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Physalis angulata var. villosa, rich in withanolides, has been used as a traditional Chinese medicine for many years. To date, few extensive molecular studies of this plant have been conducted. In the present study, the plastome of P. angulata var. villosa was sequenced, characterized and compared with that of other Physalis species, and a phylogenetic analysis was conducted in the family Solanaceae. The plastome of P. angulata var. villosa was 156,898 bp in length with a GC content of 37.52%, and exhibited a quadripartite structure typical of land plants, consisting of a large single-copy (LSC, 87,108 bp) region, a small single-copy (SSC, 18,462 bp) region and a pair of inverted repeats (IR: IRA and IRB, 25,664 bp each). The plastome contained 131 genes, of which 114 were unique and 17 were duplicated in IR regions. The genome consisted of 85 protein-coding genes, eight rRNA genes and 38 tRNA genes. A total of 38 long, repeat sequences of three types were identified in the plastome, of which forward repeats had the highest frequency. Simple sequence repeats (SSRs) analysis revealed a total of 57 SSRs, of which the T mononucleotide constituted the majority, with most of SSRs being located in the intergenic spacer regions. Comparative genomic analysis among nine Physalis species revealed that the single-copy regions were less conserved than the pair of inverted repeats, with most of the variation being found in the intergenic spacer regions rather than in the coding regions. Phylogenetic analysis indicated a close relationship between Physalis and Withania. In addition, Iochroma, Dunalia, Saracha and Eriolarynx were paraphyletic, and clustered together in the phylogenetic tree. Our study published the first sequence and assembly of the plastome of P. angulata var. villosa, reported its basic resources for evolutionary studies and provided an important tool for evaluating the phylogenetic relationship within the family Solanaceae.

We employed both metabolomic and transcriptomic approaches to explore the accumulation patterns of physalins, flavonoids and chlorogenic acid in Physalis angulata and revealed the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Physalis angulata L. is an annual Solanaceae plant with a number of medicinally active compounds. Despite the potential pharmacological benefits of P. angulata, the scarce genomic information regarding this plant has limited the studies on the mechanisms of bioactive compound biosynthesis. To facilitate the basic understanding of the main chemical constituent biosynthesis pathways, we performed both metabolomic and transcriptomic approaches to reveal the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Untargeted metabolome analysis showed that most physalins, flavonoids and chlorogenic acid were significantly upregulated. Targeted HPLC-MS/MS analysis confirmed variations in the contents of two important representative steroid derivatives (physalins B and G), total flavonoids, neochlorogenic acid, and chlorogenic acid between MeJA-treated plants and controls. Transcript levels of a few steroid biosynthesis-, flavonoid biosynthesis-, and chlorogenic acid biosynthesis-related genes were upregulated, providing a potential explanation for MeJA-induced active ingredient synthesis in P. angulata. Systematic correlation analysis identified a number of novel candidate genes associated with bioactive compound biosynthesis. These results may help to elucidate the regulatory mechanism underlying MeJA-induced active compound accumulation and provide several valuable candidate genes for further functional study.

Taxus stem barks can be used for extraction of paclitaxel. However, the composition of taxoids across the whole stem and the stem tissue-specificity of paclitaxel biosynthesis-related enzymes remain largely unknown. We used cultivated Taxus media trees for analyses of the chemical composition and protein of major stem tissues by an integrated metabolomic and proteomic approach, and the role of TmMYB3 in paclitaxel biosynthesis was investigated. The metabolomic landscape analysis showed differences in stem tissue-specific accumulation of metabolites. Phytochemical analysis revealed that there is high accumulation of paclitaxel in the phloem. Ten key enzymes involved in paclitaxel biosynthesis were identified, most of which are predominantly produced in the phloem. The full-length sequence of TmMYB3 and partial promoter sequences of five paclitaxel biosynthesis-related genes were isolated. Several MYB recognition elements were found in the promoters of TBT, DBTNBT and TS. Further in vitro and in vivo investigations indicated that TmMYB3 is involved in paclitaxel biosynthesis by activating the expression of TBT and TS. Differences in the taxoid composition of different stem tissues suggest that the whole stem of T. media has potential for biotechnological applications. Phloem-specific TmMYB3 plays a role in the transcriptional regulation of paclitaxel biosynthesis, and may explain the phloem-specific accumulation of paclitaxel.

