Search

Jingru Ma,1 Yuling Tai,2 Mengmeng Fan,3 Zhansheng Wang4 1Department of Cardiology, The Second Affiliated Hospital, Shenyang Medical College, Shenyang, 110035, People’s Republic of China; 2Department of Cardiology, Tongliao City Hospital, Tongliao, 028000, People’s Republic of China; 3Department of Cardiology, Xinxiang Central Hospital, Xinxiang, 453000, People’s Republic of China; 4Department of Cardiology, Fourth People’s Hospital of Shenyang, Shenyang, 110034, People’s Republic of ChinaCorrespondence: Jingru MaThe Second Affiliated Hospital of Shenyang Medical College, No. 64 Qishan West Road, Huanggu District, Shenyang, 110035, Liaoning Province, People’s Republic of ChinaTel + 86-18002453999Email majingru00@126.comBackground: Cardiac rehabilitation (CR) has been associated with improved cardiac function in cardiovascular diseases. Our aim was to explore the factors associated with cardiac function and CR.Methods: This prospective cohort study had 473 STEMI patients admitted for primary percutaneous coronary intervention (PCI) who were divided into a CR group (group A, n = 104) and a non-CR group (group B, n = 369) based on whether they could complete CR. Patients’ clinical features, such as age, hyperlipidemia, family history of premature coronary heart disease (FHPCHD), smoking history, body mass index (BMI, kg/m2), number of diseased vessels, arrhythmia during PCI, N-terminal pro–B-type natriuretic peptide (NT-proBNP), left ventricular ejection fraction (LVEF) and major adverse cardiac events (MACEs) at 6 months after PCI were compared. Then, the clinical characteristics of patients were further analyzed according to those with MACEs (n = 78) and those without MACEs (n = 395).Results: After CR of 6 months, NT-proBNP levels (p = 0.027), 6-MWD (meter, P = 0.000), LVEF (P = 0.000) were significantly improved in group A compared to group B, but not for SMWA (P = 0.875). Multivariate analysis indicated that even though patients in group A (OR 3.06, 95% CI 1.132– 8.274, p = 0.03) have a higher incidence of hyperlipidemia, their MACEs (OR 0.191, 95% CI 0.038– 0.961, p = 0.05) at 6 months were significantly lower than in group B, mainly because the average patient low age (< 65 years, OR 0.917, 95% CI 0.859– 0.979, p = 0.01) and significant improvement of 6-MWD (OR 7.999, 95% CI 4.342– 14.737, P = 0.00) and the LVEF at 6 months (OR 1.112, 95% CI 1.072– 1.154, p = 0.00). Further analysis based on the MACES outcomes showed that there were 6 factors associated with the occurrence of MACEs, they were age > 65 years (OR 1.032, 95% CI 1.009– 1.009, p = 0.007), smoking history (OR 0.485, 95% CI 0.238– 0.989, p = 0.046), education level (OR 2.646, 95% CI 1.370– 5.108, p = 0.004), 6-MWD (OR 1.688, 95% CI 1.104– 2.811, p = 0.044), LVEF (OR 0.958, 95% CI 0.926– 0.991, p = 0.013) and CR (OR 6.271, 95% CI 2.236– 17.590, p = 0.000).Conclusion: CR, including exercise rehabilitation, is a beneficial option to reduce MACEs in STEMI patients treated with primary PCI.Keywords: rehabilitation, ST-elevation myocardial infarction, percutaneous coronary intervention, major adverse cardiac events

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

German chamomile and Roman chamomile are the two most widely known chamomile species due to the medicinal properties of volatile compounds from their flowers. We determined the volatile compound content of different organs of these two chamomiles, and found that main volatile compounds in German chamomile were terpenoids and those in Roman chamomile were esters. Furthermore, 24 tissues from two chamomiles were sequenced and analyzed by gene co-expression network. The results showed higher terpene synthase expression levels and more modules correlated with sesquiterpenoids in German chamomile, which may explain its high sesquiterpenoid content. In both chamomiles, unigenes in volatile compound-correlated modules were significantly enriched in pathways related to plant-pathogen interactions and circadian rhythm, demonstrating that volatile compounds of chamomiles are influenced by these factors. There were ten times more unigenes related to plant-pathogen interactions in German chamomile than in Roman chamomile, which indicates German chamomile has higher resistance to pathogens.

