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In this study, a high value rare sugar D-tagatose was synthesized using recyclable magnetic catalysts. A magnetic cross-linked enzyme aggregate (mCLEA) of L-arabinose isomerase (L-AI) was developed for the transformation of D-galactose into D-tagatose. Similarly, a combined magnetic cross-linked enzyme aggregate (Combi-mCLEA) of dual enzyme L-AI and β-galactosidase (β-Gal) was also prepared for the synthesis of D-tagatose from lactose in a cascade reaction. To accomplish this, L-AI alone and together with β-Gal was stabilized in magnetic nanoparticles (MNPs) and precipitated by an appropriate precipitating agent and then cross-linked by glutaraldehyde under optimal conditions. Immobilized catalysts were characterized by scanning electron microscopy, confocal laser scanning microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. Results suggested that the immobilized catalysts retained a maximum initial activity and achieved equilibrium level conversion in 24 h of enzyme catalysis. The main advantage of the developed magnetic catalysts was their recovery using an external magnet after completion of the reaction. Additionally, mCLEA and Combi-mCLEA showed reusability potential for more than 10 consecutive batch cycles of rare sugar D-tagatose synthesis from D-galactose and lactose, respectively. Thus, these immobilized magnetic catalysts have shown promising catalytic potential for the synthesis of D-tagatose.

A dual-enzyme metal-organic hybrid crystal was constructed through self-assembling of manganese phosphate embedded with β-galactosidase and L-arabinose isomerase for facile synthesis of rare sugar D-tagatose. The synthesized crystal-like hierarchical system (MnHC@β-Gal+L-AI) was extensively characterized for structural features and catalytic reactions. The results indicated that upon immobilization onto the hybrid crystal, the activity of β-galactosidase and L-arabinose iomerase was enhanced by a factor of 1.6- and 1.5-fold, respectively. The developed MnHC@β-Gal+L-AI exhibited excellent efficiency with a net equilibrium level conversion of low-cost substrate whey lactose (100%) into D-glucose (∼50%), D-galactose (∼25%), and D-tagatose (∼25%). In addition, the fabricated hybrid crystals displayed cofactor regeneration ability. Therefore, the developed hybrid system was observed to be efficiently reused more than 5 times in a batch level conversion. Hence, the developed dual-enzyme-based hybrid crystal provides a platform for direct transformation of whey lactose into rare sugar D-tagatose.

Long-term storage of tubers in refrigerated and non-refrigerated storage induces sprouting and other degradative quality changes, thus making them unacceptable for consumption. There are already known effects of packaging materials on storability of fresh produce while optimization of package with respect to temperature for shelf life enhancement of tubers is still needed in the case of deficient storage conditions. Therefore, we investigated the combined effect on post-harvest quality parameters of various packaging materials, viz. polypropylene (PP), perforated polypropylene (PP(P)), low-density polyethylene (LDPE), perforated low-density polyethylene (LDPE(P)), brown gunny sacks (BS) and white nylon gunny sacks (WS), in refrigerated storage temperature of 8 °C and non-refrigerated storage temperature of 25 °C. Sprout and spoilage appearance, weight loss, firmness, colour difference, sucrose, reducing sugars, vitamin C, total phenolic content and DPPH-antioxidant activity and overall quality index were evaluated in stored potato tubers. Lower weight loss in LDPE and PP bags was observed along with the higher retention of total phenolics and antioxidant activity of tubers. The presence of perforations resulted in higher firmness of potatoes at both the temperatures. Colour changes were determined by the temperature effects rather than packaging. The study also established some physicochemical quality indicators based on the cut-off points produced from overall quality index evaluated using the sensory attributes of stored potatoes. Overall, PP(P), LDPE(P), BS and WS can be recommended for long-term storage, while PP and LDPE can be used as an option for short-term storage.

The present study aims to develop functional composite edible films using varied film formulations containing pectin (P), corn flour (CF), beetroot powder (BP), orange peel powder + sodium alginate (OPS), muesli root powder (MRP) and rice flour (RF) along with their structural, mechanical and functional characterization. Strong intermolecular attachment evolved between starch and pectin molecules due to hydrogen bonding of their hydrophilic groups, confirmed through scanning electron micrographs and FTIR spectra. Also, P-CF ratio (1:1) might retained some crystalline regions of starch which has significantly enhanced structural integrity and functionality of films in terms of tensile strength (TS), water solubility (WS), water vapour permeability (WVP) and thermal stability. In accordance with Herschel-Bulkley model, the predicted consistency index and flow behaviour values were affected by film formulation and the model was of good fit with R2 = 0.81 (PC3), R2 = 0.74 (PCB3) and R2 = 0.99 (for rest formulations). Although, higher proportions of hydrophilic polymers (P, OPS & BP) due to higher molecular mobility resulted in less rigid, highly soluble, moisture permeable and thermally unstable films. Furthermore, RF films possessed cracks over the surface and insoluble fractions due to insufficient starch gelatinization which decreased their TS and barrier properties. The ATR-FTIR spectra confirmed the presence of functional groups corresponding to starch and pectin in range of 500–4000 cm−1. The variation among different films underlies only in peak intensities due to some molecular changes. Overall, the films made of P-CF ratio (1:1) were more functional and structurally stable as compared to rest of the films, thus, they would make a better alternative for synthetic packaging.

