•Subcellular localization of Fny2b on microtubules was confirmed by co-localizing agroinfiltration.•De-polymerization effect on microtubules after drug treatments affected solubility characteristics of Fny2b.•Nuclear localization ability of each 2b protein form different strains were significantly different.•PGsNLS oligo nucleotides was verified as an effective nuclear localization signal.Cucumber mosaic virus (CMV) 2b protein, a gene silencing suppressor, shows cytoplasmic localization and predominantly nuclear localization due to its two nuclear localization signals (NLSs) regions. We used a series of fluorescent organelle markers and two cytoskeleton markers co-localized with GFP tagged Fny2b (the 2b protein encoded by the CMV Fny strain) fusion protein transiently expressed in leaf epidermal cells of Nicotiana benthamiana by agroinfiltration. Subcellular localization of Fny2b on microtubules was confirmed by perfectly merging with mCherry-MAP (Microtubule-associated protein) marker. High consistency of de-polymerization effect on GFP-Fny2b and microtubules after oryzalin treatment imaged by confocal scanning microscopy was an evidence of Fny2b microtubule localization. Western blot analysis showed that drug treatment affected solubility characteristics of Fny2b, however, oryzalin treatment did not affect its gene silencing suppression function. We need further research to investigate the effect of 2b protein subcellular distribution on its function. We also investigated the nuclear localization ability of transiently expressed GFP-GUS protein fused with various strains of 2b protein. Nuclear localization ability analysis of each 2b protein was obtained in this paper, which is expected to provide an experimental basis to further analyze the correlation between nuclear localization and its predicted nuclear localization signals function. Finally, we successfully verified the great capability of PGsNLS oligo nucleotides as an effective nuclear localization signal could localize macromolecular protein in nucleus.

Prunella vulgaris, a widely-used medicinal perennial herb, contains various active compounds and has multifaceted medicinal activities. As the index component in P. vulgaris, rosmarinic acid (RA) is a typical phenolic acid, with significant anti-inflammatory, anti-oxidant, anti-tumour, anti-viral, and anti-microbial properties. To better understand the RA biosynthetic pathway in P. vulgaris, we isolated and cloned the cDNA sequences of putative RA synthase (PvRAS) and cytochrome P450 monooxygenase (PvCYP98A101), two important enzymes catalysing RA biosynthesis. Sequence analysis revealed that PvRAS contained an open reading frame (ORF) of 1305 bp encoding a 435-amino-acid residue belonging to the BAHD acyltransferase family, and PvCYP98A101 contained an ORF of 1530 bp encoding a 510-amino-acid residue, as a member of the CYP98A family. The deduced PvRAS and PvCYP98A101 amino acid sequences shared high similarity with other putative/known RASs and CYPs, respectively. Quantitative real-time PCR analysis showed that constitutive expression of PvRAS and PvCYP98A101 was much higher in roots than in leaves, stems, or spikes. Further analysis indicated that PvRAS was localised in the cytosol and nucleus, whilst PvCYP98A101 existed as a membrane protein in the endoplasmic reticulum. These results highlight the RA biosynthesis pathway in P. vulgaris and, provide useful information to engineer natural products.

•An improved HPLC method was developed to simultaneously quantify saponins in Sanqi.•The best method for saponins extraction from Sanqi root was also obtained.•Accumulation of five main saponins in Sanqi was studied for the first time.•September in the third year was the best time to harvest Sanqi root.•Removing flower bud in the third year increases the content of saponins.The root of Panax notoginseng is a famous traditional Chinese medicine used to stop bleeding, and to decrease inflammation and heart disease, in which saponins are the major active components. The current methods for saponins quantification are unsatisfactory to separate multi-components in short time. An improved, simple, and reliable HPLC method with potential for wide use was developed to simultaneously quantify five major saponins extracted from P. notoginseng. Five saponins, including ginsenoside Rg1, Re, Rb1, Rd and notoginsenoside R1 were well separated within 54 min. The best program for saponins extraction was that 0.2 g samples was soaked by 8 ml 70% methanol with overnight immersion, and extracted by ultrasonic bath for 1 h. P. notoginseng had been cultivated hundreds years as an industrial crop in China and the accumulative characteristics of five main saponins were first reported in this study. During three growing seasons, root was always the main organ to accumulate saponins when compared with aboveground part. During three growing seasons, the total content of five saponins was above 5% in the root of P. notoginseng cultivating more than one year. Among the accumulation of five saponins, ginsenoside Rg1 and Rb1 in root had higher content, and occupied larger proportion of total. September in the third year was the best harvest time due to the highest content of five major saponins in roots at that time. Besides, removing the flower bud in the third year would significantly increase the content of saponins in root of P. notoginseng.