Pigeonpea (

Lilium lancifolium is an important economic crop in Huoshan county of Anhui province, China. Continuous cropping obstacles serious affect the yield and quality of L. lancifolium. At present, the effect of the continuous cropping of L. lancifolium on soil fungal community structure is not clear. In this study, Illumina MiSeq was used to study the fungi of the rhizosphere soil associated with L. lancifolium subjected to three treatments: no continuous cropping, continuous cropping for 3 years, and continuous cropping for 5 years. The results showed that continuous cropping of L. lancifolium could increase the fungal richness and diversity in the rhizosphere to different degrees. Ascomycota was the dominant phylum, and its abundance increased after continuous cropping. In addition, the abundance of beneficial fungi, such as Chaetomium, decreased, and the abundance of harmful fungi, such as Fusarium and Colletotrichum, greatly increased with the duration of continuous cropping. Overall, continuous cropping changed the composition of soil fungal communities, reduced the abundance of beneficial fungi, and increased the abundance of harmful fungi. Thus, continuous cropping increased the potential for soil-borne diseases and endangered the bulb growth of L. lancifolium.

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea (Camellia sinensis) and oil tea (Camellia oleifera) and found it to be higher in the roots than in other tissues in both species. The theanine content was significantly higher in tea than oil tea. To explore the regulatory mechanisms of theanine accumulation, we identified genes involved in theanine biosynthesis by RNA-Seq analysis and compared theanine-related modules. Moreover, we cloned theanine synthase (TS) promoters from tea and oil tea plants and found that a difference in TS expression and cis-acting elements may explain the difference in theanine accumulation between the two species. These data provide an important resource for regulatory mechanisms of theanine accumulation in tea plants.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future., Design Type(s)sequence assembly objective • sequence annotation objectiveMeasurement Type(s)whole genome sequencing assay • BAC • transcription profiling assayTechnology Type(s)DNA sequencing • RNA sequencingFactor Type(s)developmental stageSample Characteristic(s)Camellia sinensis var. sinensis • leaf • apical bud • stem • flower • fruit • root Machine-accessible metadata file describing the reported data (ISA-Tab format)

Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

Background Matricaria recutita (German chamomile) and Chamaemelum nobile (Roman chamomile) belong to the botanical family Asteraceae. These two herbs are not only morphologically distinguishable, but their secondary metabolites – especially the essential oils present in flowers are also different, especially the terpenoids. The aim of this project was to preliminarily identify regulatory mechanisms in the terpenoid biosynthetic pathways that differ between German and Roman chamomile by performing comparative transcriptomic and metabolomic analyses. Results We determined the content of essential oils in disk florets and ray florets in these two chamomile species, and found that the terpenoid content in flowers of German chamomile is greater than that of Roman chamomile. In addition, a comparative RNA-seq analysis of German and Roman chamomile showed that 54% of genes shared > 75% sequence identity between the two species. In particular, more highly expressed DEGs (differentially expressed genes) and TF (transcription factor) genes, different regulation of CYPs (cytochrome P450 enzymes), and rapid evolution of downstream genes in the terpenoid biosynthetic pathway of German chamomile could be the main reasons to explain the differences in the types and levels of terpenoid compounds in these two species. In addition, a phylogenetic tree constructed from single copy genes showed that German chamomile and Roman chamomile are closely related to Chrysanthemum nankingense. Conclusion This work provides the first insights into terpenoid biosynthesis in two species of chamomile. The candidate unigenes related to terpenoid biosynthesis will be important in molecular breeding approaches to modulate the essential oil composition of Matricaria recutita and Chamaemelum nobile.

