Multifaceted plant growth-promoting traits of indigenous rhizospheric microbes against Phomopsis theae, a causal agent of stem canker in tea plants.

Phomopsis canker is one of the major devastating stem diseases that occur in tea plants caused by the fungal pathogen Phomopsis theae. Rapid development of this disease leads to a capital loss in the tea industry which demands an ecofriendly disease management strategy to control this aggressive pathogen. A total of 245 isolates were recovered from the tea rhizosphere and screened for in vitro plant growth promoting (PGP) traits and antagonism against P. theae. Among them, twelve isolates exhibited multifarious PGP traits including phytohormones, siderophore, hydrogen cyanide, salicylic acid production, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and antifungal activity. In vitro studies on morphological, biochemical, and phylogenetic analyses classified the selected isolates as Pseudomonas fluorescens (VPF5), Bacillus subtilis (VBS3), Streptomyces griseus (VSG4) and Trichoderma viride (VTV7). Specifically, P. fluorescens VPF5 and B. subtilis VBS3 strains showed the highest level of PGP activities. On the other hand, VBS3 and VTV7 strains showed higher biocontrol efficacy in inhibiting mycelia growth and spore germination of P. theae. A detailed investigation on hydrolytic enzymes produced by antagonistic strains, which degrade the fungus cell wall, revealed that highest amount of chitinase and ß-1,3- glucanase in VTV7 and VBS3 strains. Further, the key antifungal secondary metabolites from these biocontrol agents associated with suppression of P. theae were identified using gas chromatography mass spectrometry. The above study clearly recognized the specific traits in the isolated microbes, which make them good candidates as plant growth-promoting rhizobacteria (PGPR) and biocontrol agents to improve plant growth and health. However, greenhouse trials and field application of these beneficial microbes is required to further confirm their efficacy for the management of stem canker in tea cultivation.

Theaflavin-Containing Black Tea Extract: A Potential DNA Methyltransferase Inhibitor in Human Colon Cancer Cells and Ehrlich Ascites Carcinoma-Induced Solid Tumors in Mice.

Tea is the most popularly consumed beverage in the world. Theaflavin and thearubigins are the key bioactive compounds of black tea that have anticarcinogenic properties as reported in several studies. However, the epigenetic potential of these compounds has not yet been explored. DNA methyltransferase (DNMT) enzymes induce methylation of DNA at cytosine residues and play a significant role in epigenetic regulation and cancer therapy. The present study has explored the role of black tea as a DNMT inhibitor in the prevention of cancer. Herein, the effect of theaflavin has been studied in colon cancer cell line (HCT-116) and EAC-induced solid tumors in mice. It was found that theaflavin prevented cell proliferation and inhibited tumor progression as well. In silico study showed that theaflavin interacted with DNMT1 and DNMT3a enzymes and blocked their activity. Theaflavin also decreased DNMT activity In Vitro and In Vivo as evident from the DNMT activity assay. Results of immunohistochemistry revealed that theaflavin reduced DNMT expression in the tumors of mice. Taken together, our findings showed that theaflavin has a potential role as a DNMT inhibitor in HCT-116 cell line and EAC induced solid tumors in mice.

Pharmacokinetic, toxicokinetic, and bioavailability studies of epigallocatechin-3-gallate loaded solid lipid nanoparticle in rat model.

Epigallocatechin-3-gallate (EGCG), derived from green tea, is an active phytochemical against many types of cancer, cardiovascular, neurological and inflammatory diseases. However, its pharmaceutical activity is limited due to low bioavailability and chemical instability. To overcome these limitations, we fabricated spherical, EGCG loaded solid lipid nanoparticles (SLN-EGCG) as an oral delivery system. The SLN-EGCG showed a hydrodynamic diameter of 300.2 ± 3.8 nm with the drug encapsulation efficiency of 81 ± 1.4%. Additionally, a slow and sustained release of EGCG was noted. Mathematical modeling of release kinetic data suggested that the SLN-EGCG followed the Higuchi model and released EGCG via fickian diffusion method. The data on pharmacokinetic parameters indicated significantly improved bioavailability and protection of EGCG from degradation due to encapsulation into SLN. The SLN-EGCG did not show any acute or sub-chronic toxicity when compared with free EGCG in the rat model. Together these data supported the hypothesis that SLN-EGCG is capable of enhancing the bioavailability and stability of EGCG and can be used as an alternative system for oral administration of EGCG.

Improved bioavailability and pharmacokinetics of tea polyphenols by encapsulation into gelatin nanoparticles.

The authors prepared surface modified (with polyelectrolyte layers), tea polyphenols (TPP) encapsulated, gelatin nanoparticles (TPP-GNP) and characterised them. The size of the spherical nanoparticles was ∼50 nm. Number of polyelectrolyte layers and incubation time influenced the encapsulation efficiency (EE); highest EE was noted in nanoparticles with six polyelectrolyte layers (TPP-GNP-6L) incubated for 4 h. TPP released from TPP-GNP-6L in simulated biological fluids indicated protection and controlled release of TPP due to encapsulation. Mathematical modelling indicated anomalous type as a predominant mode of TPP release. TPP-GNP-6L exhibited enhanced pharmacokinetics in rabbit model compared with free TPP. The area under the concentration-time curve and mean residence time were significantly higher in TPP-GNP-6L compared with free TPP which provide an evidence of higher bioavailability of TPP due to encapsulation. The authors demonstrated that encapsulation of TPP into GNPs favoured slow and sustained release of TPP with improved pharmacokinetics and bioavailability thereby can prolong the action of TPP.

Positive regulation of biochemical parameters by tea polyphenol encapsulated solid lipid nanoparticles at in vitro and in vivo conditions.

