Time-series transcriptome provides insights into the gene regulation network involved in the icariin-flavonoid metabolism during the leaf development of Epimedium pubescens.

Herba Epimedii (Epimedium) leaves are rich in prenylated flavonol glycosides (PFGs) with high medicinal value. However, the dynamics and regulatory network of PFG biosynthesis remain largely unclear. Here, we combined metabolite profiling (targeted to PFGs) and a high-temporal-resolution transcriptome to elucidate PFGs' regulatory network in Epimedium pubescens and identified key candidate structural genes and transcription factors (TFs) involved in PFG accumulation. Chemical profile analysis revealed that PFG content was quite different between buds and leaves and displayed a continuous decline with leaf development. The structural genes are the determinant reasons, and they are strictly regulated by TFs under temporal cues. We further constructed seven time-ordered gene co-expression networks (TO-GCNs) of PFG biosynthesis genes (including EpPAL2, EpC4H, EpCHS2, EpCHI2, EpF3H, EpFLS3, and EpPT8), and three flavonol biosynthesis routines were then predicted. The TFs involved in TO-GCNs were further confirmed by WGCNA analysis. Fourteen hub genes, comprising 5 MYBs, 1 bHLH, 1 WD40, 2 bZIPs, 1 BES1, 1 C2H2, 1 Trihelix, 1 HD-ZIP, and 1 GATA were identified as candidate key TFs. The results were further validated by TF binding site (TFBS) analysis and qRT-PCR. Overall, these findings provide valuable information for understanding the molecular regulatory mechanism of PFGs biosynthesis, enriching the gene resources, which will guide further research on PFG accumulation in Epimedium.

Response mechanism of growth and gypenosides content for Gynostemma longipes cultivated at two altitude habitats to fine root morphological characteristics.

Introduction: Fine roots are the critical functional organs of plants to absorb water and nutrients from the soil environment, while the relation between fine root morphological characteristics and yield & quality has received less attention for medicinal plants. Methods: Therefore, we investigated the relationship between fine root morphological characteristics and biomass & gypenosides content. We explored the primary environmental drivers of fine root indicators for Gynostemma longipes from three provenances cultivated at two altitude habitats. Results: At the end of the growing season, compared with the low-altitude habitat, the underground biomass of G. longipes in the high-altitude habitat increased significantly by 200%~290% for all three provenances. The response of gypenosides content to different altitude habitats varied with provenance and plant organs. The biomass of G. longipes strongly depended on the fine root characteristic indicators (P < 0.001), fine root length density, and fine root surface area. Our results also showed that the harvest yield of G. longipes could be effectively increased by promoting the growth of fine roots per unit leaf weight (P < 0.001, R2 = 0.63). Both fine root length density and fine root surface area had strong positive correlations with soil nutrient factors (R2 > 0.55) and a strong negative correlation with soil pH (R2 > 0.48). In a word, the growth of G. longipes is strongly controlled by the fine root morphological characteristics through the response of fine roots to soil nutrient factors and pH. Discussion: Our findings will help to deepen the understanding of the root ecophysiological basis driven by soil factors for the growth and secondary metabolites formation of G. longipes and other medicinal plants under changing habitat conditions. In future research, we should investigate how environmental factors drive plant morphological characteristics (e.g., fine roots) to affect the growth & quality of medicinal plants over a longer time scale.

[Rapid prediction of flavonoid content in Epimedium sagittatum by infrared spectroscopy].

