Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

Theanine metabolism and transport in tea plants (Camellia sinensis L.): advances and perspectives.

Theanine, a tea plant-specific non-proteinogenic amino acid, is the most abundant free amino acid in tea leaves. It is also one of the most important quality components of tea because it endows the "umami" taste, relaxation-promoting, and many other health benefits of tea infusion. Its content in tea leaves is directly correlated with the quality and price of green tea. Theanine biosynthesis primarily occurs in roots and is transported to new shoots in tea plants. Recently, great advances have been made in theanine metabolism and transport in tea plants. Along with the deciphering of the genomic sequences of tea plants, new genes in theanine metabolic pathway were discovered and functionally characterized. Theanine transporters were identified and were characterized on the affinity for: theanine, substrate specificity, spatiotemporal expression, and the role in theanine root-to-shoot transport. The mechanisms underlying the regulation of theanine accumulation by: cultivars, seasons, nutrients, and environmental factors are also being rapidly uncovered. Transcription factors were identified to be critical regulators of theanine biosynthesis. In this review, we summarize the progresses in theanine: biosynthesis, catabolism, and transport processes. We also discuss the future studies on theanine in tea plants, and application of the knowledge to crops to synthesize theanine to improve the health-promoting quality of non-tea crops.

Nitrogen-Regulated Theanine and Flavonoid Biosynthesis in Tea Plant Roots: Protein-Level Regulation Revealed by Multiomics Analyses.

Theanine and flavonoids (especially proanthocyanidins) are the most important and abundant secondary metabolites synthesized in the roots of tea plants. Nitrogen promotes theanine and represses flavonoid biosynthesis in tea plant roots, but the underlying mechanism is still elusive. Here, we analyzed theanine and flavonoid metabolism in tea plant roots under nitrogen deficiency and explored the regulatory mechanism using proteome and ubiquitylome profiling together with transcriptome data. Differentially expressed proteins responsive to nitrogen deficiency were identified and found to be enriched in flavonoid, nitrogen, and amino acid metabolism pathways. The proteins responding to nitrogen deficiency at the transcriptional level, translational level, and both transcriptional and translational levels were classified. Nitrogen-deficiency-responsive and ubiquitinated proteins were further identified. Our results showed that most genes encoding enzymes in the theanine synthesis pathway, such as CsAlaDC, CsGDH, and CsGOGATs, were repressed by nitrogen deficiency at transcriptional and/or protein level(s). While a large number of enzymes in flavonoid metabolism were upregulated at the transcriptional and/or translational level(s). Importantly, the ubiquitylomic analysis identified important proteins, especially the hub enzymes in theanine and flavonoid biosynthesis, such as CsAlaDC, CsTSI, CsGS, CsPAL, and CsCHS, modified by ubiquitination. This study provided novel insights into the regulation of theanine and flavonoid biosynthesis and will contribute to future studies on the post-translational regulation of secondary metabolism in tea plants.

Shading Promoted Theanine Biosynthesis in the Roots and Allocation in the Shoots of the Tea Plant (Camellia sinensis L.) Cultivar Shuchazao.

Shading was thought as an effective approach to increase theanine in harvested tea shoots. Previous studies offered conflicting findings, perhaps since the integration of theanine metabolism and transport in different tissues was not considered. Theanine is synthesized primarily in the roots and is then transported, via the vascular system, to new vegetative tissues. Here, we found that theanine increased in the stem, was reduced in the leaf, and remained stable in the roots, under shading conditions. Notably, in tea roots, shading significantly increased ethylamine and activated the theanine biosynthesis pathway and theanine transporter genes. Furthermore, shading significantly increased the expression of theanine transporter genes, CsAAP2/4/5/8, in the stem, while decreasing the expression of CsAAP1/2/4/5/6 in the leaf, in accordance with shading effects on theanine levels in these tissues. These findings reveal that shading of tea plants promotes theanine biosynthesis and allocation in different tissues, processes which appear to involve the theanine biosynthesis pathway enzymes and AAP family of theanine transporters.

