were validated by qRT-PCR, resulting in a good agreement with the Pearson R ranging from 0.83 to 1. The study provided a comprehensive flavonoid biosynthesis and regulatory network of golden buckwheat.

Integrated microRNA and transcriptome profiling reveal key miRNA-mRNA interaction pairs associated with seed development in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat seed development is an extremely complex process involving many gene regulatory pathways. MicroRNAs (miRNAs) have been identified as the important negative regulators of gene expression and performed crucial regulatory roles in various plant biological processes. However, whether miRNAs participate in Tartary buckwheat seed development remains unexplored. RESULTS: In this study, we first identified 26 miRNA biosynthesis genes in the Tartary buckwheat genome and described their phylogeny and expression profiling. Then we performed small RNA (sRNA) sequencing for Tartary buckwheat seeds at three developmental stages to identify the miRNAs associated with seed development. In total, 230 miRNAs, including 101 conserved and 129 novel miRNAs, were first identified in Tartary buckwheat, and 3268 target genes were successfully predicted. Among these miRNAs, 76 exhibited differential expression during seed development, and 1534 target genes which correspond to 74 differentially expressed miRNAs (DEMs) were identified. Based on integrated analysis of DEMs and their targets expression, 65 miRNA-mRNA interaction pairs (25 DEMs corresponding to 65 target genes) were identified that exhibited significantly opposite expression during Tartary buckwheat seed development, and 6 of the miRNA-mRNA pairs were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) and ligase-mediated rapid amplification of 5' cDNA ends (5'-RLM-RACE). Functional annotation of the 65 target mRNAs showed that 56 miRNA-mRNA interaction pairs major involved in cell differentiation and proliferation, cell elongation, hormones response, organogenesis, embryo and endosperm development, seed size, mineral elements transport, and flavonoid biosynthesis, which indicated that they are the key miRNA-mRNA pairs for Tartary buckwheat seed development. CONCLUSIONS: Our findings provided insights for the first time into miRNA-mediated regulatory pathways in Tartary buckwheat seed development and suggested that miRNAs play important role in Tartary buckwheat seed development. These findings will be help to study the roles and regulatory mechanism of miRNAs in Tartary buckwheat seed development.

Mapping QTLs for 1000-grain weight and genes controlling hull type using SNP marker in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum), an important pseudocereal crop, has high economic value due to its nutritional and medicinal properties. However, dehulling of Tartary buckwheat is difficult owing to its thick and tough hull, which has greatly limited the development of the Tartary buckwheat processing industry. The construction of high-resolution genetic maps serves as a basis for identifying quantitative trait loci (QTLs) and qualitative trait genes for agronomic traits. In this study, a recombinant inbred lines (XJ-RILs) population derived from a cross between the easily dehulled Rice-Tartary type and Tartary buckwheat type was genotyped using restriction site-associated DNA (RAD) sequencing to construct a high-density SNP genetic map. Furthermore, QTLs for 1000-grain weight (TGW) and genes controlling hull type were mapped in multiple environments. RESULTS: In total, 4151 bin markers comprising 122,185 SNPs were used to construct the genetic linkage map. The map consisted of 8 linkage groups and covered 1444.15 cM, with an average distance of 0.35 cM between adjacent bin markers. Nine QTLs for TGW were detected and distributed on four loci on chromosome 1 and 4. A major locus detected in all three trials was mapped in 38.2-39.8 cM region on chromosome 1, with an LOD score of 18.1-37.0, and explained for 23.6-47.5% of the phenotypic variation. The genes controlling hull type were mapped to chromosome 1 between marker Block330 and Block331, which was closely followed by the major locus for TGW. The expression levels of the seven candidate genes controlling hull type present in the region between Block330 and Block336 was low during grain development, and no significant difference was observed between the parental lines. Six non-synonymous coding SNPs were found between the two parents in the region. CONCLUSIONS: We constructed a high-density SNP genetic map for the first time in Tartary buckwheat. The mapped major loci controlling TGW and hull type will be valuable for gene cloning and revealing the mechanism underlying grain development and easy dehulling, and marker-assisted selection in Tartary buckwheat.

