Integration of Metabolomics and Transcriptomics Reveal the Mechanism Underlying Accumulation of Flavonols in Albino Tea Leaves.

Albino tea plants (Camellia sinensis) have been reported to possess highly inhibited metabolism of flavonoids compared to regular green tea leaves, which improves the quality of the tea made from these leaves. However, the mechanisms underlying the metabolism of catechins and flavonols in albino tea leaves have not been well elucidated. In this study, we analyzed a time series of leaf samples in the greening process from albino to green in a thermosensitive leaf-color tea mutant using metabolomics and transcriptomics. The total content of polyphenols dramatically decreased, while flavonols (such as rutin) were highly accumulated in albino leaves compared to in green leaves. After treatment with increasing environment temperature, total polyphenols and catechins were increased in albino mutant tea leaves; however, flavonols (especially ortho-dihydroxylated B-rings such as rutin) were decreased. Meanwhile, weighted gene co-expression network analysis of RNA-seq data suggested that the accumulation of flavonols was highly correlated with genes related to reactive oxygen species scavenging. Histochemical localization further demonstrated that this specific accumulation of flavonols might be related to their biological functions in stress tolerance. These findings suggest that the temperature-stimulated accumulation of total polyphenols and catechins in albino mutant tea leaves was highly induced by enhanced photosynthesis and accumulation of its products, while the initial accumulation and temperature inhibition of flavonols in albino mutant tea leaves were associated with metabolism related to oxidative stress. In conclusion, our results indicate that the biosynthesis of flavonoids could be driven by many different factors, including antioxidation and carbon skeleton storage, under favorable and unfavorable circumstances, respectively. This work provides new insights into the drivers of flavonoid biosynthesis in albino tea leaves, which will further help to increase tea quality by improving cultivation measures.

A quantitative metabolomics assay targeting 14 intracellular metabolites associated with the methionine transsulfuration pathway using LC-MS/MS in breast cancer cells.

The methionine transsulfuration pathway plays an important role in some fundamental biological processes, such as redox and methylation reactions. However, quantitative analysis of the majority of intracellular metabolites is rather challenging. In this study, we developed a simple, fast and reliable method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous detection of 14 methionine-related metabolites, including methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), cystathionine (Cysta), cysteine (CYS), glutathione (GSH), dimethylglycine (DMG), betaine, serine, folic acid (FA), dihydrofolic acid (DHF), tetrahydrofolic acid (THF) and 5-methyltetrahydrofolic acid (5-MTHF), in MCF-7 and MDA-MB-231 breast cancer cells. By taking advantage of a surrogate matrix, the linearity, sensitivity, precision, accuracy, stability, matrix effect, recovery, dilution integrity and carryover of the established method were evaluated and validated. This method enabled the precise measurement of methionine-related metabolites both in cells and in the medium and was successfully applied to profile these metabolites involved in the methionine transsulfuration pathway. The data showed that cystine deprivation or excessive supplementation with cystine had a marked impact on methionine metabolism, in addition to its effects on intracellular CYS and GSH levels, indicating that the methionine transsulfuration pathway was dependent on intracellular cystine levels. The established method provides a reliable way to target metabolomics for the quantitative determination of intracellular metabolites in the methionine transsulfuration pathway, which can greatly facilitate the understanding of the mechanisms involved in methylation and redox homeostasis in cellular metabolomic studies.

Glutamate dehydrogenase isogenes CsGDHs cooperate with glutamine synthetase isogenes CsGSs to assimilate ammonium in tea plant (Camellia sinensis L.).

Glutamate dehydrogenase (GDH) is a central enzyme in nitrogen metabolism, assimilating ammonia into glutamine or deaminating glutamate into α-oxoglutarate. Tea (Camellia sinensis L.) plants assimilate ammonium efficiently, but the role of CsGDH in ammonium assimilation remains unclear. We confirmed that tea has three GDH isogenes: CsGDH1-3. Bioinformatic analysis showed that CsGDH1 encodes the ß-GDH subunit, CsGDH2/3 encode the α-GDH subunit, and their proteins all feature an NADH-specific motif. CsGDH1 is mainly expressed in mature leaves and roots, CsGDH3 is mainly expressed in new shoots and roots, and CsGDH2 has the highest expression level in flowers compared to the other five tissues. Expression patterns of CsGDHs and glutamine synthetase isogenes (CsGSs) under different ammonium concentrations suggested that CsGDHs cooperate with CsGSs to assimilate ammonium, especially under high ammonium conditions. Inhibition of GS and its isogenes resulted in significant induction of CsGDH3 in roots and CsGDH2 in leaves, indicating their potential roles in ammonium assimilation. Moreover, CsGDHs transcripts were highly abundant in chlorotic tea leaves, in constrast to those of CsGSs, suggesting that CsGDHs play a vital role in ammonium assimilation in chlorotic tea mutant. Altogether, our circumstantial evidence that CsGDHs cooperate with CsGSs in ammonium assimilation provides a basis for unveiling their functions in tea plants.