Taxus trees are major natural sources for the extraction of taxol, an anti-cancer agent used worldwide. Taxus media is a dioecious woody tree with high taxol yield. However, the sexually dimorphic accumulation of taxoids in T. media is largely unknown. Our study revealed high accumulation of taxoids in female T. media trees using a UPLC–MS/MS method. Thereafter, many differential metabolites and genes between female and male T. media trees were identified using metabolomic and transcriptomic analyses, respectively. Most of the taxol-related genes were predominantly expressed in female trees. A female-specific R2R3-MYB transcription factor gene, TmMYB39, was identified. Furthermore, bimolecular fluorescence complementation and yeast two-hybrid assays suggested the potential interaction between TmMYB39 and TmbHLH13. Several taxol biosynthesis-related promoter sequences were isolated and used for the screening of MYB recognition elements. The electrophoretic mobility shift assay indicated that TmMYB39 could bind to the promoters of the GGPPS, T10OH, T13OH, and TBT genes. Interaction between TmMYB39 and TmbHLH13 transactivated the expression of the GGPPS and T10OH genes. TmMYB39 might function in the transcriptional regulation of taxol biosynthesis through an MYB-bHLH module. Our results give a potential explanation for the sexually dimorphic biosynthesis of taxol in T. media.

Penicillium digitatum is the most common cause of postharvest decay in citrus fruits around the world. Previous studies revealed that the bZIP gene family plays crucial roles in development, stress adaptation, and pathogenicity in fungi. However, little is known about the bZIP genes in P. digitatum. In this study, we systematically identified the bZIP family in 23 Penicillium species and analyzed their evolutionary relationships. We found that gene loss and gene duplication shaped the evolution of the Penicillium bZIP family. P. digitatum experienced 3 bZIP gene loss events, but with no gene duplication. We subsequently characterized the biological functions of one important member, PdatfA in P. digitatum by constructing the deletion mutant. Results showed that ΔPdatfA exhibited a moderate growth defect, reduced pigmentation, and slightly increased resistance to fungicides iprodione and fludioxonil. However, ΔPdatfA displayed similar rot symptoms to that of the wild-type. The ΔPdatfA mycelia were not affected in response to oxidative stress while its conidia showed enhanced resistance due to the upregulation of catalases. Our results provide new insights into the evolution and functions of the bZIP gene family in Penicillium.

Additional file 2: Figure S1. Gene map of the P. alkekengi var. franchetii chloroplast genome. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The darker gray in the inner circle indicates the GC content, and the lighter gray indicates the AT content. The inner circle also indicates that the chloroplast genome contains two copies of the inverted repeat (IRA and IRB), a large single-copy region (LSC) and a small single-copy region (SSC). The map was constructed using OrganellarGenomeDRAW. Figure S2. Gene map of the P. minima chloroplast genome. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The darker gray in the inner circle indicates the GC content, and the lighter gray indicates the AT content. The inner circle also indicates that the chloroplast genome contains two copies of the inverted repeat (IRA and IRB), a large single-copy region (LSC) and a small single-copy region (SSC). The map was constructed using OrganellarGenomeDRAW. Figure S3. Gene map of the P. pubescens chloroplast genome. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The darker gray in the inner circle indicates the GC content, and the lighter gray indicates the AT content. The inner circle also indicates that the chloroplast genome contains two copies of the inverted repeat (IRA and IRB), a large single-copy region (LSC) and a small single-copy region (SSC). The map was constructed using OrganellarGenomeDRAW. Figure S4. Amino acid frequencies in the chloroplast genomes of five Physalis species. Figure S5. Neighbor-joining (NJ) tree based on the complete chloroplast genome sequences of 36 species of Solanaceae. Numbers above branches indicate bootstrap support, and circled by the red dotted lines are the five Physalis species.