The aim of the study was to develop a tomato-based beverage and to investigate the effect of thermal (pasteurization) and non-thermal (ultrasonication, ultra-violet (UV), atmospheric cold plasma (ACP)) processing on the quality of a tomato-based beverage. The ultrasonication and ACP processing for 10 min put significant effect on L* and a* color values of the tomato beverage. All the processing techniques applied degraded ascorbic acid but maximum retention (95%) of ascorbic acid was found in ACP processed beverage. Furthermore, ACP processing of 10 min also had significant (p ≥ 0.05) effect on bioactive compounds like chlorogenic acid, sinapic acid and gallic acid in comparison to thermal and other non-thermal processing. Moreover, ACP for 10 min and ultrasonication for 15 min was found equivalent to thermal processing in microbial reduction and ACP processing for 10 min was found equivalent to pasteurization in reduction of yeast and mold count. Both thermal and non-thermal processing had insignificant (p ≥ 0.05) effects on pH, TSS and sugars of developed beverage. In summary, ACP processing for 10 min has emerged as the best processing method for retaining as well as improving the quality of the developed beverage.

Geranyl geranyl pyrophosphate synthase (GGPPS) is known to form an integral subunit of the heteromeric GPPS (geranyl pyrophosphate synthase) complex and catalyzes the biosynthesis of monoterpene in plants. Picrorhiza kurrooa Royle ex Benth., a medicinally important high altitude plant is known for picroside biomolecules, the monoterpenoids. However, the significance of heteromeric GPPS in P. kurrooa still remains obscure. Here, transient silencing of PkGGPPS was observed to reduce picroside-I (P-I) content by more than 60% as well as picroside-II (P-II) by more than 75%. Thus, PkGGPPS was found to be involved in the biosynthesis of P-I and P-II besides other terpenoids. To unravel the mechanism, small subunit of GPPS (PkGPPS.SSU) was identified from P. kurrooa. Protein-protein interaction studies in yeast as well as bimolecular fluorescence complementation (BiFC) in planta have indicated that large subunit of GPPS PkGPPS.LSUs (PkGGPPS1 and PkGGPPS2) and PkGPPS.SSU form a heteromeric GPPS. Presence of similar conserved domains such as light responsive motifs, low temperature responsive elements (LTRE), dehydration responsive elements (DREs), W Box and MeJA responsive elements in the promoters of PkGPPS.LSU and PkGPPS.SSU documented their involvement in picroside biosynthesis. Further, the tissue specific transcript expression analysis vis-a-vis epigenetic regulation (DNA methylation) of promoters as well as coding regions of PkGPPS.LSU and PkGPPS.SSU has strongly suggested their role in picroside biosynthesis. Taken together, the newly identified PkGPPS.SSU formed the heteromeric GPPS by interacting with PkGPPS.LSUs to synthesize P-I and P-II in P. kurrooa.

The aim of this study was to investigate the hurdle effect of combining atmospheric cold plasma (ACP) with hydrothermal treatment on ascorbic acid, individual polyphenolic compounds, total phenolic content, and microbial inactivation of strawberry juice. Strawberry juice was treated with ACP for 10 and 15 min using dielectric barrier discharge at 60 kV with the input voltage of 260 V. The ascorbic acid concentration was retained maximum only in ACP treatment followed by ACP + hydrothermal treatment. Furthermore, ACP treatment for 10 min coupled with hydrothermal treatment resulted in the higher concentration of gallic acid, epigallocatechin, phloretin, naringin, hyprin, and 4-O-caffeoylquinic acid with respect to control ( p

The present study aimed to enhance the shelf life of tomatoes at pink-red stage through pectin (P), corn flour (CF) and beetroot powder (B) based coatings. PCF11 coating was resulted in significant enhancement of shelf life and quality retention of tomato in terms of physiological weight loss (PWL), decay per cent, ripening index, respiration rate, hue angle, firmness and DPPH antioxidant activity. Lower amounts of glucose and fructose quantified through HPLC indicated lesser cell wall components degradation in tomatoes coated with P and PCF11. Edible coatings also protected loss of phenolic acids like gallic and p-coumaric acid in tomato. All three coatings P, PCF11 and PCF11B in tomatoes showed higher retention of gloss and overall flavour scores. Further, the minimum shrinkage (18%) in tomatoes coated with PCF11 on 30th day evinced the uniformity and little hydrophobicity of pectin-corn flour-based coatings as observed through z-average particle size (d.nm)/WVP of coating solutions. Principal component analysis revealed significant correlation of several treatments and storage effects on tomatoes during shelf life study. The least separation among different points of PCF11 in score plot showed least deviation at subsequent storage intervals, revealing retention of quality of tomatoes during storage.