•Two wild Panax species markedly increased genetic diversity of Panax notoginseng.•Variable ITS sites (5.9%) among 3 species were more than 0.05% in P. notoginseng.•Panax vietnamensis had a closer relationship with P. notoginseng than Panax stipuleanatus.In order to evaluate whether the two wild species, Panax vietnamensis (from Vietnam) and Panax stipuleanatus (from primeval forest, Yunan Province) could markedly increase the genetic diversity of cultivated Panax notoginseng (Wenshan, Yunnan Province), both start codon targeted (SCoT) markers and internal transcribed spacer (ITS) DNA barcode were firstly employed in this genus. A total of 173 amplification bands were generated by 16 selected SCoT primers, in which 153 (89.5%) were polymorphic. Nei's gene-diversity indicated that the genetic diversity of three species (h = 0.16 and I = 0.27) was obviously higher than that of P. notoginseng (h = 0.09). Similarly, 38 different ITS sites out of 639 (5.9%) were detected among three species, but only one was different within 22 samples of P. notoginseng. Analysis of molecular variance (AMOVA) showed a greater proportion of genetic diversity existed within (61.3%) rather than among (38.7%) groups at genus level. In addition, P. vietnamensis had a closer relationship with P. notoginseng than P. stipuleanatus. These results would be significant for increasing the genetic diversity of P. notoginseng population by hybridization with P. vietnamensis and P. stipuleanatus, thus obtaining more varieties for future cultivar breeding and germplasm resources management.

Sour jujube (Zizyphus jujuba Mill. var. spinosus (Bge.) Hu) has gained considerable attention for its adaptation to drought prone environments. To characterize the physiological and biochemical basis of this drought adaptation, the effects of drought stress on Sour jujube seedlings were investigated in a greenhouse. Two contrasting populations were employed in our study, which were from the wet (YL) and dry (SB) climatic regions in the Loess Plateau of China. Results showed that SB exhibited lower water consumption and growth inhibition, but higher water use efficiency than YL under drought stress, indicating that growth of the wet-climate population is more sensitive to drought stress. SB exhibited higher non-photochemical quenching (NPQ) during progressive soil drying, higher photochemical quenching (qP) during the sustained water supply stage, and higher ΔF/\(F_{\rm m}^\prime\) and qP during a re-watering period than YL. These results further indicate that the dry-climate population possesses better PSII efficiency under adverse conditions. YL showed larger increases in the production rate of superoxide anions, hydrogen peroxide, and hydroxyl radicals than SB during the progressive soil drying stage, indicating that SB suffered from less oxidative damage than YL. Antioxidant enzymes including catalase, ascorbate peroxidase, peroxidase and glutathione reductase, and antioxidants including carotenoids, flavonoids and proline; when these interact, they contribute greatly to the antioxidant capacity of the dry-climate population. Taken together, the better photosynthetic potential and antioxidant capacity contribute to the better performance of Sour jujube from the dry-climate, providing useful information for understanding the drought tolerance mechanisms of Sour jujube.

Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway inSaliva miltiorrhizahairy roots.SPXmay be involved in this process.Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

Salvia castanea Diels f. tomentosa Stib. is an endemic medicinal plant distributed in China, and the notably high content of tanshinone IIA in the root is proven effective for the therapy of heart diseases. Hairy root induction of this Salvia species was inoculated with Agrobacterium rhizogenes strain ATCC 15834. Transformed hairy root was cultured in 6,7-V liquid medium for growth kinetics assessment and elicitation. An S curve was present in the hairy root cultures based on the fresh and dry weights with an interval of 3 days. An optimum concentration of the applied elicitors (15 μM Ag+, 200 μM methyl jasmonate, and 200 mg l−1 yeast extract elicitor) benefitted both the growth status and tanshinone accumulation in the hairy root cultures. Tanshinone IIA contents were mostly stimulated 1.8-fold and 1.99-fold compared with the control by Ag+ and methyl jasmonate elicitation, respectively. Yeast extract dramatically enhanced dry mass accumulation, while it promoted cryptotanshinone content of 2.84 ± 0.33 mg g−1 dry weight at most in the hairy root cultures. Selected elicitors diversely influenced tanshinone accumulation in the time courses of hairy root cultures within 7 days. Furthermore, transcripts of selected genes in the tanshinone biosynthetic pathway were remarkably upregulated with elicitation. Yeast extract elicitor heightened 13.9-fold of isopentenyl diphosphate isomerase expression level at 12 h, while it increased 16.7-fold of geranylgeranyl diphosphate synthase transcript at 24 h compared with that of the control, which was more effective than Ag+ and methyl jasmonate. This study provided a convenient hairy root culture system of S. castanea Diels f. tomentosa Stib. for tanshinone production for the first time.