Epimedii Folium is a well-known Chinese herbal medicine with the effect of nourishing kidney and strengthening Yang. Its main active ingredients are flavonoids. In this study, 60 samples of Epimedium sagittatum were collected for the determination of total flavonoids(TF) including the total amount of epimedin A, epimedin B, epimedin C, and icariin(abbreviated as ABCI) specified in the Chinese Pharmacopoeia as well as rhamnosylicariside Ⅱ and icariside Ⅱ. The calibration parameters of "first derivativemultiva-riate scattering correction in 1 900-650 cm~(-1) band(4-point smoothing)" and "first derivativestandard normal variable correction in 4 000-650 cm~(-1) full band(4-point smoothing)" were confirmed respectively. The quantitative model was established via Fourier infrared spectroscopy plus attenuated total reflection(FTIR-ATR) accessory combined with partial least squares(PLS) method and then used to predict the flavonoid content of 11 validation sets. The average prediction accuracy for ABCI in calibration set and validation set was 98.985% and 96.087%, respectively. The average prediction accuracy for TF in calibration set and validation set was 98.998% and 94.771%, respectively. These results indicated that FTIR-ATR combined with PLS model could be used for rapid prediction of flavonoid content in E. sagittatum, with the prediction accuracy above 94.7%. The establishment of this method provides a new solution for the detection of a large number of E. sagittatum samples.

A Novel 3-O-rhamnoside: 2″-O-xylosyltransferase Responsible for Terminal Modification of Prenylflavonol Glycosides in Epimedium pubescens Maxim.

Prenylated flavonol glycosides in Epimedium plants, as key medicinal components, are known to have great pharmaceutical activities for human health. Among the main prenylated flavonol glycosides, the modification mechanism of different sugar moieties is still not well understood. In the current study, a novel prenylated flavonol rhamnoside xylosyltransferase gene (EpF3R2″XylT) was cloned from E. pubescens, and the enzymatic activity of its decoding proteins was examined in vitro with different prenylated flavonol rhamnoside substrates and different 3-O-monosaccharide moieties. Furthermore, the functional and structural domains of EpF3R2″XylT were analyzed by bioinformatic approaches and 3-D protein structure remodeling. In summary, EpF3R2″XylT was shown to cluster with GGT (glycosyltransferase that glycosylates sugar moieties of glycosides) through phylogenetic analysis. In enzymatic analysis, EpF3R2″XylT was proven to transfer xylose moiety from UDP-xylose to prenylated flavonol rhamnoside at the 2″-OH position of rhamnose. The analysis of enzymatic kinetics showed that EpF3R2″XylT had the highest substrate affinity toward icariin with the lowest Km value of 75.96 ± 11.91 mM. Transient expression of EpF3R2″XylT in tobacco leaf showed functional production of EpF3R2″XylT proteins in planta. EpF3R2″XylT was preferably expressed in the leaves of E. pubescens, which is consistent with the accumulation levels of major prenylflavonol 3-O-triglycoside. The discovery of EpF3R2″XylT will provide an economical and efficient alternative way to produce prenylated flavonol trisaccharides through the biosynthetic approach.

The complete chloroplast genome of Epimedium trifoliolatobinatum (Koidz.) Koidz. (Berberidaceae).

Epimedium L. is an important genus in the family Berberidaceae. Epimedium trifoliolatobinatum (Koidz.) Koidz. 1939 is inhabited on the west side of the Shikoku, Japan. In this study, the first complete chloroplast genome of E. trifoliolatobinatum was assembled with Illumina paired-end sequencing data, which was 157,272 bp in length with a total GC content of 38.70%. A total of 112 unique genes were annotated, comprising 78 protein-coding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic analysis suggested that E. trifoliolatobinatum was sister to E. koreanum. The current results provided fundamental information for further conducting molecular systematics and phylogenetic research of Epimedium genus.

[Content correlation of eight phenolic acids and flavonoids in different medicinal parts of PA-type Perilla germplasms].