Nanotechnology has opened up novel applications in the field of biotechnology and crop production, as nanomaterials possess unique features such as high surface area and high reactivity as compared to their bulk counterparts. In the recent era, scientists are developing different kinds of nanoparticles (NPs) like metal-based (gold, silver, silicon dioxide, zinc oxide, titanium dioxide, etc.) and carbon-based nanomaterials (carbon nanotubes and fullerenes) to play a great role in plant growth and development. Nanomaterials can behave as “magic bullets” to deliver chemicals or genes (required for proper plant functioning) at the target plant organelles with high specificity in a controlled manner. Exposure of nanomaterials to plants induce many morphological and physiological changes based on several parameters, viz., properties of NPs, chemical composition, surface coating, size, dosage, time of exposure, and many more. However, a complete knowledge regarding the dynamics of NPs interaction with plants and their effect on growth and development is still unclear. The present chapter highlights the key potentials of NPs in plant growth and development in terms of root and shoot growth, seed germination, photosynthesis, and transpiration and summarizes the mechanism of NPs interactions with plants and plant parts. The effect of nanomaterials toxicity on plant species is also discussed.

Addition of low-methoxyl pectin (LMP) was observed to reduce whey loss, improve firmness, rheology, quality and overall liking of low-fat set-yoghurt. LMP addition increased soluble solids content and resulted in higher bacterial counts (≥10 log CFU/g), acidity, bioactivity (DPPH• & ABTS+ μM Trolox/g), and lower pH of set yoghurts. A significant (p

Picroliv, a mixture of picroside I and kutkoside isolated from rhizome of Picrorrhiza kurroa has been reported for many pharmaceutical properties such as hepatoprotective, anticholestatic, antioxidant and immune‐modulating activity. However, picroliv possessed lesser efficacy due to its poor aqueous solubility and lesser bioavailability. To find solution, picroliv was loaded into biodegradable poly lactic acid nanoparticles (PLA NPs) using solvent evaporation method. The picroliv‐loaded PLA NPs were characterised by UV–vis spectroscopy, atomic force microscopy, transmission electron microscopy, Fourier transform infrared and Zeta sizer. The size of picroliv‐loaded PLA NPs was 182 ± 20 nm. Zeta potential of picroliv‐loaded PLA NPs was −23.5 mV, indicated their good stability. In vitro picroliv release from picroliv‐loaded PLA NPs showed an initial burst release followed by slow and sustained release. The efficacy of picroliv‐loaded PLA NPs was assessed against KB cell lines. Blank PLA NPs showed no cytotoxicity on KB cells. The picroliv‐loaded PLA NPs showed more cytotoxic activity on KB cells as compared to the pure drug. Hence, the developed picroliv nanoformulation would find potential application in pharma‐sector.

Betulin (BT) is an abundant triterpene found predominantly in the bark of Himalayan birch. It is difficult to deliver it in vivo because of its low aqueous solubility. We have therefore developed novel formulations of BT for improving its solubility, bioavailability and therapeutic efficacy. Poly-d,l-lactide nanovectors (PLA NVs) were synthesized using poly(vinyl alcohol) and Lonicera japonica leaf extract (LE) as a stabiliser and named as PLA-1 NVs and PLA-2 NVs. PLA-1 NVs and PLA-2 NVs were used for the encapsulation of betulin (BT) and named as BT-En-1 and BT-En-2 NVs. The encapsulation efficiency of BT-En-1 and BT-En-2 NVs were 99.3 and 100 % respectively. Prepared nanoformulations were physically stable. An in vitro study revealed 45 % BT was released over 24 h. BT had a prolonged release from BT-En-2 NVs as compared to BT-En-1 NVs. BT-En-2 NVs had better anticancerous activity against SiHa cells than BT-En-1 NVs. Developed BT-EN-2 NVs had better biocompatibility, excellent stability and enhanced release characteristics than BT-En-1 NVs.

The present work refers to a process involving the use of dilute nitric acid pretreatment and enzymatic hydrolysis for the transformation of rice straw into simple sugars. Acid pre-treated rice straw was separated into the pulp and supernatant through centrifugation and filtration. The two fractions are then converted into simple sugars by combined action of microbes producing cellulase and laccase enzymes. These microbes were isolated from soil samples which were collected from different locations with varying altitudes, expected to harbour microbes with high-hydrolysing activity. The nitric acid pretreatment was carried out at 30 °C, 200 rpm for 72 h. After 72 h, the culture supernatants were analysed for the presence of glucose with the help of HPLC. The supernatant fraction separated after the acid pre-treated rice straw produced highest amount of glucose (205 mg/g of rice straw) upon subsequent hydrolysis with synergistic action of cellulase and laccase-producing microbes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0013-6) contains supplementary material, which is available to authorized users.

Tea (Camellia sinensis) is one of the richest sources of flavan-3-ols, an important class of flavonoids. The expression level of gene-encoded key regulatory enzymes of flavan-3-ol/anthocyanin biosynthetic pathway, dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR), has been highly correlated with the flavan-3-ol contents and antioxidant activity in tea plant. In the present study, pyramiding of CsDFR and CsANR in tobacco was achieved. However, single transgenic tobacco overexpressing either CsDFR or CsANR was documented earlier. In continuation, pyramided transgenic lines were evaluated for the possible, either same or beyond, effect on flavan-3-ol accumulation and protective ability against biotic and abiotic stresses. The pyramided transgenic lines showed early flowering and improved seed yield. The transcript levels of flavan-3-ol/anthocyanin biosynthetic pathway and related genes in pyramided transgenic lines were upregulated as compared to control tobacco plants. The accumulations of flavan-3-ols were also found to be higher in pyramided transgenic lines than control tobacco plants. In contrast, anthocyanin content was observed to be decreased in pyramided transgenic lines, while DPPH activity was higher in pyramided transgenic lines. In pyramided transgenic lines, strong protective ability against feeding by Spodoptera litura was documented. The seeds of pyramided transgenic lines were also found to have better germination rate under aluminum toxicity as compared to control tobacco plants. Interestingly, the synergistic effect of these two selected genes are not beyond from transgenic lines expressing either CsDFR and CsANR alone as published earlier in terms of flavan-3-ols accumulation. However, the unique flower color and better seed germination rate are some interestingly comparable differences that were reported in pyramided lines in relation to individual transgenic plants. In conclusion, the present results reveal an interesting dynamic between CsDFR and CsANR in modulating flavan-3-ol/anthocyanin levels and functional analysis of stacked CsDFR and CsANR transgenic tobacco lines.

