Genome-wide identification,characterization, and expression analysis of nucleotide-binding leucine-rich repeats gene family under environmental stresses in tea (Camellia sinensis)

Plants often use nucleotide-binding leucine-rich repeats (NLRs) to recognize specific virulence proteins and activate the hypersensitive response thereby defending against invaders. However, data on NLRs and the resistance mechanism of NLR protein mediation in tea plant are extremely limited. In this study, 400 and 303 CsNLRs were identified from the genomes of C. sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA), respectively. Phylogenetic analysis revealed that the numbers in CNL groups are predominant in both CSS and CSA. RNA-Seq revealed that the expression of CsNLRs is induced by Colletotrichum fructicola, cold, drought, salt stress and exogenous methyl jasmonate. The 21 CsCNLs that are highly expressed in tea plant under biotic and abiotic stresses as well as during bud dormancy and in different tissues are identified. Gene structure analysis revealed several cis-regulatory elements associated with phytohormones and light responsiveness in the promoter regions of these 21 CsCNLs.     

The Inhibition of α-Amylase by Tea Polyphenols

The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECg), (?)-epigallocatechin gallate (EGCg), (?)-catechin (C), (?)-gallocatechin (GC), (?)-catechin gallate (Cg), (?)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of α-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3.     

Worldwide core collections of tea (Camellia sinensis) based on SSR markers

Tea (Camellia sinensis (L.) O. Kuntze) is the world’s most popular beverage crop. However, to date, no core collection has been selected from worldwide germplasm resources on the basis of genotype data. In this study, we analyzed 788 tea germplasm accessions using 23 simple sequence repeat (SSR) markers. Our population structure analysis divided the germplasms into a Japanese group and an exotic group. The latter could be divided into var. sinensis and var. assamica. The genetic diversity was higher in germplasms from China, Taiwan, India, and Sri Lanka than in those from other countries, and low in germplasms from Japan. Using the number of SSR alleles as a measure of genetic diversity, we developed a core collection consisting of 192 accessions and three subcore collections with 96, 48, and 24 accessions. Although the results might be affected by marker-selection bias, the core 192 collection adequately covered the range of variation of the 788 accessions in floral morphology, and the chemical composition of first-flush leaves. These collections will be powerful tools for breeding and genetic research in tea.     

Effect of shade treatment on biosynthesis of catechins in tea plants

When tea plants were shaded with black lawn cloth for severaldays in the field, the accumulations of (—)-epicatechin,(—)-epicatechin-3-gallate, (—)-epigallocatechinand (—)-epigallocatechin-3-gallate decreased in newlydeveloping tea shoots. Radioactive tracer studies showed thatthe conversions of glucose-U-¹⁴C, shikimic acid-G-¹⁴C and phenylalanine-U-¹⁴Cinto (—)-epicatechin and (—)-epigallocatechin moietieswere depressed by the shade treatment for tea plants but theincorporation of trans-cinnamic acid-3-¹⁴C was not affected.The treatment was found to have no significant effect on theactivities of phospho-2-keto-3-deoxy-heptonate. aldolase (EC.4.1.2.15 [EC] ), 3-dehydroquinate synthase (EC. 4.6.1.3 [EC] ), 3-dehydroquinatedehydratase (EC. 4.2.1.10 [EC] ), shikimate dehydrogenase (EC. 1.1.1.25 [EC] )and trans-cinnamate 4-monooxygenase (EC. 1.14.13.11 [EC] ) in theshoots, whereas the activity of phenylalanine ammonia-lyase(EC. 4.3.1.5 [EC] ) clearly decreased. (Received March 17, 1980; )     

Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells

Green tea catechins (GTCs) including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) were shown to suppress cell growth and induce apoptosis in various cell systems in addition to their chemo-preventive effect. In this study, except EC which was inactive, green tea extract (TE) and other 3 GTCs were found to suppress the growth and induce apoptosis in human prostate cancer DU145 cells largely through an increase in reactive oxygen species formation and mitochondrial depolarization. The conclusion was supported by the fact that the profiles for different GTCs in growth suppression, apoptosis induction, ROS formation and mitochondrial depolarization are in a similar order, i.e. ECG > EGCG > EGC > EC. Although the molecular mechanisms are still not clear, apoptosis induced by GTCs is not related to the members of BCL-2 family as EGCG did not alter the expression of BCL-2, BCL-X(L) and BAD in DU145 cells.     

