Assembly and comparative analysis of the complete mitochondrial genome of Pinellia ternata.

Pinellia ternata is an important natural medicinal herb in China. However, it is susceptible to withering when exposed to high temperatures during growth, which limits its tuber production. Mitochondria usually function in stress response. The P . ternata mitochondrial (mt) genome has yet to be explored. Therefore, we integrated PacBio and Illumina sequencing reads to assemble and annotate the mt genome of P . ternata . The circular mt genome of P . ternata is 876 608bp in length and contains 38 protein-coding genes (PCGs), 20 tRNA genes and three rRNA genes. Codon usage, sequence repeats, RNA editing and gene migration from chloroplast (cp) to mt were also examined. Phylogenetic analysis based on the mt genomes of P . ternata and 36 other taxa revealed the taxonomic and evolutionary status of P . ternata . Furthermore, we investigated the mt genome size and GC content by comparing P . ternata with the other 35 species. An evaluation of non-synonymous substitutions and synonymous substitutions indicated that most PCGs in the mt genome underwent negative selection. Our results provide comprehensive information on the P . ternata mt genome, which may facilitate future research on the high-temperature response of P . ternata and provide new molecular insights on the Araceae family.

Analysis of DNA methylation in potato tuber in response to light exposure during storage.

Exposure to light induces tuber greening and the accumulation of the toxic alkaloid Solanine in potato (Solanum tuberosum L) during storage greatly reduce tuber value. While the mechanism of this greening process remains unclear, it is well understood that DNA methylation plays an important role in regulating gene expression in response to environmental conditions. In this study, methylation-sensitive amplified polymorphism was used to assess the effect of light exposure on DNA methylation during storage of potato tubers. Light-induced genome-wide DNA demethylation and the rate of DNA methylation decreased with long storage times. Following, the sequencing of 14 differentially amplified fragments and analysis using the Basic Local Alignment Search Tool, eight genomic sequences and six annotated fragment sequences were identified. The latter included ADP glucose pyrophosphorylase 1/2, chlorophyllide a oxygenase 1 (CAO1), receptor-like protein kinase HAIKU2, and repressor of GA4, all of which are involved in starch biosynthesis, chlorophyll synthesis, endosperm development, and gibberellic acid signaling, respectively. Demethylation was observed in the CpG island (-273 to -166 bp) of the CAO1 promoter in response to light, which further confirmed that the variations in genome methylation are dependent upon the light exposure and suggests a direct role for DNA methylation. Our results provide an epigenetic perspective for further exploring the mechanism of light-induced tuber greening.

UHPLC-MS-based metabolomic approach for the quality evaluation of Pinellia ternata tubers grown in shaded environments.

Pinellia ternata is a native herb in China, and its tuber is widely-used in traditional Chinese medicines. It has been identified that the shading treatment promotes tuber production during cultivation. However, the tuber quality in shaded environments is unknown, which limits the scientific cultivation of P. ternata. In this study, a metabolomics approach based on UHPLC-MS was applied to assess the metabolic components of P. ternata in response to shading. Diverse metabolites were profiled using the metabolomics approach. Then, datasets of P. ternata cultivated in natural light (control) and shaded environments were subjected to multivariate analyses. Two P. ternata tuber products were well separated by the PCA. In total, four P. ternata alkaloids with contents that were not altered by the shaded environment were detected. Metabolomic analyses further identified several organic acids [mevalonic acid, 12,13-dihydroxy-9Z-octadecenoic acid (12, 13-DiHOME), urocanic acid, and γ-aminobutyric acid] that were largely enriched in the shaded environment, which likely contributed to tuber quality and growth. This study determined that shading probably improves the quality of P. ternata tubers and laid a foundation for exploring the regulatory mechanism of the shade response in P. ternata.

[Analysis of shading on DNA methylation by MSAP in Pinellia ternata].

