Transcriptome Analysis of Cultivated and Wild Ginseng in Different Growth Years Revealed the Regulatory Mechanism of Ginsenosides

Background: As a famous Chinese medicine, ginseng has been used in the world for nearly 5,000 years. Wild ginseng is endangered due to environmental damage. Thus, cultivated ginseng is developed to replace wild ginseng. The morphological and physiological characteristics of both wild ginseng and cultivated ginseng change during growth, and the mechanism of this change is not yet understood. Results: This study performed transcriptome sequencing on the roots, stems and leaves of cultivated ginseng and wild ginseng with different growth years, exploring the effect of growth years on gene expression in ginseng. The number of DEGs in cultivated ginseng is more than that in wild ginseng. Based on the weighted gene co-expression network analysis, we found that the growth years significantly affected the gene expression of MAPK signaling pathway - plant and terpenoid backbone biosynthesis pathway in cultivated ginseng, but had no effects in wild ginseng. Furthermore, the growth years had significant effects on the genes related to ginsenoside synthesis in cultivated ginseng, and the effects were different in the roots, stems and leaves. However, it had little influence on the expression of genes related to ginsenoside synthesis in wild ginseng and no effect on leaves. These results showed wild ginseng was less affected by growth years than cultivated ginseng. Furthermore, HMGR, SS, DXS, DS, IspF, AACT, CYP450 and UGTs were related with MYB, NAC, AP2/ERF, bHLH and WRKY transcription factors. Growth years may regulate genes for ginsenoside synthesis by influencing these transcription factors, thereby affecting the content of ginsenosides. Conclusions: This study complemented the gaps in the genetic information of wild ginseng in different growth periods and different tissues and provided a new insight into the mechanism of ginsenoside regulation.

Diversity and structure of the rhizosphere microbial communities of wild and cultivated ginseng

Background The resources of wild ginseng have been reducing sharply, and it is mainly dependent on artificial cultivation in China, Korea and Japan. Based on cultivation modes, cultivated ginseng include understory wild ginseng (the seeds or seedlings of cultivated ginseng were planted under the theropencedrymion without human intervention) and farmland cultivated ginseng (grown in farmland with human intervention). Cultivated ginseng, can only be planted on the same plot of land consecutively for several years owing to soilborne diseases, which is mainly because of the variation in the soil microbial community. In contrast, wild ginseng can grow for hundreds of years. However, the knowledge of rhizosphere microbe communities of the wild ginseng is limited. Result In the present study, the microbial communities in rhizosphere soils of the three types of ginseng were analyzed by high-throughput sequencing of 16 S rRNA for bacteria and internal transcribed spacer (ITS) region for fungi. In total, 4,381 bacterial operational taxonomic units (OTUs) and 2,679 fungal OTUs were identified in rhizosphere soils of the three types of ginseng. Among them, the shared bacterial OTUs was more than fungal OTUs by the three types of ginseng, revealing fungal communities were to be more affected than bacterial communities. In addition, the composition of rhizosphere microbial communities and bacterial diversity were similar between understory wild ginseng and wild ginseng. However, higher bacterial diversity and lower fungal diversity were found in rhizosphere soils of wild ginseng compared with farmland cultivated ginseng. Furthermore, the relative abundance of Chloroflexi, Fusarium and Alternaria were higher in farmland cultivated ginseng compared to wild ginseng and understory wild ginseng. Conclusions Our results showed that composition and diversity of rhizosphere microbial communities were significantly different in three types of ginseng. This study extended the knowledge pedigree of the microbial diversity populating rhizospheres, and provided insights into resolving the limiting bottleneck on the sustainable development of P. ginseng crops, and even the other crops of Panax.