Additional file 1: Table S1. Information on the four Physalis species used in the study. Table S2. Quality control of the Illumina sequencing of chloroplast genome of Physalis species. Table S3. Relative synonymous codon usage (RSCU) in five Physalis chloroplast genomes. Table S4. Evolutionary divergence among Physalis species based on complete chloroplast genome sequences. Table S5. Universal primers for amplifying complete chloroplast genomes. Table S6. The 36 studied species belonging to 11 genera of Solanaceae, and the corresponding chloroplast whole genome GenBank accession number.

Background Physalis L. is a genus of herbaceous plants of the family Solanaceae, which has important medicinal, edible, and ornamental values. The morphological characteristics of Physalis species are similar, and it is difficult to rapidly and accurately distinguish them based only on morphological characteristics. At present, the species classification and phylogeny of Physalis are still controversial. In this study, the complete chloroplast (cp) genomes of four Physalis species (Physalis angulata, P. alkekengi var. franchetii, P. minima and P. pubescens) were sequenced, and the first comprehensive cp genome analysis of Physalis was performed, which included the previously published cp genome sequence of Physalis peruviana. Results The Physalis cp genomes exhibited typical quadripartite and circular structures, and were relatively conserved in their structure and gene synteny. However, the Physalis cp genomes showed obvious variations at four regional boundaries, especially those of the inverted repeat and the large single-copy regions. The cp genomes’ lengths ranged from 156,578 bp to 157,007 bp. A total of 114 different genes, 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, were observed in four new sequenced Physalis cp genomes. Differences in repeat sequences and simple sequence repeats were detected among the Physalis cp genomes. Phylogenetic relationships among 36 species of 11 genera of Solanaceae based on their cp genomes placed Physalis in the middle and upper part of the phylogenetic tree, with a monophyletic evolution having a 100% bootstrap value. Conclusion Our results enrich the data on the cp genomes of the genus Physalis. The availability of these cp genomes will provide abundant information for further species identification, increase the taxonomic and phylogenetic resolution of Physalis, and assist in the investigation and utilization of Physalis plants.

Taxaceae is the smallest family of conifers. Taxol is an efficient anticancer drug and widely used in the treatment of several cancers. However, the accumulation of taxoids as well as other metabolites can vary hugely among Taxus species. To reveal the differential accumulation of metabolites among six Taxaceae trees, including four taxol-producing (TP) and two non-taxol producing (NTP) trees, comparative metabolomic analyses were performed in the present study. In total, 3,655 metabolites were detected in all six Taxaceae trees. For taxoids, most precursors and intermediates, and end products of the intermediates of taxol, such as 3′-N-debenzoyltaxol, 3′-N-debenzoyl-2’-deoxytaxol, taxa-4(20),11(12)-dien-5α-ol, 10-deacetyl-2-debenzoylbaccatin III, taxusin and other taxusin-like metabolites highly accumulated in the TP trees. Furthermore, great variations in the content of flavonoids between the TP and NTP trees were observed. A Liquid Chromatography-Mass Spectrometer analysis was applied to determine the contents of individual flavonoids. In detail, T. media contained the highest levels of quercetin, kaempferol, apigenin, luteolin, ginkgetin and (-)-epicatechin. For hormones, abscisate and abscisic acid highly accumulated in the NTP trees, whereas gibberellic acids substantially accumulated in the TP trees. The results can contribute to understand of the mechanisms underlying taxol biosynthesis and promote comprehensive resource utilization of the Taxaceae trees.