Additional file 8: Supplementary Figure S1. COG Functional Classification of German chamomile (A) and Roman chamomile (B). Figure S2. KEGG Functional Classification of German chamomile (A) and Roman chamomile (B). Figure S3. Validation of candidate unigenes in the German chamomile (A) and Roman chamomile (B) transcriptomes by qRT-PCR. Gene expression levels were determined by qRT-PCR. Transcription levels are indicated as the mean (2ΔCt) ± SD. Figure S4. Cluster and correlation analyses between German chamomile and Roman chamomile. Figure S5. Fully open flowers of German chamomile (A) and Roman chamomile (B) dissected to show the disc and ray florets.

Additional file 1. Essential oil compounds present in disk and ray florets of German chamomile and Roman chamomile.

We describe a novel analytical method for quantification of free amino acids in tea using variable mobile phase pH, elution gradient and column temperature of reversed-phase high-performance liquid chromatography (RP-HPLC). The study of mobile phase pH 5.7 was chosen to simultaneous quantification of 19 free amino acids in tea, while it improved maximum resolution of glutamine, histidine and theanine. Elution gradient was adapted for enhancing the solution of free amino acids, mainly because of adjustment of mobile phase A and B. The column temperature of 40 °C was conducive to separate free amino acids in tea. The limit of detection (LOD) and limit of quantitation (LOQ) of this method were in the range of 0.097–0.228 nmol/mL and 0.323–0.761 nmol/mL, respectively. The relative standard deviation of intraday and interday ranged in 0.099–1.909% and 3.231–7.025%, respectively, indicating that the method was reproducible and precise, while recovery ranged between 81.06–112.78%, showing that the method had an acceptable accuracy. This method was applied for the quantification of free amino acids in six types of tea. Multivariate analysis identified serine, glutamine, theanine and leucine as the most influencing factor for classify among analyzed sample.

Background The leaves of tea plants (Camellia sinensis) are used to produce tea, which is one of the most popular beverages consumed worldwide. The nutritional value and health benefits of tea are mainly related to three abundant characteristic metabolites; catechins, theanine and caffeine. Weighted gene co-expression network analysis (WGCNA) is a powerful system for investigating correlations between genes, identifying modules among highly correlated genes, and relating modules to phenotypic traits based on gene expression profiling. Currently, relatively little is known about the regulatory mechanisms and correlations between these three secondary metabolic pathways at the omics level in tea. Results In this study, levels of the three secondary metabolites in ten different tissues of tea plants were determined, 87,319 high-quality unigenes were assembled, and 55,607 differentially expressed genes (DEGs) were identified by pairwise comparison. The resultant co-expression network included 35 co-expression modules, of which 20 modules were significantly associated with the biosynthesis of catechins, theanine and caffeine. Furthermore, we identified several hub genes related to these three metabolic pathways, and analysed their regulatory relationships using RNA-Seq data. The results showed that these hub genes are regulated by genes involved in all three metabolic pathways, and they regulate the biosynthesis of all three metabolites. It is notable that light was identified as an important regulator for the biosynthesis of catechins. Conclusion Our integrated omics-level WGCNA analysis provides novel insights into the potential regulatory mechanisms of catechins, theanine and caffeine metabolism, and the identified hub genes provide an important reference for further research on the molecular biology of tea plants. Electronic supplementary material The online version of this article (10.1186/s12864-018-4999-9) contains supplementary material, which is available to authorized users.

Amino acids are constituents of proteins, precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. However, the amino acid transport in tea plant is rarely reported. In this study, six cationic amino acid transporter (CAT) family genes were cloned. Phylogenetic analysis categorized these CsCATs into four subgroups. These CsCATs all contain the 12–14 transmembrane domains and the conserved CAT motifs. Their expression was tissue-specific, with higher expression levels in root and stem and correlated to the abundances of key free amino acids such as Theanine. Some CsCATs expression responded to some abiotic stress conditions and to the exogenous application of theanine (Thea), glutamine or ethylamine hydrochloride, an ethylamine precursor for Thea biosynthesis. Our results indicated that the CsCATs expression is regulated by amino acid contents and is sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants.