Salvia miltiorrhiza, a traditional Chinese medicinal herb, contains two groups of bioactive components: lipid-soluble tanshinones and water-soluble phenolic acids. Many researchers have recently focused on elicitation of secondary metabolites to improve their production. In this paper, folic acid was used as an elicitor to stimulate phenolics production in S. miltiorrhiza hairy root cultures. The results showed that folic acid use was effective for eliciting rosmarinic acid (RA), salvianolic acid B (SAB), caffeic acid (CA), and total phenolics from S. miltiorrhiza hairy roots and that 50 μM is the most effective folic acid concentration for elicitation. Roots treated with 50 μM folic acid showed 97.6, 76.7, and 47 % increases in RA, SAB, and CA contents, respectively, relative to the control. The expression of three key genes involved in SAB biosynthesis (i.e., PAL, TAT, and RAS) were upregulated by folic acid. In conclusion, we report for the first time that folic acid is an effective elicitor of phenolics production in S. miltiorrhiza hairy roots. Our results suggest that folic acid is a potential elicitor that may improve phenolic acid production in medicinal plants.

Salvia miltiorrhiza (Chinese name: dānshēn) is used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. The tanshinones represent the most important biological active class of compounds present in dānshēn extracts. They are synthesized via either the cytoplasmic mevalonate or the plastidial 2-C-methyl-D-erythritol-4-phosphate pathway. Here, we summarize recent discoveries regarding the mechanisms underlying tanshinone biosynthesis and how the process is regulated. Tanshinone accumulation in planta is affected by a range of elicitors and by the composition of the culture medium. Its production in hairy root cultures can be enhanced by the over-expression of genes encoding 1-deoxy-D-xylulose 5-phosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranyl diphosphate synthase and allene oxide cyclase. The pathway leading to the biosynthesis of the tanshinone precursors miltiradiene and ferruginol, has been engineered in yeast.

Salvia miltiorrhiza, a popular traditional Chinese medicine, is widely used for treatments in cardiotonic disease. Tanshinones are a group of bioactive ingredients in S. miltiorrhiza. In this study, Ce3+ was used as an elicitor to enhance tanshinones production in S. miltiorrhiza hairy roots. The results showed that contents of dihydrotanshinone I (DTI) and cryptotanshinone (CT) were significantly enhanced by 50 μmol/L Ce3+, and reached to 0.875 mg/g and 0.271 mg/g, respectively. However, tanshinone II A (TIIA) and tanshinone I (TI) contents were significantly decreased to 59% and 62% of the control. Simultaneously, expressions of genes (HMGR, DXR, DXS1, DXS2 and GGPPS) involved in tanshinone biosynthesis were upregulated by Ce3+. Responses of DXS1, DXS2 and GGPPS to Ce3+ treatments were later than HMGR and DXR. We speculated that branch pathways of DTI and CT biosynthesis were probably different from TIIA and TI. This work will help us understand biosynthetic mechanism of tanshinones in plants.Presume pathway of tanshinones biosynthesis

•We select 53 different morphological Salvia miltiorrhiza accessions to assess their genetic diversity.•We examine the genetic diversity of morphology-based S. miltiorrhiza accessions based on morphology, ISSR and SRAP markers.•There exists a great genetic diversity among different morphological S. miltiorrhiza accessions.•The genetic variation enlarges the genetic base for the collection, conservation of S. miltiorrhiza germplasm.Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.