Biochemical Evaluation of Triploid Progenies of Diploid × Tetraploid Breeding Populations of Camellia for Genotypes Rich in Catechin and Caffeine

To verify the quality of triploid varieties of Camellia tea species at the secondary metabolite level, we tested caffeine and catechin profiles of 97 F₁ segregating progenies in two breeding populations with a common tetraploid parent and diploid parents of two geographic and varietal origins. Catechin and caffeine levels of the triploid progenies were quantified and compared against their diploid parent. Some of the progenies showed better performance than their diploid parent. Most of the progenies of the diploid C. sinensis × tetraploid cross showed heterosis for caffeine and EGCG. Progenies of the C. assamica subsp. lasiocalyx × tetraploid cross showed heterosis for +C, EC, EGC, and TC. The genomic contributions of the diploid parent seem to be the main factor in the variation between the two populations. Our studies showed quantitative enhancement of some of the quality-related parameters in tea, providing a platform to refocus on this classical breeding approach for developing quality cultivars in tea.     

Inhibitory effect of tea polyphenols on histamine and leukotriene B4 release from rat peritoneal exudate cells

Summary The effect of tea polyphenols on the release of chemical mediators, histamine and leukotriene B₄ (LTB₄), from rat peritoneal exudate cells (PEC) was studied. Among polyphenols, (−)-epigallocatechin gallate (EGCG) most strongly inhibited the histamine release from the cells stimulated with a calcium ionophore, A23187 or compound 48/80. Though (+)-catechin (C) and (−)-epicatechin (EC) had no effect, (−)-epigallocatechin (EGC) and (−)-epicatechin gallate (ECG) moderately inhibited the histamine release. Similarly, EGCG, ECG, and EGC inhibited LTB₄ release from PEC, whereas C and EC were not effective. The magnitude of the inhibitory effect on the release of these mediators of tea polyphenols was in the order of EGCG>ECG>EGC. These results indicated an important role of the triphenol structure in the inhibitory activity. Therefore, the possible antiallergic effect of tea polyphenols can be expected.     

Molecular cloning of phenylalanine ammonia-lyase cDNA and classification of varieties and cultivars of tea plants (Camellia sinensis) using the tea PAL cDNA probe

Tea (Camellia sinensis) phenylalanine ammonia-lyase (PAL) cDNA was cloned using labelled rice PAL cDNA as a probe. The PAL genes of the tea plant were investigated by restriction fragment length polymorphism (RFLP) analysis using tea PAL cDNA. PAL genetic variation in tea plants was much larger than predicted due to the presence of various hybridized fragments in the Assam hybrids, which are hybrids between C. sinensis var assamica and var sinensis. On the other hand, hybridized band patterns of Japanese green tea cultivars belonging to var sinensis could be divided into five groups. Furthermore, a short-length PAL probe, about 280 bp including the 3 untranslated sequence, detected 3 DNA fragments of different lengths, which were named A, B and D. An experiment tracing the PAL gene heredity showed that A, B and D fragments were inherited according to the Mendelian monogenic ratio. Therefore, PAL genes identifiable by A, B and D fragments are multiple alleles, and the PAL gene is present as a single gene in the tea haploid genome. It was also clear that five groups of Japanese green tea cultivars were characterized by the composition of these PAL fragments. From RFLP analysis using tea PAL cDNA, we succeeded in distinguishing Assam hybrids and Japanese green tea cultivars with high and low catechin content, respectively, and in grouping Japanese green tea at the cultivar level.     

