Genetic diversity and population divergence of Leonurus japonicus and its distribution dynamic changes from the last interglacial to the present in China.

BACKGROUND: Leonurus japonicus, a significant medicinal plant known for its therapeutic effects on gynecological and cardiovascular diseases, has genetic diversity that forms the basis for germplasm preservation and utilization in medicine. Despite its economic value, limited research has focused on its genetic diversity and divergence. RESULTS: The avg. nucleotide diversity of 59 accessions from China were 0.00029 and hotspot regions in petN-psbM and rpl32-trnL(UAG) spacers, which can be used for genotype discrimination. These accessions divided into four clades with significant divergence. The four subclades, which split at approximately 7.36 Ma, were likely influenced by the Hengduan Mountains uplift and global temperature drop. The initial divergence gave rise to Clade D, with a crown age estimated at 4.27 Ma, followed by Clade C, with a crown age estimated at 3.39 Ma. The four clades were not showed a clear spatial distribution. Suitable climatic conditions for the species were identified, including warmest quarter precipitation 433.20 mm ~ 1,524.07 mm, driest month precipitation > 12.06 mm, and coldest month min temp > -4.34 °C. The high suitability distribution showed contraction in LIG to LGM, followed by expansion from LGM to present. The Hengduan Mountains acted as a glacial refuge for the species during climate changes. CONCLUSIONS: Our findings reflected a clear phylogenetic relationships and divergence within species L. japonicus and the identified hotspot regions could facilitate the genotype discrimination. The divergence time estimation and suitable area simulation revealed evolution dynamics of this species and may propose conservation suggestions and exploitation approaches in the future.

Untargeted Metabolomic Analysis and Chemometrics to Identify Potential Marker Compounds for the Chemical Differentiation of Panax ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. japonicus var. major.

The Panax L. genus is well-known for many positive physiological effects on humans, with major species including P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. japonicus var. major, the first three of which are globally popular. The combination of UPLC-QTOF-MS and chemometrics were developed to profile "identification markers" enabling their differentiation. The establishment of reliable biomarkers that embody the intrinsic metabolites differentiating species within the same genus is a key in the modernization of traditional Chinese medicine. In this work, the metabolomic differences among these five species were shown, which is critical to ensure their appropriate use. Consequently, 49 compounds were characterized, including 38 identified robust biomarkers, which were mainly composed of saponins and contained small amounts of amino acids and fatty acids. VIP (projection variable importance) was used to identify these five kinds of ginseng. In conclusion, by illustrating the similarities and differences between the five species of ginseng with the use of an integrated strategy of combining UPLC-QTOF-MS and multivariate analysis, we provided a more efficient and more intelligent manner for explaining how the species differ and how their secondary metabolites affect this difference. The most important biomarkers that distinguished the five species included Notoginsenoside-R1, Majonoside R1, Vinaginsenoside R14, Ginsenoside-Rf, and Ginsenoside-Rd.

Arbuscular mycorrhiza alters the nutritional requirements in Salvia miltiorrhiza and low nitrogen enhances the mycorrhizal efficiency.

Salvia miltiorrhiza Bunge (danshen in Chinese) is one of the most important medicinal cash crops in China. Previously, we showed that arbuscular mycorrhizal fungi (AMF) can promote S. miltiorrhiza growth and the accumulation of bioactive compounds. Fertilization may affect mycorrhizal efficiency, and appropriate doses of phosphate (P) and nitrogen (N) fertilizers are key factors for obtaining mycorrhizal benefits. However, the optimal fertilization amount for mycorrhizal S. miltiorrhiza remains unclear. In this study, we studied the effects of AMF on the growth and bioactive compounds of S. miltiorrhiza under different doses (low, medium, and high) of P and N fertilizer. The results showed that the mycorrhizal growth response (MGR) and mycorrhizal response of bioactive compounds (MBC) decreased gradually with increasing P addition. Application of a low (N25) dose of N fertilizer significantly increased the MGR of mycorrhizal S. miltiorrhiza, and a medium (N50) dose of N fertilizer significantly increased the MBC of phenolic acids, but decreased the MBC of tanshinones. Our results also showed that the existence of arbuscular mycorrhiza changes nutrient requirement pattern of S. miltiorrhiza. P is the limiting nutrient of non-mycorrhizal plants whereas N is the limiting nutrient of mycorrhizal plants.

