Robin Joshi

Differential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins

Tea [Camellia sinensis (L.) O. Kuntze] leaves are a major source of epicatechin (EC) and its gallolyl derivatives epicatechin gallate, epigallocatechin and epigallocatechin gallate, collectively known as epicatechins (ECs). Epicatechins are important factors determining tea quality, and they also possess many medicinal properties. To gain further information about the regulation of the biosynthesis of ECs, we cloned the gene encoding anthocyanidin reductase from tea (CsANR) by first quantifying changes in the concentrations of ECs in response to drought, gibberellic acid (GA(3)), abscisic acid (ABA) and wounding treatments, followed by differential display of mRNAs and analysis of those bands exhibiting a change in expression paralleling the treatment-induced changes observed in the EC data. Analysis of 133 bands yielded a partial cDNA of CsANR that was later cloned to the full length by rapid amplification of the cDNA ends. The full-length CsANR (Accession No. AY641729) comprised 1233 bp with an ORF of 1014 bp (from 79 to 1092 bp) encoding a polypeptide of 337 amino acids. Expression of CsANR in an Escherichia coli expression vector yielded a functional protein that catalyzed the conversion of cyanidin to EC in the presence of NADPH. Analysis of ECs and gene expression in leaves at different developmental stages and across five tea clones exhibiting variable concentrations of ECs revealed a positive correlation between concentration of ECs and CsANR expression. Expression of CsANR was down-regulated in response to drought, ABA and GA(3) treatments and up-regulated in response to wounding.

Isolation, purification and characterization of antioxidative peptide of pearl millet (Pennisetum glaucum) protein hydrolysate

Pearl millet (Pennisetum glaucum) is a rich source of protein, used for present study to hydrolyze protein, peptide separation and its functional activity. Antioxidative bioactive peptide was successfully identified from pearl millet using trypsin enzyme. Different antioxidative potential of isolated peptide were assessed based on activity of DPPH radical, ABTS radical, hydroxyl radical, Fe(2+) chelating ability and reducing power. Bioactive peptide separated by gel-filtration chromatography, showed the higher antioxidant activity as tested by different free radicals. The activity of pearl millet protein hydrolysate fraction was found for DPPH assay (67.66%), ABTS assay (78.81%), Fe(2+) chelating ability (51.20%), hydroxyl assay (60.95%) and reducing power (0.375nm) was further purified using reversed-phase UFLC and subjected to matrix assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) for sequential identification of the peptide. The sequence SDRDLLGPNNQYLPK was identified as antioxidant peptide.

Caffeine Biosynthesis and Degradation in Tea [Camellia sinensis (L.) O. Kuntze] is under Developmental and Seasonal Regulation

To study caffeine biosynthesis and degradation, here we monitored caffeine synthase gene expression and caffeine and allantoin content in various tissues of four Camellia sinensis (L.) O. Kuntze cultivars during non-dormant (ND) and dormant (D) growth phases. Caffeine synthase expression as well as caffeine content was found to be higher in commercially utilized tissues like apical bud, 1st leaf, 2nd leaf, young stem, and was lower in old leaf during ND compared to D growth phase. Among fruit parts, fruit coats have higher caffeine synthase expression, caffeine content, and allantoin content. On contrary, allantoin content was found lower in the commercially utilized tissues and higher in old leaf. Results suggested that caffeine synthesis and degradation in tea appears to be under developmental and seasonal regulation.

Analysis of the essential oil of large cardamom (Amomum subulatum Roxb.) growing in different agro-climatic zones of Himachal Pradesh, India.

BACKGROUND The aim of the present study was to investigate variations in the chemical composition of the essential oil from seeds of large cardamom grown at different altitudes in Himachal Pradesh, India. The composition of the essential oil was determined by gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O). RESULTS The oil components showed qualitative and quantitative variations in the composition. GC and GC-MS analysis led to the identification of 55 compounds representing 98% of total oil. Major components in the oil were 1,8-cineole, α-terpineol, DL-limonene, nerolidol, 4-terpineol, δ-terpineol, δ-3-carene, β-myrcene, germacrene D, α-terpinene and longifolenaldehyde. The oil yields obtained were 9.8-19.5 g kg(-1). Cardamom oil from Himachal Pradesh was found to contain new compounds, viz. 4-terpineol, δ-3-carene, trans-sabinene hydrate, 1-phellandrene, α-terpinene, bicyclo-germacrene, isopinocarveol and ledenoxid-II. α-Terpenyl acetate, the major constituent of small cardamom, was also detected in the oil of large cardamom grown in Himachal Pradesh. Application of aroma extract dilution analysis revealed 35 compounds having aroma impact with the flavour dilution factor ranging from 2 to 1024, and 34 of these compounds were identified. The five most intense aromatic components are dl-limonene, 1,8-cineole, β-myrcene, α-pinene, α-basabolol. This is the first time that the characterisation of odour-active compounds has been carried out on large cardamom. CONCLUSION The presence of 4-terpineol, δ-3-carene, trans-sabinene hydrate, 1-phellandrene, α-terpinene, 1-terpineol, bicyclogermacrene, isopinocarveol, ledenoxid-II, longifolenaldehyde and α-terpenyl acetate make the aroma of the oil different from large cardamom oil of Sikkim and could offer potential as a new food flavour.