Our study found that except Novosphingobium resinovorum (B5) Salvia miltiorrhiza root endophytic bacteria Pseudomonas brassicacearum sub sp. neoaurantiaca (B1), Rhizobium radiobacter (B2), Pseudomonas thivervalensis (B3), Pseudomonas frederiksbergensis (B4) significantly improved the activity of key enzymes 3-hydroxy-3-methyglutary1-CoA reductase and 1-deoxy-d-xylulose-5-phosphate synthase in the biosynthetic pathway of tanshinones. Specifically, HMGR activity with B1 treatment increased 2.1-fold that of control, 1-deoxy-d-xylulose-5-phosphate synthase activity with B2 treatment increased 5.0-fold that of control, which caused a significant increase in tanshinone content in the hairy roots. The dihydrotanshinone I and cryptotanshinone content under B1 treatment increased 19.2-fold and 11.3-fold, respectively, and total tanshinone content increased 3.7-fold that of control. The five endophytic bacteria B1, B2, B3, B4 and B5 all significantly decreased phenylalanine ammonia-lyase and tyrosine aminotransferase activity in hairy roots, of which, B3 treatment decreased phenylalanine ammonia-lyase activity by 46.2 %, and B2 treatment decreased tyrosine aminotransferase activity by 44.7 % compared with the control. Each of the five endophytic bacteria decomposed rosmarinic acid and salvianolic acid B, which caused a significant decrease in rosmarinic acid and salvianolic acid B content in hairy roots, with B2 treatment decreasing rosmarinic acid and salvianolic acid B content by 94.5 and 89.0 %, respectively, compared with the control. The five endophytic bacteria also inhibited the growth of S. miltiorrhiza hairy roots, of which, B2 and B4 treatment decreased hairy root biomass by 55.2 and 51.3 %, respectively, compared with the control, while hairy roots promoted the growth of B4 and B5 and inhibited the growth of B1 and B3.

The fermentation broth and mycelium pellet of Streptomyces pactum Act12 (Act12) may promote the accumulation of soluble sugar when added to Salvia miltiorrhiza hairy roots, increasing the accumulation level by as much as 23.20 % compared with the control; it may also inhibit the accumulation of soluble protein in the hairy roots, decreasing it by as much as 17.96 % compared with the control. The ACT12 also has a certain promotional effect on the growth of hairy root at an appropriate concentration of elicitors and upregulates the expression of genes 3-hydroxy-3-methyglutary1-CoA reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), and geranylgeranyl diphosphate synthase (GGPPS). Among these effects, that of the HMGR gene expression is as high as 33.66 times that of the control, indicating that the test Streptomyces pactum may efficiently adjust the secondary metabolism of S. miltiorrhiza at the level of gene transcription, thereby greatly increasing the accumulation level of tanshinone in the hairy roots; among which, the cryptotanshinone levels increased most significantly, as much as 33.63 times that of the control, and the total tanshinone levels were 12.61 times that of the control.

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of various diseases. Phenolic acids in S. miltiorrhiza, as important effective compounds, have become a new research focus in plant secondary metabolism in recent years. This review summarizes the recent advances in the regulation of water-soluble phenolic acid biosynthesis in S. miltiorrhiza via regulators at molecular level, such as the phenylalanine ammonia-lyase gene (PAL), cinnamic acid 4-hydroxylase gene (C4H), 4-coumarate-CoA ligase gene (4CL), tyrosine aminotransferase gene (TAT), 4-hydroxyphenylpyruvate reductase gene (HPPR), 4-hydroxyphenylpyruvated dioxygenase gene (HPPD), hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase-like gene (RAS-like), and v-myb avian myeloblastosis viral oncogene homolog 4 gene (MYB4), and production of anthocyanin pigmentation 1 gene (AtPAP1), and via regulators at cell level, such as methyl jasmonate, salicylic acid, abscisic acid, polyamines, metal ions, hydrogen peroxide (H2O2), ultraviolet-B radiation, and yeast elicitor.

Prunus subgenus Padus is a group with a wide distribution in temperate eastern Asia and eastern North America with one species extending to Europe and one to Central America. Phylogenetic relationships of subgenus Padus were reconstructed using sequences of nuclear ribosomal ITS, and plastid ndhF gene, and rps16 intron and rpl16 intron. Prunus subgenus Padus is shown to be polyphyletic. Taxa of subgenus Padus and subgenus Laurocerasus are highly intermixed in both the ITS and the plastid trees. The results support two disjunctions between eastern North America and Eurasia within the Padus group. One disjunction is between Prunus virginiana of eastern North America and P. padus of Eurasia, estimated to have diverged at 2.99 (95 % HPD 0.59–6.15)–4.1 (95 % HPD 0.63–8.59) mya. The other disjunction is between P. serotina and its Asian relatives. The second disjunction may have occurred earlier than the former one, but the age estimate is difficult due to the unresolved phylogenetic position of the P. serotina complex.