Nanocomposites of plant cellulose nanocrystals (CNCs) were developed by binding model proteins BSA and HSA onto CNCs by physical adsorption and chemical conjugation methods The spectroscopy and microscopy studies confirmed the protein binding onto CNCs. Phosphate buffer saline (pH=4.0, 7.4) and simulated gastric and intestinal fluids (SGF/SIF; pH=1.1/6.5) showed maximum protein release of ∼62% over a period of time. The released proteins were found to retain both structural integrity as well as≥90% of bioactivity. Further, these cytocompatible nanocomposites showed ∼58-85% cholesterol release from HUVEC whereas no selectivity was observed for HCAEC. It is speculated that due to the presence of combination of shuttles (albumins) and sinks (CNCs and albumins), these prepared nanocomposites with increased cholesterol effluxing ability may serve as a potential candidate for future biomedical applications in pharmaceuticals.

Purple coloured tea shoot clones have gained interest due to high content of anthocyanins in addition to catechins. Transcript expression of genes encoding anthocyanidin reductase (ANR), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), flavonol synthase (FLS) and leucoantho cyanidin reductase (LAR) enzymes in three new purple shoot tea clones compared with normal tea clone showed higher expression of CsDFR, CsANR, CsANS and lower expression of CsFLS and CsLAR in purple shoot clones compared to normal clone. Expression pattern supported high content of anthocyanins in purple tea. Four anthocyanins (AN1–4) were isolated and characterized by UPLC-ESI-QToF-MS/MS from IHBT 269 clone which recorded highest total anthocyanins content. Cyanidin-3-O-β-d-(6-(E)-coumaroyl) glucopyranoside (AN2) showed highest in vitro antioxidant activity (IC50 DPPH = 25.27 ± 0.02 μg/mL and IC50 ABTS = 10.71 ± 0.01 μg/mL). Anticancer and immunostimulatory activities of cyanidin-3-glucoside (AN1), cyanidin-3-O-β-d-(6-(E)-coumaroyl) glucopyranoside (AN2), delphinidin-3-O-β-d-(6-(E)-coumaroyl) glucopyranoside (AN3), cyanidin-3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-glucopyranoside (AN4) and crude anthocyanin extract (AN5) showed high therapeutic perspective. Anthocyanins AN1–4 and crude extract AN5 showed cytotoxicity on C-6 cancer cells and high relative fluorescence units (RFU) at 200 μg/mL suggesting promising apoptosis induction activity as well as influential immunostimulatory potential. Observations demonstrate potential of purple anthocyanins enriched tea clone for exploitation as a nutraceutical product.

In diabetes, hyperglycemic state immensely hinders the wound healing. Here, nanobiocomposites (NCs) developed by impregnation of in situ prepared silver nanoparticles in the matrix of bamboo cellulose nanocrystals were investigated for their ability to hasten the progress of healing events in streptozotocin induced diabetic mice model. Wounds treated with topically applied NCs (hydrogels) showed full recovery (98-100%) within 18days post wounding in contrast to the various control groups where incomplete healing (88-92%) was noticed. Biochemical estimations documented a marked decrease in the levels of pro-inflammatory cytokines IL-6 and TNF-α leading to decreased inflammation in NCs treated mice. Significantly increased expression of collagen and growth factors (FGF, PDGF, VEGF) upon NCs treatment resulted in improved re-epithelialization, vasculogenesis and collagen deposition as compared to control groups. Hence, developed nanobiocomposites showcased potential to serve as highly effective and biocompatible wound dressings for diabetic patients.

This work deals with the development of an improved process for xylitol production from corn cob hydrolysate by biotechnological routes emphasizing the detoxification of corncob acid hydrolysate. The acid hydrolysate obtained by acid hydrolysis of corn cob was concentrated and detoxified by activated charcoal, membrane process and ion exchange resin process. The resultant partially purified corncob hydrolysate was used in fermentation. The fermentation of acid hydrolysate containing 56.5 g/L xylose was carried out in a 14 L fermenter at pH 4.5 for 48 h with 150 rpm stirring rate at 30 °C. A xylitol yield of 62% was achieved from the partially purified acid hydrolysate medium during fermentation using Candida tropicalis MTCC 6192. The purity of xylitol was increased to 92–94% upon downstream processing of carbonation, subsequently ion exchange process and activated charcoal.

