Evaluation of Angelica decursiva reference genes under various stimuli for RT-qPCR data normalization

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the RT-qPCR (quantitative real-time reverse transcription polymerase chain reaction) study of coumarins. This RT-qPCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the RT-qPCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of RT-qPCR for an accurate and far-reaching gene expression analysis in this medicinal plant.

Authentication of Three Source Spices of Arnebiae Radix Using DNA Barcoding and HPLC

Arnebia decumbens (Vent.) Coss. et Kralik, A. euchroma (Royle) Johnst and A. guttata Bunge, three commonly used traditional Chinese medicinal plants have been widely used for the clinical treatment of inflammatory diseases caused by fungal, bacterial, oxidation, and other related pathogens. However, precise identification at the similar species level is usually challenging due to the influence of the source of medicinal materials, traditional ethnic medicine and medicinal habits. Here we developed a comprehensive and efficient identification system for three source spices of Arnebiae Radix based on DNA barcoding and HPLC fingerprinting. A total of 599 samples from thirty-five wild populations were collected and identified by using DNA barcodes of ITS2 regions, and the chemotypes of seven naphthoquinoneswere revealed by HPLC quantitative analysis including principal component analysis and hierarchical clustering analysis. Our results showed that the ITS2 sequences can distinguish three source spices of Arnebiae Radix from adulterants. However, it was difficult to identify them by HPLC-specific chromatograms combined with chemometric analysis. These results indicated that DNA barcoding was a more powerful method than HPLC fingerprinting for the identification of related species that were genetically similar. DNA barcoding analysis could be a promising and reliable tool to accurately confirm the identities of medicinal materials, especially for those whose sources are multiple and difficult to be identified by conventional chromatography.

Changxun Fang. Pioneers of Allelopathy: Wenxiong Lin

Wenxiong Lin did research for > 30 years in plant molecular ecology and agro-ecology, rice allelopathy and continuous monoculture problems/soil sickness of Chinese medicinal plants. His 20 Patents and most publications (about 100) have focussed on (i). Gene regulation of allelochemicals biosynthesis in allelopathic rice, (ii). allelopathic inhibitory effects on weeds, (iii). continuous monoculture problems/soil sickness in medicinal plants and (iv). the influence of allelochemicals and monoculture practice on the rhizospheric micro-environment. He helped in development of microbial fertilizer to alleviate the autotoxicity problem in continuous cropping of Radix pseudostellariae and Rehmannia glutinosa, etc. He had been the President of Asian Allelopathy Society, and got the outstanding achievement from the platform of International Allelopathy Foundation. His student, Dr. Changxun Fang got Rice Award and Grodzinsky Award respectively in the 6th and 8th World Congress of International Allelopathy Society. His elucidation of the molecular ecological mechanism of rice allelopathy was awarded the II Class Prize of Fujian Provincial Scientific and Technological Progress Award in 2007. His researches to find the mechanism of development of continuous cropping obstacle/soil sickness in R. pseudostellariae and its remedy was awarded the I Class Prize of Fujian Provincial Scientific and Technological Progress Award in 2020.

Investigations of metallic elements and phenolics in Chinese medicinal plants

Traditional Chinese Medicines (TCM) can be contaminated with heavy metals, and therefore, the aim of this study is to analyze the Fe, Mn, Zn, Cu, Cd, Pb, Cr, and phenolic compounds contents in TCM plants used against civilization diseases. Metals were determined by flame atomic absorption spectroscopy (FAAS) for Fe, Mn, Zn, and Cu and inductively coupled plasma-optical emission spectroscopy (ICP-OES) for Pb, Cd, and Cr. The total phenolic, flavonoid, and phenolic acid contents were determined by HPLC and UV/vis spectrometry. The contents of the studied elements were highest in Radix Rehmanniae, whereas lowest in Fructus Lycii and Fructus Crataegi. The studied metals were assayed in the decreasing order: Fe, Zn, Mn, Cu, Cr, Pb, and Cd. Radix Rehmanniae Glutinosae Preparata showed the lowest phenolic composition, while Fructus Lycii showed the richest content. Principal component analysis (PCA) revealed that the contents of ferulic acid, caffeic acid, rutin, and Cu, Cr, and Cd were among the most important factors responsible for the differentiation between the investigated medicinal plants. Cluster analysis (CA) showed that the TCM samples originating from the same botanical plant species were often found in the same cluster, which confirms the similar level of studied elements determined within the samples.

Transcriptomic analysis reveals the GRAS family genes respond to gibberellin in Salvia miltiorrhiza hairy roots

Background Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants with high medicinal value. Gibberellins are growth-promoting phytohormones that regulate numerous growth and developmental processes in plants. However, their role on the secondary metabolism regulation has not been investigated. Results In this study, we found that gibberellic acid (GA) can promote hairy roots growth and increase the contents of tanshinones and phenolic acids. Transcriptomic sequencing revealed that many genes involved in the secondary metabolism pathway were the GA-responsive. After further analysis of GA signaling pathway genes, which their expression profiles have significantly changed, it was found that the GRAS transcription factor family had a significant response to GA. We identified 35 SmGRAS genes in S. miltiorrhiza, which can be divided into 10 subfamilies. Thereafter, members of the same subfamily showed similar conserved motifs and gene structures, suggesting possible conserved functions. Conclusions Most SmGRAS genes were significantly responsive to GA, indicating that they may play an important role in the GA signaling pathway, also participating in the GA regulation of root growth and secondary metabolism in S. miltiorrhiza.