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea (

Background Matricaria recutita (German chamomile) and Chamaemelum nobile (Roman chamomile) belong to the botanical family Asteraceae. These two herbs are not only morphologically distinguishable, but their secondary metabolites – especially the essential oils in flowers are also different, especially the terpenoids. The aim of this project was to preliminarily reveal differential regulatory mechanisms in terpenoids biosynthetic pathway between German and Roman chamomile by performing comparative transcriptomic and metabolomics analyses. Results We determined the content of essential oil in disk florets and ray florets in these two chamomiles, and found that the terpenoids content of German chamomile is greater than that of Roman chamomile. In addition, comparisons between German and Roman chamomile were studied by RNA-Seq, which showed that 54% of genes shared >75% sequence identity between the two species. In particular, more highly expressed DEGs (differentially expressed genes) and TFs (transcription factors), different regulation of CYPs (cytochrome P450 enzymes) and rapid evolution of the downstream enzymes in terpenoids biosynthetic pathways in German chamomile, and maybe the reasons result to differences in the types and levels of terpenoids compounds in these two chamomiles. In addition, the phylogenetic tree of single copay genes showed that German chamomile and Roman chamomile had a high identity to C. nankingense. Conclusion This work provided first insights into the terpenoids biosynthesis of chamomiles, and the candidate unigenes related to terpenoids biosynthesis can serve as important information for molecular breeding approaches to modulate the essential oil composition.

Background Alternative splicing (AS) regulates mRNA at the post-transcriptional level to change gene function in organisms. However, little is known about the AS and its roles in tea plant (Camellia sinensis), widely cultivated for making a popular beverage tea. Results In our study, the AS landscape and dynamics were characterized in eight tissues (bud, young leaf, summer mature leaf, winter old leaf, stem, root, flower, fruit) of tea plant by Illumina RNA-Seq and confirmed by Iso-Seq. The most abundant AS (~ 20%) was intron retention and involved in RNA processes. The some alternative splicings were found to be tissue specific in stem and root etc. Thirteen co-expressed modules of AS transcripts were identified, which revealed a similar pattern between the bud and young leaves as well as a distinct pattern between seasons. AS events of structural genes including anthocyanidin reductase and MYB transcription factors were involved in biosynthesis of flavonoid, especially in vegetative tissues. The AS isoforms rather than the full-length ones were the major transcripts involved in flavonoid synthesis pathway, and is positively correlated with the catechins content conferring the tea taste. We propose that the AS is an important functional mechanism in regulating flavonoid metabolites. Conclusion Our study provides the insight into the AS events underlying tea plant’s uniquely different developmental process and highlights the important contribution and efficacy of alternative splicing regulatory function to biosynthesis of flavonoids. Electronic supplementary material The online version of this article (10.1186/s12870-018-1497-9) contains supplementary material, which is available to authorized users.

German chamomile (Matricaria chamomilla L.) is one of the most ancient medicinal species in the world and terpenoids from their flowers have important medicinal value. We cloned three sesquiterpene synthase genes, McGDS1, McGDS2 and McGDS3, and performed sequence alignment and phylogenetic analysis. The encoded proteins possess three conserved structural features: an RRxxxxxxxxW motif, an RxR motif, and a DDxxD motif. McGDS1, McGDS2 and McGDS3 were confirmed to be (E)-farnesene synthase, germacrene D synthase, and germacrene A synthase, respectively. Subcellular localization revealed diffuse GFP reporter-gene signals in the cytoplasm and nucleus. qPCR indicated that McGDS1, McGDS2 and McGDS3, were more highly expressed in young flowers than in old flowers and the expression was highly correlated with amounts of the end-product essential oils ((E)-β-farnesene, germacrene D and β-elemene), with coefficients of 0.76, 0.83 and 0.68, respectively. We also established a transformation system for chamomile hairy roots. The overexpression of McGDS1, McGDS2 and McGDS3 resulted in γ-muurolene accumulation in hairy roots. The activity of three aphid alarm pheromones here forms the molecular basis for the study of the biosynthesis and regulation of volatile terpenes. Transformation of chamomile hairy roots provides a simple system in which to study terpene biosynthesis in chamomile.

