Comprehensive Dissection of Metabolic Changes in Albino and Green Tea Cultivars.

Albino tea cultivars are special mutants of tea plants with white or yellow leaf color. In this study, three albino tea cultivars, including 'Anji Baicha', 'Huangjinya', and 'Baijiguan', and two green tea cultivars, 'Longjing 43' and 'Fuding Dabaicha', were applied to metabolite profiling by gas chromatography-mass spectrometry and ultraperformance liquid chromatography-mass spectrometry. Multivariate analyses revealed significantly different metabolite phenotypes in leaves among albino cultivars and green cultivars. The differential metabolite-related pathways included galactose metabolism, tryptophan metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. For the young leaves of albino cultivars, the sugar (sorbitol and erythrose) and amino acid (mainly proline, isoleucine, ornithine, aspartic acid, threonine, and valine) concentrations increased, whereas gallocatechin and epigallocatechin gallate concentrations decreased. These results reveal the divergence in metabolic profiling between tea plant cultivars with different leaf colors. With the development of leaves, the concentrations of flavonoids increased largely in the older leaves of albino cultivars.

Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in 'Anji Baicha' (Camellia sinensis).

BACKGROUND: The new shoots of the albino tea cultivar 'Anji Baicha' are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. 'Anji Baicha' metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized. RESULTS: We used RNA-sequencing to analyze 'Anji Baicha' leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher ß-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of 'Anji Baicha' tea plants. CONCLUSIONS: Our study presents the results of transcriptomic and biochemical analyses of 'Anji Baicha' tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Conservation and sustainable use of medicinal plants: problems, progress, and prospects.

Medicinal plants are globally valuable sources of herbal products, and they are disappearing at a high speed. This article reviews global trends, developments and prospects for the strategies and methodologies concerning the conservation and sustainable use of medicinal plant resources to provide a reliable reference for the conservation and sustainable use of medicinal plants. We emphasized that both conservation strategies (e.g. in situ and ex situ conservation and cultivation practices) and resource management (e.g. good agricultural practices and sustainable use solutions) should be adequately taken into account for the sustainable use of medicinal plant resources. We recommend that biotechnical approaches (e.g. tissue culture, micropropagation, synthetic seed technology, and molecular marker-based approaches) should be applied to improve yield and modify the potency of medicinal plants.

Transcriptomic Analysis of Tea Plant Responding to Drought Stress and Recovery.

Tea plant (Camellia sinensis) is an economically important beverage crop. Drought stress (DS) seriously limits the growth and development of tea plant, thus affecting crop yield and quality. To elucidate the molecular mechanisms of tea plant responding to DS, we performed transcriptomic analysis of tea plant during the three stages [control (CK) and during DS, and recovery (RC) after DS] using RNA sequencing (RNA-Seq). Totally 378.08 million high-quality trimmed reads were obtained and assembled into 59,674 unigenes, which were extensively annotated. There were 5,955 differentially expressed genes (DEGs) among the three stages. Among them, 3,948 and 1,673 DEGs were up-regulated under DS and RC, respectively. RNA-Seq data were further confirmed by qRT-PCR analysis. Genes involved in abscisic acid (ABA), ethylene, and jasmonic acid biosynthesis and signaling were generally up-regulated under DS and down-regulated during RC. Tea plant potentially used an exchange pathway for biosynthesis of indole-3-acetic acid (IAA) and salicylic acid under DS. IAA signaling was possibly decreased under DS but increased after RC. Genes encoding enzymes involved in cytokinin synthesis were up-regulated under DS, but down-regulated during RC. It seemed probable that cytokinin signaling was slightly enhanced under DS. In total, 762 and 950 protein kinases belonging to 26 families were differentially expressed during DS and RC, respectively. Overall, 547 and 604 transcription factor (TF) genes belonging to 58 families were induced in the DS vs. CK and RC vs. DS libraries, respectively. Most members of the 12 TF families were up-regulated under DS. Under DS, genes related to starch synthesis were down-regulated, while those related to starch decomposition were up-regulated. Mannitol, trehalose and sucrose synthesis-related genes were up-regulated under DS. Proline was probably mainly biosynthesized from glutamate under DS and RC. The mechanism by which ABA regulated stomatal movement under DS and RC was partly clarified. These results document the global and novel responses of tea plant during DS and RC. These data will serve as a valuable resource for drought-tolerance research and will be useful for breeding drought-resistant tea cultivars.

Identification and expression profiling of the auxin response factors (ARFs) in the tea plant (Camellia sinensis (L.) O. Kuntze) under various abiotic stresses.