Transcriptome Analysis of Cultivated and Wild Ginseng in Different Growth Years Revealed the Regulatory Mechanism of Ginsenosides

Background: As a famous Chinese medicine, ginseng has been used in the world for nearly 5,000 years. Wild ginseng is endangered due to environmental damage. Thus, cultivated ginseng is developed to replace wild ginseng. The morphological and physiological characteristics of both wild ginseng and cultivated ginseng change during growth, and the mechanism of this change is not yet understood. Results: This study performed transcriptome sequencing on the roots, stems and leaves of cultivated ginseng and wild ginseng with different growth years, exploring the effect of growth years on gene expression in ginseng. The number of DEGs in cultivated ginseng is more than that in wild ginseng. Based on the weighted gene co-expression network analysis, we found that the growth years significantly affected the gene expression of MAPK signaling pathway - plant and terpenoid backbone biosynthesis pathway in cultivated ginseng, but had no effects in wild ginseng. Furthermore, the growth years had significant effects on the genes related to ginsenoside synthesis in cultivated ginseng, and the effects were different in the roots, stems and leaves. However, it had little influence on the expression of genes related to ginsenoside synthesis in wild ginseng and no effect on leaves. These results showed wild ginseng was less affected by growth years than cultivated ginseng. Furthermore, HMGR, SS, DXS, DS, IspF, AACT, CYP450 and UGTs were related with MYB, NAC, AP2/ERF, bHLH and WRKY transcription factors. Growth years may regulate genes for ginsenoside synthesis by influencing these transcription factors, thereby affecting the content of ginsenosides.Conclusions: This study complemented the gaps in the genetic information of wild ginseng in different growth periods and different tissues and provided a new insight into the mechanism of ginsenoside regulation.

Genetic Diversity and Variety Identification of Panax Species Based on EST-SSR Markers

Background: The roots of Panax species are widely used in the East because of their high medicinal and economic value. They are similar in plant morphology and chemical composition, but have quite differences in medicinal properties and efficacy, therefore, genetic diversity and variety identification of Panax species is particularly important. Methods: We screened 7 Simple Sequence Repeat (SSR) markers from expressed sequence tags (ESTs) database of Panax species in NCBI. Using these markers test SSR polymorphism in Panax species. Results: Seven SSR markers could successfully identify Panax ginseng, Panax quinquefolium, Panax notoginseng, and their commercial products. Among three ginseng varieties, garden ginseng, forest ginseng, and wild ginseng, the polymorphism of EST-SSR markers decreased gradually, which may be related to age and environment. Two pairs of EST-SSR primers can specifically identify three ginseng cultivars. The phylogenetic relationships analysis showed that Panax ginseng and Panax quinquefolium were closer than Panax notoginseng. Compared with wild ginseng, the relationship between the garden ginseng and the forest ginseng was closer. Conclusion: SSR molecular markers have high repeatability and can be used as reliable molecular markers for genetic diversity and variety identification of Panax species.

Production of minor ginsenosides by combining Stereum hirsutum and cellulase

Minor ginsenosides (MGs) (include ginsenoside F2, Compound K, PPT, etc), which are generally not produced by ginseng plants naturally, are obtained by deglycosylation of major ginsenosides. However, the conventional processes used to produce deglycosylated ginsenosides focus on the use of intestinal microorganisms for transformation. In this study, an edible and medicinal mushroom Stereum hirsutum JE0512 was screened from 161 β-glucosidase-producing soil microorganisms sourced from wild ginseng using the plate coloration method. Furthermore, JE0512 was used for the production of CK from ginseng extracts (GE) in solid-state fermentation (SSF) using 20 g corn bran as substrate, 4 g GE, and 20% inoculation volume, and the results showed that the highest CK content was 29.13 mg/g. After combining S. hirsutum JE0512 with cellulase (Aspergillus niger), the MGs (F2, CK, and PPT) content increased from 1.66 to 130.79 mg/g in the final products. Our results indicate that the Stereum genus has the potential to biotransform GE into CK and the combination of S. hirsutum JE0512 and cellulase could pave the way for the production of MGs from GE.