Background Plants of the genus Taxus have attracted much attention owing to the natural product taxol, a successful anti-cancer drug. T. fuana and T. yunnanensis are two endangered Taxus species mainly distributed in the Himalayas. In our study, an untargeted metabolomics approach integrated with a targeted UPLC-MS/MS method was applied to examine the metabolic variations between these two Taxus species growing in different environments. Results The level of taxol in T. yunnanensis is much higher than that in T. fuana, indicating a higher economic value of T. yunnanensis for taxol production. A series of specific metabolites, including precursors, intermediates, competitors of taxol, were identified. All the identified intermediates are predominantly accumulated in T. yunnanensis than T. fuana, giving a reasonable explanation for the higher accumulation of taxol in T. yunnanensis. Taxusin and its analogues are highly accumulated in T. fuana, which may consume limited intermediates and block the metabolic flow towards taxol. The contents of total flavonoids and a majority of tested individual flavonoids are significantly accumulated in T. fuana than T. yunnanensis, indicating a stronger environmental adaptiveness of T. fuana. Conclusions Systemic metabolic profiling may provide valuable information for the comprehensive industrial utilization of the germplasm resources of these two endangered Taxus species growing in different environments.

Cutleaf groundcherry ( Physalis angulata L.) is an annual plant with a number of medicinal ingredients. However, studies about the secondary metabolism of P. angulata are very limited. An integrated metabolome and proteome approach was used to reveal the variations in the metabolism associated with bioactive compounds under methyl-jasmonate (MeJA) treatment. Application of MeJA to the hairy roots could significantly increase the accumulation of most active ingredients. A targeted approach confirmed the variations in physalins D and H between MeJA treatment and the controls. Increases in the levels of a number of terpenoid backbone biosynthesis and steroid biosynthesis related enzymes, cytochrome P450 monooxygenases and 3β-hydroxysterioid dehydrogenase might provide a potential explanation for the MeJA-induced active ingredient synthesis. Our results may contribute to a deeper understanding of the regulation mechanism underlying the MeJA-induced active compound accumulation in P. angulata.

Figure S4. The coefficient of variation of the metabolites from T. yunnanensis and T. fuana. (DOCX 91 kb)

Figure S3. The top 20 largest metabolic categories of all identified metabolites. (DOCX 351 kb)

Figure S1. The total ion chromatograms of all the samples. (DOCX 531 kb)

Figure S2. The m/z width (a) and retention time width (b) of our MS data. (DOCX 611 kb)

Figure S5. The principal component analysis of the data from these two Taxus species. (DOCX 64 kb)

Figure S6. The statistical analysis of the DAMs between T. fuana and T. yunnanensis. (DOCX 15 kb)

Some researchers suggest that many plant species or extracts have medicinal value; in particular, some have great antitumor potential. Rhizoma curcuma is the dried roots ofCurcuma phaeocaulis, C. kwangsinensis and C. wenyujing in Chinese herbal medicine and the essential oil from it has been shown to inhibit tumor cell growth mainly due to its elemene component. Elemene has marked the characteristic of wide spectrum of antineoplastic activity and low adverse toxicity, especially β-elemene. Commercialized elemene injection is produced by R. curcuma, and applied as a national second-class new drug of antitumor in China.Literature reveals that many other plant species contain β-elemene or isomers in their essential oils from plant or parts of plant, and may represent a potential alternative to R. curcuma in the elemene processing industry and for use in antineoplastic medicine. This review summarizes the list of plants studied for their constituents of essential oils, and forty-four plant species are given much attention. Key words: Elemene, plant species, medicine, Rhizoma curcuma.