Mikania micrantha invasion enhances the carbon (C) transfer from plant to soil and mediates the soil C utilization through altering microbial community.

Exotic plant invasion alters the structure and coverage of terrestrial vegetation and affects the carbon (C) stocks in ecosystems. Previous studies have shown the increases in the C stocks with increasing invasive plants, but these results remain contentious. Soil microbial communities are usually altered by plant invasion, which potentially influences the C cycling underground. We hypothesized that the plant invasion-caused dynamic changes in soil microbes would lead to the corresponding change in soil C accumulation. Using greenhouse experiments we simulated different invader intensities through varying the relative abundance of invasive species Mikania micrantha and its co-occurring native species Paederia scandens. By analyzing 13C-phospholipid fatty acid we found the invasive M. micrantha assimilated more 13C and transferred faster the fixed 13C through different tissues to soils, as compared to native P. scandens. Soil microbial components, i.e., i15:0, 16:0, 10Me16:0, 18:1w9c and 18:2w6,9 were mainly using the photo-assimilated 13C. In addition, we found a hump-shaped relationship between soil net 13C accumulate rate and rhizosphere microbial biomass, indicating that the soil C accumulation may be either enhanced or reduced in invaded ecosystems, depending on microbe abundance.

Transcriptome Analysis Reveals Key Seed-Development Genes in Common Buckwheat (Fagopyrum esculentum).

Seed development is an essential and complex process, which is involved in seed size change and various nutrients accumulation, and determines crop yield and quality. Common buckwheat (Fagopyrum esculentum Moench) is a widely cultivated minor crop with excellent economic and nutritional value in temperate zones. However, little is known about the molecular mechanisms of seed development in common buckwheat (Fagopyrum esculentum). In this study, we performed RNA-Seq to investigate the transcriptional dynamics and identify the key genes involved in common buckwheat seed development at three different developmental stages. A total of 4619 differentially expressed genes (DEGs) were identified. Based on the results of Gene Ontology (GO) and KEGG analysis of DEGs, many key genes involved in the seed development, including the Ca2+ signal transduction pathway, the hormone signal transduction pathways, transcription factors (TFs), and starch biosynthesis-related genes, were identified. More importantly, 18 DEGs were identified as the key candidate genes for seed size through homologous query using the known seed size-related genes from different seed plants. Furthermore, 15 DEGs from these identified as the key genes of seed development were selected to confirm the validity of the data by using quantitative real-time PCR (qRT-PCR), and the results show high consistency with the RNA-Seq results. Taken together, our results revealed the underlying molecular mechanisms of common buckwheat seed development and could provide valuable information for further studies, especially for common buckwheat seed improvement.

Global transcriptome analysis and identification of genes involved in nutrients accumulation during seed development of rice tartary buckwheat (Fagopyrum Tararicum).

Tartary buckwheat seeds are rich in various nutrients, such as storage proteins, starch, and flavonoids. To get a good knowledge of the transcriptome dynamics and gene regulatory mechanism during the process of seed development and nutrients accumulation, we performed a comprehensive global transcriptome analysis using rice tartary buckwheat seeds at different development stages, namely pre-filling stage, filling stage, and mature stage. 24 819 expressed genes, including 108 specifically expressed genes, and 11 676 differentially expressed genes (DEGs) were identified. qRT-PCR analysis was performed on 34 DEGs to validate the transcriptome data, and a good consistence was obtained. Based on their expression patterns, the identified DEGs were classified to eight clusters, and the enriched GO items in each cluster were analyzed. In addition, 633 DEGs related to plant hormones were identified. Furthermore, genes in the biosynthesis pathway of nutrients accumulation were analyzed, including 10, 20, and 23 DEGs corresponding to the biosynthesis of seed storage proteins, flavonoids, and starch, respectively. This is the first transcriptome analysis during seed development of tartary buckwheat. It would provide us a comprehensive understanding of the complex transcriptome dynamics during seed development and gene regulatory mechanism of nutrients accumulation.

[Studies on flash extraction methods of Arnebia euchroma].

