The complete chloroplast genome of Mussaenda pubescens and phylogenetic analysis.

The chloroplast (cp) genome sequence of Mussaenda pubescens, a promising resource that is used as a traditional medicine and drink, is important for understanding the phylogenetic relationships among the Mussaenda family and genetic improvement and reservation. This research represented the first comprehensive description of the morphological characteristics of M. pubescens, as well as an analysis of the complete cp genome and phylogenetic relationship. The results indicated a close relationship between M. pubescens and M. hirsutula based on the morphological characteristics of the flower and leaves. The cp was sequenced using the Illumina NovaSeq 6000 platform. The results indicated the cp genome of M. pubescens spanned a total length of 155,122 bp, including a pair of inverted repeats (IRA and IRB) with a length of 25,871 bp for each region, as well as a large single-copy (LSC) region and a small single-copy (SSC) region with lengths of 85,370 bp and 18,010 bp, respectively. The results of phylogenetic analyses demonstrated that species within the same genus displayed a tendency to group closely together. It was suggested that Antirhea, Cinchona, Mitragyna, Neolamarckia, and Uncaria might have experienced an early divergence. Furthermore, M. hirsutula showed a close genetic connection to M. pubescens, with the two species having partially overlapping distributions in China. This study presents crucial findings regarding the identification, evolution, and phylogenetic research on Mussaenda plants, specifically targeting M. pubescens.

Metabolomics reveals the importance of metabolites in Mussaenda pubescens for antioxidant properties and quality traits.

Mussaenda pubescens (Mp) is a valuable medicinal plant that has traditionally been used for medicinal purposes or as a tea substitute. However, there are few studies on the comprehensive and dynamic evaluation of Mp metabolites. This study used an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach and biochemical analysis to investigate substance changes in leaves at three different stages and elucidate the relationship between metabolites and antioxidant capacity. The findings showed that Mp leaves contained 957 metabolites, the majority of which were phenolic acids, lipids, and terpenoids. The metabolite profiling of Mp leaves was significantly influenced by their growth and development at different stages. A total of 317 differentially accumulated metabolites (DAMs) were screened, including 150 primary metabolites and 167 secondary metabolites, with 202 DAMs found in bud leaf vs. tender leaf, 54 DAMs in tender leaf vs. mature leaf, and 254 DAMs in bud leaf vs. mature leaf. Total phenolics, flavonoids, and anthocyanin concentrations decreased as Mp leaves grew and developed, whereas terpenoids increased significantly. The secondary metabolites also demonstrated a positive correlation with antioxidant activity. Phenolics, flavonoids, terpenoids, and anthocyanins were the primary factors influencing the antioxidant activity of leaves. These findings provide new insights into the metabolite formation mechanism, as well as the development and utilization of Mp tea.

The Changes in Metabolites, Quality Components, and Antioxidant Activity of Tea (Camellia sinensis) Infected with Exobasidium vexans by Applying UPLC-MS/MS-Based Widely Targeted Metabolome and Biochemical Analysis.

Blister blight infection with Exobasidium vexans is one of the most destructive foliar diseases that seriously affect the quality and yield of tea. This research investigated the metabolite changes of healthy and infected leaves on tea cultivar 'Fuding Dabaicha' and further explored the potential antimicrobial substances against E. vexans infection. In total, 1,166 compounds were identified during the entire course of an infection, among which 73 different common compounds were significantly accumulated involved in the important antimicrobial substances of flavonoids and phenolic acids, including kaempferol (3,5,7,4'-tetrahydroxyflavone), kaempferol-3-O-sophoroside-7-O-glucoside, phloretin, 2,4,6-trihydroxybenzoic acid, galloylprocyanidin B4, and procyanidin C1 3'-O-gallate, which indicated that these metabolites might positively dominate resistance to E. vexans. Furthermore, relevant biological pathways, such as the flavone and flavonol biosynthesis, flavonoid biosynthesis, and phenylpropane pathways, were more closely related to resistance to E. vexans. Additionally, total flavonoids, phenolics, alkaloids, and terpenoids contributing to antimicrobial and antioxidant capacity were significantly altered during four different infection periods, especially the Leaf_S2 stage (the second stage of infection), in which the most concentration accumulated. The leaves affected by E. vexans infection at the second stage had the relatively highest antioxidant activity. Accordingly, this study provides a theoretical support for and comprehensive insights into the effects on the metabolite changes, tea quality components, and antioxidant activity of blister blight caused by E. vexans.

Duyun compound green tea extracts regulate bile acid metabolism on mice induced by high-fat diet.

Duyun compound green tea (DCGT) is a healthy beverage with lipid-lowering effect commonly consumed by local people, but its mechanism is not very clear. We evaluated the effect of DCGT treatment on bile acids (BA) metabolism of mice with high-fat diet (HFD) - induced hyperlipidaemia by biochemical indexes and metabolomics and preliminarily determined the potential biomarkers and metabolic pathways of hyperlipidaemia mice treated with DCGT as well as investigated its lipid-lowering mechanism. The results showed that DCGT treatment could reduce HFD - induced gain in weight and improve dyslipidaemia. In addition, a total of ten types of BA were detected, of which seven changed BA metabolites were observed in HFD group mice. After DCGT treatment, glycocholic acid, tauroursodeoxycholic acid and taurochenodeoxycholic acid were significantly down-regulated, while hyodeoxycholic acid, deoxycholic acid and chenodeoxycholic acid were markedly up-regulated. These results demonstrated that DCGT treatment was able to make the BA metabolites in the liver of hyperlipidaemia mice normal and alleviate hyperlipidaemia by regulating the metabolites such as glycocholic acid, tauroursodeoxycholic acid and taurochenodeoxycholic, as well as the BA metabolic pathway and cholesterol metabolic pathway involved.

Compound green tea (CGT) regulates lipid metabolism in high-fat diet induced mice.

This work aims to study the effect of compound green tea (CGT) on liver lipid metabolism in mice based on metabolomics of liquid chromatography-mass spectrometry (LC-MS), and preliminarily identify potential biomarkers and pathways of action by using a metabonomic network database to explore the lipid-lowering effect of CGT. In this study, forty mice were randomly divided into four groups: compound tea treatment group (DH), high-fat model control group (NK), normal control group (CK) and positive drug group (YK). After a month of different interventions, the mice were weighed and the blood lipid indexes were detected. In addition, differential liver metabolites were monitored by using LC-MS. The results showed that CGT and positive drug treatment were able to decrease body weight, liver coefficient, TC, TG and LDL levels of obese mice, while increasing HDL levels. Among the 110 compounds obtained, 54 metabolites were significantly altered in the four comparisons. More importantly, 15 remarkably downregulated metabolites involved in Lysopc 16:1, Lysopc 18:1, and Lysopc 18:2 were found in the DH group when the mice were treated with CGT; meanwhile, the positive drug Xuezhikang was able to significantly downregulate 14 compounds, including (±)18-HEPE, and 6 keto-PGF1α, compared with the NK group. Besides, KEGG enrichment analysis also revealed the important metabolic pathways, such as linoleic acid metabolism, Biosynthesis of unsaturated fatty acids, and α-linolenic acid metabolism, were related to fatty acid metabolism. These results suggested that CGT could regulate the lipid metabolism in the liver of hyperlipidemia mice, and may regulate 54 potential biomarkers in mice through a related metabolic pathway to make them return to a normal state and improve the disorder of lipid metabolism.

Heterologous Expression and Characterization of Tea (Camellia sinensis) Polyamine Oxidase Homologs and Their Involvement in Stresses.

Polyamine oxidase (PAO) is a key enzyme maintaining polyamine homeostasis, which affects plant physiological activities. Until now, the gene members and function of PAOs in tea (Camellia sinenesis) have not been fully identified. Here, through the expression in Escherichia coli and Nicotiana benthamiana, we identified six genes annotated as CsPAO in tea genome and transcriptome and determined their enzyme reaction modes and gene expression profiles in tea cultivar 'Yinghong 9'. We found that CsPAO1,2,3 could catalyze spermine, thermospermine, and norspermidine, and CsPAO2,3 could catalyze spermidine in the back-conversion mode, which indicated that the precursor of γ-aminobutyric acid might originate from the oxidation of putrescin but not spermidine. We further investigated the changes of CsPAO activity with temperature and pH and their stability. Kinetic parameters suggested that CsPAO2 was the major PAO modifying polyamine composition in tea, and it could be inactivated by ß-hydroxyethylhydrazine and aminoguanidine. Putrescine content and CsPAO2 expression were high in tea flowers. CsPAO2 responded to wound, drought, and salt stress; CsPAO1 might be the main member responding to cold stress; anoxia induced CsPAO3. We conclude that in terms of phylogenetic tree, enzyme characteristics, and expression profile, CsPAO2 might be the dominant CsPAO in the polyamine degradation pathway.

Metabolomics Analysis Reveals the Effects of Compound Fuzhuan Brick Tea (CFBT) on Regulating Dyslipidemia and Metabolic Disorders in Mice Induced by High-Fat Diet.

BACKGROUND: It is well known that obesity induced by high-fat diet (HFD) poses a serious threat to people's health. Fuzhuan brick tea, one of the most popular beverages, is reported to possess a significant effect on regulating lipid metabolism, attributed to its many bioactive ingredients. However, the efficacy and mechanism of compound Fuzhuan brick tea (CFBT) made from Fuzhuan brick tea and other six Chinese herbal medicines are still not well defined. METHODS: Sixty mice were divided into six groups: normal control group (CK), high-fat model group (NK), positive control group with anti-hyperlipidemic drug (YK), CFBT at low-(FL), medium-(FM) and high-(FH) dosage. Intervening for 30 days, conventional indexes analysis combined with metabolomics were performed to evaluate the changes in biochemical indexes and liver metabolic profiles in mice submitted to HFD. RESULTS: CFBT treatment was able to ameliorate obesity, serum biochemical parameters, antioxidant activity and hepatic steatosis. In addition, significant alterations in the liver tissue metabolic profiles were observed, with most of these associated with inflammation, glucose and lipid metabolism. CONCLUSIONS: This study provides evidence that consumption of CFBT is capable of preventing dyslipidemia, reducing weight gain, restoring liver injury, as well as improving metabolic disorders.

Integration of Metabolome and Transcriptome Reveals the Relationship of Benzenoid-Phenylpropanoid Pigment and Aroma in Purple Tea Flowers.

Tea (Camellia sinensis) flowers are normally white, even though the leaves could be purple. We previously discovered a specific variety with purple leaves and flowers. In the face of such a phenomenon, researchers usually focus on the mechanism of color formation but ignore the change of aroma. The purple tea flowers contain more anthocyanins, which belong to flavonoids. Meanwhile, phenylalanine (Phe), derived from the shikimate pathway, is a precursor for both flavonoids and volatile benzenoid-phenylpropanoids (BPs). Thus, it is not clear whether the BP aroma was attenuated for the appearance of purple color. In this study, we integrated metabolome and transcriptome of petals of two tea varieties, namely, Zijuan (ZJ) with white flowers and Baitang (BT) with purple flowers, to reveal the relationship between color (anthocyanins) and aroma (volatile BPs). The results indicated that in purple petals, the upstream shikimate pathway promoted for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) was elevated. Among the increased anthocyanins, delphinidin-3-O-glucoside (DpG) was extremely higher; volatile BPs, including benzyl aldehyde, benzyl alcohol, acetophenone (AP), 1-phenylethanol, and 2-phenylethanol, were also enhanced, and AP was largely elevated. The structural genes related to the biosynthesis of volatile BPs were induced, while the whole flavonoid biosynthesis pathway was downregulated, except for the genes flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), which were highly expressed to shift the carbon flux to delphinidin, which was then conjugated to glucoside by increased bronze-1 (BZ1) (UDP-glucose: flavonoid 3-O-glucosyltransferase) to form DpG. Transcription factors (TFs) highly related to AP and DpG were selected to investigate their correlation with the differentially expressed structural genes. TFs, such as MYB, AP2/ERF, bZIP, TCP, and GATA, were dramatically expressed and focused on the regulation of genes in the upstream synthesis of Phe (DAHPS; arogenate dehydratase/prephenate dehydratase) and the synthesis of AP (phenylacetaldehyde reductase; short-chain dehydrogenase/reductase), Dp (F3'H; F3'5'H), and DpG (BZ1), but inhibited the formation of flavones (flavonol synthase) and catechins (leucoanthocyanidin reductase). These results discovered an unexpected promotion of volatile BPs in purple tea flowers and extended our understanding of the relationship between the BP-type color and aroma in the tea plant.

Effect of Duyun Compound Green Tea on Gut Microbiota Diversity in High-Fat-Diet-Induced Mice Revealed by Illumina High-Throughput Sequencing.

Intake of a high-fat diet (HFD) is closely related to disorders of the intestinal microbiota, which plays a key role in the pathogenesis of obesity. Duyun compound green tea, an ancient Chinese drink, is widely consumed to reduce weight, although the mechanism is not clear. In this study, 50 mice were randomly divided into 5 groups: normal control group (CK), HFD model control group (NK), positive control group with medicine (YK), low-dose compound tea group (DL), and high-dose compound tea group (DH). After 4 weeks of intervention, the feces of mice were taken under sterile conditions and evaluated using Illumina high-throughput sequencing technology. The results showed that the diversity of intestinal microbiota was the highest in the CK group, the lowest in the NK group, and relatively increased in the compound tea treatment group. Second, there were differences in intestinal microbiota in each group, among which the beneficial bacteria in the intestinal tract of the CK group were higher than those in the other groups, while the beneficial bacteria in each compound tea treatment group were more abundant than those in the NK group, in which harmful bacteria in the intestinal tract were found to be the highest. These results suggest that compounds in tea may be able to attenuate imbalances of intestinal microbiota induced by poor diet, acting as a therapeutic agent in obesity or other diseases associated with gut dysbiosis.

Chloroplast genome structure and phylogenetic position of Lophatherum gracile.

Lophatherum gracile is distributed in south China, Japan and South Asia, and it is wild in the valley, stream, woodland, forest edge and gully edge. In this study, the complete chloroplast genome sequence of Lophatherum gracile was successfully obtained using Illumina sequencing. The full length of the chloroplast genome length was 137,749 bp with a typical quadripartite structure: one large single copy (LSC) region (80,610 bp), one small single copy (SSC) region (12,429 bp), and a pair of inverted repeats (IRs) (22,355 bp each). The GC content of this genome was 38.64%. The whole genome contained 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis indicated that Lophatherum gracile was closely related to Cenchrus americanus and Cenchrus longispinus.

Compound Fu brick tea modifies the intestinal microbiome composition in high-fat diet-induced obesity mice.

Compound Fu Brick Tea (CFBT), which is from Duyun city in China, is a traditional Chinese dark tea, Fu Brick Tea, mixed with six herbal medicine. It is consumed by local people for reducing weight, but the mechanism is not clear. The disorder of intestinal microbiome caused by long-term high-fat diet (HFD) is one of the inducements of obesity and related metabolic syndrome. In this study, mice were fed with HFD to establish a high-fat model. Fifty mice were randomly divided into six groups: normal control (CK), HFD model control (NK), positive control with medicine (YK), CFBT groups with low, middle, and high dose (FL, FM, FH). The V3-V4 DNA region of fecal microbiome from mouse intestine was sequenced. The results showed that the diversity of intestinal microflora was highest in CK and lowest in NK. Compared with CK, the dominant bacterium Firmicutes was increased and Bacteroidetes decreased at phylum level in NK. Compared with NK, the abundance of microbiome in CFBT groups was significantly higher and the composition was changed: Muribaculaceae, Bacteroidaceae, and Prevotellaceae increased and Lachnospiraceae decreased in CFBT groups at family level, while at the genus level, Bacteroides increased and Lactobacillus decreased. These results conclude that CFBT can increase the abundance of intestinal microbiome in mice, promote the growth of beneficial bacteria and reduce the number of pathogenic bacteria, and restore the imbalance of intestinal microbiome caused by poor diet.

Metabolome and Transcriptome Analysis Reveals Putative Genes Involved in Anthocyanin Accumulation and Coloration in White and Pink Tea (Camellia sinensis) Flower.

A variant of tea tree (Camellia sinensis (L.)) with purple buds and leaves and pink flowers can be used as a unique ornamental plant. However, the mechanism of flower coloration remains unclear. To elucidate the molecular mechanism of coloration, as well as anthocyanin accumulation in white and pink tea flowers, metabolite profiling and transcriptome sequencing was analyzed in various tea flower developmental stages. Results of metabolomics analysis revealed that three specific anthocyanin substances could be identified, i.e., cyanidin O-syringic acid, petunidin 3-O-glucoside, and pelargonidin 3-O-ß-d-glucoside, which only accumulated in pink tea flowers, and were not able to be detected in white flowers. RNA-seq and weighted gene co-expression network analysis revealed eight highly expressed structural genes involved in anthocyanin biosynthetic pathway, and particularly, different expression patterns of flavonol synthase and dihydroflavonol-4-reductase genes were observed. We deduced that the disequilibrium of expression levels in flavonol synthases and dihydroflavonol-4-reductases resulted in different levels of anthocyanin accumulation and coloration in white and pink tea flowers. Results of qRT-PCR performed for 9 key genes suggested that the expression profiles of differentially expressed genes were generally consistent with the results of high-throughput sequencing. These findings provide insight into anthocyanin accumulation and coloration mechanisms during tea flower development, which will contribute to the breeding of pink-flowered and anthocyanin-rich tea cultivars.

Comprehensive analysis of putative dihydroflavonol 4-reductase gene family in tea plant.

One identified dihydroflavonol 4-reductases (DFR) encoding gene (named as CsDFRa herein) and five putative DFRs (named as CsDFRb1, CsDFRb2, CsDFRb3, CsDFRc and CsDFRd) in tea (Camellia sinensis) have been widely discussed in recent papers concerning multi-omics data. However, except for CsDFRa, their function and biochemical characteristics are not clear. This study aims to compare all putative CsDFRs and preliminarily evaluate their function. We investigated the sequences of genes (coding and promoter regions) and predicted structures of proteins encoded, and determined the activities of heterologously expressed CsDFRs under various conditions. The results showed that the sequences of five putative CsDFRs were quite different from CsDFRa, and had lower expression levels as well. The five putative CsDFRs could not catalyze three dihydroflavonol substrates. The functional CsDFRa had the strongest affinity with dihydroquercetin, and performed best at pH around 7 and 35°C but was not stable at lower pHs or higher temperatures. Single amino acid mutation at position 141 modified the preference of CsDFRa for dihydroquercetin and dihydromyricetin, and also weakened its stability. These data suggest that only CsDFRa works in the pathway for generating anthocyanidins and catechins. This study provides new insights into the function of CsDFRs and may assist to develop new strategies to manipulate the composition of tea flavonoids in the future.

A Review on the Weight-Loss Effects of Oxidized Tea Polyphenols.

The mechanistic systems in the body through which tea causes weight loss are complex and multi-dimensional. Additionally, the bioactive components in tea such as catechins, caffeine, and products of tea polyphenol oxidation vary greatly from one major tea type to the next. Green tea has been the primary subject of consideration for investigation into the preventative health effects of tea because it contains the highest levels of phenolic compounds and retains the highest antioxidant capabilities of any major tea type. However, recent research suggests decreasing body fat accumulation has little to do with antioxidant activity and more to do with enzyme inhibition, and gut microbiota interactions. This paper reviews several different tea polyphenol-induced weight-loss mechanisms, and purposes a way in which these mechanisms may be interrelated. Our original 'short-chain fatty acid (SCFA) hypothesis' suggests that the weight-loss efficacy of a given tea is determined by a combination of carbohydrate digestive enzyme inhibition and subsequent reactions of undigested carbohydrates with gut microbiota. These reactions among residual carbohydrates, tea polyphenols, and gut microbiota within the colon produce short-chain fatty acids, which enhance lipid metabolism through AMP-activated protein kinase (AMPK) activation. Some evidence suggests the mechanisms involved in SCFA generation may be triggered more strongly by teas that have undergone fermentation (black, oolong, and dark) than by non-fermented (green) teas. We discussed the mechanistic differences among fermented and non-fermented teas in terms of enzyme inhibition, interactions with gut microbiota, SCFA generation, and lipid metabolism. The inconsistent results and possible causes behind them are also discussed.