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Tanshinones, a group of active compounds in S. miltiorrhiza, are derived from two biosynthetic pathways: the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Water stress is well known to stimulate the accumulation of secondary metabolites in plants. Reactive oxygen species (ROS) serve as important secondary messengers in water stress-induced signal transduction pathways. In this study, the effects of polyethylene glycol (PEG) and abscisic acid (ABA) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of ROS in PEG- and ABA-induced tanshinone production were further elucidated. The results showed that contents and yields of four tanshinones in S. miltiorrhiza hairy roots were significantly enhanced by 2 % PEG and 200 μM ABA. Simultaneously, the mRNA levels and activities of two key enzymes (3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase) involved in tanshinone biosynthesis were upregulated. Both PEG and ABA were able to trigger the burst of H2O2 and O2−. The PEG- and ABA-induced increases of tanshinone production, gene expression, and enzyme activity were all dramatically suppressed by two ROS scavengers, catalase and superoxide dismutase. In addition, ROS treatments resulted in a significant increase in tanshinone production. These results demonstrated that the MVA and MEP pathways were activated by PEG and ABA to stimulate tanshinone biosynthesis, and the increase of tanshinone production was probably via ROS signaling.

MicroRNAs (miRNAs) are a class of non-coding RNAs that play critical roles in post-transcriptional regulation. Their target genes are involved in a variety of biological processes such as development, metabolism, and stress response. Panicum miliaceum L. (Panicum) is an important grain crop, but, until now, no miRNAs have been identified in this plant. Using a homology search based on expressed sequence tag (EST) analysis and miRNA precursor secondary structure, a total of 43 new miRNAs were identified. The miRNAs were found to be unevenly distributed among 11 miRNA families. Target analysis using the plant small RNA target analysis server psRNATarget showed that the newly identified miRNAs can potentially regulate 68 target genes. Ten of the 11 miRNA families were annotated as involved in RNA regulation, suggesting they may play an essential role in post-transcriptional regulation in Panicum. Selected miRNAs representing eight of the families were verified by northern blotting, indicating that the prediction method that we used to identify the miRNAs was effective.

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O2− and H2O2 were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.

Technique for Order Performance by Similarity to Ideal Solution (TOPSIS) method was used to comprehensively evaluate total natural antioxidants and antioxidant activities in Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou fruits based on geographical origin. Correlations between natural antioxidants, antioxidant activities and geographical conditions were investigated. TOPSIS evaluation showed that the fruits from Ningxia possess the largest amount of natural antioxidants and antioxidant activities among other samples. Correlation analyses indicated that both altitude and annual precipitation exert profound effects on natural antioxidant levels. Moreover, the fruits exhibited significant DNA damage protection activity and in vitro antiradical potentials. In particular, the fruits from Ningxia, Gansu and Shaanbei which were grown in the semi-arid regions of loess plateaus showed fairly higher antioxidant activities. To a certain degree, this study concludes that the fruits in arid harsh and high-altitude areas can accumulate higher levels of natural antioxidants and display stronger antioxidant activities.

Two new eudesmane derivatives were isolated from the leaves and flowers of Verbesina virginica, along with the known 6-O-β-E-p-coumaroyl-4α-hydroxyeudesmane (1). Their structures were determined as 6-O-β-Z-p-coumaroyl-4α-hydroxyeudesmane (2) and 6-O-α-E-p-coumaroyl-1β-4α-dihydroxyeudesmane (3) by spectroscopic methods.

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.

This study investigated the induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata (Thunb.) Berit (Araceae). We planned to use this material in future studies related to the mass production of medicinally valuable compounds and regulation of alkaloid metabolism. Different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), kinetin (Kin), and α-naphthaleneacetic acid (NAA) were used to induce callus and PLB formation from P. ternata tuber explants. The results showed that three physiologically distinct calluses were induced by different combinations of 2,4-D, 6-BA, and Kin used in this study. The calluses differed in color, texture, differentiation status, and alkaloid content. The alkaloid content of the three calli types ranged from 0.0175% to 0.0293%. In comparison, the alkaloid content of field-grown tubers was 0.0072%. Many reports have indicated that 2,4-D suppresses the biosynthesis of secondary metabolites; however, our results show that 2,4-D promoted alkaloid production in Pinellia calluses. The combination of NAA + 6-BA induced PLB formation. The PLB alkaloid content of 0.0321% was 1.1 to 1.8 times higher than the alkaloid content of the calluses and 4.5 times higher than the field-grown tubers. In conclusion, the induction of calluses and PLBs with alkaloid content greater than that of field-grown tubers indicates the potential use of these tissue culture materials for bioprocessing alkaloids from P. ternata and for the study of alkaloid metabolism.