Diversity and structure of the rhizosphere microbial communities of wild and cultivated ginseng

Background Different plant species, even different plant varieties, will promote different combinations of microbial communities related to them. Here, the objective was to explore the differences in the rhizosphere microbial communities in the wild ginseng, farmland cultivated ginseng and understory wild ginseng. The rhizosphere soil was obtained from three type of ginsengs, namely wild ginseng (WDG), farmland cultivated ginseng (CDG) and understory wild ginseng (LXG) (all ginsengs grown in the field). The 16S rRNA gene and internal transcribed spacer (ITS) region were analyzed to investigate the diversity and structure of the microbial community. Result We found the fungal communities were more influenced bacterial communities. There were differences in the microbial community composition under three types of ginsengs. Moreover, higher bacterial diversity and lower fungal diversity in CDG compared with WDG. Changes in rhizosphere microbial community composition and diversity of WDG and CDG may be caused by domestication. Furthermore, the relative abundance of potential phytopathogens, Chloroflexi, Fusarium and Alternaria were higher in CDG compared to WDG and LXG. This may be related to the fact that cultivated ginseng has a short life cycle and is susceptible to disease. Conclusion We found differences in the rhizosphere microbial community of the three types of ginsengs, and the abundance of pathogenic microorganisms is significantly different. This result provided insights into the underlying mechanisms of ginseng planting and disease resistance.

Curcubinoyl flavonoids from wild ginseng adventitious root cultures

Wild ginseng (Panax ginseng) adventitious root cultures were prepared by elicitation using methyl jasmonate and investigated further to find new secondary metabolites. Chromatographic fractionation of wild ginseng adventitious root cultures led to the isolation of eleven compounds. The chemical structures of isolated compounds were identified as four known flavanone derivatives (1–4), one new curcubinoyl derivative, jasmogin A (5) and six new curcubinoyl-flavanone conjugates, jasmoflagins A-F (6–11) by extensive spectroscopic analysis. Newly isolated curcubinoyl derivatives showed inhibitory activity against lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. Therefore, our present study suggested that elicitor stimulated plant cell cultures might contribute to the production of new metabolites.

177 Saponins, Including 11 New Compounds in Wild Ginseng Tentatively Identified via HPLC-IT-TOF-MSn, and Differences among Wild Ginseng, Ginseng under Forest, and Cultivated Ginseng

Wild ginseng (W-GS), ginseng under forest (F-GS, planted in mountain forest and growing in natural environment), and cultivated ginseng (C-GS) were compared via HPLC-DAD and HPLC-IT-TOF-MSn. A total of 199 saponins, including 16 potential new compounds, were tentatively identified from 100 mg W-GS (177 saponins in W-GS with 11 new compounds), F-GS (56 saponins with 1 new compound), and C-GS (60 saponins with 6 new compounds). There were 21 saponins detected from all the W-GS, F-GS, and C-GS. Fifty saponins were only detected from W-GS, including 23 saponins found in ginseng for the first time. Contents of ginsenosides Re (12.36–13.91 mg/g), Rh1 (7.46–7.65 mg/g), Rd (12.94–12.98 mg/g), and the total contents (50.52–55.51 mg/g) of Rg1, Re, Rf, Rb1, Rg2, Rh1, and Rd in W-GS were remarkably higher than those in F-GS (Re 1.22–3.50 mg/g, Rh1 0.15–1.49 mg/g, Rd 0.19–1.49 mg/g, total 5.69–18.74 mg/g), and C-GS (Re 0.30–3.45 mg/g, Rh1 0.05–3.42 mg/g, Rd 0.17–1.68 mg/g, total 2.99–19.55 mg/g). Contents of Re and Rf were significantly higher in F-GS than those in C-GS (p < 0.05). Using the contents of Re, Rf, or Rb1, approximately a half number of cultivated ginseng samples could be identified from ginseng under forest. Contents of Rg1, Re, Rg2, Rh1, as well as the total contents of the seven ginsenosides were highest in ginseng older than 15 years, middle–high in ginseng between 10 to 15 years old, and lowest in ginseng younger than 10 years. Contents of Rg1, Re, Rf, Rb1, Rg2, and the total of seven ginsenosides were significantly related to the growing ages of ginseng (p < 0.10). Similarities of chromatographic fingerprints to W-GS were significantly higher (p < 0.05) for F-GS (median: 0.824) than C-GS (median: 0.745). A characteristic peak pattern in fingerprint was also discovered for distinguishing three types of ginseng. Conclusively, wild ginseng was remarkably superior to ginseng under forest and cultivated ginseng, with ginseng under forest slightly closer to wild ginseng than cultivated ginseng. The differences among wild ginseng, ginseng under forest, and cultivated ginseng in saponin compositions and contents of ginsenosides were mainly attributed to their growing ages.