Decoding and analysis of organelle genomes of Indian tea (Camellia assamica) for phylogenetic confirmation

The NCBI database has >15 chloroplast (cp) genome sequences available for different Camellia species but none for C. assamica. There is no report of any mitochondrial (mt) genome in the Camellia genus or Theaceae family. With the strong believes that these organelle genomes can play a great tool for taxonomic and phylogenetic analysis, we successfully assembled and analyzed cp and mt genome of C. assamica. We assembled the complete mt genome of C. assamica in a single circular contig of 707,441 bp length comprising of a total of 66 annotated genes, including 35 protein-coding genes, 29 tRNAs and two rRNAs. The first ever cp genome of C. assamica resulted in a circular contig of 157,353 bp length with a typical quadripartite structure. Phylogenetic analysis based on these organelle genomes showed that C. assamica was closely related to C. sinensis and C. leptophylla. It also supports Caryophyllales as Superasterids.     

An investigation into the altered binding mode of green tea polyphenols with human serum albumin on complexation with copper

Green tea is rich in several polyphenols, such as (?)-epicatechin-3-gallate (ECG), (?)-epigallocatechin (EGC), and (?)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86?±?0.72?×?10⁴ M^?1 (K _ECG-HSA), 4.22?±?0.15?×?10⁴ M^?1 (K _{ECG-Cu(II)-HSA}), and 9.51?±?0.31?×?10⁴ M^?1 (K _{EGCG-Cu(II)-HSA}) at 293?K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.     

RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-India type)

An efficient in vitro propagation method using enhanced axillary branching cultures produced plants from nodal explants of three mature, elite tea clones: diploid UPASI 26 and UPASI 27 (2n=2x=30) representing Camellia sinensis (China type) and triploid UPASI 3 (2n=3x=45) representing C. assamica ssp. assamica (Assam-India type). The genetic fidelity of the micropropagated plants of these three tea clones was assessed by analysing their nuclear, mitochondrial (mt), and chloroplast (cp) genomes using multiple molecular DNA markers. A total of 465, 446 and 462 genetic loci were produced with RFLP, RAPD and ISSR fingerprinting in the micropropagated plants and the corresponding mother plant of C. sinensis clone U (UPASI) 26, and C. assamica ssp. assamica clones U3 and U27, respectively. RFLP fingerprinting was performed using six restriction endonuclease digests and 14 mt and cp gene probes in 84 enzyme-probe combinations. For PCR fingerprinting, 50 RAPD and SSR primers were used for amplifications. The micropropagated plants of both the U3 and U27 clones revealed complete stability in the 462 and 446 genetic loci analysed. In comparison, 36 (7.7%) of the 465 loci were polymorphic among micropropagated plants of the U26 clone. The observed polymorphic loci were not restricted to a particular genome (nuclear or organellar), although a relatively low (7.43%) level of polymorphism was observed in the nuclear as compared to the mt genome (16.3%). ISSR fingerprinting (12.8%) detected more polymorphic loci than RAPD fingerprinting (4.28%). No polymorphism was observed in the cp genome of the micropropagated plants of the three tea clones. The rigorous screening of nuclear and two organellar genomes has demonstrated, for the first time, subtle genetic variation at the DNA sequence level in organized meristem-derived micropropagated plants of tea. Clearly, this is another example demonstrating that organized meristem cultures are not always genetically true-to-type. The genomic changes in tea clones are genotype dependent rather than culture condition dependent.     

Effect of green tea powder (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L. cv. Benifuuki) particle size on <Emphasis Type="Italic">O</Emphasis>-methylated EGCG absorption in rats; The Kakegawa Study