The extraction of functional components from radix of Arnebia euchroma was optimized using orthogonal design based on the extraction yields of shikonin, and hydroxyl-naphthoquinone pigments. The data processing was carried out with the multiple guidelines grading method for optimizing the extraction condition. Compared with the traditional method (refluxing and ultrasonic extraction), the flash extraction method was more efficient The optimal conditions were as follows: 95% ethanol extract 3 times with 90 s for each. Under these conditions, the extraction yields of shikonin, and hydroxyl-naphthoquinone pigments were 93.16%, 93.89%, respectively, and the dry extract rate was 5.16%. In conclusion, the result showed that the flash extraction technology was appropriate, stable and feasible.

[Pharmacokinetics of scutellarin and its derivant scutellarin ethyl ester in rats].

To develop a HPLC method for determination of the concentration of scutellarin and scutellarin ethyl ester and their pharmacokinetics were also compared. 104 mg kg-1of scutellarin or 114. 5 mg kg-1 scutellarin ethyl ester were given at single dose by oral gavarge. Blood samples were collected from the jugular vein. Plasma concentration was measured by HPLC. The pharmacokinetic parameters were calculated with Winnonlin program. The plasma concentration-time profile of scutellarin and scutellarin ethyl ester were both fitted with non-compartment model and both were double peaks. The main pharmacokinetic parameters of scutellarin and scutellarin ethyl ester were as follows: Tmax Cmax and AUC0-t for scutellarin were (6 +/- 1.26) h, (321.55 +/-48.31) microg L-1 and (2 974 +/-753) h micro.g L-1; for scutellarin ethyl ester, Tmax, Cmax and AUC0-t were 0.5 h, (1 550.82 +/-219.75) +/- microg L- and (6 407 +/- 399) h microg L-1. The speed ingested into the blood of scutellarin ethyl ester was faster than scutellarin, and the bioavailability of scutellarin ethyl ester was two times higher than scutellarin.

[Observation on therapeutic effect of electroacupuncture plus blood-letting puncture and cupping combined with diet intervention for treatment of acute gouty arthritis].

OBJECTIVE: To explore a more effective therapy for acute gouty arthritis. METHODS: Sixty cases were randomly divided into an observation group and a control group, 30 cases in eachgroup. On the basis of diet intervention, the observation group was treated with electroacupuncture at local points combined with blood-letting puncture and cupping, and the control group with oral administration of Probenecid. Their therapeutic effects were ob served. RESULTS: The effective rate was 96.7% in the observation group which was better than 86.7% in the control group (P < 0.01). After treatment, blood uric acid decreased significantly in the two groups (both P < 0.01), the observed group being lower than the control group (P < 0.01). CONCLUSION: On the basis of diet intervention, electroacupuncture plus blood-letting puncture and cupping is a better therapy for acute gouty arthritis.

[Effect of 5-fluorouracil in combination with Astragalus membranaceus on amino acid metabolism in mice model of gastric carcinoma].

OBJECTIVE: To study the effect of 5-fluorouracil-FU in combination with astragalus membranaceus(AM) on amino acid metabolism in mice model of gastric carcinoma induced by 3-methylcholanthrene(MC). METHODS: Mice gastric carcinoma models were established by 3-methylcholanthrene induction and randomly divided into different groups, and received 5-FU treatment (group A) 5-FU plus AM (group B), 5-FU plus a high dose of AM(group C), no treatment (group D). Normal mice were used as control (group N). Free amino acid in the tumor specimens were examined. RESULTS: The levels of free Valine, Methionine, Leucine, Arginine and cystine in the tumor specimens in group D were significantly higher than that in group N(P< 0.05). The levels of free serine in group A, B, C, D were significantly higher than that in group N. The levels of free glutamic acid in group A, B were significantly higher than that in group N(P< 0.05). The levels of free proline in group C, D were significantly higher than that in group P, N(P< 0.05). CONCLUSIONS: The increasing levels of free serine and proline in tumor specimens in gastric cancer mice model reveals metabolic disturbance of amino acid. 5-FU plus astragalus membranaceus can decrease the level of free glutamic acid in the mice models, and inhibit tumor growth.