Chromosome-level genome assembly of Salvia miltiorrhiza with orange roots uncovers the role of Sm2OGD3 in catalyzing 15,16-dehydrogenation of tanshinones.

Salvia miltiorrhiza is well known for its clinical practice in treating heart and cardiovascular diseases. Its roots, used for traditional Chinese medicine materials, are usually brick-red due to accumulation of red pigments, such as tanshinone IIA and tanshinone I. Here we report a S. miltiorrhiza line (shh) with orange roots. Compared with the red roots of normal S. miltiorrhiza plants, the contents of tanshinones with a single bond at C-15,16 were increased, whereas those with a double bond at C-15,16 were significantly decreased in shh. We assembled a high-quality chromosome-level genome of shh. Phylogenomic analysis showed that the relationship between two S. miltiorrhiza lines with red roots was closer than the relationship with shh. It indicates that shh could not be the mutant of an extant S. miltiorrhiza line with red roots. Comparative genomic and transcriptomic analyses showed that a 1.0 kb DNA fragment was deleted in shh Sm2OGD3m. Complementation assay showed that overexpression of intact Sm2OGD3 in shh hairy roots recovered furan D-ring tanshinone accumulation. Consistently, in vitro protein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone, 15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA, tanshinone I and 1,2-dihydrotanshinone I, respectively. Thus, Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis. The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.

Comparative transcriptomic analysis reveals genes regulating the germination of morphophysiologically dormant Paris polyphylla seeds during a warm stratification.

We previously analyzed the expression of genes associated with Paris polyphylla var. yunnanensis seed maturation and dormancy release; however, we were unable to clarify the relationship between gene expression levels and these processes. To reveal the molecular mechanisms underlying P. polyphylla var. yunnanensis seed dormancy release during a warm stratification, the transcriptomes of dormant and germinating P. polyphylla var. yunnanensis seeds were separately analyzed by RNA sequencing and were also compared with the transcriptomes of stem-leaf and root tissues harvested during the seed maturation stage. The RNA sequencing of five tissues generated 234,331 unigenes, of which 10,137 (4.33%) were differentially expressed among the analyzed tissues. The 6,619 unigenes whose expression varied among mature dormant, sprouted, and germinated seeds included 95 metabolic and 62 signaling genes related to abscisic acid, gibberellin, auxin, brassinosteroid, cytokinin, ethylene, jasmonic acid and salicylic acid. Additionally, 243 differentially expressed genes were annotated as known seed dormancy/germination-related genes. Among these genes, 109 were regulated by hormones or involved in hormone signal transduction. Finally, 310 transcription factor unigenes, including 71 homologs of known seed dormancy/ germination-related genes, were observed to be differentially expressed during a warm stratification. These results confirm that multiple hormones and transcription factors influence P. polyphylla var. yunnanensis seed dormancy release and germination during a warm stratification. This study identified candidate genes (e.g., ABI5) that should be cloned and functionally characterized regarding their effects on the release of P. polyphylla var. yunnanensis seed morphophysiological dormancy.

Quality evaluation of Panax quinquefolium from different cultivation regions based on their ginsenoside content and radioprotective effects on irradiated mice.

Ginsenosides are one of major types of bioactive compounds in American ginseng (AG) and utilized to assess the quality of various AG samples. The contents of ginsenosides showed cultivation region-related variation, which is possibly associated with AG's pharmacological effect difference. Therefore, to reveal the quality difference of AGs in different cultivation regions, AG samples from seven cultivation regions were evaluated via analyzing their contents of nine ginsenosides and the biochemical parameters in AG-treated irradiated mice. Pre-administration of AG decoctions could reversely modulate the irradiation-induced changes of antioxidant enzymatic activity, cytokine level and hormone level in irradiated mice, which demonstrated that AG had the radioprotective effects due to its antioxidative, immunomodulatory and anti-inflammatory properties. However, this radioprotection effect varied among different cultivation regions of AGs. Collectively, Beijing and Canada-cultivated AGs had the best radioprotection. Heilongjiang and Jilin-originated AGs had the similar pharmacological effects while USA, Shandong and Shaanxi-grown AGs had closer pharmacological effects. This biochemical measurements-based PCA and heatmap clustering of AGs from seven cultivation regions was nearly consistent with ginsencoside content- and the previous serum metabolome-based analyses. However, the pearson correlation analysis revealed that only Rb3 and Rd were significantly correlated with some of assayed biochemical parameters in irradiated mice pretreated with different cultivation regions of AG extracts.