Recently a novel approach has been developed for the synthesis of biodegradable nanoparticles (NPs) using plant leaf extracts. Following this approach, quercetin loaded poly lactic acid (PLA) NPs have been synthesised using Syzygium cumini leaf extract as surfactant/stabiliser. In the current study, developed biocompatible nanoformulation of PLA-quercetin was used for the first time to see its effect on shelf life of perishable fruits/vegetables like tomato. Nanoformulation treated tomatoes were evaluated for their total weight loss, total phenolic content, lycopene content, total sugar content, and quercetin content in comparison to untreated tomatoes. Developed nanoformulation was found to be effective in extending the shelf life of tomatoes by at least 10 d at room temperature relative to untreated and pure quercetin treated controls. Importantly, nutritional quality parameters were also maintained along with shelf life of tomatoes upon treatment with nanoformulation. Hence, the PLA-quercetin nanoformulation would be very useful to improve the shelf life of various perishable fruits and vegetables.

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.

Three protocols viz. Trichloroacetic Acid (TCA)-ace tone, phenol and multi-detergent were compared for the extraction of proteins from horsegram ( Macrotyloma uniflorum ) which is less studied yet nutritionally and economically very important pulse crop. Almost equal quantity of proteins was extracted by all the three protocols. However, qual ity of protein from phenol method was superior to that from other protocols. Bands in One-Dimensional (1-D) and Two-Dimensional Electrophoresis (2DE) of phenol protocol were reproducible and better . We report here a modified and common protocol for the extraction of proteins for proteomic analys is. The developed protocol is applicable not only t o other economically important leguminous but also no n-leguminous crops.

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.

Flavonoids are widespread throughout the plant kingdom and present in different parts of plants. Tea (Camellia sinensis) is well known for very high content of flavonoids especially flavan-3-ols antioxidants and is an aluminium (Al) accumulator plant. Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be regulatory enzymes of flavonoid biosynthetic pathway. In this study, cDNA encoding DFR (CsDFR) and ANR (CsANR) from tea were overexpressed individually in tobacco to check their influence on accumulation of flavonoid contents and antioxidant potential in roots of transgenic tobacco. Root morphological features, such as total volume and the number of lateral roots were improved in CsDFR and CsANR overexpressing tobacco plants relative to control tobacco plants. Both types of transgenic showed higher content of flavonoids and proanthocyanidins and lower content of anthocyanins in the roots compared to roots of control tobacco. Among flavan-3-ols, only epigallocatechin was observed in the roots and its content was higher in CsDFR and CsANR overexpressing tobacco as compared to control tobacco. Expression of genes encoding various other enzymes of flavonoid pathway like Phenylalanine ammonia-lyase, Chalcone isomerase, Flavanol synthase and Anthocyanin synthase was increased in roots of CsDFR and CsANR overexpressing tobacco plants as compared to control tobacco. The antioxidant potential of root portion of CsDFR and CsANR transgenic tobacco plants was found to be increased as indicated by enhanced total free radical scavenging activity and tolerance against Al toxicity. Taken together, these changes in roots of CsDFR and CsANR transgenic tobacco provided tolerance to aluminum toxicity.

An innovative approach was adopted where in situ synthesized silver nanoparticles (AgNPs) from leaf extract mediated reduction of AgNO3 were simultaneously impregnated into the matrix of cellulose nanocrystals (CNCs) isolated from Dendrocalamus hamiltonii and Bambusa bambos leaves, for formation of nanobiocomposites (NCs) in film and ointment forms. Here, use of plant CNCs was chosen as an alternate to bacterial cellulose for wound dressings. NCs possessing water absorption capacity and strong antibacterial activity showed synergistic effect on in vivo skin wound healing and documented faster and significant wound closure in treated mice. NCs exhibited lesser inflammation and early vasculogenesis at day 3 coupled with increased fibroblasts and collagen content at day 8 leading to faster neo-epithelization by day 14. Highly effective, biocompatible, and easy to apply NCs wound dressings (ointment and films) containing low amounts of Ag (0.05±0.01wt%) are potential candidates for effective skin repair.

The aim of this study was to evaluate the effect of flavonoids quercetin and epicatechin on the transcript expression and activity of antioxidant enzymes. Tobacco seedlings were exposed to 50 and 100 µM quercetin and epicatechin. The transcript expression level and the activity analysis of various antioxidant enzymes were monitored in their root and shoot. Interestingly, 50 µM epicatechin and 100 µM quercetin exposures were found to increase the expression of genes encoding antioxidant enzymes in shoot. In tobacco root, only GST and GPx expression were increased with 50 µM epicatechin and 100 µM quercetin exposures. Activity assay of all the enzymes showed similar trend to that of the transcript expression in shoot tissue. While in root, except CAT and SOD other enzymes activity also showed similar trend to that of expression pattern. Results have suggested the possible regulation of antioxidant enzymes by these two flavonoids at transcriptional and post-transcriptional level. Additionally, appropriate levels of such flavonoids seem to be essential for such regulations.

Stevia rebaudiana, a perennial herb from the Asteraceae family, is known to the scientific world for its sweetness and steviol glycosides (SGs). SGs are the secondary metabolites responsible for the sweetness of Stevia. They are synthesized by SG biosynthesis pathway operating in the leaves. Most of the genes encoding the enzymes of this pathway have been cloned and characterized from Stevia. Out of various SGs, stevioside and rebaudioside A are the major metabolites. SGs including stevioside have also been synthesized by enzymes and microbial agents. These are non-mutagenic, non-toxic, antimicrobial, and do not show any remarkable side-effects upon consumption. Stevioside has many medical applications and its role against diabetes is most important. SGs have made Stevia an important part of the medicinal world as well as the food and beverage industry. This article presents an overview on Stevia and the importance of SGs.