The gene encoding a thermophilic group 3 alcohol dehydrogenase from Thermotoga lettingae TMO was cloned and over-expressed in Escherichia coli. The full-length DNA sequence of TlADH was 1086-bp encoding a polypeptide of 361 amino acids. Comparative and bioinformatic analysis revealed that TlADH showed high similarity to group 3 ADHs from thermophilic and mesophilic alcohol dehydrogenases, but relatively highest similarity to T. maritime 1, 3-propanediol dehydrogenase. The optimal pH-values of butanol oxidation and butylaldehyde reduction of TlADH were 11.9 and 6.0, respectively. Kinetic parameters of the enzymes showed TlADH preferred NADP+ to NAD+ as a cofactor. TlADH can catalyze a range of primary alcohols oxidation, while it was inactive towards branched-chain alcohols and cyclitol. TlADH also can reduce aldehydes to corresponding alcohols, including aliphatic and aromatic aldehydes. It showed higher activity of aldehyde reduction than that of alcohol oxidation that may be relative to reactive aldehyde detoxification in cell metabolism. In the case of external ions addition we found a 5.23-fold increase in reaction activity by the adding of 1 mM MnCl2. T. lettinga ADH was cloned and overproduced in a mesophilic heterologous expression system, and the recombinant enzyme was characterized. It will be helpful to understand more about the physiological role of group 3 alcohol dehydrogenase. Key words: Group 3 ADH, bioinformatic analysis, recombinant expression, thermophilic,catalytic properties.

Supercritical fluid extraction (SFE) is one of the most commonly used extraction techniques in the course of analysis or preparation. It is environmentally friendly and has some advantages over other conventional extraction methods. This review covers the recent developments of SFE in the extraction of essential oils from the plant materials during the period 2005 to 2011, in particular some factors influencing SFE extraction yield, its characteristics and applications. Key words: Supercritical fluid extraction, essential oils, review.

Zhaowu Zeng1, Guanglin Zhou1, Xiaoli Wang2, Eric Zhijian Huang1, Xiaori Zhan1, Jun Liu1, Shuling Wang1, Anming Wang1, Haifeng Li1, Xiaolin Pei1, Tian Xie11Research Center for Biomedicine and Health, Hangzhou Normal University, Hangzhou, Zhejiang, China; 2Yichun University of Jiangxi Province, Yichun, Jiangxi, ChinaAbstract: The objective was to develop an elemene oil/water (o/w) microemulsion and evaluate its characteristics and oral relative bioavailability in rats. Elemene was used as the oil phase and drug, polysorbate 80 as a surfactant along with ethanol, propylene glycol, and glycerol as the cosurfactants. The microemulsion was prepared by mixing method, or ultrasonication method in an ultrasonic bath. Its three-dimensional response surface diagram was drawn by Mathcad software. The microemulsion was characterized by visual observation, cross-polarized microscopy, size, zeta potential, acidity, viscosity, and surface tension measurement. The drug content and entrapment efficiency were determined by ultra fast liquid chromatography (UFLC) and liquid surface method. Blood was drawn from rats at different time points after oral administration of an elemene microemulsion or a commercial elemene emulsion for measurement of the drug in plasma by UFLC to establish the pharmacokinetic parameters and relative bioavailability. The elemene microemulsion as a clarified and isotropic system containing 1% elemene (w/v), 5% ethanol (v/v), 15% propylene glycol (v/v), 15% glycerol (v/v), and 5% polysorbate 80 (w/v), was characterized as (57.7 ± 2.8) nm in size, 0.485 ± 0.032 in polydispersity index, (3.2 ± 0.4) mv in zeta potential, (5.19 ± 0.08) in pH, 6 mpa•s in viscosity, (31.8 ± 0.3) mN•m-1 in surface tension, (8.273 ± 0.018) mg•mL-1 in content of ß-elemene, and (99.81 ± 0.24)% in average entrapment efficiency. The area under the concentration-time curves from 0 h to 24 h (AUC0→24h) of the elemene microemulsion and commercial elemene emulsion were integrated to be 3.092 mg•h•L-1 and 1.896 mg•h•L-1 respectively, yielding a relative bioavailability of 163.1%. The present study demonstrates the elemene microemulsion as a new formulation with ease of preparation, high entrapment efficiency, excellent clarity, good stability, and improved bioavailability.Keywords: elemene, microemulsion, relative bioavailability