An HPLC method has been developed for the fingerprinting and quantitative analysis of cardiotonic pills. A standard fingerprint containing 11 common peaks at three wavelengths was constructed from ten batches of pills to evaluate batch-to-batch consistency. In addition, the amounts of three marker compounds were also determined to evaluate the quality of the quantitative analysis. Chromatographic fingerprints at three wavelengths, along with the content of three marker compounds were found to be suitable for quality assessment. Fufang Danshen Pian (cardiotonic tablets), a traditional dosage form is produced from three kinds of Chinese medicinal herbs for the prevention and treatment of angina pectoris and coronary heart disease. Fufang Danshen Pian mainly contained an additional group of liposoluble components besides salvianolic acid B from Salvia miltiorrhiza Bunge while cardiotonic pills only contained water-soluble components. Therefore, the fingerprint accompanied with marker compounds can be used to assess the quality of the cardiotonic pills.

The growth and water metabolism of three common shrubs on the Loess Plateau were studied under soil with different water contents. Results showed that water consumption of those species decreased with the increase in drought stress, and water consumptions of these shrubs were different: Forsythia suspensa was the greatest, and Syringa oblata was the lowest. The growth rate of new branches and leaf area of three species were the fastest under adequate soil water conditions, and were the lowest under severe drought. Under the same water conditions, the growth of F. suspensa was the fastest while that of S. oblata was the slowest. The water content, proline and chlorophyll content of different species changed with the increase in soil water stress. The leaf water content of Periploca sepium and F. suspensa was obviously higher than that of S. oblata, while the leaf proline content of F. suspensa and S. oblata was lower than that of P. sepium. The ratio leaf chlorophyll a: b of F. suspensa and S. oblata decreased with the decrease in soil water content. Although these three shrubs had different mechanisms in response to drought stress, they all had higher drought resistance and could adapt to the drought condition on the Loess Plateau. This paper provided some bases for choosing tree species on the Loess Plateau.

Rosmarinic acid and salvianolic acid B are two important phenolic compounds with therapeutic properties in Salvia miltiorrhiza Bunge. The biosynthesis of rosmarinic acid is initiated by two parallel pathways, namely the phenylpropanoid pathway and the tyrosine-derived pathway. Salvianolic acid B is a structural dimer of rosmarinic acid and is believed to be derived from rosmarinic acid. In the current study, methyl jasmonate (MeJA) and hyphal extracts from fungi were used as elicitors to examine the relationship between enzymes in the two parallel pathways and accumulation of phenolic compounds in S. miltiorrhiza hairy root cultures. The results showed that accumulations of rosmarinic acid, salvianolic acid B and total phenolics were enhanced by MeJA while suppressed by fugal extracts. Responses of enzymes in the tyrosine-derived pathway, at both the gene transcript and enzyme activity levels, showed a better consistency with alterations of phenolic compounds content after the two elicitors treated. Our study implied that compared with enzymes in the phenylpropanoid pathway, enzymes in the tyrosine-derived pathway are more correlated to rosmarinic acid and salvianolic acid B biosynthesis in S. miltiorrhiza hairy roots.