Anthocyanins and flavan-3-ols are distributed widely in plants and synthesized by a common biosynthetic pathway. Anthocyanin reductase (ANR) represents branching-point enzyme of this pathway converting anthocyanidins to flavan-3-ols. Since tea contains highest amount of flavonoids, a cDNA encoding anthocyanin reductase from tea (CsANR) was overexpressed in transgenic tobacco to check the influence on anthocyanin and flavan-3-ols. The transgenic tobacco was confirmed by genomic PCR and expression of transgene was analyzed through semiquantitative PCR. Interestingly flowers of transgenic tobacco were light pink/white in color instead of dark pink in wild tobacco, documenting the decrease in anthocyanins content. Upon measurement, flower anthocyanin content was found to be lesser. While flavan-3-ols (epicatechin and epigallocatechin) contents were increased in leaf tissue of transgenic lines. The expressions of other endogenous flavonoid biosynthetic pathway genes in different floral parts (sepal, petal, stamen, and carpel) of CsANR overexpressing tobacco as well as wild tobacco were analyzed. The transcript levels of PAL and CHI genes were downregulated, while transcript levels of F3H, FLS, CHS, ANR1, and ANR2 genes were upregulated in all floral parts of CsANR transgenic plants compared to wild tobacco. The expressions of DFR and ANS genes were also spatially modulated in different floral parts due to overexpression of CsANR. Thus, CsANR overexpression increased flavan-3-ols and decreased anthocyanin content by modulating the expressions of various flavonoid biosynthetic pathway genes in flower of tobacco. These changes might be responsible for the observed pollen tube in the pollens of CsANR overexpressing transgenic tobacco when they were still in the anther before pollination.

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 lm) 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.

Tea is a product made up from topmost part of the plant Camellia sinensis. This part includes bud, first leaf (next to bud), second leaf, and stem (spanning from bud to second leaf). Tea is the second most consumed beverage in the world, well ahead of coffee, beer, wine, and carbonated soft drinks. Clinical studies have demonstrated that one of the harmful effects of tea over consumption, at least in sensitive peoples is due to its caffeine content. In view of this, major points discussed in this article are the following: i) a brief overview on tea and its biochemical composition, ii) health effects of tea drink, iii) caffeine metabolism and its functions, iv) possible strategies for caffeine reduction in tea, and v) feasibility of tea improvement through biotechnological approaches.

The response of Camellia sinensis (L.) O. Kuntze cultivars Chinary and Assamica to Cu and Al stresses was investigated. Exposure to 100 µM CuSO4 or 100 µM AlCl3 led to accumulation of reactive oxygen species (ROS) more in Assamica than in Chinary. Proline content was higher in Chinary compared to Assamica, while chlorophyll and protein contents decreased upon Cu and Al exposure in both the cultivars. Expression of glutathione biosynthetic enzymes γ-glutamylcysteinyl synthetase (γ-ECS) and glutathione synthetase (GSHS) was elevated. Phytochelatin synthase (PCS), an enzyme involved in phytochelatins synthesis by using glutathione as a substrate was up-regulated at its transcript level more in Chinary than in Assamica. These results suggest that Chinary could be more tolerant than Assamica.

Adverse effect of caffeine consumption has been well documented in animals and in human beings. However, here we studied the influence of caffeine exposure on seedling growth of Arabidopsis and tobacco plants. Retardation in the seedling growth of these plants was observed when grown on MS medium plates containing 1 mM caffeine and their growth retarded further upon increasing the concentration of caffeine to 5 mM. Retardation in seedling size including both root and shoot size, yellowing and decrease in chlorophyll content of seedlings upon caffeine treatment indicated that caffeine exposure induced early senescence in plants. Therefore, the influence of caffeine exposure on transcript expression and activity of Rubisco in tobacco and Arabidopsis seedlings was monitored. Caffeine exposure has been found to decrease the expression and activity of Rubisco in both the plants. Hence, this study documents that caffeine exposure retarded seedling growth and one reason for this could be its negative effect on Rubisco.

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.

Glutamine synthetase is a very important enzyme of ammonium assimilation in plants. Here, we report on the regulation of a cytosolic glutamine synthetase (CsGS) from Camellia sinensis (L.) O. Kuntze during developmental stages and light/dark conditions on the utilization of nitrate and ammonia. The CsGS expression levels decreased during dormancy compared to non-dormancy phase of growth. Different leaf positions present different ages of the leaf and CsGS expression level was highest in apical bud (youngest leaf) and lower in mature fourth leaf, suggesting transcriptional regulation of CsGS during developmental stages. The CsGS enzyme activity showed similar trend to that of expression during developmental stages. The nitrate, ammonium and total amino acid contents were increased upon exposure to both N-sources during light and dark conditions. During light conditions, expression of CsGS in apical bud increased upon exposure to both N-sources nitrate and ammonium. While during dark conditions expression was increased only by ammonium and nitrate had no influence. Exposure to both N-sources also showed enhancing effect on CsGS enzyme activity during light. Under dark, ammonium application increased CsGS enzyme activity and nitrate had inhibitory effect on the activity. Results suggest the transcriptional regulation of CsGS on N-utilization during light/dark conditions.