We describe a novel analytical method for quantification of free amino acids in tea using variable mobile phase pH, elution gradient and column temperature of reversed-phase high-performance liquid chromatography (RP-HPLC). The study of mobile phase pH 5.7 was chosen to simultaneous quantification of 19 free amino acids in tea, while it improved maximum resolution of glutamine, histidine and theanine. Elution gradient was adapted for enhancing the solution of free amino acids, mainly because of adjustment of mobile phase A and B. The column temperature of 40 °C was conducive to separate free amino acids in tea. The limit of detection (LOD) and limit of quantitation (LOQ) of this method were in the range of 0.097–0.228 nmol/mL and 0.323–0.761 nmol/mL, respectively. The relative standard deviation of intraday and interday ranged in 0.099–1.909% and 3.231–7.025%, respectively, indicating that the method was reproducible and precise, while recovery ranged between 81.06–112.78%, showing that the method had an acceptable accuracy. This method was applied for the quantification of free amino acids in six types of tea. Multivariate analysis identified serine, glutamine, theanine and leucine as the most influencing factor for classify among analyzed sample. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-018-3366-9) contains supplementary material, which is available to authorized users.

Matricaria recutita L. has been known as an important medicinal plant for centuries. The main active components are sesquiterpenes. Squalene synthase (SQS) is an enzyme that plays an important role in regulation of isoprenoid biosynthesis. In this study, the full-length cDNA of the M. recutita SQS gene was cloned and analyzed for the first time. The clone, designated MrSQS (Matricaria recutita L. squalene synthase gene), consisted of 1580 bp, including a 1230-bp-coding sequence for a predicted protein of 409 amino acids with 46.8 kDa molecular weight. MrSQS shows higher homology to the SQS from Artemisia annua than to SQSs from other plants based on its amino acid sequence. Recombinant MrSQS, produced in a bacterial expression system, showed squalene synthase activity in vitro. In M. recutita, the expression level of MrSQS is determined by real-time quantitative PCR that was highest in disk florets and lowest in stems. These results provide basis and foundation for the study and development of regulation of valuable products from M. recutita L.

Significance A high-quality genome assembly of Camellia sinensis var. sinensis facilitates genomic, transcriptomic, and metabolomic analyses of the quality traits that make tea one of the world’s most-consumed beverages. The specific gene family members critical for biosynthesis of key tea metabolites, monomeric galloylated catechins and theanine, are indicated and found to have evolved specifically for these functions in the tea plant lineage. Two whole-genome duplications, critical to gene family evolution for these two metabolites, are identified and dated, but are shown to account for less amplification than subsequent paralogous duplications. These studies lay the foundation for future research to understand and utilize the genes that determine tea quality and its diversity within tea germplasm., Tea, one of the world’s most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Camellia sinensis (L.) Kuntze and Camellia oleifera C. Abel (Theaceae) are closely related perennial woody shrubs, but the accumulation of metabolites and gene expression patterns are quite different between these two species. In order to understand the mechanisms behind the accumulation and biosynthesis of tea-specific secondary metabolites and the key genes that regulate their target pathways, 1-year-old clone cuttings of C. sinensis and C. oleifera were grafted in both directions, and self-grafted C. sinensis were used as controls. The transcriptomes and metabolomes of leaves and roots from the grafts were analyzed. We found that 1375 unigenes were up-regulated in the leaves of the CS-CO grafts (C. sinensis scion, C. oleifera stock), while 2437 unigenes were down-regulated. OPLS-DA models established for 7230 and 3223 mass spectra peaks were obtained in the positive and negative modes by LC-MS detection. Association analysis of the secondary metabolism pathways was performed, and the relative gene expressions of 14 genes from the transcriptome screening were verified by qRT-PCR. Among the differential metabolites screened and identified, we found that the relative levels of theanine and caffeine decreased significantly, and that many of the genes in these metabolic pathways were also down-regulated. In contrast, the levels of flavonoids apparently increased, and the expression of related genes in the flavonoid biosynthetic pathway were mostly up-regulated.

