Characterization of the physiological parameters, effective components, and transcriptional profiles of Polygonum multiflorum Thunb. Under pH stress.

Polygonum multiflorum Thunb. is a traditional Chinese medicine with extensive distribution and robust adaptability, but comprehensive research on its acid and alkali resistance is presently lacking. This study aimed to analyze the effects of 5 months of continuous pH stress on the physiological and photosynthetic parameters of P. multiflorum, and the content of effective components. Results revealed that pH stress significantly influenced the normal growth, physiological functions, and photosynthetic indicators of P. multiflorum. At soil pH 4.5, the tubers of P. multiflorum exhibited the highest levels of 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-d-glucoside (THSG) and total anthraquinones at 5.41% and 0.38%, respectively. However, increased soil pH significantly reduced the content of THSG and total anthraquinones. Reference-free transcriptome analysis was further conducted on P. multiflorum treated at pH 4.5 and 9.5, generating a total of 47,305 unigenes with an N50 of 2118 bp, of which 31,058 (65.65%) were annotated. Additionally, 2472 differentially expressed genes (DEGs) were identified. Among them, 17 DEGs associated with the biosynthesis of THSG and anthraquinones were screened. A comprehensive analysis of differential gene expression and effective component content demonstrated a significant positive correlation between the content of effective components and the 14 DEGs' expression but a negative correlation with soil pH. This study highlighted the influence of varying soil pH values on the effective component content of P. multiflorum. Specific acidic conditions proved beneficial for the synthesis and accumulation of THSG and total anthraquinones in P. multiflorum, thereby enhancing the quality of the medicinal material.

Study on the effect of processing methods on the total polyphenol, 2,3,5,4'-tetrahydroxystilben-2-O-ß-D-glucoside, and physcion contents in Fallopia multiflora Thunb. Haraldson root

Abstract This study investigated the changes in the ingredients in Fallopia multiflora Thunb. Haraldson (FMT) root after processing it with different methods such as soaking, stewing, and steaming or combined methods. The total polyphenol, 2,3,5,4'-tetrahydroxystilben-2-O-ß-D-glucoside (THSG), and physcion contents in FMT products after processing were determined using high-performance liquid chromatography (HPLC) and ultraviolet-visible (UV-VIS) methods. The results demonstrated that the processing method and time significantly affected the contents of polyphenol, THSG, and physcion. The physcion and total polyphenol content increased or decreased during processing depending upon the processing time, while the THSG content gradually decreased with an increase in the processing time. The content of physcion (a substance that can cause liver toxicity) was analysed, and the suitable conditions for processing of the FMT products were determined as initial soaking in rice swill for 24 h and subsequent stewing with black beans and water for 12 h

Determination of the species status of Fallopia multiflora, Fallopia multiflora var. angulata and Fallopia multiflora var. ciliinervis based on morphology, molecular phylogeny, and chemical analysis.

Relationships among Fallopia multiflora (Thunb.) Haraldson., F. multiflora var. angulata (S. Y. Liu) H. J. Yan, Z. J. Fang & Shi Xiao Yu., and F. multiflora var. ciliinervis (Nakai) Yonekura & H. Ohashi. were determined based on macroscopic and microscopic morphology, molecular phylogeny, and chemical analysis. The macroscopic and microscopic morphologies of root tubers or rhizomes, stems, and leaves were compared among the three taxa. The content of 11 chemical components (catechin, polydatin, stilbene glucoside, emodin, emodin-8-O-ß-D-glucopyranoside, rhein, chrysophanol, aloe-emodin, quercetin, physcion, and resveratrol) in the three taxa was determined by HPLC, and the chemical diversity was further evaluated by principal component and hierarchical cluster analyses. Molecular phylogenies were mapped using two chloroplast markers (matK and the psbA-trnH intergenic region) and a nuclear ribosomal marker [internal transcribed spacer 2 (ITS2) region]. Analyses of macroscopic and microscopic morphological characteristics revealed that the subterranean organs of F. multiflora and F. multiflora var. angulata are root tubers, whereas those of F. multiflora var. ciliinervis are rhizomes. In the phylogenetic trees, F. multiflora and F. multiflora var. angulata were clustered into a clade based on the combine matK + psbA-trnH sequence, with neighbour-joining, maximum likelihood, and Bayesian inference bootstrap support values of 99, 85, and 0.99, respectively. In addition, there were obvious differences in the chemical compositions of F. multiflora, F. multiflora var. angulata and F. multiflora var. ciliinervis. The root tubers of F. multiflora contain higher levels of stilbene glucoside and catechin, but lower levels of polydatin and anthraquinone compounds. In contrast to F. multiflora, the rhizomes of F. multiflora var. ciliinervis contain higher levels of polydatin and anthraquinone compounds, but lack stilbene glucoside. The content of all 11 assessed components was lower in F. multiflora var. angulata than in F. multiflora and F. multiflora var. cillinervis. Principal component and hierarchical cluster analyses revealed that F. multiflora and F. multiflora var. angulata individuals were clustered into a single clade, whereas F. multiflora var. ciliinervis individuals were clustered into a single clade separate from that containing F. multiflora and F. multiflora var. angulata individuals. On the basis of the results of our morphological, molecular phylogeny, and chemical analyses, we tentatively conclude that F. multiflora var. ciliinervis is an independent species, whereas F. multiflora var. angulata should be considered as a variety of F. multiflora.

Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum.

Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

Deep sequencing reveals transcriptome re-programming of Polygonum multiflorum thunb. roots to the elicitation with methyl jasmonate.

The phytohormone methyl jasmonate (MeJA) has been successfully used as an effective elicitor to enhance production of stilbenoid which is induced in plants as a secondary metabolite possibly in defense against herbivores and pathogens. However, the mechanism of MeJA-mediated stilbenoid biosynthesis remains unclear. Genomic information for Polygonum multiflorum Thunb. (P. multiflorum) is currently unavailable. To obtain insight into the global regulation mechanism of MeJA in the steady state of stilbene glucoside production (26 h after MeJA elicitation), especially on stilbene glucoside biosynthesis, we sequenced the transcriptomes of MeJA-treated and untreated P. multiflorum roots and obtained more than 51 million clean reads, from which 79,565 unigenes were obtained by de novo assembly. 56,972 unigenes were annotated against databases including Nr, Nt, Swiss-Prot, KEGG and COG. 18,677 genes expressed differentially between untreated and treated roots. Expression level analysis indicated that a large number of genes were associated with plant-pathogen interaction, plant hormone signal transduction, stilbenoid backbone biosynthesis, and phenylpropanoid biosynthesis. 15 known genes involved in the biosynthesis of stilbenoid backbone were found with 7 genes showing increased transcript abundance following elicitation of MeJA. The significantly up (down)-regulated changes of 70 genes in stilbenoid biosynthesis were validated by qRT-PCR assays and PCR product sequencing. According to the expression changes and the previously proposed enzyme functions, multiple candidates for the unknown steps in stilbene glucoside biosynthesis were identified. We also found some genes putatively involved in the transcription factors. This comprehensive description of gene expression information could greatly facilitate our understanding of the molecular mechanisms of MeJA-mediated stilbenoid biosynthesis in P. multiflorum roots. Our results shed new light on the global regulation mechanism by which MeJA regulates the physiology of P. multiflorum roots and is helpful to understand how MeJA elicits other plant species besides P. multiflorum.

Transcriptome changes in Polygonum multiflorum Thunb. roots induced by methyl jasmonate.

Transcriptome profiling has been widely used to analyze transcriptomic variation in plants subjected to abiotic or biotic stresses. Although gene expression changes induced by methyl jasmonate (MeJA) have been profiled in several plant species, no information is available on the MeJA-triggered transcriptome response of Polygonum multiflorum Thunb., a species with highly valuable medicinal properties. In this study, we used transcriptome profiling to investigate transcriptome changes in roots of P. multiflorum seedlings subjected to a 0.25 mmol/L-MeJA root-irrigation treatment. A total of 18 677 differentially expressed genes (DEGs) were induced by MeJA treatment, of which 4535 were up-regulated and 14 142 were down-regulated compared with controls. These DEGs were associated with 125 metabolic pathways. In addition to various common primary and secondary metabolic pathways, several secondary metabolic pathways related to components with significant pharmacological effects were enriched by MeJA, including arachidonic acid metabolism, linoleic acid metabolism, and stilbenoid biosynthesis. The MeJA-induced transcriptome changes uncovered in this study provide a solid foundation for future study of functional genes controlling effective components in secondary metabolic pathways of P. multiflorum.