Tea leaves are major source of catechins—antioxidant flavonoids. Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is one of the important enzymes that catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key “late” step in the biosynthesis of catechins. This manuscript reports characterization of DFR from tea (CsDFR) that comprised 1,413 bp full-length cDNA with ORF of 1,044 bp (115–1,158) and encoding a protein of 347 amino acids. Sequence comparison of CsDFR with earlier reported DFR sequences in a database indicated conservation of 69–87% among amino acid residues. In silico analysis revealed CsDFR to be a membrane-localized protein with a domain (between 16 and 218 amino acids) resembling the NAD-dependent epimerase/dehydratase family. The theoretical molecular weight and isoelectric point of the deduced amino sequence of CsDFR were 38.67 kDa and 6.22, respectively. Upon expression of CsDFR in E. coli, recombinant protein was found to be functional and showed specific activity of 42.85 nmol min−1 mg protein−1. Expression of CsDFR was maximum in younger rather than older leaves. Expression was down-regulated in response to drought stress and abscisic acid, unaffected by gibberellic acid treatment, but up-regulated in response to wounding, with concomitant modulation of catechins content. This is the first report of functionality of recombinant CsDFR and its expression in tea.

The aim of this study was to monitor the influence of proline and betaine exposure on antioxidant and methylglyoxal (MG) detoxification system during cold stress in Camellia sinensis (L.) O. Kuntze. Cold stress enhanced MG and lipid peroxidation levels in tea bud (youngest topmost leaf). This increase was resisted upon the exposure of tea bud to proline and betaine. Exposure of tea bud with proline and betaine also help in maintaining thiol/disulfide ratio during cold stress. Proline exposure enhanced glutathione-S-transferase and glutathione reductase (GR) activity, while betaine exposure increased only GR activity during cold stress. Furthermore, effect of proline/betaine was studied on glyoxalase pathway enzymes that are involved in MG detoxification and comprise of two enzymes glyoxalase I and glyoxalase II. Both proline and betaine showed protective effect on glyoxalase I and activating effect on glyoxalase II during cold stress in tea bud. This investigation, therefore, suggest that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.

Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan 3-ols or catechins. Apart from being responsible for tea quality, these compounds have medicinal properties. Flavanone 3-hydroxylase (F3H) is an abundant enzyme in tea leaves that catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. We report a full-length cDNA sequence of F3H from tea (CsF3H Accession no. AY641730). CsF3H comprised 1365 bp with an open reading frame of 1107 nt (from 43 to 1149) encoding a polypeptide of 368 amino acids. Expression of CsF3H in an expression vector in Escherichia coli yielded a functional protein with a specific activity of 32 nmol min(-1) mg protein(-1). There was a positive correlation between the concentration of catechins and CsF3H expression in leaves of different developmental stages. CsF3H expression was down-regulated in response to drought, abscisic acid and gibberellic acid treatment, but up-regulated in response to wounding. The concentration of catechins paralleled the expression data. Exposure of tea shoots to 50-100 microM catechins led to down-regulation of CsF3H expression suggesting substrate mediated feedback regulation of the gene. The strong correlation between the concentration of catechins and CsF3H expression indicates a critical role of F3H in catechin biosynthesis.

Glutamine synthetase (GS) showed highest expression and activity in bud (youngest topmost leaf) of Camellia sinensis, lower in older leaves, while lowest activity in stem and roots. GS expression and activity was increased by ammonium and nitrate and also by cadmium and salt stress but decreased by copper, aluminum, drought, cold and heat stress.

Glyoxalase II, one of the enzymes of the glyoxalase pathway, cDNA cloned from rice (OsglyII) consists of 1623 nucleotides with an open reading frame of 1010 bp encoding a polypeptide of 336 amino acids and an estimated isoelectric point of 8.08. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of approximately 37 kDa. Catalytic parameters of the protein were determined using S-D-lactoylglutathione as a thioester substrate. The K(m) (61 microM) and K(cat) (301 s(-1)) values were lower than those reported for Arabidopsis, human and yeast and showed pH optima at 7.2. The E. coli overexpressing OsglyII were able to grow on higher concentration of methylglyoxal. Transcript analysis in rice showed that OsglyII gene expression is stimulated within 15 min in response to various abiotic stresses as well as treatment with abscisic acid or salicylic acid. This multistress response of OsglyII gene documents its future utility in developing tolerance to various stresses in crop plants.

Glutathione, a tripeptide with sulfhydryl (-SH) group is a very crucial compound primarily involved in redox balance maintenance of the cellular environment. In this study, we monitored the influence of Cd exposure on the transcript levels of glutathione metabolic genes in bud tissues, the youngest leaf, of Camellia sinensis L. In addition, some physiochemical parameters were also studied. Cd exposure decreased chlorophyll and protein contents, while increase was observed in lipid peroxidation upon Cd treatments. These changes were found to be concentration and duration dependent, indicating the occurrence of oxidative stress upon Cd exposure. The transcript levels of glutathione biosynthetic genes viz. �- glutamylcysteine synthetase (�-ECS) and glutathione synthetase (GSHS) increased upon Cd exposure. Furthermore, transcript levels of glutathione reductase (GR), an enzyme involved in reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), also showed upregulation on Cd exposure. However, the transcript levels of glutathione-S-transferase (GST), an enzyme involved in forming metal–GSH complex and help in sequestration of high levels of metal ions to vacuole, did not show any change on Cd treatment. This study document that Cd exposure induces oxidative stress in Camellia sinensis and the upregulation in transcript levels of glutathione metabolic genes except GST have suggested the role of these enzymes in the protection of plants from high level Cd exposure. # 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 368–374, 2007.