Tea polyphenols (TPPs) comprise preventive and therapeutic potentials against cancer, cardiovascular and neurological disorders. Chemical instability of TPP which leads to low bioavailability is the major constrain to its use as therapeutic agent. The authors prepared TPP encapsulated solid lipid nanoparticles (TPP-SLNs) to increase its stability and bioefficacy. Comparison of Fourier transformed infrared spectra of unloaded SLN, free TPP and TPP-SLN indicated encapsulation of TPP. Sustained release of TPP from TP-SLN was observed. TPP-SLN showed prolonged free radical scavenging activity compared with free TPP indicating protection of TPP. TPP-SLN showed activation of Caspases-9 and -3 cascades in breast cancer cell line (Michigan cancer foundation (MCF)-7) at in vitro conditions. Biochemical parameters were altered in Ehrlich ascetic carcinoma (EAC) cell bearing mice compared with normal (uninduced) mice which were ameliorated significantly by oral feeding of TPP-SLN. Oral administration (pre- and post-treated) of TPP-SLN in EAC bearing mice resulted in significant increase of plasma haemoglobin, glucose, superoxide dismutase and catalase when compared with EAC bearing control mice. Other biochemical parameters (cholesterol, bilirubin, triglyceride, urea, total protein, alanine aminotransferase, alkaline phosphatase and aspertate transaminase were significantly decreased on oral administration (pre- and post-treated) of TPP-SLN in EAC bearing mice.

Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG).

Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) enforce an overwhelming social and economic burden on society. They are primarily characterized through the accumulation of modified proteins, which further trigger biological responses such as inflammation, oxidative stress, excitotoxicity and modulation of signalling pathways. In a hope for cure, these diseases have been studied extensively over the last decade to successfully develop symptom-oriented therapies. However, so far no definite cure has been found. Therefore, there is a need to identify a class of drug capable of reversing neural damage and preventing further neural death. This review therefore assesses the reliability of the neuroprotective benefits of epigallocatechin-gallate (EGCG) by shedding light on their biological, pharmacological, antioxidant and metal chelation properties, with emphasis on their ability to invoke a range of cellular mechanisms in the brain. It also discusses the possible use of nanotechnology to enhance the neuroprotective benefits of EGCG.

Molecular cloning, computational and expression analysis of anthocyanidin reductase in tea (Camellia sinensis).

Tea [Camellia sinensis (L.) O. Kuntze] is one of the most popular non-alcoholic beverages rich in phenolic compounds, which includes epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and catechin (C). Anthocyanidin reductase (ANR) is responsible for catechin biosynthesis in plants, and analysis of its protein sequences and structures will be valuable for further research in the field. We have screened our dormant bud-specific complementary DNA (cDNA) library and reported 1,322-bp cDNA encoding CsANR. Analysis of the sequence revealed the presence of 1,011-bp open reading frame with coding capacity for a polypeptide of 337 amino acids, flanked by 1,123- and 196-bp 5' and 3' untranslated regions, respectively. Theoretical molecular weight (MW) and isoelectric point (pI) of the deduced ANR protein were predicted (using ProtParam) to be 36.4 kDa and 6.54. For the first time, we have reported 3D model of ANR from C. sinensis. Quality of the predicted model was analysed with PROCHECK analysis. Molecular docking of modelled ANR revealed similar binding pockets for both substrates and products. Expression analyses of CsANR and accumulation pattern of catechins were observed to be varied with developmental age of tissue and seasonal condition. Variation in accumulation pattern of catechins and its fractions was found to be correlated with expression pattern of ANR.

Susceptibility against grey blight disease-causing fungus Pestalotiopsis sp. in tea (Camellia sinensis (L.) O. Kuntze) cultivars is influenced by anti-oxidative enzymes.

Reactive oxygen species (ROS) production is the first level of response by a host during stress. Even though the ROS are toxic to cell, when present in a limited amount, they act as a signalling molecule for the expression of defence-related genes and later are scavenged by either enzymatic or non-enzymatic mechanisms of the host. The different anti-oxidative enzymes like glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APO), peroxidase (POD) and polyphenol oxidase (PPO) were estimated, and their activities were compared between infected and healthy leaves of the tolerant and susceptible cultivars of tea. The infected leaves of the susceptible cultivars registered higher amount of enzyme activity when compared with the tolerant cultivars. The study reveals that the more anti-oxidative enzymes, the more susceptible the cultivar will be.

Analysis of dormant bud (Banjhi) specific transcriptome of tea (Camellia sinensis (L.) O. Kuntze) from cDNA library revealed dormancy-related genes.

Bud dormancy is of ecological and economical interest due to its impact on tea (Camellia sinensis (L.) O. Kuntze) plant growth and yield. Growth regulation associated with dormancy is an essential element in plant's life cycle that leads to changes in expression of large number of genes. In order to identify and provide a picture of the transcriptome profile, cDNA library was constructed from dormant bud (banjhi) of tea. Sequence and gene ontology analysis of 3,500 clones, in many cases, enabled their functional categorization concerning the bud growth. Based on the cDNA library data, the putative role of identified genes from tea is discussed in relation to growth and dormancy, which includes morphogenesis, cellular differentiation, tropism, cell cycle, signaling, and various metabolic pathways. There was a higher representation of unknown processes such as unknown molecular functions (65.80 %), unknown biological processes (62.46 %), and unknown cellular components (67.42 %). However, these unknown transcripts represented a novel component of transcripts in tea plant bud growth and/or dormancy development. The identified transcripts and expressed sequence tags provides a valuable public resource and preliminary insights into the molecular mechanisms of bud dormancy regulation. Further, the findings will be the target of future expression experiments, particularly for further identification of dormancy-related genes in this species.

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 ß-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-protein interaction studies for identifying its potential role in intracellular communication and its physiological significance in tea.

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line.

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.