[Changes of transport sugar content in different organs of Rehmannia glutinosa].

Raffinose series oligosaccharides are the transport and storage sugars of many plants, Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, medicinal parts ist he roots. Root and tuber of R. glutinosa contains stachyose, raffinose and other oligosaccharides, but the study about the process of growth and development of other organs in the non-structural changes in sugar content is rare.In this study, leaves, stems and roots of R. glutinosa were used as materials to analyze the diurnal variation and the changes of sugar content of sucrose, raffinose and stachyose in different organs of R. glutinosa. The results showed that the content of sucrose in R. glutinosa leaves gradually increased from seedling stage.However, the content of stachyose did not change much at the early stage of growth, and the stachyose rapidly increased at the later stage of growth. The raffinose content gradually decreased throughout the growing season, young leaves of R. glutinosa have higher ability to sucrose synthesis than mature leaves, while mature leaf has higher raffinose and stachyose synthesis ability than young leaves. Sucrose and stachyose content in stem gradually increased, while there was little change in raffinose content. The content of raffinose and stachyose in root increased rapidly from the beginning of fast growing period, while the content of sucrose did not change much. The content of sucrose in leaves of R. glutinosa did not change much at day and night, while the daily changes of raffinose and stachyose contents were very obvious. The contents of raffinose and stachyose in daytime were higher than those at night. The content of raffinose in root and stem was not changed much, but the change of stachyose in root, stem and leaf was very obvious, especially in stem and leaf. In summary, the leaf is the main synthetic organ of raffinose, leaves, stems and roots are stachyose synthesis organ. Sucrose, raffinose and stachyose are the major transport forms of carbohydrates in R. glutinosa.

Comparison of Serum Metabolite Changes of Radiated Mice Administered with Panax quinquefolium from Different Cultivation Regions Using UPLC-Q/TOF-MS Based Metabolomic Approach.

Chemometric analysis of bioactive compounds revealed that American ginsengs (AGs) from different cultivation regions of China had a difference in quality, which indicates their possible pharmacological difference. A UPLC-Q/TOF-MS-based untargeted metabolomic approach was used to uncover serum metabolite changes in radiated mice pre-administered with AG root decoctions from seven cultivation regions and to further assess their quality difference. OPLS-DA revealed that 51 metabolites (ESI−) and 110 (ESI⁺) were differentially expressed in sera between the control and the radiated model mice. Heatmap analysis further revealed that AG could not reverse most of these radiation-altered metabolites, which indicates dietary supplement of AG before cobalt radiation had the weak potential to mediate serum metabolites that were altered by the sub-lethal high dose radiation. In addition, 83 (ESI−) and 244 (ESI⁺) AG altered metabolites were detected in radiated mice under radiation exposure. Both OPLS-DA on serum metabolomes and heatmap analysis on discriminant metabolites showed that AGs from different cultivation regions differentially influenced metabolic alterations in radiated mice, which indicates AGs from different cultivation regions showed the pharmacological difference in modulation of metabolite changes. AGs from Shandong, Shanxi, and Beijing provinces had more similar pharmacological effects than AGs from USA, Canada, Jilin, and Heilongjiang. Finally, 28 important potential biomarkers were annotated and assigned onto three metabolic pathways including lipid, amino acid, and energy metabolisms.

[Influence of plant growth regulater on yield and quality of Salvia miltiorrhiza].

The study is aimed to investigate the effect of plant growth regulators on yield and quality of the Salvia miltiorrhiza. The plant growth regulators was spraying on Salvia plants in July or August in field experiment, then the yield, ingredient content and the antioxidant activity were determined. The results showed that plant growth regulator 'Zhuanggenling' could increase the yield of Salvia with root-planting by 38.45%. Plant growth regulator 'Duoxiaozuo' could increase the yield of Salvia with seedling planting by 14.19%. Both plant growth regulator significantly reduced the antioxidant activity of Salvia in vitro, but they had no significant effect on active ingredient contents.

[Study on distribution and dynamic accumulation of catalpol and total iridoid in fresh Rehmannia glutinosa].