Effects of shading on lignin biosynthesis in the leaf of tea plant (Camellia sinensis (L.) O. Kuntze).

Shading can effectively reduce photoinhibition and improve the quality of tea. Lignin is one of the most important secondary metabolites that play vital functions in plant growth and development. However, little is known about the relationship between shading and xylogenesis in tea plant. To investigate the effects of shading on lignin accumulation in tea plants, 'Longjing 43' was treated with no shading (S0), 40% (S1) and 80% (S2) shading treatments, respectively. The leaf area and lignin content of tea plant leaves decreased under shading treatments (especially S2). The anatomical characteristics showed that lignin is mainly distributed in the xylem of tea leaves. Promoter analysis indicated that the genes involved in lignin pathway contain several light recognition elements. The transcript abundances of 12 lignin-associated genes were altered under shading treatments. Correlation analysis indicated that most genes showed strong positive correlation with lignin content, and CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation under 40% and 80% shading treatments. The results showed that shading may have an important effect on lignin accumulation in tea leaves. This work will potentially helpful to understand the regulation mechanism of lignin pathway under shading treatment, and provide reference for reducing lignin content and improving tea quality through shading treatment in field operation.

Identification and expression analysis of caffeoyl-coenzyme A O-methyltransferase family genes related to lignin biosynthesis in tea plant (Camellia sinensis).

Tea plant, an economically important crop, is used in producing tea, which is a non-alcoholic beverage. Lignin, the second most abundant component of the cell wall, reduces the tenderness of tea leaves and affects tea quality. Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) involved in lignin biosynthesis affects the efficiency of lignin synthesis and lignin composition. A total of 10 CsCCoAOMTs were identified based on tea plant genome. Systematic analysis of CCoAOMTs was conducted for its physicochemical properties, phylogenetic relationships, conserved motifs, gene structure, and promoter cis-element prediction. Phylogenetic analysis suggested that all the CsCCoAOMT proteins can be categorized into three clades. The promoters of six CsCCoAOMT genes possessed lignin-specific cis-elements, indicating they are possibly essential for lignin biosynthesis. According to the distinct tempo-spatial expression profiles, five genes were substantially expressed in eight tested tissues. Most CsCCoAOMT genes were expressed in stems and leaves in three tea plant cultivars 'Longjing 43,' 'Anjibaicha,' and 'Fudingdabai' by RT-qPCR detection and analysis. The expression levels of two genes (CsCCoAOMT5 and CsCCoAOMT6) were higher than those of the other genes. The expression levels of most CsCCoAOMT genes in 'Longjing 43' were significantly higher than that those in 'Anjibaicha' and 'Fudingdabai.' Correlation analysis revealed that only the expression levels of CsCCoAOMT6 were positively correlated with lignin content in the leaves and stems. These results lay a foundation for the future exploration of the roles of CsCCoAOMTs in lignin biosynthesis in tea plant.

Cytosolic ascorbate peroxidase 1 modulates ascorbic acid metabolism through cooperating with nitrogen regulatory protein P-II in tea plant under nitrogen deficiency stress.

Nitrogen (N) element is essential nutrient, and affect metabolism of secondary metabolites in higher plants. Ascorbate peroxidase (APX) plays an important role in ascorbic acid (AsA) metabolism of tea plant. However, the roles of cytosolic ascorbate peroxidase 1 (CsAPX1) in AsA metabolism under N deficiency stress in tea plant remains unclear in detail. In this work, nitrogen regulatory protein P-II (CsGLB1) and CsAPX1 were identified by isobaric tags for relative and absolute quantitation (iTRAQ) from tea plant. The cell growth rates in transgenic Escherichia coli overexpressing CsAPX1 and CsGLB1 were higher than empty vector under N sufficiency condition. Phenotype of shoots and roots, AsA accumulation, and expression levels of AtAPX1 and AtGLB1 genes were changed in transgenic Arabidopsis hosting CsAPX1 under N deficiency stress. These findings suggested that cytosolic CsAPX1 acted a regulator in AsA accumulation through cooperating with GLB1 under N deficiency stress in tea plant.