Increase in flavan-3-ols by silencing flavonol synthase mRNA affects the transcript expression and activity levels of antioxidant enzymes in tobacco.

Flavonoids are plant secondary metabolites widespread throughout the plant kingdom involved in many physiological and biochemical functions. Amongst the flavonoids, flavan-3-ols (catechin and epicatechin) are known for their direct free radical scavenging activity in vitro, but studies on their antioxidant potential and interaction with antioxidant enzymes in vivo are lacking. Here, the flavonoid pathway was engineered by silencing a gene encoding flavonol synthase (FLS) in tobacco to direct the flow of metabolites towards production of flavan-3-ols. FLS silencing reduced flavonol content 17-53%, while it increased catechin and epicatechin content 51-93% and 18-27%, respectively. The silenced lines showed a significant increase in expression of genes for dihydroflavonol reductase and anthocyanidin synthase, a downstream gene towards epicatechin production, with no significant change in expression of other genes of the flavonoid pathway. Effects of accumulation of flavan-3-ols in FLS silenced lines on transcript level and activities of antioxidant enzymes were studied. Transcripts of the antioxidant enzymes glutathione reductase (GR), ascorbate peroxidase (APx), and catalase (CAT) increased, while glutathione-S-transferase (GST), decreased in FLS silenced lines. Enhanced activity of all the antioxidant enzymes was observed in silenced tobacco lines. To validate the affect of flavan-3-ols on the antioxidant system, in vitro experiments were conducted with tobacco seedlings exposed to two concentrations of catechin (10  and 50 μm) for 2 days. In vitro exposed seedlings produced similar levels of transcripts and activity of antioxidant enzymes as FLS silenced seedlings. Results suggest that flavan-3-ols (catechin) might be increasing activity of GR, Apx and CAT by elevating their mRNAs levels. Since these enzymes are involved in scavenging of reactive oxygen species, this strategy would help in tailoring crops for enhanced catechin production as well as making them tolerant to oxidative stresses.

Approaches to prepare tfc polyamide membranes by coating diamine during, and/or post formation of asymmetric membranes and their performances

Thin film composite membranes were prepared by using1,3 - phenylene diamine in the gelation bath as well as in coating conditions. The cross - sectional view of the membranes shows different patterns for membranes prepared in a water - bath (Mem - I) and a diamine - containing water bath (Mem - II). The thin film composite membranes prepared using a diamine bath and coating (Mem - III) had improved salt rejecting ability but low volume flux. The performances of the salt rejection follows the order Na2SO4 > NaCl > MgSO4> MgCl2, as expected. The salt separation performances of the membranes made with higher concentration of 1,3 phenylene diamine follow the order Mem - IIIb > Mem - I > Mem - IIb. For membranes made with low concentration of 1,3 - phenylene diamine, the salt separation follows the same trend, (Mem - IIIa > Mem - IIa).

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Histochemical evaluation of catechins in PEG stressed transgenic tea plants using catechin-specific-diazotized sulfanilamide reagent

Abstract We investigated the applicability of catechin-specific-reagent (CSR) for histochemical evaluation of catechins. The diazotized arylamine moiety in CSR reacts specifically with the A-ring of catechins to yield a golden yellow complex. This makes it highly specific for spectrophotometric quantification of catechins. Therefore, microtome cut sections of untransformed and osmotin-expressing transgenic leaves and stem of tea were stained with CSR. We found catechins in the form of golden yellow globules. The catechin globules increased in the structurally intact and highly turgid cells of osmotin expressing transgenic tea plants after stress treatment with 20% PEG; by contrast, the cells in non-transgenic plants accumulated fewer catechin globules. Spectrophotometric quantification of catechins also confirmed higher levels in transgenics compared to untransformed plants. We found elevated accumulation of catechins in stress tolerant cells of tea leaves.

Comparative proteome analysis of Picrorhiza kurrooa Royle ex Benth. in response to drought

Abstract Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) is an important medicinal herb of western Himalayan region and has been used to treat various diseases and disorders. Over-harvesting and lack of cultivation has led to

Germplasm appraisal of western Himalayan tea: a breeding strategy for yield and quality improvement

Western Himalayan tea represents the status of Geographical Indication in the form of “Kangra Tea” which is unique to the tea grown world over due to its delicate flavor and high quality. The tea germplasm resources of western Himalayan region include selections from the commercial tea estates, abandoned tea gardens of Kangra valley and elite tea clones of CSIR-IHBT, Palampur. Data were recorded for different morphological traits contributing to yield and the biochemical parameters were evaluated using standard HPLC procedures. Multivariate clustering of the data differentiated the germplasm resources into distinct groups based on comparisons among the clusters for leaf size and biochemical parameters. Leaf size differentiated the tea accessions into six phenotypic groups, while shoot density which is an important yield parameter was observed to be independent of leaf size. On the basis of total catechin and caffeine contents, tea accessions were differentiated into nine groups of which Group I in comparison with other groups, recorded the highest total catechin content, moderate to high caffeine level and high astringency factor (AF). Epicatechin gallate (ECG) had a significantly high correlation with AF, implying that high levels of ECG are critical along with corresponding levels of Epigallocatechin gallate (EGCG) for the production of Theaflavin 3,3′digallate (TFDG) which is an important quality constituent providing astringency and briskness to black tea liquor. Sustainability of tea breeding programme can be achieved through organization of germplasm resources and by utilizing elite tea genotypes in breeding programme.