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The aim of this study was to investigate the chemical differences between genunine Isodon japonica and its adulterants. A linear ion trap liquid chromatography with tandem mass spectrometry analytical method has been developed for the identification and quantification of 14 major diterpenoids in I. japonica. Data acquisition was multiple reaction monitoring transitions mode followed by an information- dependent acquisition using the enhanced product ion (EPI) scan in a single run. The target compounds were further identified and confirmed using an EPI spectral library. Overall validation of the assay was carried out including linearity, accuracy, precision, limits of detection and quantification. The results demonstrated that the method was selective, sensitive and reliable. The determination results of 21 batches of I. japonica and adulterants were then analyzed and differentiated by hierarchical clustering analysis. [ABSTRACT FROM AUTHOR]

An ultra performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UPLC–MS-MS) method was developed for analyzing and identifying the constituents of 11 compounds including berberine, epiberberine, berberrubine, jatrorrhizine, coptisine, palmatine, evodiamine, rutaecarpine, limonin, paeoniflorin and albiflorin in Wuji pill (WJ pill), a traditional Chinese medicine. The chromatographic separation was performed on a C18 column and the mobile phase was composed of water (0.1% formic acid and 2 mmol ammonium acetate) and methanol with a linear gradient elution. The detection was performed by multiple reaction monitoring mode, using electrospray ionization in the positive ion mode. The total run time was 14 min. The calibration curveswere linear with all correlation coefficients higher than 0.9987 in the tested range. The intra- and interday precisions were no more than 4.9%, and the average recoveries were from 92.4 to 107.8% with the relative standard deviations no more than 7.8%. The developed method was successfully employed to analyze five batches of WJ pill samples. This is the first time to establish a method for the quality control of WJ pill to ensure the safety and efficacy in clinical applications effectively. [ABSTRACT FROM AUTHOR]

Sharp eyespot disease (primarily caused by the pathogen Rhizoctonia cerealis) and freezing stress are important yield limitations for the production of wheat (Triticum aestivum). Here, we report new insights into the function and underlying mechanisms of an ethylene response factor (ERF) in wheat, Pathogen-Induced ERF1 (TaPIE1), in host responses to R. cerealis and freezing stresses. TaPIEl-overexpressing transgenic wheat exhibited significantly enhanced resistance to both R. cerealis and freezing stresses, whereas TaPIEl-underexpressing wheat plants were more susceptible to both stresses relative to control plants. Following both stress treatments, electrolyte leakage and hydrogen peroxide content were significantly reduced, and both proline and soluble sugar contents were elevated in TaPIEl-overexpressing wheat, whereas these physiological traits in TaPIEI-underexpressing wheat exhibited the opposite trend. Microarray and quantitative reverse transcription-polymerase chain reaction analyses of TaPIE1-overexpressing and -underexpressing wheat plants indicated that TaPIE1 activated a subset of defense- and stress-related genes. Assays of DNA binding by electrophoretic mobility shift and transient expression in tobacco (Nicotiana tabacum) showed that the GCC boxes in the promoters of TaP1El-activated genes were essential for transactivation by TaPIE1. The transactivation activity of TaP1E1 and the expression of TaPIEl-activated defense- and stress-related genes were significantly elevated following R. cerealis, freezing, and exogenous ethylene treatments. TaPIEl-mediated responses to R. cerealis and freezing were positively modulated by ethylene biosynthesis. These data suggest that TaPIE1 positively regulates the defense responses to R. cerealis and freezing stresses by activating defense- and stress-related genes downstream of the ethylene signaling pathway and by modulating related physiological traits in wheat. [ABSTRACT FROM AUTHOR]

Wheat sharp eyespot, primarily caused by a soil-borne fungus Rhizoctonia cerealis, has become one of the most serious diseases of wheat in China. In this study, an ethylene response factor (ERF) gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the T. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERF1 is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmai12, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down from the T0 to T4 generations. Quantitative reverse transcription-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the expression levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-dependent signal pathway, such as a chitinase gene and a β-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ethylene-dependent pathway. [ABSTRACT FROM PUBLISHER]