Identification and Analysis of Genes Involved in Auxin, Abscisic Acid, Gibberellin, and Brassinosteroid Metabolisms Under Drought Stress in Tender Shoots of Tea Plants.

Endogenous phytohormones auxin (indole-3-acetic acid [IAA]), abscisic acid (ABA), gibberellin (GA3), and brassinosteroid (BR) play a role in responses to drought stress in higher plants. Tea plant is one of the major economic corps worldwide. The tender shoots of tea plants are the main source for tea production. The effects of drought stress on endogenous IAA, ABA, GA3, and BR metabolisms in tender shoots of tea plants need to be illustrated. In this study, a total of 17 IAA-related genes, 17 ABA-related genes, 18 GA3-related genes, and 8 BR-related genes were identified under drought stress in tender shoots of tea plants, respectively. By using a combination of phytohormone determination, phylogenetic tree construction and sequence analysis, gene expression profiles, functional classification, Kyoto encyclopedia of genes and genomes enrichment, and distribution of genes analysis, we have demonstrated that IAA, ABA, GA3, and BR metabolisms might participate in the regulation of the response to drought stress in tender shoots of tea plants. The expression level of CsLYCE negatively correlated with ABA accumulation under drought stress. Our findings could shed new light on the effects of drought stress on the IAA, ABA, GA3, and BR metabolisms in tender shoots of tea plants.

[Effects of Marsdenia tenacissima extract on proliferation and apoptosis of hematologic neoplasm cell line cells].

OBJECTIVE: To investigate the effects of the extract from Marsdensia tenacissima on proliferation and apoptosis of human hematologic neoplasm cell line cells. METHODS: Raji, NB4 and K562 cells were treated in vitro with different concentrations of the extract from Marsdensia tenacissima, including different ethanol elution components and C21 steroidal saponin monomer compounds, for different periods. Tumor cell proliferation was measured by MTT colorimetric assay and its apoptosis was determined by the flow cytometry (FCM). RESULTS: Firstly, with higher concentrations, 100 microg/mL and 200 microg/mL, 70% ethanol eluate from Marsdensia tenacissima inhibited the proliferation of Raji, NB4 and K562 cells significantly, in a dose and time dependent manner, compared with 30% and 50% ethanol elution components from Marsdensia tenacissima (P < 0.05). Secondly, four C21 steroidal saponin monomer compounds, tenacissosides B,C,I and marsdenoside K, also inhibited the proliferation of Raji, NB4 and K562 cells in vitro significantly, in a dose and time dependent manner, compared with that of control group (P < 0.05). Among them, tenacissoside C showed the strongest inhibition effects on proliferation of these cells under all experimental conditions compared with the other three C21 steroidal saponin monomer compounds (P < 0.05). Furthermor, the IC50 of tenacissosides C on proliferation of Raji, NB4 and K562 cells were 64.1 micromol/L, 70.4 micromol/L and 105.8 micromol/L, respectively. Finally, after Raji, NB4 and K562 cells were treated with 98.4 micromol/L tenacissoside C for 24 h and 48 h, the early apoptosis rates and late apoptosis rates of these tumor cells increased markedly, compared with the control group (P < 0.05). CONCLUSION: The extract from Marsdensia tenacissima, including different ethanol elution components and C21 steroidal saponin monomer compounds, may inhibit the proliferation of some human hematologic neoplasm cell line cells and induce these tumor cells apoptosis in vitro, especially tenacissoside C, one of the C21 steroidal saponin monomer compounds, showed the strongest effects on proliferation of these tumor cells when compared with other ones, with the strongest inhibition activities on human Burkitt's lymphoma cell line Raji cells.