Pinellia ternata is a medicinal herb of Araceae, and its tubers are used as medicines. It is a common Chinese herbal medicine in China and has a large market demand. When exposing to strong light intensity and high temperature during the growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade can effectively delay sprout tumble formation and increase its yield, however the relevant regulation mechanism is unclear. DNA methylation, as a self-modifying response to environmental changes, is often involved in the regulation of plant growth and development. In this study, P. ternata grown under natural light and 90% shading were selected as the control group and the experimental group for genomic DNA methylation analysis by using methylate sensitive amplification polymorphism(MSAP). The results showed that a total of 617 loci were detected with 20 pairs of primers, of which 311 were in the natural light group and 306 in the shading group. The methylation sites in the light and shading groups accounted for 58.2% and 71.57%, respectively, and the methylation ratios in the methylation sites were 27.65% and 29.41%, respectively, indicating that shading significantly induced the genome DNA methylation of P. ternata. Compared to the natural light group, shading promoted 32.51% of the genes methylation, while inducing 16.25% gene demethylation. This study reveals the DNA methylation variation of P. ternata under shading conditions, which lays a preliminary theoretical foundation for further analysis of the mechanism of shading regulation of P. ternata growth from epigenetic level.

Nanoceria restrains PM2.5-induced metabolic disorder and hypothalamus inflammation by inhibition of astrocytes activation related NF-κB pathway in Nrf2 deficient mice.

Increasing studies demonstrated that air pollution (PM2.5) plays a significant role in metabolic and neurological diseases. Unfortunately, there is no direct testimony of this, and yet the molecular mechanism by which the occurrence remains unclear. In this regard, we investigated the role of NF-κB and Nrf2 signaling in PM2.5-induced metabolic disorders and neuroinflammation, and further confirmed whether Nrf2 deficiency promoted PM2.5-induced inflammatory response by up regulating astrocytes activation and nerve injury via modulating NF-κB signaling pathways. Present results found that, indeed, PM2.5 challenges results in glucose tolerance, insulin resistance, dysarteriotony, peripheral inflammation, nerve injury and hypothalamus oxidative stress through astrocytes activation related NF-κB pathway in Nrf2 deficient mice. Moreover, in vitro study, we confirmed that activated astrocytes induced by PM2.5 were involved in pathogenesis of hypothalamic inflammation, which were significantly associated with NF-κB signaling. Nanoceria as potential anti-inflammatory and anti-oxidant stress biomaterial has gained increasing attention. Moderate nanoceria treatment is able to restrain PM2.5-induced metabolic syndrome and inflammation. Inhibition of astrocytes activation related NF-κB and enhancement of Nrf2 by cerium oxide were observed in vivo and in vitro, suggesting cerium oxide inhibited hypothalamic inflammation and nerve injury by altering hypothalamic neuroendocrine alterations and decreasing glial cells activation. In addition, NF-κB inhibitor pyrollidine dithiocarbamate (PDTC) treated primary astrocytes directly determined Nrf2 pathway could be up regulated by dose-dependent nanoceria. These results suggest a new therapeutic approach or target to protect against air pollution related diseases by cerium oxide treatment.

Metabolic Analysis of Medicinal Dendrobium officinale and Dendrobium huoshanense during Different Growth Years.

Metabolomics technology has enabled an important method for the identification and quality control of Traditional Chinese Medical materials. In this study, we isolated metabolites from cultivated Dendrobium officinale and Dendrobium huoshanense stems of different growth years in the methanol/water phase and identified them using gas chromatography coupled with mass spectrometry (GC-MS). First, a metabolomics technology platform for Dendrobium was constructed. The metabolites in the Dendrobium methanol/water phase were mainly sugars and glycosides, amino acids, organic acids, alcohols. D. officinale and D. huoshanense and their growth years were distinguished by cluster analysis in combination with multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Eleven metabolites that contributed significantly to this differentiation were subjected to t-tests (P<0.05) to identify biomarkers that discriminate between D. officinale and D. huoshanense, including sucrose, glucose, galactose, succinate, fructose, hexadecanoate, oleanitrile, myo-inositol, and glycerol. Metabolic profiling of the chemical compositions of Dendrobium species revealed that the polysaccharide content of D. huoshanense was higher than that of D. officinale, indicating that the D. huoshanense was of higher quality. Based on the accumulation of Dendrobium metabolites, the optimal harvest time for Dendrobium was in the third year. This initial metabolic profiling platform for Dendrobium provides an important foundation for the further study of secondary metabolites (pharmaceutical active ingredients) and metabolic pathways.