This paper documents the engineering of Arabidopsis thaliana for the ectopic over-expression of SrKA13H (ent-kaurenoic acid-13 hydroxylase) cDNA from Stevia rebaudiana. HPLC analysis revealed the significant accumulation of steviol (1-3 μg g(-1) DW) in two independent transgenic Arabidopsis lines over-expressing SrKA13H compared with the control. Independent of the steviol concentrations detected, both transgenic lines showed similar reductions in endogenous bioactive gibberellins (GA1 and GA4). They possessed phenotypic similarity to gibberellin-deficient mutants. The reduction in endogenous gibberellin content was found to be responsible for dwarfism in the transgenics. The exogenous application of GA3 could rescue the transgenics from dwarfism. The hypocotyl, rosette area, and stem length were all considerably reduced in the transgenics. A noteworthy decrease in pollen viability was noticed and, similarly, a retardation of 60-80% in pollen germination rate was observed. The exogenous application of steviol (0.2, 0.5, and 1.0 μg ml(-1)) did not influence pollen germination efficiency. This has suggested that in planta formation of steviol was not responsible for the observed changes in transgenic Arabidopsis. Further, the seed yield of the transgenics was reduced by 24-48%. Hence, this study reports for the first time that over-expression of SrKA13H cDNA in Arabidopsis has diverted the gibberellin biosynthetic route towards steviol biosynthesis. The Arabidopsis transgenics showed a significant reduction in endogenous gibberellins that might be responsible for the dwarfism, and the abnormal behaviour of pollen germination and seed set.

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.

Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61–67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.

Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

PLA nanoparticles (NPs) were prepared via green route using turmeric (Curcuma longa) extract (TE) as biostabiliser/biosurfactant. Of the 29 formulations, two formulations of TE synthesized PLA NPs were evaluated for encapsulation and controlled release of well known antioxidant and less bioavailable molecules curcumin and quercetin. Size of curcumin loaded PLA NPs synthesized using 0.8 mg/ml PLA (C-En-D) and 0.1 mg/ml PLA (C-En-P) were 203±77 and 110±44 nm, respectively. However, quercetin loaded PLA NPs synthesized at 0.8 mg/ml (Q-En-D) and 0.1mg/ml (Q-En-P) PLA concentrations were 170±95 and 102±31 nm, respectively. The encapsulation efficiency of curcumin loaded PLA (C-En-D and C-En-P) NPs as well as quercetin loaded PLA (Q-En-D and Q-En-P) NPs was found ∼95%. In vitro release study of C-En-D, C-En-P, Q-En-D and Q-En-P NPs showed initial burst release followed by slow and sustained release. C-En-D NPs and Q-En-D NPs showing maximum in vitro release (∼100%) were evaluated for cytotoxicity. Blank PLA NPs, C-En-D and Q-En-D NPs were found to be safe against normal human leukocytes up to 2 mg/ml dose. Both C-En-D and Q-En-D NPs showed anticancer activity against A549 cell line. But Q-En-D NPs showed better anticancer activity than C-En-D NPS on A549 cells. While blank PLA NPs did not possess anticancer activity. TE extract stabilized PLA NPs were non-toxic, biocompatible and safe to normal human leukocytes. Such technology will be better, effective and safer in use for anticancer as well as other biological application.

BackgroundSteviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins.Methodology/principal findingsRNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes.ConclusionsSrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll.

Steviol glycoside and gibberellin biosynthetic routes are known as divergent branches of a common origin in Stevia. A UDP-glycosyltransferase encoded by SrUGT74G1 catalyses the conversion of steviolbioside into stevioside in Stevia rebaudiana leaves. In the present study, transgenic Arabidopsis thaliana overexpressing SrUGT74G1 cDNA from Stevia were developed to check the probability of stevioside biosynthesis in them. However, stevioside accumulation was not evident in transgenics. Also, the transgenic Arabidopsis showed no change in GA3 content on SrUGT74G1 overexpression. Surprisingly, significant accumulation of catechin was noticed in transgenics. The transgenics showed a considerable increase in shoot length, root length and rosette area. An increase in free radical scavenging activity of transgenics was noticed. Moreover, the seed yield of transgenics was also increased by 6–15 % than control. Additionally, variation in trichome branching pattern on leaf surface of transgenics was observed. The trichome branching pattern was also validated by exogenous catechin exposure (10, 50, 100 ng ml−1) to control plants. Hence, present study reports the probable role of SrUGT74G1 from Stevia in catechin accumulation of transgenic Arabidopsis thaliana. Thus, detailed study in present perspective has revealed the role of Stevia SrUGT74G1 gene in trichome branching pattern, improved vegetative growth, scavenging potential and seed yield by catechin accumulation in transgenic Arabidopsis.