Rapid Discovery of the Potential Toxic Compounds in Polygonum multiflorum by UHPLC/Q-Orbitrap-MS-Based Metabolomics and Correlation Analysis

The dry roots of Polygonum multiflorum (PM), involving both the raw and processed materials, are widely used as the traditional Chinese medicine for treating various diseases in China. Hepatotoxicity has been occasionally reported in patients who consume PM. Unfortunately, no definite criteria are currently available regarding the processing technology of PM for reduction the toxicity. In this work, we aimed to investigate the variations of PM metabolite profiles induced by different processing technologies by UHPLC/Q-Orbitrap-MS and multivariate statistical analysis, and to discover the potential toxic compounds by correlating the cytotoxicity of L02 cell with the contents of metabolites in raw and processed PM samples. We could identify two potential toxic compounds, emodin-8-O-glucoside and torachrysone-O-hexose, which could be selected as the toxic markers to evaluate different processing methods. The results indicated all processed PM samples could decrease the cytotoxicity on L02 cell. The best processing technology for PM process was to steam PM in black soybean decoction (BD-PM) for 24 h

Global Analysis of UDP Glucose Pyrophosphorylase (UDPGP) Gene Family in Plants: Conserved Evolution Involved in Cell Death

UDP glucose pyrophosphorylase (UDPGP) family genes have been reported to play essential roles in cell death or individual survival. However, a systematic analysis on UDPGP gene family has not been performed yet. In this study, a total of 454 UDPGP proteins from 76 different species were analyzed. The analyses of the phylogenetic tree and orthogroups divided UDPGPs into three clades, including UDP-N-acetylglucosamine pyrophosphorylase (UAP), UDP-glucose pyrophosphorylase (UGP, containing UGP-A and UGP-B), and UDP-sugar pyrophosphorylase (USP). The evolutionary history of the UDPGPs indicated that the members of UAP, USP, and UGP-B were relatively conserved while varied in UGP-A. Homologous sequences of UGP-B and USP were found only in plants. The expression profile of UDPGP genes in Oryza sativa was mainly motivated under jasmonic acid (JA), abscisic acid (ABA), cadmium, and cold treatments, indicating that UDPGPs may play an important role in plant development and environment endurance. The key amino acids regulating the activity of UDPGPs were analyzed, and almost all of them were located in the NB-loop, SB-loop, or conserved motifs. Analysis of the natural variants of UDPGPs in rice revealed that only a few missense mutants existed in coding sequences (CDSs), and most of the resulting variations were located in the non-motif sites, indicating the conserved structure and function of UDPGPs in the evolution. Furthermore, alternative splicing may play a key role in regulating the activity of UDPGPs. The spatial structure prediction, enzymatic analysis, and transgenic verification of UAP isoforms illustrated that the loss of N- and C-terminal sequences did not affect the overall 3D structures, but the N- and C-terminal sequences are important for UAP genes to maintain their enzymatic activity. These results revealed a conserved UDPGP gene family and provided valuable information for further deep functional investigation of the UDPGP gene family in plants

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

<p>Abstract</p> <p>Background</p> <p><it>Bupleurum chinense </it>DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of <it>B. chinense</it>, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.</p> <p>Results</p> <p>One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A <it>de novo </it>assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the <it>Bupleurum </it>genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel <it>Bupleurum </it>genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (<it>P450</it>s) and 102 glycosyltransferases (<it>GT</it>s) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 <it>P450</it>s and 7 uridine diphosphate <it>GT</it>s (<it>UGT</it>s) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two <it>P450</it>s and three <it>UGT</it>s were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with <it>β-AS </it>in methyl jasmonate-treated adventitious roots and on their similar expression patterns with <it>β-AS </it>in various <it>B. chinense </it>tissues.</p> <p>Conclusions</p> <p>A collection of high-quality ESTs for <it>B. chinense </it>obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of <it>B. chinense </it>and other <it>Bupleurum </it>species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the <it>P450</it>s and <it>UGT</it>s, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.</p

The perceiving and disseminating of the conception of primary level Chinese language curriculum reform of the textbookwriters, teacher trainers and teachers in China (a case study)

published_or_final_versionEducationDoctoralDoctor of Philosoph

The Different Metabolic Responses of Resistant and Susceptible Wheats to Fusarium graminearum Inoculation