Iridoid glycosides were the main active ingredient of Rehmannia glutinosa, of which catalpol has the highest content. This work will provide theoretical basis for metabolic study and cultivation of iridoids on the basis of the dynamic accumulation of catalpol and total iridoids in the growth of R. glutinosa. The samples of rehmannia 85-5 were gathered in the same filed from July to October. The contents of catalpol and total iridoid glycosides were measured by HPLC and specteophotometric, respectively. The results showed that youngest leaves had the higher content of catalpol and total iridoid glyosides than that of the other two leaf ages in the same growth stage from July to September, while their content of catalpol and total iridoid glycosides were all decreased as the growth of leaves of R. glutinosa. The content of catalpol didn't differ significantly from July to September, whereas it has significantly increased in October in the three leaf stage. In the same stage, the wider the root diameter is, the higher content of the effective components are. In August and September, the total iridoid glycosides have the fastest accumulation. The content of catalpol was increased as the accumulation of total iridoid glycosides.

[Pharmacodynamic differences between hangmaidong and chuanmaidong based on metabonomics].

To evaluate the differences of Ophiopogonjaponicus from different cultivations, the metabolomics based method was conducted to compare the effects of Hangmaidong and Chuanmaidong (Chinese name) on plasma endogenous metabolites of normal rats. Data were collected by ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS), and were analyzed by multivariate statistical method, such as Principal Component Analysis and Orthogonal Signal Correction Partial Least Square Discriminant Analysis. Results revealed that the plasma metabolites profiling of low and middle dose group of Chuanmaidong were similar to the control group, but different from the high dose group obviously. Meanwhile the high, middle and low dose groups of Hangmaidong were different from control group notably, and the difference is dose dependent. Lysophosphatidylcholines, the possible endogenous metabolites which contribute to the classification most significantly, are closely related to cardiovascular system diseases. Compared with the group of Chuanmaidong, Hangmaidong has greater impact on the plasma metabolic profiling of normal rats. Hangmaidong and Chuanmaidong showed significant differences pharmacodynamically.

The complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza.

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

[Growth and development of fruit and seed of Panax quinquefolium].

OBJECTIVE: To understand the embryo after-ripening phenomenon of Panax quinquefolium, the growth and development process of fruit and seed was investigated in this study. METHOD: The growth and development characteristics of fruit and seed were obtained by field observation, the morphological changes were measured with a vernier caliper, paraffin section was used as well. RESULT AND CONCLUSION: The plant reached the most flowering numbers in 5-8 d after initial blooming time and the lag phase of embryo occurred in about 70 d. The size, fresh and dry weight of fruit and seed were all reached maximum in fruit maturation period. As the result showed, the development of seed was stopped at torpedo form of embryos, this conclusion can be applied to explore the morphological after-ripening mechanism of P. quinquefolium seed.

[Effects of seedling quality on growth of Rehmannia glutinosa and oligosaccharide content].

OBJECTIVE: To study the effect of seedling quality on growth, yield and quality of Rehmannia glutinosa at harvest and build a basis for its GAP. METHOD: The seedling quality of R. glutinosa in main producing regions was surveyed to understand the current status of seedling quality. Field experiments with different varieties and seedling quality were conducted to measure dry matter accumulation with different growth of R. glutinosa and oligosaccharide content, and economic yield at harvest. RESULT: The seedling was randomly selected by farmers in R. glutinosa producing regions. Seedling quality could significantly improve on seedling emergence rate, and promote seedling growth, especially with early stage R. glutinosa, finally increase yield at harvest. At harvest, 63% and 50% of yield with A and B seedling could be improved for variety of 85-5, and 50% and 47% of yield could be increased for variety of Beijing No. 1, compared to the C seeding. CONCLUSION: In cultivation, the seedlings with the diameter > 1.5 cm should be transplanted firstly.

Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza.

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.

[Study on seed quality of Achyranthes bidentata].

OBJECTIVE: To study the seed quality of Achyranthes bidentata from different sources and establish a standard of seed quality grading. METHOD: Seed weight and moisture content determination, germination test, purity analysis were carried out by 100 grains method, high-temperature drying, double deck filter paper and GB/T354. 3-Seed Testing for Crops methods respectively. Eight collections were planted in several field plots. RESULT: The 1 000 grains weight of 33 collections is 0.69 to 3.54 g with a variance coefficient 24.83%, the germination rate between 19.02% to 99% with a variance coefficient 38.24%. Seed moisture content and purity showed no significantly difference. K-means cluster analysis showed that 33 collections were divided into 3 grades: the first grade including 23 collections, the second and third grade including 5 collections respectively. Eight collections showed different field growth and yield characters. CONCLUSION: There are significant differences in seed quality of A. bidentata, so the classification management is needed in medicinal materials market. Germination test is an effective method before cultivating.