In this study, 10 PA-type Perilla germplasms were selected to detect the content of two phenolic acids, i.e., rosmarinic acid(RA) and caffeic acid(CA), and six flavonoids, including scutellarin-7-O-diglucuronoside(SDG), luteolin-7-O-diglucuronoside(LDG), apigenin-7-O-diglucuronoside(ADG), scutellarin-7-O-glucuroside(SG), luteolin-7-O-glucuroside(LG), and apigenin-7-O-glucuroside(AG) in leaves, stems, and fruits. The total content of phenolic acids and flavonoids in leaves was 3.991-12.028 mg·g~(-1) and 12.309-25.071 mg·g~(-1), respectively, which was much higher than that in stems(0.586-2.015 mg·g~(-1) and 0.879-1.413 mg·g~(-1), respectively) and fruits(0.004-2.222 mg·g~(-1) and 0.651-1.936 mg·g~(-1), respectively). RA was detected in five fruit samples, and RA content between leaves and fruits showed a significant negative correlation in the other five samples. For flavonoids, only LG and LDG could be detected in stems, and SG and SDG were not detected in fruits, while other flavonoids were not detected in some samples. The content of total flavonoids and LG in leaves and fruits was significantly positively correlated, and the content of LG in stems and fruits was significantly positively correlated. In 10 stem samples, seven met the standard that the content of RA in the stem should be not less than 0.1% specified in the Chinese Pharmacopoeia(2020 edition). Only one fruit sample reached the standard of RA content in the fruit not less than 0.25% specified in the Chinese Pharmacopoeia.

The complete chloroplast genome of Epimedium qingchengshanense G. Y. Zhong et B. L. Guo (Berberidaceae), an endangered species endemic to China.

Epimedium qingchengshanense G. Y. Zhong & B. L. Guo is an endangered species with high ornamental value and medicinal value in China. In this study, we reported the first complete chloroplast (cp) genome of E. qingchengshanense. The whole cp genome of E. qingchengshanense is 159,087 bp in length, comprising a pair of inverted repeat regions (IRs) (27,709 bp) that are separated by a large single-copy (LSC) region (86,607 bp) and a small single-copy (SSC) region (17,062 bp). The circular genome contains 112 unique genes, of which 78 are protein-coding genes, 30 tRNA, and 4 rRNA genes. Phylogenetic analysis shows that E. qingchengshanense has a closer relationship with other Epimedium species.

The complete chloroplast genome of Epimedium mikinorii Stearn. (Berberidaceae).

Epimedium mikinorii is a vulnerable species in the Epimedium genus of Berberaceae. Here, we sequenced the complete chloroplast genome of E. mikinorii, which is 157,136 bp in length, and is a typical quadripartite circular molecule composed of two inverted repeats (IRs) of 25,896 bp for each, a large single-copy region (LSC) of 88,395 bp, and a small single-copy region (SSC) of 16,949 bp. The complete chloroplast genome of E. mikinorii contains 134 genes, including 83 protein-coding genes, 38 tRNA genes, 8 rRNA genes, and 5 pseudogenes. Phylogenetic analysis showed that E. mikinorii was closely related to E. dolichostemon.

The complete chloroplast genome of Epimedium brevicornu (Berberidaceae), a traditional Chinese medicinal herb.

Epimedii Folium has been used as a common traditional Chinese medicine for more than 2000 years in China. In this study, we assembled the complete chloroplast (cp) genome of Epimedium brevicornu. The whole cp genome of E. brevicornu is 158,658 bp in length, comprising a pair of inverted repeat (IR) regions (27,699 bp) separated by a large single copy (LSC) region (86,558 bp) and a small single copy (SSC) region (16,702bp). The E. brevicornu cp genome contains 129 genes, of which 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis shows that E. brevicornu is closely clustered with E. wushanense, E. lishihchenii, and E. sagittatum. The published E. brevicornu chloroplast genome will provide useful information for the phylogenetic and evolutionary study on Epimedium family of Berberidaceae.

The complete chloroplast genome of Epimedium elachyphyllum Stearn (Berberidaceae), an endangered species endemic to China.

Epimedium elachyphyllum, which belongs to Berberidaceae, is only distributed in Guizhou province of China. In this study, the complete chloroplast (cp)genome of E. elachyphyllum was sequenced and assembled. The circular genome is 157,201 bp in length, which comprises of a large single-copy region (LSC, 88,519 bp), a small single-copy region (SSC, 17,042 bp), and a pair of inverted repeat regions (IRa and IRb, 25,820 bp). The genome of E. elachyphyllum contains 112 unique genes, of which 78 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis based on 18 complete cp genome sequences indicated that E. elachyphyllum was closely related to E. dolichostemon.

