Plant Growth-Promoting Rhizobacterial Strain-Mediated Induced Systemic Resistance in Tea (Camellia sinensis (L.) O. Kuntze) Through Defense-Related Enzymes Against Brown Root Rot and Charcoal Stump Rot

Induction of systemic resistance in host plants through microbes and their bioactive metabolites are attaining popularity in modern agricultural practices. In this regard, individual application of two strains of Pseudomonas, RRLJ 134 and RRLJ 04, exhibited development of induced systemic resistance in tea plants against brown root rot and charcoal stump rot under split root experiments. The experimental findings also confirmed that the cuttings treated with fungal test pathogen and plant growth-promoting rhizobacteria (PGPR) strains survived longer as compared with pathogen–alone-treated cuttings. The enzyme level studies revealed that the presence of PGPR strains reduced the viscosity loss of cellulose and pectin by both the pathogens to a significant level. The activity of defense-related enzymes like l-phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase were also recorded higher in tea cuttings treated with PGPR strains in presence of pathogen. Crude bioactive metabolites isolated from these strains also showed in vitro antagonism against the test pathogens besides reducing the number of diseased plants under gnotobiotic conditions. These findings confirm the utilization of these two strains for induction of systemic resistance against two major root diseases in tea plants under plantation conditions.     

Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of ??-actin gene. A total of 377 and 720 clones with insert size >250?bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59?%) or hypothetical functions (23 and 18?%) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.     

Structural and Docking Studies of a Nucleoside Diphosphate Kinase 1 (CsNDPK1) from Tea [Camellia sinensis (L.) O. Kuntze]

Nucleoside diphosphate kinase (NDPK, EC 2.7.4.6) is a housekeeping gene, which functions in the general homeostasis of cellular nucleoside triphosphate (NTP) pools. Among the various NDPK isoforms, cytosolic NDPK1 has been shown to be the main NDPK isoform in plants, accounting for more than 70?% of total NDPK activity in plants. For the first time, a full-length cDNA (697?bp), designated as CsNDPK1 was cloned from tea leaves and consisted of a 448-bp open reading frame (ORF) encoding a 147-amino-acid polypeptide with calculated molecular mass of 16.1?kDa and a pI of 6.3. Homology modeling of CsNDPK1 shows that the presented tea NDPK1 also contains several motifs, binding and catalytic sites which are highly conserved among other NDPKs. Docking studies of CsNDPK1 with its substrates (NTPs) are discussed in detail. In summary, we describe a reliable model of CsNDPK1 that can be used in structure-based protein?Cprotein interaction studies for identifying its potential role in intracellular communication and its physiological significance in tea.     

Gibberellin Increases the Bud Yield and Theanine Accumulation in Camellia sinensis (L.) Kuntze

Tea (Camellia sinensis) is one of the most important cash crops in the world. Theanine, as an important amino acid component in tea, is a key quality index for excellent tea quality and high economic value. People increase theanine accumulation in tea mainly through the application of nitrogen fertilizer, shading and pruning. However, these methods are not effective. In this study, we treated tea buds with a 100 μM solution of GA₃ containing 1‰ tween-20, investigated the effects of GA₃ on theanine accumulation, bud yield, chlorophyll fluorescence parameters and expression level of theanine biosynthesis pathway genes in tea plant by qPCR, LC-MS/MS etc. Results showed that change trends of theanine and GA₃ was extremely positively correlated with each other. Exogenous GA₃ upregulated the expression level of theanine biosynthesis pathway genes, caused an increase of theanine content (mg·g^-1) by 27% in tea leaves compared with Mock, and accelerated the germination of buds and elongation of shoots, which lead to a significant increase of tea yield by 56% (w/w). Moreover, the decrease of chlorophyll contents, photochemical quenching coefficient (qP) and relative electron transport rate (rETR) under GA₃ treatment suggested that GA₃ reduced photosynthesis in the tender tea leaves, indicating that the decline of carbon assimilation in tea plants was conducive to the nitrogen metabolism, and it was beneficial to the accumulation of theanine. This study provided a new technical and theoretical support for the precise control of tea quality components and phenophase.     

Sensory-Guided Identification and Characterization of Kokumi-Tasting Compounds in Green Tea (Camellia sinensis L.)

The chemical substances responsible for the kokumi taste of green tea infusion are still unclear. Here, we isolated the kokumi compound-containing fractions from green tea infusion through ultrafiltration, and the major kokumi compounds were characterized as γ-Glu-Gln and γ-Glu-Cys-Gly (GSH) through ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS). The results indicated that peptides and amino acids were essential compounds in the kokumi-enriched fractions for conducting the sense of kokumi. L-theanine had an enhancing effect on the kokumi taste of green tea infusion, which was confirmed in the sensory reconstitution study. Thus, peptides, especially γ-Glu-Gln and GSH, are the major kokumi compounds in green tea infusion, which has the potential of improving the flavor of tea beverages.     

