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.

Two-phase dual-signal-readout immunosensing platform based on multifunctional carbon nano-onions for ovarian cancer biomarker detection.

In order to detect early tumor markers and gain valuable time for treatment, there is an urgent need to develop a fast, cheap, and ultrasensitive multi-reading sensing platform. Herein, a solid/liquid two-phase dual-output biosensor was explored based on a sensitized sonochemiluminescence (SCL) strategy and a multifunctional carbon nano-onion (CNO) probe. It is clear that ultrasonic radiation caused the formation of hydroxyl radicals (˙OH), triggering the SCL signal of the emitter lucigenin (Luc2+). Meanwhile, titanium carbide nanodots and ethanol were used to enhance the SCL signal, and an astonishingly linear enhancement of the SCL intensity was produced with increasing ethanol concentration. More importantly, the CNOs, with their excellent photothermal properties and adsorption capacity, can output both the temperature signal and an enhanced SCL strength from the solid-liquid phase. Through inter-calibration of the signals from the two-phases, this biosensor shows excellent analytical performance for the detection of the ovarian cancer biomarker, human epididymis-specific protein 4, from 10-5 to 10 ng mL-1 with a low detection limit of 3.3 fg mL-1. This work not only provides a novel two-phase signal-output mode that broadens the scope of multiperformance joint applications of CNOs, but also enriches the quantitative detection of point-of-care testing.

Modular and Noncontact Wireless Detection Platform for Ovarian Cancer Markers: Electrochemiluminescent and Photoacoustic Dual-Signal Output Based on Multiresponse Carbon Nano-Onions.

An electrochemiluminescent (ECL)-photoacoustic (PA) dual-signal output biosensor based on the modular optimization and wireless nature of a bipolar electrode (BPE) was constructed. To further simplify the detection process, the BPE structure was designed as three separate units: anode ECL collection, cathode catalytic amplification, and intermediate functional sensing units. Specifically, the anode unit was placed with Eosin Yellow, a cheap and effective ECL reagent, and the cathode unit was a laser-induced polyoxometalate-graphene electrode, which was helpful to enhance the anode ECL signal. The intermediate functional sensing unit consisted of a temperature-sensitive conductive film. Further, using a carbon nano-onion nanocomposite with excellent absorption performance in the near-infrared region as a signal tag not only leads to changes in the electrical conductivity of the film through heat transfer and thus affects the ECL signal but also produces a strong PA response. With this design, PA and ECL signals can be output simultaneously. This work not only realizes multiple modularization processes in the design of sensors but also implements the diversification of signal output modes, which will enrich the joint research field of ECL detection technology and other new detection methods.

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.

Theanine transporters are involved in nitrogen deficiency response in tea plant (Camellia sinensis L.).

Nitrogen in soil directly influences the production and quality of tea. However, high nitrogen application in tea plantation leads to soil acidification and environmental pollution. Studies in model plants showed that plasma membrane localized amino acid transporter can regulate the distribution of amino acids to enhance nitrogen use efficiency. Our recent study identified six CsAAPs as transporters for theanine, a unique and most abundant non-proteinaceous amino acid in tea plant. In this work, we found these theanine transporters can also transport Glutamine, Glutamate, aspartate, alanine and γ-aminobutyric acid. Tissue-specific expression analyses showed that CsAAP1, CsAAP5 and CsAAP6 mainly expressed in leaves, CsAAP8 in root, CsAAP4 and CsAAP2 in stem. Furthermore, the expression of these CsAAPs was induced by nitrogen deficiency in a tissue-specific manner. Subcellular localization analyses showed that CsAAP1, CsAAP2 and CsAAP6 location were in the plasma membrane and endoplasmic reticulum. Taken together, these results suggested theanine transporters are involved in nitrogen deficiency response probably by mediating amino acid transport from roots to new shoots and from source to sink tissues in tea plants.

Theanine transporters identified in tea plants (Camellia sinensis L.).

Theanine, a unique non-proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H+ -dependent. The tissue-specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root-to-bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root-to-shoot delivery in the tea plant.