[Comparison of different methods for isolating total RNA from bulblet of Fritillaria anhuiensis].

OBJECTIVE: To optimize a simple and effective method for total RNA extraction from bulblet of Fritillaria anhuiensis. METHOD: Four methods, i. e. guanidine isothiocyanate, bentonite, modified SDS/phenol and the RNAiso plus, were used to extract total RNA from bulblet of F. anhuiensis. Then the results of the extraction were compared and analyzed by electrophoresis detection and RT-PCR verification. RESULT: The total RNA extracted by bentonite method were clear and no dispersion, the integrity of the RNA was well, and there was no obvious contamination with DNA and other impurities, was suitable for RT-PCR test. CONCLUSION: The bentonite method is quick, economic, and efficient for total RNA extraction from bulblet of F. anhuiensis.

[Genetic transformation of Pinellia ternata with Agrobacterium tumefaciens-mediated sHSP genes].

OBJECTIVE: To establish an efficient genetic transformation system of Pinellia ternata. METHOD: With petioles from test-tube seedlings of P. ternata as explants, Agrobacterium tumefaciens mediation method was adopted to explore the effect of phenolic substances, A. tumefaciens's concentration, infection time, pre-incubation time and co-cultivation time on genetic transformation efficiency of P. ternata. RESULT AND CONCLUSION: The genetic transformation efficiency could be effectively enhanced by infecting in A. tumefaciens culture containing AS 40 mg x L(-1) for 15 min for three days. The petioles were put into the differentiation medium containing 150 mg x L(-1) Kan and 350 mg x L(-1) Carb to screening and cultivation. After around 30 days, microtubers could be observed at both sides of the petioles. Gus staining and PCR verification on the regenerated plants showed that the exogenous gene sHSP had been integrated into genome of P. ternata.

[Study on optimization of induction system of test-tube tuberous roots from leaves of Rehmannia glutinosa].

OBJECTIVE: To study the effect of sucrose and plant growth substances of different concentrations on the induction of test-tube tuberous roots of Rehmannia glutinosa, in order to establish an efficient system for the induction of test-tube tuberous roots from leaves of R. glutinosa. METHOD: Leaves from test-tube seedlings of 85-5 R. glutinosa were used as explants. After rooting induction, they were transferred to medium with orthogonal design for inducing test-tube tuberous roots of R. glutinosa. RESULT AND CONCLUSION: NAA played a significant role in induction of test-tube tuberous roots of R. glutinosa, followed by sucrose and 6-BA. With leaves from test-tube seedlings as the explants, the optimal medium for inducing test-tube tuberous roots of R. glutinosa was MS + BA 3.0 mg x L(-1) + NAA 0.1 mg x L(-1) + sucrose 7%. The study provides an efficient induction system for studies on artificial seeds and secondary metabolism with test-tube tuberous roots of R. glutinosa.

[Study on optimization of SRAP-PCR reaction system for Pinellia ternata in Suzhou].

OBJECTIVE: To investigate the optimization system of SRAP-PCR molecular marker technology in the analysis on Pinellia ternata. METHOD: SRAP-PCR reaction system for P. ternata was optimized by L16 (5(4)) orthogonal design with five elements (dNTPs, Mg2+, the template DNA, primers, Taq enzyme) and four standards. RESULT: The most suitable forward primer for SRAP for Pinellia ternata was 5'-TGAGTCCAAACCGGAAG-3', while the reverse primer was 5'-GACTGCGTACGAATTACG-3'. The optimized reaction system contained 70 ng DNA template, 0.9 micromol x L(-1) primer, 0.20 mmol x L(-1) dNTP s, 1.5 - 2.0 mmol x L(-1) Mg2+, and 2 U Taq enzyme. CONCLUSION: SRAP-PCR system for P. ternata is established to lay a foundation for future construction of SRAP genetic map of P. ternata.

[Effect of low temperature on cell synchronization division in suspension culture cells of Pinellia ternata].