Correlation among Metabolic Changes in Tea Plant Camellia sinensis (L.) Shoots,Green Tea Quality and the Application of Cow Manure to Tea Plantation Soils

Traditionally, the supplement of organic manure in tea plantations has been a common approach to improving soil fertility and promoting terroir compounds, as manifested by the coordinated increase in yield and quality for the resulting teas. However, information regarding the effect of organic manure in the metabolome of tea plants is still inadequate. The metabolite profiles of tea shoots applied with cow manure, urea or no fertilizer were studied using gas chromatography–mass spectrometry (GC–MS). In total, 73 metabolites were detected, and the modulated metabolites included mainly amino acids, organic acids and fatty acids. In particular, glutamine, quinic acid and proline accumulated more in tea shoots in soils treated with cow manure, but octadecanoic acid, hexadecanoic acid and eicosanoic acid were drastically reduced. Pearson correlation analysis indicated that organic acids and amino acids in tea shoots were the two major metabolite groups among the three treatments. The analysis of metabolic pathways demonstrated that the cow manure treatment significantly changed the enrichment of pathways related to amino acids, sugars and fatty acids. Sensory evaluation showed that the quality of green teas was higher when the plants used to make the tea were grown in soil treated with cow manure rather than urea during spring and late summer. The results indicated that the application of cow manure in soils changed the metabolic characteristics of tea shoots and improved the qualities of the resulting teas.     

Effect of Steeped Black Tea (Camellia sinensis (L.) var. assamica) on Immunoglobulin Titer and Lymphocyte Proliferation in Responses to Hepatitis B Vaccine in Mice

Effect of steeped black tea (Camellia sinensis (L.) var. assamica) on immunoglobulin titer (IgM and IgG) and lymphocyte proliferation in responses to hepatitis B vaccine inBALB/c mice has been investigated. The study was divided into two stages i.e. the determination of immunoglobulin titer and the test of lymphocytes proliferation. In the first stage mice were divided into 5 groups each consisting of 5 mice. Group I, II and III was given steeped black tea respectively with a dose of 600 mg/kg bw; 1.2 g/kg bw and 2.4 g/kg bw. Group IV was given Stimuno^® with a dose of 6.5 mg/kg bw, and group V was given aquadest as negative control. All groups were induced by hepatitis B vaccine on day-0 (after 7 days of acclimatization). Serum was taken on day-14 and 21 for measurement of IgM titer and IgG, respectively. In the second stage, the mice were grouped as in the first stage, then all groups were induced by hepatitis B vaccine at day-0 and day-7. On day-27 lymphocyte was isolated and then tested for the growth and proliferation of lymphocytes. The results of this study showed that the steeped black tea has an effect in increasingIgM and IgG titer of BALB/cmice induced by hepatitis B vaccine, where the most effective dose was 1.2 g/kg bw. Steeped black tea also could increase lymphocytes proliferation in mice BALB/cinduced by hepatitis B vaccine, where the most effective dose was 1.2 g/kg bw.     

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.     

Full Validation of a Simple Method for Determination of Catechins and Caffeine in Brazilian Green Tea (Camellia sinensis var. assamica) Using HPLC

This paper describes the validation of an HPLC method for the assay of a green tea brew. The method employs a RP-18 column with water:methanol:ethyl acetate elution and UV detection at 280 nm. Specificity was evaluated using a photodiode array detector. The validation data showed that the assay is specific, accurate, precise, and reproducible for determination of six catechins and caffeine simultaneously. The response was linear over a range of 37–185 μg mL^?1 for caffeine, 99–500 μg mL^?1 for (?)-epigallocatechin (EGC), 20–100 μg mL^?1 for (+)-catechin (C), 30–150 μg mL^?1 for (?)-epicatechin (EC), 150–800 μg mL^?1 for (?)-epigallocatechin gallate (EGCG), 20–105 μg mL^?1 for (?)-gallocatechin gallate (GCG) and 40–205 μg mL^?1 for (?)-epicatechin gallate (ECG) (r > 0.9999 for all compounds). The range of recoveries was 96.12–110.48% according to substances. The RSD values for intra- and inter-day precision studies were <2.07 and <6.65%, respectively. The composition of samples assayed suggests that the summer is the best season for extract a major content of EGCG and caffeine. This assay can be readily utilized as quality controlled method for major green tea compounds.     