Tea polyphenols, e.g., (-)-epigallocatechin-3-O-(3-O-methyl gallate (EGCG3”Me), (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-O-gallate (ECG), and (-)-epicatechin (EC), are believed to be responsible for the beneficial effects of tea. ‘Benifuuki’, a tea (Camellia sinensis L.) cultivar grown in Japan, is rich in the anti-allergic molecule epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me). Pulverized Benifuuki green tea powder (BGP) is more widely distributed than leaf tea in Japan. Japanese people mix their pulverized tea with water directly, whereas it is common to drink leaf tea after extraction. However, few studies of the effects of BGP particle size on polyphenol bioavailability have been performed. This study was conducted to investigate the absorption of catechins in rats after the intragastric administration of Benifuuki green tea. Therefore, we assessed the plasma concentrations of catechins following the ingestion of BGP with different mean particle sizes (2.86, 18.6, and 76.1 μm) or Benifuuki green tea infusion (BGI) as a control in rats. The bioavailabilities of EGCG3”Me, EGCG, ECG, EGC, and EC were analyzed after the oral administration of a single dose of Benifuuki green tea (125 mg/rat) to rats. The plasma concentrations of tea catechins were determined by HPLC analysis combined with of electrochemical detection (ECD) using a coulometric array. The AUC (area under the drug concentration versus time curve; min μg/mL) of ester-type catechins (EGCG3”Me, EGCG, and ECG) for the BGP 2.86 μm were significantly higher than those in the infusion and 18.6 and 76.1 μm BGP groups, but the AUC of free-type catechins (EGC and EC) showed no differences between these groups. Regarding the peak plasma level of EGCG3”Me adjusted for intake, BGP 2.86 μm and BGI showed higher values than the BGP 18.6 and 76.1 μm groups, and the peak plasma levels of the other catechins displayed the same tendency. The present study demonstrates that the bioavailability of ester-type catechins (EGCG and ECG) can be improved by reducing the particle size of green tea, but the plasma level of EGCG3”Me in the BGI group was similar to that in the BGP 2.86 μm group. This result suggests that drinking Benifuuki green tea with a particle size of around 2 μm would deliver the anti-allergic EGCG3”Me and the anti-oxidant EGCG efficiently.     

Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG> or = EGCG>EC> or = (+)-C>EGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.     

Free radicals generated during oxidation of green tea polyphenols: Electron paramagnetic resonance spectroscopy combined with density functional theory calculations

Electron paramagnetic resonance spectroscopy and density functional theory calculations have been used to investigate the redox properties of the green tea polyphenols (GTPs) (?)-epigallocatechin gallate (EGCG), (?)-epigallocatechin (EGC), and (?)-epicatechin gallate (ECG). Aqueous extracts of green tea and these individual phenols were autoxidized at alkaline pH and oxidized by superoxide anion (O₂^?) radicals in dimethyl sulfoxide. Several new aspects of the free radical chemistry of GTPs were revealed. EGCG can be oxidized on both the B and the D ring. The B ring was the main oxidation site during autoxidation, but the D ring was the preferred site for O₂^? oxidation. Oxidation of the D ring was followed by structural degradation, leading to generation of a radical identical to that of oxidized gallic acid. Alkaline autoxidation of green tea extracts produced four radicals that were related to products of the oxidation of EGCG, EGC, ECG, and gallic acid, whereas the spectra from O₂^? oxidation could be explained solely by radicals generated from EGCG. Assignments of hyperfine coupling constants were made by DFT calculations, allowing the identities of the radicals observed to be confirmed.     

Glycosides from Breynia fruticosa and Breynia rostrata

Glycosides, 3-acetyl-(?)-epicatechin 7-O-β-glucopyranoside (1), 3-acetyl-(?)-epicatechin 7-O-(6-isobutanoyloxyl)-β-glucopyranoside (2), 3-acetyl-(?)-epicatechin 7-O-[6-(2-methyl-butanoyloxyl)]-β-glucopyranoside (3), (5Z)-6-[5-(2-hydroxypropan-2-yl)-2-methyl-tetrahydrofuran-2-yl]-3-methylhexa-1,5-dien-3-O-β-glucopyranoside (4), hydroquinone O-[6-(3-hydroxyisobutanoyl)]-β-galactopyranoside (5), 4-(4-O-β-glucopyranosyl-phenoxy)-1-O-β-glucopyranosyl-1,3-benzenediol (6), 7,8-erythro-dihydroxy-3,4,5-trimethoxy-phenyl-propane8-O-β-glucopyranoside (7), 6,7-dimethylbenzofuranol 5-O-β-xylopyranosyl-(1 → 6)-β-glucopyranoside (8), along with 30 known glycosides, were isolated from Breynia fruticosa and Breynia rostrata (Euphorbiaceae). Their structures were determined on the basis of spectroscopic analysis and chemical methods.