The complete chloroplast genome of Epimedium xichangense Y. J. Zhang (Berberidaceae).

Epimedium xichangense, a critically endangered herb with limited population, mainly distributes in Sichuan province, China. In our study, we obtained the complete chloroplast genome of E. xichangense with a length of 158,955 bp, including a large single copy region of 86,478 bp, small single copy region of 17,027 bp, and a pair of inverted repeat regions of 27,725 bp. The GC content in the whole chloroplast genome of E. xichangense is 38.81%. Among the 112 unique genes in the circular genome, 30 tRNA, four rRNA and 78 protein-coding genes were successfully annotated. We constructed the Maximum likelihood (ML) tree with 26 species, and came to the conclusion that E. xichangense was phylogenetically closely related to E. acuminatum and E. chlorandrum.

The complete chloroplast genome of Epimedium enshiense B. L. Guo et Hsiao (Berberidaceae).

The genus of Epimedium belongs to Berberidaceae family, which is famous for their medicinal and ornamental value. In recent years, Epimedium has attracted increasing attention due to their medicinal and nutritive value. In this research, we reported the complete chloroplast (cp) genome of Epimedium enshiense. The complete chloroplast of this species is 157,076 bp in length, including a pair of invert repeat regions (IRS) (25,833 bp) that is divided by a large single copy area (LSC) (88,340 bp) and a small single copy area (SSC) (17,070 bp). The circular chloroplast genome of E. enshiense contains 112 unique genes, composing of 78 protein-coding genes, 30 tRNA, and four rRNA genes. Phylogenetic analysis indicates that E. enshiense has a closer relationship with E. dolichostmon.

The complete chloroplast genome of Epimedium pudingense (Berberidaceae), a narrowly distributed plant species in China.

Epimedium L. is the largest herbaceous genus of Berberidaceae which comprises more than 60 species. Epimedium pudingense is a rare plant species only narrowly inhabited in the Puding County in the Guizhou Province of China. Here, we first report the complete chloroplast genome of E. pudingense assembled from Illumina short-read sequencing data. The chloroplast genome of E. pudingense was 157,325 bp in length, with a total GC content of 36.11%. A total of 112 unique genes were identified, among which are 78 protein-coding genes, 30 tRNA genes and four rRNA genes. The phylogenetic analysis revealed that E. pudingense closely related to E. elachyphyllum. Our study will provide useful fundamental data for further phylogenetic and evolutionary studies of the Epimedium genus.

[Regional characteristic analysis of alkaloids and chlorogenic acid in wild Phellodendri Amurensis Cortex].

In the present research, 674 wild medicinal material samples of Phellodendri amurensis Cortex were collected from 31 sampling sites in the whole distribution of its original plant Phellodendron amurense. The samples were collected under the premise that the stem diameter of sampling plant, sampling position and time were controlled. And the sampling sites were set at the interval of a latitude. The content of 6 kinds of active ingredients, palmatine chloride, berberine hydrochloride, phellodendrine chloride, jatrorrhizine hydrochloride, magnoflorine, chlorogenic acid, etc in the medicinal material samples were determined, and the results showed that the content of most active ingredients in the medicinal materials showed significant differences due to the difference of sampling sites. Among them, the medicinal materials from Liaoning region had the highest content of active ingredients, followed by Beijing and Jilin regions, and that from Heilongjiang region had the lowest content. The study has important directive significance to the exploration of environmental factors for the formation of active constituent and artificial planting regionalization of high quality Phellodendri amurensis Cortex.

[Evaluation for heavy metal pollution of soil and herb from main producing area of Phellodendron amurense in China].

In order to evaluate the heavy metal potential pollution of soil and medicinal materials in main producing area of Phellodendron amurense, we collected 32 soil samples and 32 herb samples from northeast and north of China covering four provinces. In this study, the detection of heavy metal contents was conducted by ICP emission spectroscopy and atomic fluorescence spectrometry. The results showed that the soil from all areas of Ph amurense generally reached the national standard. As, Hg, Cr, Cd, Pb and Cu content of herb samples met the requirtment of the national standard except Hg content exceeding standard slight in a few samples. The reason of excessive Hg was the ability of Hg accumulation in Ph. amurense and atmospheric environment was polluted. So, national standard and Good Agricultural Practice (GAP) must be carried out severely in Ph. amurense resources production.