Clustering dendrogram of samples based on gene expression. (PDF 99 kb)

Figure S1. Hierarchical cluster tree showing co-expression modules identified by WGCNA. Figure S2. The proposed pathway related to flavonoid biosynthesis. Black and red numbers in brackets following each gene indicate the number of expressed full-length and AS genes identified in eight tissues, respectively. Figure S3. Alternative spliced isoforms of theanine-related genes in tea plant. The orange solid dot indicate the targeted AS transcripts identified in specific tissues. Figure S4. Alternatively spliced isoforms of caffeine-related genes in tea plant. Figure S5. An unrooted neighbor joining phylogenetic tree constructed from 12 amino acid sequences of MYBs identified in C. sinensis and other species. The CsMYB genes are highlighted by a solid purple circle. The bold font indicates the CsMYBs which were identified as alternatively spliced genes. Figure S6. Validation of AS isoforms using independent samples by RT-PCR. The AS isoforms were verified by RT-PCR in four tissues (bud, young leaf, root and flower) and each tissue was performed with three biological replicates. Lane 1, 500 bp DNA maker. Lane 2, 6, 10 and 14, full-length transcripts of corresponding genes. Lane 3, 4 and 5, AS isforms in bud. Lane 7, 8 and 9, AS isoforms in young leaf. Lane 11, 12 and 13, AS isoforms in root. Lane 15, 16 and 17, AS isoforms in flower. Table S1. Statistics of different AS events obtained from the Illumina libraries generated in this study. Table S2. Statistic analysis of characterization of identified AS genes in different tissues. Table S3. Statistics of different AS events obtained from the specific modules based on WGCNA analysis. Table S4. Statistical analysis of characterized AS genes identified in different tissues. The solid dot indicated the targeted AS transcripts identified in specific tissues. Table S5. Primers used in this study (application details of primers are described in the materials and method. (PDF 1036 kb)

Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.

Characteristic secondary metabolites, including flavonoids, theanine and caffeine, in the tea plant (

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.

Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of Camellia sinensis. Moreover, we identified 94 full-length transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting full-length transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in Camellia sinensis.

Lilium lancifolium Thunb. is a Chinese traditional plant with various health benefits. In this study, we purified and characterized the water-soluble polysaccharide fraction (LLP-1A) of L. lancifolium. We also investigated the in vitro immune-enhancing activity of LLP-1A in macrophages and the underlying molecular mechanism. Results showed that LLP-1A was mainly composed of mannose and glucose at a molar ratio of 1.77:1, and its molecular weight was approximately 78.61kDa. The Fourier transform-infrared spectra of LLP-1A also revealed typical polysaccharide characteristics: the presence of uronic acid, pyranose rings and β-glycosidic bonds. With regard to its effects on macrophages, LLP-1A enhanced phagocytic activity and induced the NO production in a dose-dependent manner. Further, it induced expression of the cytokines interleukin-6, monocyte chemotactic protein 1, tumor necrosis factor-α and interleukin-1β. With regard to the molecular mechanism, LLP-1A increased protein expression of Toll-like receptor 4 and phosphorylation of the inhibitor of nuclear factor kappa-B kinase, inhibitor of NF-κB, and nuclear factor-kappa B in RAW 264.7 cells. Therefore, the data suggest that LLP-1A significantly upregulated the expression of immune reactive cytokines in RAW 264.7 macrophages through the TLR4-mediated NF-κB signal pathway. Thus, LLP-1A may have immunomodulatory functions that may prove beneficial for the treatment of immune-related diseases.

The genus Camellia (C.) contains many species, including C. sinensis, C. assamica, and C. taliensis, C. gymnogyna and C. tachangensis. The polyphenols of C. sinensis and C. assamica are flavan-3-ols monomers and their dimers and trimmers. However, the biosynthesis of procyanidins in Camellia genus remains unclear. In the present study, a comparative chemical analysis of flavan-3-ols, flavan-3-ols glycoside and procyanidins was conducted by high performance liquid chromatography (HPLC) and liquid chromatography diode array detection coupled with triple-quadrupole mass-spectrometry (LC-DAD-QQQ-MS). The results showed that C. tachangensis had a significant higher contents of (-)-epicatechin (EC) and (-)-epigallocatechin (EGC) compared with C. sinensis (p C. sinensis. LC-DAD-MS/MS indicated that the main secondary metabolites of C. tachangensis were non-galloylated catechins, procyanidin dimers and trimers. Furthermore, (-)-epicatechin glucose (EC-glucose) and (-)-epigallocatechin glucose (EGC-glucose) were also abundant in C. tachangensis. A correlation analysis of EC-glucose and procyanidins dimers was conducted in five Camellia species. The levels of EC-glucose were closely related to the procyanidin dimers content. Thus, it was suggested that EC-glucose might be an important substrate for the biosynthesis of procyanidins.

Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length (FL) splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of C. sinensis. Moreover, we identified 94 FL transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting FL transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in C. sinensis.

Tea is one of the most popular beverages across the world and is made exclusively from cultivars of Camellia sinensis. Many wild relatives of the genus Camellia that are closely related to C. sinensis are native to Southwest China. In this study, we first identified the distinct genetic divergence between C. sinensis and its wild relatives and provided a glimpse into the artificial selection of tea plants at a genome-wide level by analyzing 15,444 genomic SNPs that were identified from 18 cultivated and wild tea accessions using a high-throughput genome-wide restriction site-associated DNA sequencing (RAD-Seq) approach. Six distinct clusters were detected by phylogeny inferrence and principal component and genetic structural analyses, and these clusters corresponded to six Camellia species/varieties. Genetic divergence apparently indicated that C. taliensis var. bangwei is a semi-wild or transient landrace occupying a phylogenetic position between those wild and cultivated tea plants. Cultivated accessions exhibited greater heterozygosity than wild accessions, with the exception of C. taliensis var. bangwei. Thirteen genes with non-synonymous SNPs exhibited strong selective signals that were suggestive of putative artificial selective footprints for tea plants during domestication. The genome-wide SNPs provide a fundamental data resource for assessing genetic relationships, characterizing complex traits, comparing heterozygosity and analyzing putatitve artificial selection in tea plants.

Echinacea purpurea has become a commonly used herbal medicine because it contains various biologically active compounds, such as cichoric acid. Extraction conditions of cichoric acid from the flowers of E. purpurea L. were investigated through an orthogonal design of L9 (34) assay. For purii¬cation, the extract was adsorbed on an AB-8 macroporous resin and eluted with 50% ethanol, then extracted with ethyl acetate four times, resulting in a residue containing 60.23% cichoric acid. Tyrosinase inhibition property of cichoric acid extracts was tested compared with arbutin by Spectrophotometer method. The extraction was optimized by adding 40% ethanol at a ratio of 1:15 (g/mL) to extract three times at 70i‚°C for 1 h. The cichoric acid extracts from E. purpurea was found to have significant tyrosinase inhibition activity in broad range of concentration (≥10 mg/mL). Key words: Echinacea purpurea L., cichoric acid; extraction, orthogonal design, antimicrobial activity.

Primers used for qRT-PCR. (DOCX 18Â kb)

Echinacea purpurea L. is a medicinal plant originally from North America. It has become a commonly used herbal medicine worldwide because it contains various biologically active compounds. This study was designed to investigate the anti-inflammatory effects of essential oils from E. purpurea in both mice and rats. The extract was obtained from flower of E. purpurea by steam distillation. The anti-inflammatory potential was evaluated in vivo by using different animal models such as xylene-induced mouse ear edema, egg-white-induced rat paw edema, and cotton-induced granuloma tissue proliferating inflammation in mice. The serial dosages were used in vivo: the low dosage, the medium dosage and the high dosage. The low, medium and high dosages of extracts produced inhibitions of 39.24%, 47.22% and 44.79% respectively in the ear edema induced by xylene when compare with the control group. Only the high dosage group showed statistically significant inhibition (48.51%) of paw edema formation induced three hours by egg white compared with the control group (P0.01). Moreover, the granulation formation was also significantly reduced the most by 28.52% in the high dose groups compared with the control group (P0.05). The pro-inflammatory cytokines such as IL-2, IL-6 and TNF-α in the blood were reduced in the treated groups. The essential oils from extracts of E. purpurea have anti-inflammatory effects.