Heterogeneous Genetic Diversity Estimation of a Promising Domestication Medicinal Motherwort Leonurus Cardiaca Based on Chloroplast Genome Resources.

Leonurus cardiaca has a long history of use in western herbal medicine and is applied for the treatment of gynaecological conditions, anxiety, and heart diseases. Because of its botanical relationship to the primary Chinese species, L. japonicus, and extensive medical indications that go beyond the traditional indications for the Chinese species, it is a promising medicinal resource. Therefore, the features of genetic diversity and variability in the species have been prioritized. To explore these issues, we sequenced the chloroplast genomes of 22 accessions of L. cardiaca from different geographical locations worldwide using high-throughput sequencing. The results indicate that L. cardiaca has a typical quadripartite structure and range from 1,51,236 bp to 1,51,831 bp in size, forming eight haplotypes. The genomes all contain 114 distinct genes, including 80 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Comparative analysis showed abundant diversity of single nucleotide polymorphisms (SNPs), indels, simple sequence repeats (SSRs) in 22 accessions. Codon usage showed highly similar results for L. cardiaca species. The phylogenetic and network analysis indicated 22 accessions forming four clades that were partly related to the geographical distribution. In summary, our study highlights the advantage of chloroplast genome with large data sets in intraspecific diversity evaluation and provides a new tool to facilitate medicinal plant conservation and domestication.

Elucidation of metabolite isomers of Leonurus japonicus and Leonurus cardiaca using discriminating metabolite isomerism strategy based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry.

The isomer structural discrimination is a significant challenge in metabolome analysis based on ultrahigh performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS). In this study, a new discriminating metabolite isomerism strategy is proposed to elucidate the metabolome, especially the isomers, of Leonurus japonicus and Leonurus cardiaca. This strategy consists of three steps. First, the metabolite biosynthesis pathways are constructed based on a home-built compound database to rapidly profile the compounds of interest using the multiple diagnostic product ions (DPIs) screening analysis and binary comparison based on SUMPRODUCT function. Second, the fragmentation patterns (e.g. the high-resolution DPIs, DPI ratios) and chromatographic elution order are defined based on scattered reference chromatographic and mass spectrometry data, calculated lipophilicity parameters, molecular hydrogen bond analysis, and chemical reference standards. Finally, all discovered isomerisms are mapped with the defined applicable rules and the isomers are identified conveniently. Using this strategy, a total of 257 compounds were tentatively characterized, including 212 potential novel compounds and 67 pairs of cis-, trans-, and positional isomers of flavonoids, phenylethanoid glycosides, glucaric acids, novel quinic acids, and esters of fatty acids. Moreover, 56 characteristic markers were identified to discriminate these two herbal medicines. This strategy may significantly improve the efficiency and reliability of identifying isomers found in metabolite biosynthesis pathways.

Effects of different doses of cadmium on secondary metabolites and gene expression in Artemisia annua L.

This study aims to elucidate the underlying molecular mechanisms of artemisinin accumulation induced by Cd. The effects of different Cd concentrations (0, 20, 60, and 120 µmol/L) on the biosynthesis of Artemisia annua L. were examined. Intermediate and end products were quantified by HPLC-ESI-MS/MS analysis. The expression of key biosynthesis enzymes was also determined by qRT-PCR. The results showed that the application of treatment with 60 and 120 µmol/L Cd for 3 days significantly improved the biosynthesis of artemisinic acid, arteannuin B, and artemisinin. The concentrations of artemisinic acid, arteannuin B, and artemisinin in the 120 µmol/L Cd-treated group were 2.26, 102.08, and 33.63 times higher than those in the control group, respectively. The concentrations of arteannuin B and artemisinin in 60 µmol/L Cd-treated leaves were 61.10 and 26.40 times higher than those in the control group, respectively. The relative expression levels of HMGR, FPS, ADS, CYP71AV1, DBR2, ALDH1, and DXR were up-regulated in the 120 µmol/L Cd-treated group because of increased contents of artemisinic metabolites after 3 days of treatment. Hence, appropriate doses of Cd can increase the concentrations of artemisinic metabolites at a certain time point by up-regulating the relative expression levels of key enzyme genes involved in artemisinin biosynthesis.