Carbon isotope discrimination (Δ) has been repeatedly reported to positively correlate with grain yield in wheat grown under post-anthesis stress environments. Several hypotheses have been put forward to explain this association. However, there is few reported direct evidence clarified the relationship between Δ and grain yield, which is the objective of the present work. The relationships between Δ and grain yield (GY), harvest index (HI), ash content (ma), specific stem dry weight (SSDW) and carbohydrate in stem, were studied in a collection of 20 bread wheat cultivars (landraces, released cultivars and advanced lines) in Yinchuan of the Ningxia region (Northwest of China) in three successive years (2006, 2007 and 2008). Relationships between GY, HI, stem specific dry weight, accumulation and mobilization of stem reserved carbohydrate and Δ were analyzed. The effects of year, sampling time and genotypes on measured traits were significant. Positive correlations between grain yield and Δ were noted in 2007 and 2008 when stress occurred after anthesis, but not in 2006 when soil water condition was nearly optimal. Significant and positive correlations among maLm, grain Δ and harvest index were found. Stem water-soluble carbohydrate content (SWSCC) and specific dry weight 7 days after anthesis was positively associated with Δ. Remobilization efficiency of stem water-soluble carbohydrate and total carbohydrate (the sum of water-soluble carbohydrate and starch) was also found to be significantly and positively correlated to Δ. There were negative relationships between ΔGm, ΔSm, and SSDW at maturity. In conclusion, Δ may predict yield when irrigation does not meet crop's water requirement and wheat experiences a slight water stress after anthesis in central region of Ningxia. Δ or maLm is related to the efficiency of carbon partitioning to the grain. Basal mature stem Δ seems to be an integrated character reflecting both early carbohydrate assimilation and dry matter remobilization to grain during grain-filling.

Salvia miltiorrhiza Bunge is one of the most important and popular plant of the traditional Chinese medicine (TCM), but Salvia castanea Diels f. tomentosa Stib and Salvia miltiorrhiza Bunge f. alb have also been reported to have the same therapeutic effects as S. miltiorrhiza. To better distinguish between these species, the phytochemical profiles of three Salvia species were investigated by liquid chromatography. All the Salvia species were good sources of tanshinones, with the contents of phenolics being high in S. miltiorrhiza and S. miltiorrhiza f. alb, but not in S. castanea Diels f. tomentosa Stib. These results pave the way for a better phytotherapy exploitation of these plants.Graphical abstractDownload full-size imageS. miltiorrhiza, S. miltiorrhiza f. alb and S. castanea Diels f. tomentosa Stib. were good sources of tanshinones, with the contents of phenolics being high in S. miltiorrhiza and S. miltiorrhiza f. alb, but not in S. castanea Diels f. tomentosa Stib.

BackgroundBlind and excessive application of fertilizers was found during the cultivation of Panax notoginseng in fields, as well as increase in root rot disease incidence.MethodsBoth “3414” application and orthogonal test designs were performed at Shilin county, Yunnan province, China, for NPK (nitrogen, phosphorus, and potassium) and mineral fertilizers, respectively. The data were used to construct the one-, two-, and three-factor quadratic regression models. The effect of fertilizer deficiency on root yield loss was also analyzed to confirm the result predicted by these models. A pot culture experiment was performed to observe the incidence rate of root rot disease and to obtain the best range in which the highest yield of root and saponins could be realized.ResultsThe best application strategy for NPK fertilizer was 0 kg/667 m2, 17.01 kg/667 m2, and 56.87 kg/667 m2, respectively, which can produce the highest root yield of 1,861.90 g (dried root of 100 plants). For mineral fertilizers, calcium and magnesium fertilizers had a significant and positive effect on root yield and the content of four active saponins, respectively. The severity of root rot disease increased with the increase in soil moisture. The best range of soil moisture varied from 0.56 FC (field capacity of water) to 0.59 FC, when the highest yield of root and saponins could be realized as well as the lower incidence rate of root disease.ConclusionThese results indicate that the amount of nitrogen fertilizer used in these fields is excessive and that of potassium fertilizer is deficient. Higher soil moisture is an important factor that increases the severity of the root rot disease.

This study investigated the regulation of ascorbate and glutathione metabolism by jasmonic acid (JA) in Agropyron cristatum leaves under water stress induced by 10% PEG 6000. The results showed that JA level, the transcript levels and activities of APX, GR, MDHAR, DHAR, GalLDH and γ-ECS, and the contents of AsA, GSH, total ascorbate and total glutathione were increased by water stress. Above increases except for total glutathione content and the transcript level and activity of γ-ECS were suppressed by application of JA biosynthesis inhibitor ibuprofen (IBU), a lipoxygenase inhibitor, for 12 h before water stress treatment. Application of JA to IBU-inhibited plants prevented the reduction in the transcript levels and activities of GalLDH, APX, GR, DHAR and MDHAR, and the contents of AsA, GSH, total ascorbate induced by IBU under water stress. Meanwhile, pretreatment with IBU increased the malondialdehyde content and electrolyte leakage of plants and these increases were suppressed by application of JA under water stress. Our results suggested that water stress-induced JA is a signal that leads to the regulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance in A. cristatum.