Effects of Hydrogen Peroxide on In Vitro Cultures of Tea (Camellia sinensis L.) Grown in the Dark and in the Light: Morphology,Content of Malondialdehyde,and Accumulation of Various Polyphenols

Tea plants (Camellia sinensis L.) are phenol-accumulating crops that are widely used for public health. The healing effect of tea leaf products is due to the biosynthesis of such phenolic compounds (PCs) as flavans, which have P-vitamin capillary-strengthening activity. Due to their limited habitat and the value of their specialized metabolites of a phenolic nature, a promising approach is to establish in vitro cultures from them that retain the ability to form PCs, which is characteristic of ex vivo tea plants. The aim of this study was to investigate the effect of exogenic H₂O₂ (0.01 mM; 0.1 mM; 1 mM) on the growth, morphology, degree of stress response, and accumulation of various phenolic compounds in tea plant callus cultures of different ages (24 or 36 days) grown under different cultivation conditions (darkness or light). According to the results obtained, the H₂O₂ effect on tea callus cultures of different ages did not cause changes in their morphophysiological characteristics, both after 2 h of exposure (rapid response of callus culture, RRCC) and after 48 h (delayed response of callus culture, DRCC). The determination of the malondialdehyde (MDA) content, which serves as an indicator of changes in the level of lipid peroxidation (LPO) and the presence of stress responses in plant cells, indicated either its maintenance at the control level, a decrease, or an increase. All these effects depended on the growth conditions of the tea callus cultures (darkness or light), their age, the duration of exposure (rapid or delayed response), and the H₂O₂ concentration. Similar trends were noted for the total content of PCs as well as the amount of flavans, proanthocyanidins (soluble and insoluble forms), and lignin. The plant cell responses reflected changes in its adaptation programs, when specialized metabolites act as a target for the action of H₂O₂, thereby contributing to an increase in their resistance.     

Mechanism of Fenpropathrin Resistance in Red Spider Mite, <Emphasis Type="Italic">Oligonychus coffeae</Emphasis> (Acarina: Tetranychidae), Infesting Tea [<Emphasis Type="Italic">Camellia sinensis</Emphasis> L. (O. Kuntze)]

Red spider mite (RSM), Oligonychus coffeae (Nietner) (Acarina: Tetranychidae), has gained special attention in view of their widespread occurrence as a pest on tea [Camellia sinensis L. (O. Kuntze)]. The development of acaricide (fenpropathrin) resistance has been screened in field populations (FPs) of RSMs from different tea-growing regions of south India and compared with a laboratory-susceptible population (SP) based on toxicity bioassay, detoxifying enzyme activities, analysis of acetylcholine esterase gene (AChE, 2064 bp), and their expression pattern using semiquantitative RT-PCR. The increased resistance ratio (RR, 1.39 to 2.13) in LC₅₀ of fenpropathrin observed in field populations of RSM provides a baseline for screening the development of resistance to fenpropathrin. This resistance developed due to hyperexpression of detoxifying enzymes, i.e., esterase (RR of 1.43 to 2.53) and glutathione S-transferase (RR of 1.11 to 1.86), and overexpression of AChE gene at 1.4 to 2.7-fold. These results necessitate molecular studies and warrant the continuous monitoring of acaricide susceptibility and resistance pattern in order to analyze the usefulness of AChE gene as target for developing alternate pest control strategies and management of pesticide resistance in tea ecosystem.     

Characterization of aroma active compounds in fruit juice and peel oil of Jinchen sweet orange fruit (Citrus sinensis (L.) Osbeck) by GC-MS and GC-O

Gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) were used to determine the aromatic composition and aroma active compounds of fruit juice and peel oil of Jinchen sweet orange fruit. Totals of 49 and 32 compounds were identified in fruit juice and peel oil, respectively. GC-O was performed to study the aromatic profile of Jinchen fruit juice and peel oil. A total of 41 components appeared to contribute to the aroma of fruit juice and peel oil. Twelve components were the odorants perceived in both samples. The aromatic compositions of fruit juice were more complex than that of peel oil. Ethyl butanoate, beta-myrcene, octanal, linalool, alpha-pinene, and decanal were found to be responsible for the aromatic notes in fruit juice and peel oil. Nineteen components have been perceived only in the juice and ten compounds were described as aromatic components of only the peel oil by the panelists. These differences lead to the different overall aroma between fruit juice and peel oil.     

Detection and partial sequence of phytochrome genes in the ferns Anemia phyllitidis (L.)Sw (Schizaeaceae) and Dryopteris filix-mas L. (Polypodiaceae) by using polymerase-chain reaction technology.

