Time-series transcriptomic analysis reveals novel gene modules that control theanine biosynthesis in tea plant (Camellia sinensis).

Theanine (thea) is a unique non-protein amino acid in tea plant (Camellia sinensis) and one of the most important small molecular compounds for tea quality and health effects. The molecular mechanism that maintains thea biosynthesis is not clear but may be reflected in complicated biological networks as other secondary metabolites in plants. We performed an integrative transcriptomic analysis of tea seedlings bud and leave over the time-course of ethylamine (EA) treatment that activated thea pathway. We identified 54 consistent differentially expressed genes (cDEGs, 25 upregulated and 29 downregulated) during thea activation. Gene Ontology (GO) functional enrichment analysis of upregulated genes and downregulated genes showed that they may function as a cascade of biological events during their cooperative contribution to thea biosynthesis. Among the total cDEGs, a diversity of functional genes (e.g., enzymes, transcription factors, transport and binding proteins) were identified, indicating a hierarchy of gene control network underlying thea biosynthesis. A gene network associated with thea biosynthesis was modeled and three interconnected gene functional modules were identified. Among the gene modules, several topologically important genes (e.g., CsBCS-1, CsRP, CsABC2) were experimentally validated using a combined thea content and gene expression analysis. Collectively, we presented here for the first time a comprehensive landscape of the biosynthetic mechanism of thea controlled by a underling gene network, which might provide a theoretical basis for the identification of key genes that contribute to thea biosynthesis.

The complete chloroplast genome of Akebia trifoliata (Lardizabalaceae), a traditional herb in China.

Akebia trifoliata, commonly known as 'Bayuezha' in China, has been widely used as traditional Chinese medicinal herbs with a long history. In the present study, the complete chloroplast genome of A. trifoliata was sequenced using Illumina high-throughput sequencing approach. The length of the complete chloroplast genome is 157,952 bp with 38.7% GC content. It contains 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis indicated that A. trifoliata was closely related to another Lardizabalaceae species, Akebia quinata, which further confirms traditional species classification.

The tea plant reference genome and improved gene annotation using long-read and paired-end sequencing data.

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.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality.

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.

Transcriptomic and phytochemical analysis of the biosynthesis of characteristic constituents in tea (Camellia sinensis) compared with oil tea (Camellia oleifera).

BACKGROUND: Tea plants (Camellia sinensis) are used to produce one of the most important beverages worldwide. The nutritional value and healthful properties of tea are closely related to the large amounts of three major characteristic constituents including polyphenols (mainly catechins), theanine and caffeine. Although oil tea (Camellia oleifera) belongs to the genus Camellia, this plant lacks these three characteristic constituents. Comparative analysis of tea and oil tea via RNA-Seq would help uncover the genetic components underlying the biosynthesis of characteristic metabolites in tea. RESULTS: We found that 3,787 and 3,359 bud genes, as well as 4,042 and 3,302 leaf genes, were up-regulated in tea and oil tea, respectively. High-performance liquid chromatography (HPLC) analysis revealed high levels of all types of catechins, theanine and caffeine in tea compared to those in oil tea. Activation of the genes involved in the biosynthesis of these characteristic compounds was detected by RNA-Seq analysis. In particular, genes encoding enzymes involved in flavonoid, theanine and caffeine pathways exhibited considerably different expression levels in tea compared to oil tea, which were also confirmed by quantitative RT-PCR (qRT-PCR). CONCLUSION: We assembled 81,826 and 78,863 unigenes for tea and oil tea, respectively, based on their differences at the transcriptomic level. A potential connection was observed between gene expression and content variation for catechins, theanine and caffeine in tea and oil tea. The results demonstrated that the metabolism was activated during the accumulation of characteristic metabolites in tea, which were present at low levels in oil tea. From the molecular biological perspective, our comparison of the transcriptomes and related metabolites revealed differential regulatory mechanisms underlying secondary metabolic pathways in tea versus oil tea.

[Effect of several physiochemical factors on cell growth and isoflavone accumulation of Pueraria lobata cell suspension culture].

OBJECTIVE: To illustrate the effects of several physiochemical factors on cell growth and isoflavone accumulation of Pueraria lobata cell suspension cultures. METHOD: High performance liquid chromatography and plant tissue culture were applied. RESULT AND CONCLUSION: Cell growth and isoflavone accumulation were significantly stimulated in P. lobata cell suspension cultures by the increase of the sucrose concentration. Maintaining the pH value at the range over 5. 4 to 5. 8 was most suitable for isoflavone accumulation in P. lobata cell suspension cultures. Cell dried weight and isoflavone accumulation decreased sharply with the increase of the treated concentration of active carbon, while XAD-4 significantly stimulated cell growth and isoflavone accumulation.

Separation and determination of isoflavonoids in several kudzu samples by high-performance capillary electrophoresis (HPCE).

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from root, stem, leaf, callus and cell samples, is very important for the best, safest and most efficacious use of kudzu as a medicinal plant, and for the studies on quantitative analysis in the secondary metabolism of isoflavonoids. In this paper, a simple, rapid and precise high-performance capillary electrophoresis (HPCE) method with diode array detection (DAD) has been developed for separation and determination of isoflavonoids in several kudzu samples. The isoflavonoids could be well separated within 15 min in a 40 cm length capillary at a separation voltage of 15kV in a 30 mmol L(-1) borax buffer (pH9.29), and this proposed method demonstrated excellent reproducibility and accuracy with relative standard deviations of less than 5% for isoflavonoid content (n = 5) of different kudzu samples. The relationship between peak areas and isoflavone concentrations, in a specified working range with linear response, was determined by first-order polynomial regression over the range 0.05-0.5 mg mL(-1) for puerarin and 2.5-50 microg mL(-1) for 3'-methoxypuerarin, daidzin and daidzein, respectively, and quantitative evaluation of those four main isoflavonoid components was determined by ultraviolet absorption at lambda = 192 nm. The differences were also illustrated by comparison of the determination of isoflavonoid components from kudzu root, stem, leaf samples and plant tissue cultures in vitro.

Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.