Natural Resource Monitoring of Rheum tanguticum by Multilevel Remote Sensing.

Remote sensing has been extensively applied in agriculture for its objectiveness and promptness. However, few applications are available for monitoring natural medicinal plants. In the paper, a multilevel monitoring system, which includes satellite and aerial remote sensing, as well as ground investigation, was initially proposed to monitor natural Rheum tanguticum resource in Baihe Pasture, Zoige County, Sichuan Province. The amount of R. tanguticum from images is M = S*ρ and S is vegetation coverage obtained by satellite imaging, whereas ρ is R. tanguticum density obtained by low-altitude imaging. Only the R. tanguticum which coverages exceeded 1 m(2) could be recognized from the remote sensing image because of the 0.1 m resolution of the remote sensing image (called effective resource at that moment), and the results of ground investigation represented the amounts of R. tanguticum resource in all sizes (called the future resource). The data in paper showed that the present available amount of R. tanguticum accounted for 4% to 5% of the total quantity. The quantity information and the population structure of R. tanguticum in the Baihe Pasture were initially confirmed by this system. It is feasible to monitor the quantitative distribution for natural medicinal plants with scattered distribution.

Comprehensive analysis of alternative splicing in Digitalis purpurea by strand-specific RNA-Seq.

Digitalis purpurea (D. purpurea) is one of the most important medicinal plants and is well known in the treatment of heart failure because of the cardiac glycosides that are its main active compounds. However, in the absence of strand specific sequencing information, the post-transcriptional mechanism of gene regulation in D. purpurea thus far remains unknown. In this study, a strand-specific RNA-Seq library was constructed and sequenced using Illumina HiSeq platforms to characterize the transcriptome of D. purpurea with a focus on alternative splicing (AS) events and the effect of AS on protein domains. De novo RNA-Seq assembly resulted in 48,475 genes. Based on the assembled transcripts, we reported a list of 3,265 AS genes, including 5,408 AS events in D. purpurea. Interestingly, both glycosyltransferases and monooxygenase, which were involved in the biosynthesis of cardiac glycosides, are regulated by AS. A total of 2,422 AS events occurred in coding regions, and 959 AS events were located in the regions of 882 unique protein domains, which could affect protein function. This D. purpurea transcriptome study substantially increased the expressed sequence resource and presented a better understanding of post-transcriptional regulation to further facilitate the medicinal applications of D. purpurea for human health.

[Quality variation and ecotype division of Panax quinquefolium in China].