The objective of this study was to determine the effect of two endophytic bacterial elicitors (Pseudomonas sp. and Enterobacter sp.) on the production of alkaloids in protocorm-like bodies (PLBs) of Pinellia ternata Breit. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9–166%, inosine production by 2–33%, and trigonelline production by 114–1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells (co-culture treatment). Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. These results suggest that Pseudomonas sp. and Enterobacter sp. could increase alkaloid yield from P. ternata under field or tissue culture conditions. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants. Additional study needs to be done to determine if these endophytic bacteria could be used to produce alkaloids in the fermentation industry.

•The transcript of CHS was suppressed in Salvia miltiorrhiza hairy root cultures.•The transgenic lines and controls were treated with SA elicitor.•The substrate flow was directed to phenolic acids pathway after the CHS silencing.•Combining CHS silencing and SA treatment is a new way to produce phenolic acids.Salvia miltiorrhiza is an important traditional Chinese herb in China and some other Asian countries. Phenolic acids are bioactive compounds in S. miltiorrhiza, the biosynthesis of which shares the general phenylpropanoid pathway with flavonoids. Chalcone synthase (CHS) is the entry point enzyme of the flavonoid pathway and plays a crucial role in flavonoid biosynthesis. In this study, the transcript of CHS was suppressed through RNAi-mediated silencing, and salicylic acid (SA) was added to the CHS silencing lines and wild-type lines as elicitor to investigate effects of the genetic modification and/or elicitor treatment on the production of phenolic acids in S. miltiorrhiza hairy roots. The results showed that the silencing of CHS enhanced contents of phenolic acids and decreased the accumulation of total flavonoids. The contents of phenolic acids in the SA treated CHS silencing lines were much higher than that in the CHS silencing lines and the SA treated wild-type lines. Our results imply that the substrate flow is directed to the phenolic acids pathway when the flavonoid pathway is interrupted, and combining the genetic modification and elicitor treatment is a new way to improve the production of phenolic acids in S. miltiorrhiza hairy root cultures.

This study investigated the role of the protein kinase MEK1/2 in the regulation of ascorbate and glutathione content by jasmonic acid in Agropyron cristatum leaves. The results showed that JA induced increases in the transcript levels and activities of APX, GR, MDHAR, DHAR, GalLDH and γ-ECS, the contents of AsA, GSH, total ascorbate and total glutathione, and the ratios of AsA/DHA and GSH/GSSG, and reduced the EGSSG/2GSH. The increases, except for the transcript level and activity of γ-ECS and the reduction in EGSSG/2GSH, were all suppressed by pre-treatment with the MEK1/2 inhibitors PD98059 and U0126. The results of western blot analyses showed that JA induced increases in the phosphorylation level of MEK1/2. Our results suggest that JA could induce the activation of MEK1/2 by increasing the phosphorylation level, which, in turn, resulted in the up-regulation of ascorbate and glutathione content in A. cristatum leaves.

Recently, an important topic of research has been how climate change is seriously threatening the sustainability of agricultural production. However, there is surprisingly little experimental data regarding how elevated temperature and CO2 will affect the growth of medicinal plants and production of bioactive compounds. Here, we comprehensively analyzed the effects of elevated CO2 and temperature on the photosynthetic process, biomass, total sugars, antioxidant compounds, antioxidant capacity, and bioactive compounds of Gynostemma pentaphyllum. Two different CO2 concentrations [360 and 720 μmol mol−1] were imposed on plants grown at two different temperature regimes of 23/18 and 28/23 °C (day/night) for 60 days. Results show that elevated CO2 and temperature significantly increase the biomass, particularly in proportion to inflorescence total dry weight. The chlorophyll content in leaves increased under the elevated temperature and CO2. Further, electron transport rate (ETR), photochemical quenching (qP), actual photochemical quantum yield (Yield), instantaneous photosynthetic rate (Photo), transpiration rate (Trmmol) and stomatal conductance (Cond) also increased to different degrees under elevated CO2 and temperature. Moreover, elevated CO2 increased the level of total sugars and gypenoside A, but decreased the total antioxidant capacity and main antioxidant compounds in different organs of G. pentaphyllum. Accumulation of total phenolics and flavonoids also decreased in leaves, stems, and inflorescences under elevated CO2 and temperature. Overall, our data indicate that the predicted increase in atmospheric temperature and CO2 could improve the biomass of G. pentaphyllum, but they would reduce its health-promoting properties.