[The quality of Salvia miltforthiza from Beijing suburbs with different growing modes].

OBJECTIVE: Study the yield and quality of Salvia miltforthiza in the poplars and apple trees intercropping system. METHODS: Assay the yield, heavy metals, organochlorine pesticides and active components of Salvia miltforthiza in different intercropping systems are different. RESULTS: The contents of Cd exceeds the allowed figure seriously though other heavy metals such as Pb, As, Hg, Cu fell in the standard range. The contents of organochlorine pesticides of different Salvia miltforthiza fell in the standard range. The active components of biennial Salvia miltforthiza intercropping with apple trees, including Tanshinone II A and Salvianolic acid B complied with the quality standards of Pharmacopoeia. CONCLUSION: Soil fertility, heavy metals of intercropping system and planting years should be considered in order to ensure the quality and stability of Chinese medicine.

[Study on seed testing for Salvia miltiorrhiza].

OBJECTIVE: To establish a seed testing methods for Salvia miltiorrhiza. METHOD: Referring to the International Seed Testing Rules made by ISTA and the Seed Testing for Crops (GB/T3543. 1-1995) issued by China. RESULT: The seeds are selected by winnowing; the seed purity is about 50%-60%; 100 grain weight is used to determine the quality of the seed; the seed moisture content is determined by air drying, the drying hour is 3 h. Seed viability is tested by TFC method.

[Comparative analysis of character on germplasm resources of Rehmannia glutinosa].

OBJECTIVE: To compare difference in character between wild germplasm and cultivar of Rehmannia glutinosa Libosch. METHOD: Field test and statistical analysis were applied. RESULT: The results showed that the plant height and leave weight of individual plant in cultivar were decreased significantly comparing to wild germplasm, and the output was increased significantly. The leave length was reduced. The leave width, the catalpol content in leave and polysaccharides and reducing sugar content in cultivar was increased not significantly. Whereas the catalpol content and the water extract content in cultivar were equal to wild germplasm. CONCLUSION: The plant height and leave weight of individual plant of R. glutinosa was decreased significantly in cultivar, but the active compounds content not changed obviously.

[Analysis of genetic diversity of wild Rehmannia glutinosa by using RAPD and ISSR markers].

OBJECTIVE: To analyze the genetic diversity of wild Rehmannia glutinosa and evaluate and compare random amplified polymorphic DNA (RAPD) and inter sample sequence repeat (ISSR) for analysis of R. glutinosa accessions. METHOD: Two molecular markers, RAPD and ISSR were used for analyzing 55 wild R. glutinosa accessions. RESULT: Average 16.00 and 19.08 bands were amplified by RAPD primers and ISSR primers respectively, and the percentage of polymorphic bands were 89.58% and 94.32% respectively; Fifty-five R. glutinosa accessions categorized into 7 clusters were identified by unweighted pair-group method, arithmetic average (UPGMA) method. CONCLUSION: A high level of genetic diversity of wild Rehmannia glutinosa was displayed at DNA level, and genetic diversity coefficient of R. glutinosa from different production areas was 0.63-0.98, and ISSR marker can detect higher genetic diversity of R. glutinosa germplasms than RAPD marker.

[Study on seed determination practice in Polygala tenuifolia].

OBJECTIVE: To control the seed quality market, a study on the seed determination practice in Polygala tenuifolia was carried out. METHOD: By studying the thousand grain weight moisture content, viability, genuineness, purity and germination percentage, some indices of seeds were fixed to the standards. The seed determination practice in P. tenuifolia was established. RESULT AND CONCLUSION: The practice could be utilized to control the seed quality of P. tenuifolia.

[Studies on diversity of quality of Forsythia suspensa].

OBJECTIVE: To study on diversity of quality of Forsythia suspense collected from different regions. METHOD: The hundred-seed weight of shucks was analyzed by the method of hundred grain mass. The thousand-seed weight of seeds was analyzed by the method of thousand grain mass. The contents of the active components in shucks and seeds were determined by HPLC. RESULT: The hundred-seed weight of shucks, the thousand-seed weight of seeds and the contents of the active components in the shucks and seeds from different regions were significantly different. CONCLUSION: The quality of F. suspense from different regions is not consistant.