Phytochrome controls several developmental steps during formation and differentiation of the fern gametophyte, including spore germination, morphogenesis of the gametophyte or differentiation of the sexual cells. To obtain information about the amino acid sequence and the regulation of phytochrome expression at the gene level, two degenerated oligonucleotides against well conserved amino acid regions were designed after an optimal alignment of the known phytochrome sequences. These primers were tested against DNA isolated from Arabidopsis thaliana, and the polymerase-chain reaction (PCR) products were cloned and sequenced. The DNA fragment produced with this method proved to be identical with a phytochrome-A-gene fragment from A. thaliana, and hence this fragment was used in further experiments to prove whether amplified DNA from fern species contains phytochrome-like DNA. With this procedure we successfully detected and cloned gene fragments both from gametophytes of Anemia phyllitidis and Dryopteris filix-mas, cultured for 7 days under vegetative conditions. In addition, poly(A)+ RNA was prepared from 7-day-old gametophytes of A. phyllitidis, induced to differentiate antheridia under generative conditions. This poly(A)+ RNA was transcribed into complementary DNA and used together with both phytochrome specific primers in a PCR experiment. We thereby obtained another DNA fragment. These data strongly suggest that A. phyllitidis has at least two phytochrome genes, and that at least one of them is expressed in light-grown gametophytes.     

Essential oil yield and quality of methyl eugenol rich Ocimum tenuiflorum L.f. (syn. O. sanctum L.) grown in south India as influenced by method of harvest

A field experiment carried out during 2001--2002 under semi-arid conditions of Hyderabad, India investigated the effect of three different methods of harvesting at full bloom stage, on essential oil yield and quality of methyl eugenol rich sacred/holy basil (Ocimum tenuiflorum L.f.; Lamiaceae). The harvest methods were: harvesting of primary branches, secondary branches and shoot biomass cut at 30 cm above ground level. Four harvests at 102, 192, 287 and 360 days after transplanting of the crop were taken in 1 year in each method of harvest. Harvesting of secondary branches led to maximum plant height and number of secondary branches per plant compared to harvesting of primary branches or shoot biomass cut at 30 cm above ground during second, third and fourth harvests. On the contrary, secondary branch harvest gave least biomass yield in all the four harvests. But due to higher essential oil content, secondary branch harvest gave 25.2 and 15.4% higher total (sum total of all four harvests) essential oil yield (kg/ha per year) over primary branches and shoot biomass cut at 30 cm above ground methods of harvesting, respectively. A similar treatment difference was observed in respect of oil composition studied in the first harvest. Harvesting shoot biomass at 30 cm above ground produced oil containing highest amount of methyl eugenol. The content of methyl eugenol decreased in the order of shoot biomass cut at 30 cm above ground > primary branch > secondary branch treatments. A reverse trend was observed, however, in respect of (E)-cinnamyl acetate, eugenol and beta-elemene constituents of the oil. Little variability was, however, observed among the treatments in respect of 24 other constituents of the oils.     

Cumulated double bond systems as ligands : X. N-sulfinylaniline complexes of rhodium(I) and iridium(I) of the type [MCl(PR3)2L] in which L is coordinated via the π-NS and via the sulfur atom respectively; crystal and molecular structure of [RhCl(P-i-Pr3)2(4-MeC6H4NSO)]; determination of the 1J(103Rh15N) by 15N NMR spectroscopy

Each of the compounds [MCl(Pr₃)₂(ArylNSO)] (M = Rh^I, Ir^I; R = i-Pr, Cy: Aryl = C₆H₅, 4-MeC₆H₄, 4-ClC₆H₄, 2,4,6-Me₃C₆H₂ appears to exist as two isomers both in the solid state and in solution. The molecular and single crystal structure of one of the isomers of [RhCl(P-i-Pr)₃)₂(4-Me₆H₄NSO)] shows that the N-sulfinylaniline ligand is in the cis-configuration and coordinated to the rhodium atom via the sulfur-atom. The ligand lies in a plane which includes the rhodium atom and is in agreement with the Rh-S distance of 2.10 Å. IR results of the compounds (solid and solutions), ²¹P NMR data and ¹⁵N NMR of a ¹⁵N labelled compound, which yielded a ¹⁰³Rh¹⁵N coupling constant of 15.5 Hz, show that in the second isomer the N-sulfinylaniline ligand is probably bonded to the metal atom via the π-NS bond.The ratio of the metal-π-NS bonded isomer and the metal-S bonded isomer decreases in the order Aryl = 4-ClC₆H₄ > C₆H₅ > 4-MeC₆H₄; R = i-Pr > Cy and M = Rh > Ir. The interconversion of the two isomers is intramolecular and becomes observable on the ³¹P NMR time scale at about 40° C for M = Rh.In the case of [Ir(P-i-Pr₃)₂(4-MeC₆H₄NSO)], cyclometallation of the sul- finylaniline is observed via the ortho-carbon atom, whereas cyclometallation via P-i-Pr₃ is observed when the ortho-positions are blocked by methyl groups, e.g. when L = 2,4,6-Me₃C₆H₂NSO.