Quality variation and ecotype classification of Chinese herbal medicine are important scientific problems in Daodi herbal medicine research. The diversity of natural environmental conditions has led to form unique multi-Daodi, multi-product areas that produce particular Chinese herbal medicine. China is one of three big American ginseng (Panax quinquefolium L.) producing areas worldwide, with over 300 years of application and 40 years of cultivation history. Long-term production practice has led to the formation of three big advocate produce areas in China: Northeast province, Beijing and Shandong. P. quinquefolium L. grown under certain environmental conditions will develop long-term adaptations that will lead to more stable strains (different ecotypes). P. quinquefolium L., can vary greatly in quality; however, the ecological mechanisms causing this variation are still unclear. Root samples were collected from four-year-old cultivated P. quinquefolium L. plants in the three major genuine (Daodi) American ginseng-producing areas of Northeast province, Beijing and Shandong province, China. Ultra-performance liquid chromatography was used to analyze the contents of eight ginsenosides (Rg1, Re, Rb1, Rb2, Rb3, Rc, Rd, Rg2). Data for nine ecological factors, including temperature, moisture and sunlight, were obtained from the ecological database of Geographic Information System for Traditional Chinese Medicine. Soil samples from the sampling sites were collected. Effective boron and iron, available nitrogen and potassium, as well as other trace elements and soil nutrients, were determined by conventional soil physicochemical property assay methods. Analytical methods of biostatistics and numerical taxonomy were used to divide ecotypes of the three main Panax quinquefolium L. producing areas in China based on ginsenoside content, climate, soil and other ecological factors. To our knowledge, this is the first time that ecological division of P. quinquefolium L. producing areas in China has ever been conducted. The results show that there are two chemoecotypes of P. quinquefolium L. in China: ginsenoside Rb1-Re from outside Shanhaiguan, and ginsenoside Rg2-Rd from inside Shanhaiguan. Similarly, there are two types of climatic characteristics: inside Shanhaiguan (Beijing, Shandong) and outside Shanhaiguan (Northeast). This suggests that the formation and differentiation of chemoecotypes of P. quinquefolium L. is closely related to variability of the climatic and geographical environment. Additionally, ecological variation of the three main producing areas, characteristics of two climatic ecotypes, and soil characteristics are also discussed and summarized. These results provide experimental scientific evidence of the quality variation and ecological adaptation of P. quinquefolium L. from different producing areas. They also deepen our understanding of the biological nature of Daodi P. quinquefolium L. formation, and offer novel research models for other multi-origin, multi-Daodi Chinese herbal medicines ecotypes. In addition, the results demonstrate the critical need for improving quality, appropriate ecological regionalization and promoting industrialized development of P. quinquefolium L.

[Quantitative study on ecological suitability of Chinese herbal medicine based on GIS].

The quality of Chinese herbal medicine is closely related to its producing region. In order to apply mathematical models to do a quantitative study on the suitability of Chinese herbal medicine, it is necessary to study on the ecological factors and the interpolation of climatic data, which influence the Chinese herbal medicine growth. The paper firstly studied the judgment standard of ecological index from the points of ecology and statistics, and how to calculate the optimum range values and the weight of each ecological factor. Secondly, meteorological element data is essential data in analyzing the suitable region of Chinese herbal medicine, and the spatial distribution of meteorological elements is closely related to terrain environment, so, in order to make the results close to true value by the greatest degree. The paper adopted multiple linear regression interpolation method which based on DEM. The paper distinguished the factor system of suitable region and interpolation on the point of datumization, and made a study on it about some key issues.

Transcript profiles of Panax quinquefolius from flower, leaf and root bring new insights into genes related to ginsenosides biosynthesis and transcriptional regulation.

American ginseng (Panax quinquefolius L.) has been used for a wide range of therapeutic purposes in China. The major bioactive phytochemicals responsible for this plant's pharmacological features are ginsenosides. Thus far, little is known regarding the genes involved in ginsenosides biosynthesis in this species. As a non-model plant, information about its genomes is generally not available. In this study, we generated 6678 expressed sequence tags (ESTs) from the flower, leaf and root cDNA libraries of American ginseng. Assembly of ESTs resulted in 3349 unigenes including 534 contigs (with ESTs number ranging from 2 to 52) and 2815 singletons. By analyzing the predominant transcripts within specific tissues, a gene expression pattern was obtained in a tissue-specific manner. They were assigned according to the functional classification of unigenes to broad ranges of Gene Ontology categories which include biological processes, cellular components and molecular functions. Based on blastx search results, 24 unigenes representing candidates related to ginsenosides biosynthesis were identified. Cloning and characterization of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC: 1.1.1.34), the rate-limiting enzyme in mevalonic acid pathway, demonstrated that it belonged to the plant HMGR family and was highly expressed in leaves. Putative transcription factors were detected in 63 unigenes, including zinc finger, WRKY, homeobox and MADS-box family proteins. Five hundred and eighty-eight simple sequence repeat motifs were identified, of which, dimer was the most abundant motif. These data will provide useful information on transcript profiles, gene discovery, transcriptional regulation, flower biogenesis and marker-assisted selections. The analysis and information from this study will greatly contribute to the improvement of this medicinal plant as well as of other species in the Araliaceae family, for the purpose of ensuring adequate drug resources.