Auxin response factor (ARF) proteins are a multigene family of regulators involved in various physiological and developmental processes in plants. However, their modes of action in the tea plant (Camellia sinensis) remain largely unknown. In this study, we identified 15 members of the tea ARF gene family, using the public information about C. sinensis, both in our laboratory, as well as in other laboratories, and analyzed their phylogenetic relationships, conserved domains and the compositions of the amino acids in the middle region. A comprehensive expression analysis in different tissues and organs revealed that many ARF genes were expressed in a tissue-specific manner, suggesting they have different functions in the growth and development processes of the tea plant. The expression analysis under three forms of auxin (indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, naphthylacetic acid) treatment showed that the majority of the ARF genes were down-regulated in the shoots and up-regulated in the roots, suggesting opposite action mechanisms of the ARF genes in the shoots and roots. The expression levels of most ARF genes were changed under various phytohormone and abiotic stresses, indicating the ARF gene family plays important roles in various phytohormone and abiotic stress signals and may mediate the crosstalk between phytohormones and abiotic stresses. The current study provides basic information for the ARF genes of the tea plant and will pave the way for deciphering the precise role of ARFs in tea developmental processes and breeding stress-tolerant tea varieties.

Differential Metabolic Profiles during the Albescent Stages of 'Anji Baicha' (Camellia sinensis).

'Anji Baicha' is an albino tea cultivar with white shoots at low air temperature and green shoots at high air temperature in early spring. The metabolite contents in the shoots dynamically vary with the color changes and with shoot development. To investigate the metabolomic variation during the albescent and re-greening stages, gas chromatography-mass spectrometry combined with multivariate analysis were applied to analyze the metabolite profiles in the different color stages during the development of 'Anji Baicha' leaves. The metabolite profiles of three albescent stages, including the yellow-green stage, the early albescent stage, and the late albescent stage, as well as the re-greening stage were distinguished using principal component analysis, revealing that the distinct developmental stages were likely responsible for the observed metabolic differences. Furthermore, a group classification and pairwise discrimination was revealed among the three albescent stages and re-greening stage by partial least squares discriminant analysis. A total of 65 differential metabolites were identified with a variable influence on projection greater than 1. The main differential metabolic pathways of the albescent stages compared with the re-greening stage included carbon fixation in photosynthetic organisms and the phenylpropanoid and flavonoid biosynthesis pathways. Compared with the re-greening stage, the carbohydrate and amino acid metabolic pathways were disturbed during the albescent stages. During the albescent stages, the sugar (fructofuranose), sugar derivative (glucose-1-phosphate) and epicatechin concentrations decreased, whereas the amino acid (mainly glycine, serine, tryptophan, citrulline, glutamine, proline, and valine) concentrations increased. These results reveal the changes in metabolic profiling that occur during the color changes associated with the development of the albino tea plant leaves.

Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis).

BACKGROUND: Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways. RESULTS: RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis. CONCLUSIONS: Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.

Large-Scale SNP Discovery and Genotyping for Constructing a High-Density Genetic Map of Tea Plant Using Specific-Locus Amplified Fragment Sequencing (SLAF-seq).

Genetic maps are important tools in plant genomics and breeding. The present study reports the large-scale discovery of single nucleotide polymorphisms (SNPs) for genetic map construction in tea plant. We developed a total of 6,042 valid SNP markers using specific-locus amplified fragment sequencing (SLAF-seq), and subsequently mapped them into the previous framework map. The final map contained 6,448 molecular markers, distributing on fifteen linkage groups corresponding to the number of tea plant chromosomes. The total map length was 3,965 cM, with an average inter-locus distance of 1.0 cM. This map is the first SNP-based reference map of tea plant, as well as the most saturated one developed to date. The SNP markers and map resources generated in this study provide a wealth of genetic information that can serve as a foundation for downstream genetic analyses, such as the fine mapping of quantitative trait loci (QTL), map-based cloning, marker-assisted selection, and anchoring of scaffolds to facilitate the process of whole genome sequencing projects for tea plant.

[Salvia miltiorrhiza as medicinal model plant].

Research on medicinal model organism is one of the core technologies to promote the modernization of traditional Chinese medicine (TCM). The research progress of Salvia miltiorrhiza as medicinal model plant is summarized in this paper. The genome of S. miltiorrhiza is small and its life cycle is short, as well as this plant can be stably genetically transformed. Because S. miltiorrhiza possesses the important medicinal and economic values, recently the transcriptome and genome of S. miltiorrhiza have been significantly recovered. The research prospect of S. miltiorrhiza as medicinal model plant in TCM was discussed, including biosynthesis of active components and their genetic regulation, relationship between quality of TCM and ecological environments, and selective breeding of good quality lines. Furthermore, as medicinal model plant, the construction of mutant library for S. miltiorrhiza, the genome map with high quality, and the functional genome should be investigated. Accompanying modern investigation of life sciences, the platform for medicinal model plant, S. miltiorrhiza, will be promoted to be established. It is important to develop the ethnopharmacology and new drugs around the world.