OBJECTIVE: To study the effect of low temperature on cell synchronization division in suspension culture cells of Pinellia ternata, in order to find a proper condition for cell synchronization. METHOD: The loosened calli of P. ternata were used as materials. The effect of low temperature on cell synchronization division was studied and researched in suspension culture cells by the method of orthogonal experiment, the obtained experimental data was compared and analyzed through the analysis of range and analysis of variance. RESULT: After the low temperature treatment, the cell division index was increased in various degrees, among the total results, when the temperature was set at 8 degrees C for 24 hours and then recovered for 36 hours, the effect was the best, the mitotic index was significantly increased to 11.3%. CONCLUSION: The low temperature could obviously influence the cell synchronization division in suspension culture cells of P. ternata, and the mitotic index was efficiently increased.

[Study on hydroxyproline tolerance of different cultivars of Chrysanthemum morifolium in vitro].

OBJECTIVE: To investigate hydroxyproline tolerance of Chrysanthemum morifolium plantlets included " Boju", "Huaiju", "Chuju", "Gongju" and "Hangju",and provide references basis for excellent cultivar and breeding of Ch. morifolium. METHOD: Plantlets in vitro from five kinds of Ch. morifolium were inoculated on medium added with different concentrations of hydroxyproline. Free proline in leaves from plantlets was determined, then the damage index and survival rate were compared. RESULT AND CONCLUSION: The results showed that hydroxyproline tolerance of " Boju" and "Huaiju" were superior, the survival rates and free proline of them were higher, but the damage index was inferior. The hydroxyproline tolerance of "Hangju" was the worst, and the survival rate was minimum. The survival rate of "Chuju" and "Gongju" was between "Boju" and " Hangju", and the hydroxyproline tolerance of them was also medium.

[Study on preservation of Fritillaria anhuiensis by vitrification in vitro].

OBJECTIVE: To establish cryopreservation system of shoot-tips from Fritillaria anhuiensis. METHOD: Taking vitrification as system of cryopreservation, the shoot tips with length 2-3 mm were precultured in MS medium enriched with 0.4 mol x L(-1) sucrose for 3 d. They were treated for 20 min with 60% PVS2 at 25 degrees C, and then subjected to ice-cooled vitrification solution for 60 min and transferred to 2 mL cryotubes with fresh vitrification solution (PVS2) and plunged into liquid nitrogen. After rapid thawing in 40 degrees C water bath for 1 min, shoot-tips were expelled into MS medium containing 1.2 mol x L(-1) sucrose for 20 min. Further recovery and growth took place on regeneration medium in the dark at 25 degrees C for 2 weeks, and then with light/dark cycle of 12/12 h. The genetic integrity of cryopreserved shoot-tips was identified through products of PCR with arbitrary primers. RESULT AND CONCLUSION: The highest survival rates of shoot-tips reached 79.9% by vitrification, and the regeneration rates were 52.3%. No changes were found between treated materials and untreated materials in genomic DNA.

[Effects of different factors on direct induction of microtubers from explants in Pinellia ternate].

OBJECTIVE: To study the effects of different factors on direct induction of microtubers. These factors included plant growth substances, casein hydrolysate (CH), active carbon (AC), polyethylene glycol (PEG 4000), sucrose and glucose. METHOD: Using the orthogonal design method. RESULT AND CONCLUSION: The optimal media to directly induce microtubers from leaves were MS + MET 0.5 mg x L(-1) + 6-BA 0.5 mg x L(-1) + sucrose 3% and MS+ 6-BA 0.5 mg x L(-1) + IAA 0.5 mg x L(-1) + sucrose 3%. Optimal media to directly induce microtubers from tubers were MS + 6-BA 1.0 mg x L(-1) + PEG 5% + sucrose 5% and MS+ CH 500 mg x L(-1) + MET 0.5 mg x L(-1) + 6-BA 0.5 mg x L(-1) + AC 0.5% + sucrose 5%. Media suitable for plantlet growth were MS + 6-BA 0.5 mg x L(-1) + IAA 0.5 mg x L(-1) + AC 0.5% + sucrose 5% and MS + MET 0.5 mg x L(-1) + 6-BA 0.5 mg x L(-1) + sucrose 3%.