Fusarium head blight (FHB) is a serious wheat disease caused by Fusarium graminearum (Fg) Schwabe. FHB can cause huge loss in wheat yield. In addition, trichothecene mycotoxins produced by Fg are harmful to the environment and humans. In our previous study, we obtained two mutants TPS1&minus; and TPS2&minus;. Neither of these mutants could synthesize trehalose, and they produced fewer mycotoxins. To understand the complex interaction between Fg and wheat, we systematically analyzed the metabolic responses of FHB-susceptible and -resistant wheat to ddH2O, the TPS&minus; mutants and wild type (WT) using NMR combined with multivariate analysis. More than 40 metabolites were identified in wheat extracts including sugars, amino acids, organic acids, choline metabolites and other metabolites. When infected by Fg, FHB-resistant and -susceptible wheat plants showed different metabolic responses. For FHB-resistant wheat, there were clear metabolic differences between inoculation with mutants (TPS1&minus;/TPS2&minus;) and with ddH2O/WT. For the susceptible wheat, there were obvious metabolic differences between inoculation with mutant (TPS1&minus;/TPS2&minus;) and inoculation with ddH2O; however, there were no significant metabolic differences between inoculation with TPS&minus; mutants and with WT. Specifically, compared with ddH2O, resistant wheat increased the levels of Phe, p-hydroxy cinnamic acid (p-HCA), and chlorogenic acid in response to TPS&minus; mutants; however, susceptible wheat did not. Shikimate-mediated secondary metabolism was activated in the FHB-resistant wheat to inhibit the growth of Fg and reduce the production of mycotoxins. These results can be helpful for the development of FHB-resistant wheat varieties, although the molecular relationship between the trehalose biosynthetic pathway in Fg and shikimate-mediated secondary metabolism in wheat remains to be further studied

Proteomics Analysis Reveals the Underlying Factors of Mucilage Disappearance in <i>Brasenia schreberi</i> and Its Influence on Nutrient Accumulation

Brasenia schreberi J.F. Gmel (BS) is rich in mucilage, which has diverse biological activities, and is utilized in the food and pharmaceutical industries due to its nutritional value. Proteomics analysis was employed to investigate the cause of mucilage disappearance in BS and its effect on nutrient accumulation. Among the 2892 proteins identified, 840 differentially expressed proteins (DEPs) were found to be involved in mucilage development. By comparing the expression patterns and functions and pathway enrichment, the DEPs mainly contributed to carbon and energy metabolism, polysaccharide metabolism, and photosynthesis. Our study also revealed positive correlations between mucilage accumulation and tryptophan metabolism, with high levels of indole-3-acetic (IAA) contributing to mucilage accumulation. Furthermore, environmental changes and particularly excessive nutrients were found to be detrimental to mucilage synthesis. Overall, in the absence of various stimuli in the growing environment, BS accumulates more nutrients within the plant itself instead of producing mucilage

Herbivore-Induced Callose Deposition on the Sieve Plates of Rice: An Important Mechanism for Host Resistance1[W][OA]

The brown planthopper (Nilaparvata lugens Stål; BPH) is a specialist herbivore on rice (Oryza sativa) that ingests phloem sap from the plant through its stylet mouthparts. Electronic penetration graphs revealed that BPH insects spent more time wandering over plants carrying the resistance genes Bph14 and Bph15, but less time ingesting phloem than they did on susceptible plants. They also showed that their feeding was frequently interrupted. Tests with [14C]sucrose showed that insects ingested much less phloem sap from the resistant than the susceptible plants. BPH feeding up-regulated callose synthase genes and induced callose deposition in the sieve tubes at the point where the stylet was inserted. The compact callose remained intact in the resistant plants, but genes encoding β-1,3-glucanases were activated, causing unplugging of the sieve tube occlusions in susceptible plants. Continuing ingestion led to a remarkable reduction in the susceptible plants' sucrose content and activation of the RAmy3D gene, leading to starch hydrolysis and ultimately carbohydrate deprivation in the plants. Our results demonstrate that BPH feeding induces the deposition of callose on sieve plates in rice and that this is an important defense mechanism that prevents insects from ingesting phloem sap. In response, however, the BPH can unplug sieve tube occlusions by activating β-1,3-glucanase genes in rice plants

Real-Time NMR-Based Drug Discovery to Identify Inhibitors against Fatty Acid Synthesis in Living Cancer Cells

The aberrant metabolism of fatty acids is recognized as a key driver in the development and progression of tumors. Although numerous inhibitors have been developed to target this pathway, finding drugs with high specificity that do not disrupt normal cellular metabolism remains a formidable challenge. In this manuscript, we introduced a novel real-time NMR-based drug screening technique that operates within living cells. This technique provides a direct way to putatively identify molecular targets involved in specific metabolic processes, making it a powerful tool for cell-based drug screening. Using 2-13C acetate as a tracer, combined with 3D cell clusters and a bioreactor system, our approach enables real-time detection of inhibitors that target fatty acids metabolism within living cells. As a result, we successfully demonstrate the initial application of this method in discovering traditional Chinese medicines that specifically target fatty acid metabolism. Elucidating the mechanisms behind herbal medicines remains challenging due to the complex nature of their compounds and the presence of multiple targets. Remarkably, our findings demonstrate the significant inhibitory effect of Poria cocos on fatty acid synthesis within cells, thereby illustrating the potential of this approach in analyzing fatty acid metabolism events and identifying candidate drugs that selectively inhibit fatty acid synthesis at the cellular level. Moreover, this systematic approach represents a valuable strategy for discovering the intricate effects of herbal medicine

The Bphi008a Gene Interacts with the Ethylene Pathway and Transcriptionally Regulates MAPK Genes in the Response of Rice to Brown Planthopper Feeding1[C][W][OA]

We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant’s resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP)