Genome-wide identification and expression analysis of NIN-like protein (NLP) genes: Exploring their potential roles in nitrate response in tea plant (Camellia sinensis).

NIN-like proteins (NLPs) are evolutionarily conserved transcription factors that are unique to plants and play a pivotal role in responses to nitrate uptake and assimilation. However, a comprehensive analysis of NLP members in tea plants is lacking. The present study performed a genome-wide analysis and identified 33 NLP gene family members of Camellia sinensis that were distributed unequally across 5 chromosomes. Subcellular localisation predictions revealed that all CsNLP proteins were localised in the nucleus. Conservative domain analysis revealed that all of these proteins contained conserved RWP-RK and PB1 domains. Phylogenetic analysis grouped the CsNLP gene family into four clusters. The promoter regions of CsNLPs harboured cis-acting elements associated with plant hormones and abiotic stress responses. Expression profile analysis demonstrated that CsNLP8 was significantly upregulated in roots under nitrate stress conditions. Subcellular localisation analysis found CsNLP8 localised to the nucleus. Dual-luciferase reporter assay demonstrated that CsNLP8 positively regulated the expression of a nitrate transporter gene (CsNRT2.2). These findings provide a comprehensive characterisation of NLP genes in Camellia sinensis and offer insights into the biological function of CsNLP8 in regulating the response to nitrate-induced stress.

Shading-Dependent Greening Process of the Leaves in the Light-Sensitive Albino Tea Plant 'Huangjinya': Possible Involvement of the Light-Harvesting Complex II Subunit of Photosystem II in the Phenotypic Characteristic.

The light-sensitive albino tea plant can produce pale-yellow shoots with high levels of amino acids which are suitable to process high-quality tea. In order to understand the mechanism of the albino phenotype formation, the changes in the physio-chemical characteristics, chloroplast ultrastructure, chlorophyll-binding proteins, and the relevant gene expression were comprehensively investigated in the leaves of the light-sensitive albino cultivar 'Huangjinya' ('HJY') during short-term shading treatment. In the content of photosynthetic pigments, the ultrastructure of the chloroplast, and parameters of the photosynthesis in the leaves of 'HJY' could be gradually normalized along with the extension of the shading time, resulting in the leaf color transformed from pale yellow to green. BN-PAGE and SDS-PAGE revealed that function restoration of the photosynthetic apparatus was attributed to the proper formation of the pigment-protein complexes on the thylakoid membrane that benefited from the increased levels of the LHCII subunits in the shaded leaves of 'HJY', indicating the low level of LHCII subunits, especially the lack of the Lhcb1 might be responsible for the albino phenotype of the 'HJY' under natural light condition. The deficiency of the Lhcb1 was mainly subject to the strongly suppressed expression of the Lhcb1.x which might be modulated by the chloroplast retrograde signaling pathway GUN1 (GENOMES UNCOUPLED 1)-PTM (PHD type transcription factor with transmembrane domains)-ABI4 (ABSCISIC ACID INSENSITIVE 4).

The CsHSFA-CsJAZ6 module-mediated high temperature regulates flavonoid metabolism in Camellia sinensis.

High temperatures (HTs) seriously affect the yield and quality of tea. Catechins, derived from the flavonoid pathway, are characteristic compounds that contribute to the flavour of tea leaves. In this study, we first showed that the flavonoid content of tea leaves was significantly reduced under HT conditions via metabolic profiles; and then demonstrated that two transcription factors, CsHSFA1b and CsHSFA2 were activated by HT and negatively regulate flavonoid biosynthesis during HT treatment. Jasmonate (JA), a defensive hormone, plays a key role in plant adaption to environmental stress. However, little has been reported on its involvement in HT response in tea. Herein, we demonstrated that CsHSFA1b and CsHSFA2 activate CsJAZ6 expression through directly binding to heat shock elements in its promoter, and thereby repress the JA pathway. Most secondary metabolites are regulated by JA, including catechin in tea. Our study reported that CsJAZ6 directly interacts with CsEGL3 and CsTTG1 and thereby reduces catechin accumulation. From this, we proposed a CsHSFA-CsJAZ6-mediated HT regulation model of catechin biosynthesis. We also determined that negative regulation of the JA pathway by CsHSFAs and its homologues is conserved in Arabidopsis. These findings broaden the applicability of the regulation of JAZ by HSF transcription factors and further suggest the JA pathway as a valuable candidate for HT-resistant breeding and cultivation.

Indole supplementation ameliorates MCD-induced NASH in mice.

Indole is a microbiota metabolite that functions to protect against obesity-associated non-alcoholic fatty liver disease. The present study examined the extent to which indole supplementation alleviates the severity of non-alcoholic steatohepatitis (NASH), which is the advanced form of non-alcoholic fatty liver disease. In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregations of macrophages in the liver compared with control diet-fed mice. Upon indole supplementation, the severity of MCD-induced hepatic steatosis and inflammation, as well as liver fibrosis, was significantly decreased compared with that of MCD-fed and control-treated mice. In vitro, indole treatment caused significant decreases in lipopolysaccharide-induced proinflammatory responses in hepatocytes incubated with either basal or MCD-mimicking media. However, indole treatment only significantly decreased lipopolysaccharide-induced proinflammatory responses in bone marrow-derived macrophages incubated with basal, but not MCD-mimicking media. These differential effects suggest that, relative to the responses of macrophages to indole, the responses of hepatocytes to indole appeared to make a greater contribution to indole alleviation of NASH, in particular liver inflammation. While indole supplementation decreased liver expression of desmin in MCD-fed mice, treatment of LX2 cells (a line of hepatic stellate cells) with indole also decreased the expression of various markers of hepatic stellate cell fibrogenic activation. Lastly, indole supplementation decreased intestinal inflammation in MCD-fed mice, suggesting that decreased intestinal inflammation also was involved in indole alleviation of NASH. Collectively, these results demonstrate that indole supplementation alleviates MCD-induced NASH, which is attributable to, in large part, indole suppression of hepatocyte proinflammatory responses and hepatic stellate cell fibrogenic activation, as well as intestinal proinflammatory responses.

Screening the Key Region of Sunlight Regulating the Flavonoid Profiles of Young Shoots in Tea Plants (Camellia sinensis L.) Based on a Field Experiment.

Both UV and blue light have been reported to regulate the biosynthesis of flavonoids in tea plants; however, the respective contributions of the corresponding regions of sunlight are unclear. Additionally, different tea cultivars may respond differently to altered light conditions. We investigated the responses of different cultivars ('Longjing 43', 'Zhongming 192', 'Wanghai 1', 'Jingning 1' and 'Zhonghuang 2') to the shade treatments (black and colored nets) regarding the biosynthesis of flavonoids. For all cultivars, flavonol glycosides showed higher sensitivity to light conditions compared with catechins. The levels of total flavonol glycosides in the young shoots of different tea cultivars decreased with the shade percentages of polyethylene nets increasing from 70% to 95%. Myricetin glycosides and quercetin glycosides were more sensitive to light conditions than kaempferol glycosides. The principal component analysis (PCA) result indicated that shade treatment greatly impacted the profiles of flavonoids in different tea samples based on the cultivar characteristics. UV is the crucial region of sunlight enhancing flavonol glycoside biosynthesis in tea shoots, which is also slight impacted by light quality according to the results of the weighted correlation network analysis (WGCNA). This study clarified the contributions of different wavelength regions of sunlight in a field experiment, providing a potential direction for slightly bitter and astringent tea cultivar breeding and instructive guidance for practical field production of premium teas based on light regimes.

Antiviral Effects of Green Tea EGCG and Its Potential Application against COVID-19.

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant component of catechins in tea (Camellia sinensis (L.) O. Kuntze), plays a role against viruses through inhibiting virus invasiveness, restraining gene expression and replication. In this paper, the antiviral effects of EGCG on various viruses, including DNA virus, RNA virus, coronavirus, enterovirus and arbovirus, were reviewed. Meanwhile, the antiviral effects of the EGCG epi-isomer counterpart (+)-gallocatechin-3-O-gallate (GCG) were also discussed.

The anti-allergic potential of tea: a review of its components, mechanisms and risks.

Allergy is an immune-mediated disease with increasing prevalence worldwide. Regular treatment with glucocorticoids and antihistamine drugs for allergy patients is palliative rather than permanent. Daily use of dietary anti-allergic natural products is a superior way to prevent allergy and alleviate the threat. Tea, as a health-promoting beverage, has multiple compounds with immunomodulatory ability. Persuasive evidence has shown the anti-allergic ability of tea against asthma, food allergy, atopic dermatitis and anaphylaxis. Recent advances in potential anti-allergic ability of tea and anti-allergic compounds in tea have been reviewed in this paper. Tea exerts its anti-allergic effect mainly by reducing IgE and histamine levels, decreasing FcεRI expression, regulating the balance of Th1/Th2/Th17/Treg cells and inhibiting related transcription factors. Further research perspectives are also discussed.

Study on the Accumulation Mechanism of Amino Acids during Bruising and Withering Treatment of Oolong Tea.

Amino acids are very important for oolong tea brisk-smooth mouthfeel which is mainly associated with bruising and withering treatment (BWT). In this study, metabolome and transcriptome analyses were performed to comprehensively investigate the changes in abundance of amino acids and the expression pattern of relevant genes during BWT of oolong tea manufacturing. Levels of most amino acids increased during BWT in the leaves harvested from 4 cultivars, while expression of the relevant function genes responsible for synthesis and transformation of amino acids up-regulated accordingly. Upstream hub genes including receptor-like protein kinase IKU2, serine/threonine-protein kinase PBL11, MYB transcription factor MYB2, ethylene-responsive transcription factor ERF114, WRKY transcription factor WRKY71, aspartate aminotransferase AATC, UDP-glycosyltransferase U91D1, and 4-hydroxy-4-methyl-2-oxoglutarate aldolase 2 RRAA2, were predicted to be involved in regulation of the function genes expression and the amino acids metabolism through weighted gene coexpression network analysis. A modulation mechanism for accumulation of amino acids during BWT was also proposed. These findings give a deep insight into the metabolic reprogramming mechanism of amino acids during BWT of oolong tea.

The contribution ratio of various characteristic tea compounds in antioxidant capacity by DPPH assay.

Tea is a worldwide health beneficial beverage for its antioxidant ability. 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay is a common method to measure the antioxidant capacity of tea compounds, yet the contribution ratio of various characteristic tea compounds is still unclear. high-performance liquid chromatography (HPLC) was used to examine the content of polyphenol compounds in 24 tea samples from four tea categories (green tea, white tea, oolong tea, and black tea). Based on the results of DPPH and HPLC, contribution ratio of each tea compound was analyzed by the Pearson correlation analysis and the partial least squares regression (PLSR). The Pearson correlation analysis showed that the order of correlation between the area of 13 peaks and the antioxidant ability of tea samples was x8 > x6 > x3 > x13 > x10 > x7 > x2 > x5 > x11 > x9 > x4 > x12 > x1; the regression equation fit by PLSR was Y = 47.258 - 0.760x1 + 0.287x2 - 1.484x3 - 0.569x4 + 0.674x5 + 2.257x6 + 1.698x7 + 1.389x8 - 0.188x9 + 0.467x10 + 0.297x11 + 1.314x12 + 0.963x13. We identified nine common peaks by reference standard substances: the x3 was gallic acid (GA), x4 was theobromine, x5 was catechuic acid (CA), x6 was epigallocatechin (EGC), x8 was epigallocatechin gallate (EGCG), x9 was caffeine, x10 was epicatechin (EC), x12 was epicatechin gallate (ECG), and x13 was gallocatechin gallate (GCG). Based on the study of spectrum-effect correlation, we obtain a better understanding of the antioxidant activity of complex tea polyphenols component. PRACTICAL APPLICATIONS: Identify the contribution of specific chemical compound to antioxidant activity by the coefficients in PLSR equation and provide a deeper insight into the joint effect of multiple ingredients of tea. Further, we can infer the DPPH free radical scavenging ability of a new kind of tea by the PLSR equation without chemical detection.

MicroRNAs and their targeted genes associated with phase changes of stem explants during tissue culture of tea plant.

Elucidation of the molecular mechanism related to the dedifferentiation and redifferentiation during tissue culture will be useful for optimizing regeneration system of tea plant. In this study, an integrated sRNAome and transcriptome analyses were carried out during phase changes of the stem explant culture. Among 198 miRNAs and 8001 predicted target genes, 178 differentially expressed miRNAs and 4264 potential targets were screened out from explants, primary calli, as well as regenerated roots and shoots. According to KEGG analysis of the potential targets, pathway of "aminoacyl-tRNA biosynthesis", "proteasome" and "glutathione metabolism" was of great significance during the dedifferentiation, and pathway of "porphyrin and chlorophyll metabolism", "mRNA surveillance pathway", "nucleotide excision repair" was indispensable for redifferentiation of the calli. Expression pattern of 12 miRNAs, including csn-micR390e, csn-miR156b-5p, csn-miR157d-5p, csn-miR156, csn-miR166a-3p, csn-miR166e, csn-miR167d, csn-miR393c-3p, csn-miR394, csn-miR396a-3p, csn-miR396 and csn-miR396e-3p, was validated by qRT-PCR among 57 differentially expressed phase-specific miRNAs. Validation also confirmed that regulatory module of csn-miR167d/ERF3, csn-miR156/SPB1, csn-miR166a-3p/ATHB15, csn-miR396/AIP15A, csn-miR157d-5p/GST and csn-miR393c-3p/ATG18b might play important roles in regulating the phase changes during tissue culture of stem explants.

Involvement of Salicylic Acid in Anthracnose Infection in Tea Plants Revealed by Transcriptome Profiling.

Anthracnose is a major leaf disease in tea plant induced by Colletotrichum, which has led to substantial losses in yield and quality of tea. The molecular mechanism with regards to responses or resistance to anthracnose in tea remains unclear. A de novo transcriptome assembly dataset was generated from healthy and anthracnose-infected leaves on tea cultivars "Longjing-43" (LJ43) and "Zhenong-139" (ZN139), with 381.52 million pair-end reads, encompassing 47.78 billion bases. The unigenes were annotated versus Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), National Center for Biotechnology Information (NCBI) non-redundant protein sequences (Nr), evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Swiss-prot. The number of differential expression genes (DEGs) detected between healthy and infected leaves was 1621 in LJ43 and 3089 in ZN139. The GO and KEGG enrichment analysis revealed that the DEGs were highly enriched in catalytic activity, oxidation-reduction, cell-wall reinforcement, plant hormone signal transduction and plant-pathogen interaction. Further studies by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography (HPLC) showed that expression of genes involved in endogenous salicylic acid biosynthesis and also accumulation of foliar salicylic acid are involved in the response of tea plant to anthracnose infection. This study firstly provided novel insight in salicylic acid acting as a key compound in the responses of tea plant to anthracnose disease. The transcriptome dataset in this study will facilitate to profile gene expression and metabolic networks associated with tea plant immunity against anthracnose.

De novo transcriptome assembly of fluorine accumulator tea plant Camellia sinensis with fluoride treatments.

Tea plant (Camellia sinensis) is a typical fluoride (F) hyperaccumulator enriching most F in old leaves. There is association between the risk of fluorosis and excessive consumption of teas prepared using the old leaves. It is meaningful to develop methods for controlling F levels in tea leaves. We generated a comprehensive RNA-seq dataset from tea plants grown at various F levels for different durations by hydroponics, aiming at providing information on mechanism of F metabolism in tea plant. Besides raw reads of the RNA-seq dataset, we present assembled unigenes and aligned unigenes with annotations versus the Gene Ontology (GO) databases, Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases, and Nonredundant (Nr) protein databases with low e-values. 69,488 unigenes were obtained in total, in which 40,894 were given Nr annotations.

Association of Tea Consumption with Risk of Alzheimer's Disease and Anti-Beta-Amyloid Effects of Tea.

Neurodegenerative disease Alzheimer's disease (AD) is attracting growing concern because of an increasing patient population among the elderly. Tea consumption is considered a natural complementary therapy for neurodegenerative diseases. In this paper, epidemiological studies on the association between tea consumption and the reduced risk of AD are reviewed and the anti-amyloid effects of related bioactivities in tea are summarized. Future challenges regarding the role of tea in preventing AD are also discussed.

Identification of Unequally Represented Founder Viruses Among Tissues in Very Early SIV Rectal Transmission.

Characterizing the transmitted/founder (T/F) viruses of multi-variant SIV infection may shed new light on the understanding of mucosal transmission. We intrarectally inoculated six Chinese rhesus macaques with a single high dose of SIVmac251 (3.1 × 104 TCID50) and obtained 985 full-length env sequences from multiple tissues at 6 and 10 days post-infection by single genome amplification (SGA). All 6 monkeys were infected with a range of 2 to 8 T/F viruses and the dominant variants from the inoculum were still dominant in different tissues from each monkey. Interestingly, our data showed that a cluster of rare T/F viruses was unequally represented in different tissues. This cluster of rare T/F viruses phylogenetically related to the non-dominant SIV variants in the inoculum and was not detected in any rectum tissues, but could be identified in the descending colon, jejunum, spleen, or plasma. In 2 out of 6 macaques, identical SIVmac251 variants belonging to this cluster were detected simultaneously in descending colon/jejunum and the inoculum. We also demonstrated that the average CG dinucleotide frequency of these rare T/F viruses found in tissues, as well as non-dominant variants in the inoculum, was significantly higher than the dominant T/F viruses in tissues and the inoculum. Collectively, these findings suggest that descending colon/jejunum might be more susceptible than rectum to SIV in the very early phase of infection. And host CG suppression, which was previously shown to inhibit HIV replication in vitro, may also contribute to the bottleneck selection during in vivo transmission.

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.

Food-grade Encapsulation Systems for (-)-Epigallocatechin Gallate.

(-)-Epigallocatechin gallate (EGCG) has attracted significant research interest due to its health-promoting effects such as antioxidation, anti-inflammation and anti-cancer activities. However, its instability and poor bioavailability have largely limited its efficacy and application. Food-grade materials such as proteins, carbohydrates and lipids show biodegradability, biocompatibility and biofunctionality properties. Food-grade encapsulation systems are usually used to improve the bioavailability of EGCG. In the present paper, we provide an overview of materials and techniques used in encapsulating EGCG, in which the adsorption mechanisms of food-grade systems during in vitro digestion are reviewed. Moreover, the potential challenges and future work using food-grade encapsulates for delivering EGCG are also discussed.

Effect of fluoride treatment on gene expression in tea plant (Camellia sinensis).

Tea plant is a typical fluorine (F) accumulator. F concentration in mature tea leaves is several hundred times higher than that in normal field crops. Long-term consumption of teas with high level F will increase the risks of dental and skeletal fluorosis. The mechanism of F accumulation in tea stands unclear. RNA-Seq and digital gene expression (DGE) techniques were used to investigate the effect of F on the differential expressions of transcriptome in tea plant. The results showed that F content in mature tea leaves was increased with increase in F concentration of cultural solution and duration of F treatment time. Based on comparison with data of GO, COG, KEGG and Nr databases, 144 differentially expressed unigenes with definite annotation were identified. Real-time reverse transcription PCR (qRT-PCR) was used to validate the effect of F on expression of 5 unigenes screened from the 144 unigenes. F treatment induced the expression of defense genes such as receptor-like kinases (RLKs) and U-box domain-containing protein. Based on the present study, F uptake is considered to be related to calcium-transporting ATPase, especially autoinhibited Ca2+ ATPase (ACAs) which was activated by the RLKs and worked as a carrier in uptake of F by tea plant.

Antidiabetic Effects of Tea.

Diabetes mellitus (DM) is a chronic endocrine disease resulted from insulin secretory defect or insulin resistance and it is a leading cause of death around the world. The care of DM patients consumes a huge budget due to the high frequency of consultations and long hospitalizations, making DM a serious threat to both human health and global economies. Tea contains abundant polyphenols and caffeine which showed antidiabetic activity, so the development of antidiabetic medications from tea and its extracts is increasingly receiving attention. However, the results claiming an association between tea consumption and reduced DM risk are inconsistent. The advances in the epidemiologic evidence and the underlying antidiabetic mechanisms of tea are reviewed in this paper. The inconsistent results and the possible causes behind them are also discussed.

Tea Polysaccharides and Their Bioactivities.

Tea (Camellia sinensis) is a beverage beneficial to health and is also a source for extracting bioactive components such as theanine, tea polyphenols (TPP) and tea polysaccharides (TPS). TPS is a group of heteropolysaccharides bound with proteins. There is evidence showing that TPS not only improves immunity but also has various bioactivities, such as antioxidant, antitumor, antihyperglycemia, and anti-inflammation. However, inconsistent results concerning chemical composition and bioactivity of TPS have been published in recent years. The advances in chemical composition and bioactivities of TPS are reviewed in the present paper. The inconsistent and controversial results regarding composition and bioactivities of TPS are also discussed.

Suppressive Effects of Tea Catechins on Breast Cancer.

Tea leaf (Camellia sinensis) is rich in catechins, which endow tea with various health benefits. There are more than ten catechin compounds in tea, among which epigallocatechingallate (EGCG) is the most abundant. Epidemiological studies on the association between tea consumption and the risk of breast cancer were summarized, and the inhibitory effects of tea catechins on breast cancer, with EGCG as a representative compound, were reviewed in the present paper. The controversial results regarding the role of tea in breast cancer and areas for further study were discussed.