[Cloning and expression analysis of a key device of HMGR gene involved in ginsenoside biosynthesis of Panax ginseng via synthetic biology approach].

3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), the first enzyme of mavalonic acid pathway, is one of the key devices involved in ginsenoside biosynthesis based on synthetic biology approach. The open reading frame of a novel HMGR gene from Panax ginseng (PgHMGR2) was cloned and analyzed in this study. PgHMGR2-encoding protein showed 71.6% sequence similarity to a P. ginseng HMGR in GenBank. The full-length cDNA sequence of PgHMGR2 containing 1 770 bp, which encodes 589 amino acids, was cloned by RT-PCR strategy from P. ginseng. The bioinformatic analysis showed that PgHMGR2-encoding protein contained two transmembrane regions and the HMG_CoA_reductase domain, without signal peptide. The protein sequence of PgHMGR2 had the highest sequence similarity (99%) with Panax quinquefolius HMGR (GenBank accession No. ACV65036). The expression level of PgHMGR2 was the highest in flower based on a real-time PCR analysis, followed by leaf and root, and the lowest was in stem. The result will provide a foundation for exploring the molecular function of PgHMGR2 involved in ginsenoside biosynthesis based on synthetic biology approach in P. ginseng plants.

[Genomics and synthetic biology of traditional Chinese medicine].

Traditional Chinese medicine (TCM) genomics and TCM synthetic biology are two hot fields in the TCM modernization. TCM genomics, including transcriptomics, structural genomics, genomic markers and functional genomics, aims to elucidate the biosynthetic pathways of TCM bioactive compounds and mine the related genes encoding enzymes involved in these pathways by analyzing genetic information on the original species of TCM, thus promoting the development of TCM synthetic biology, genome-assisted molecular identification and molecular breeding, and elucidation of the genetic mechanism underlying "Daodi". Genomics and related research provide us much deeper understanding of life process and improve our ability to create new life or modify the present organisms. Based on TCM genomics, TCM synthetic biology sets up a series of procedures to realize the production of TCM pharmacological active compounds in microorganism, including screening and modification of parts and devices, establishment of standardized part and device libraries, and reconstruction and modification of the biosynthetic pathway of TCM pharmacological active compounds in microorganism. TCM synthetic biology will provide a new resource of TCM pharmacological active compounds for the pharmacological study and research & development of new drugs, thus enhancing the core competitiveness of our pharmaceutical industry in the international markets.

[Investigation on treatment of fetal growth restriction by salvia injection combined with composite amino acid].

OBJECTIVE: To explore the effect of salvia injection (SI) combined with composite amino acid (CAA) in treating fetal growth restriction (FGR). METHODS: A retrospective analysis was conducted on 106 pregnant women with FGR hospitalized from January 2007 to January 2008. Patients were randomized into 2 groups equally, the treated group (53 cases) treated with SI plus CAA, and the control group treated with CAA alone, all for 7 days. The clinical effect and umbilical blood flow (S/D) in patients were observed. RESULTS: (1) The total effective rate in the treated group was 81.13%, it was 88.80% (16/18) for patients in the gestation period of 24+ -28 weeks, 80.00% (12/15) for those of 28+ -32 weeks, and 75.00 (15/20) for 32+ -36 weeks, while in the control group, the corresponding rates were 69.81%, 77.77% (14/18), 66.66% (10/15), and 65.00% (13/20), respectively. The difference between the two groups was significant (P < 0.05). (2) After treatment, S/D significantly lowered in patients of the treated group (P < 0.05, P < 0.01), no matter how long the gestation period was, but it was insignificantly changed in the control group. CONCLUSION: The combined treatment with SI and CAA on FGR could improve the condition of the fetus.

Formic acid: a rare but deadly source of carbon monoxide poisoning.

INTRODUCTION: Formic acid decomposes upon contact with strong acids producing carbon monoxide. Carbon monoxide poisoning from such a source, however, is extremely rare. CASE REPORT: A 26-year-old man committed suicide by mixing 2.5 L of formic acid and 2.5 L of sulfuric acid in three beakers and staying in a closed room. The 53-year-old father performed cardiopulmonary resuscitation on his son but soon lost consciousness. In hospital, he initially manifested coma, hypoxemia, metabolic acidosis, and a carboxyhemoglobin level of 45.8%. He was treated with hyperbaric oxygen but developed acute respiratory distress syndrome on day four despite an early improvement. He was successfully weaned from the ventilator on day 8. The 53-year-old mother felt dizziness, headache and had a carboxyhemoglobin level of 23.0%. Her symptoms improved after oxygen therapy. DISCUSSION AND CONCLUSIONS: Formic acid is a highly fatal source of carbon monoxide poisoning when mixed with sulfuric acid. In addition to the toxicities of carbon monoxide, concomitant inhalation of formic acid fumes can cause severe lung injury, which may complicate the management of carbon monoxide poisoning.