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Highlight AtROS1 overexpression increases demethylation levels of promoters and coding regions of genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways to provide salt stress tolerance in transgenic tobacco., In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants.

In this study, three plants Populus alba, Hibiscus arboreus and Lantana camara were explored for the synthesis of silver nanoparticles (SNPs). The effect of reaction temperature and leaf extract (LE) concentration of P. alba, H. arboreus and L. camara was evaluated on the synthesis and size of SNPs. The SNPs were characterised by ultra-violet-visible spectroscopy, scanning electron microscopy and atomic force microscopy. The synthesis rate of SNPs was highest with LE of L. camara followed by H. arboreus and P. alba under similar conditions. L. camara LE showed maximum potential of smaller size SNPs synthesis, whereas bigger particles were formed by H. arboreous LE. The size and shape of L. camara LE synthesised SNPs were analysed by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX). TEM analysis revealed the formation of SNPs of average size 17 ± 9.5 nm with 5% LE of L. camara. The SNPs synthesised by LE of L. camara showed strong antibacterial activity against Escherichia coli. The results document that desired size SNPs can be synthesised using these plant LEs at a particular temperature for applications in the biomedical field.

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.

The ever increasing incidences of non-healing skin wounds have paved way for many efforts on the convoluted process of wound healing. Unfortunately, the lack of relevance and success of modern wound dressings in healing of acute and diabetic wounds still remains a matter of huge concern. Here, an in situ three step approach was embraced for the development of nanocomposite (NCs) dressings by impregnating silver nanoparticles (AgNPs) onto a matrix of cellulose nanocrystals (CNCs) isolated from Syzygium cumini leaves using an environmental friendly approach. Topical application of NCs (ointments and strips) on acute and diabetic wounds of mice documented enhanced tissue repair (~99% wound closure) via decrease in inflammation; increase in angiogenesis, collagen deposition, and rate of neo-epithelialization that ultimately led to formation of aesthetically sound skin in lesser time than controls. Due to the synergistic action of CNCs (having high water uptake capacity) and AgNPs (anti-microbial agents), NCs tend to increase the expression of essential growth factors (FGF, PDGF and VEGF) and collagen while decreasing the pro-inflammatory factors (IL-6 and TNF-α) at the same time, thus accelerating healing. The results suggested the potential of these developed anti-microbial, cytocompatible and nanoporous NCs having optimized AgNPs concentration as ideal dressings for effective wound management.

This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6–8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

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.

Nanotechnology has now reached to a stage where the nanoparticles (NPs) have been in applicability in wide-ranging realms of science and technology. NPs are the materials with at least one dimension in the order of 100 nm or less. NPs display astonishing properties of high surface/volume ratio and enhanced physical, chemical, optical, and thermal properties which are extremely different than their bulk materials. The conventional methods of synthesis of nanocompounds involve the employment of physical and chemical methods, which have few drawbacks such as the requirement of toxic hazardous chemicals, energy intensive, and costly processes make it difficult to be widely implemented. To overcome these limitations, the researchers have looked forward for an easy and feasible alternative approach for the synthesis of nanocompounds. The employment of alternative biogenic route for the NP synthesis by using biological entities of unicellular living organisms such as bacteria, fungi, and actinomycetes has sought apparent attention of the scientists throughout the global earth. A greener approach interconnecting nanobiology with microbial biotechnology is responsible for the formation of NPs mediated by microbes that allow synthesis in aqueous environment, with low energy consumption and at low costs. Biosynthesis of gold, silver, copper, quantum dots, and magnetite NPs by bacteria, fungi, actinomycetes, and yeasts has been reported. In a view to form noble metal NPs of uniform shape and size, biological routes using microbial cultures at optimal temperature, pressure, and pH have been formulated. In this chapter, the main focus is given on the intracellular and extracellular approaches used for synthesis of metallic NPs by various microbial species. A detailed discussion is provided to explain the various factors which affect the synthesis of nanocompounds to further augment the growth rate of NPs as well as the mechanism of action at the cellular, biochemical, and molecular level. A great stress is given on the role of these nanocompounds in the medical field for the diagnostic and disease treatment. The potential of great biodiversity of microbial cultures as biological candidates leading to the manufacturing of NPs is needed to be fully investigated.

Nanotechnology is one of the most promising tools for the current revolution in agri-food sector. Nanotechnology approaches provide novel and innovative ways to improve crop yield and to reduce the use of harmful crop protection agents. Nanotechnology can be useful for biotic and abiotic stress tolerance of plants, lesser use of pesticides/insecticides/herbicides, nanosensors for crop protection from bacteria, disease treatment, pest control management and genetic manipulations by nano delivery vehicles. Also, nanotechnology has great advantages in food sector like detection of pesticides/toxicants in food items, development of food storage and packaging materials, enhancement of shelf life of foods, nanoencapsulation of food nutraceuticals or bioactive molecules, detection of pathogens in food materials, enhancement of food taste, color and odour. Above all, safety concerns regarding the use of nanomaterials should be of priority before their exploration for positive impact on agriculture and food.

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.

Tea quality and yield is influenced by various factors including developmental tissue, seasonal variation and cultivar type. Here, the molecular basis of these factors was investigated in three tea cultivars namely, Him Sphurti (H), TV23 (T), and UPASI-9 (U) using RNA-seq. Seasonal variation in these cultivars was studied during active (A), mid-dormant (MD), dormant (D) and mid-active (MA) stages in two developmental tissues viz. young and old leaf. Development appears to affect gene expression more than the seasonal variation and cultivar types. Further, detailed transcript and metabolite profiling has identified genes such as F3′H, F3′5′H, FLS, DFR, LAR, ANR and ANS of catechin biosynthesis, while MXMT, SAMS, TCS and XDH of caffeine biosynthesis/catabolism as key regulators during development and seasonal variation among three different tea cultivars. In addition, expression analysis of genes related to phytohormones such as ABA, GA, ethylene and auxin has suggested their role in developmental tissues during seasonal variation in tea cultivars. Moreover, differential expression of genes involved in histone and DNA modification further suggests role of epigenetic mechanism in coordinating global gene expression during developmental and seasonal variation in tea. Our findings provide insights into global transcriptional reprogramming associated with development and seasonal variation in tea.

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.

Inspired by the availability of de novo transcriptome of horse gram (Macrotyloma uniflorum) and recent developments in systems biology studies, the first ever global protein-protein interactome (PPI) map was constructed for this highly drought-tolerant legume. Large-scale studies of PPIs and the constructed database would provide rationale behind the interplay at cascading translational levels for drought stress-adaptive mechanisms in horse gram. Using a bidirectional approach (interolog and domain-based), a high-confidence interactome map and database for horse gram was constructed. Available transcriptomic information for shoot and root tissues of a sensitive (M-191; genotype 1) and a drought-tolerant (M-249; genotype 2) genotype of horse gram was utilized to draw comparative PPI subnetworks under drought stress. High-confidence 6804 interactions were predicted among 1812 proteins covering about one-fourth of the horse gram proteome. The highest number of interactions (33.86%) in horse gram interactome matched with Arabidopsis PPI data. The top five hub nodes mostly included ubiquitin and heat-shock-related proteins. Higher numbers of PPIs were found to be responsive in shoot tissue (416) and root tissue (2228) of genotype 2 compared with shoot tissue (136) and root tissue (579) of genotype 1. Characterization of PPIs using gene ontology analysis revealed that kinase and transferase activities involved in signal transduction, cellular processes, nucleocytoplasmic transport, protein ubiquitination, and localization of molecules were most responsive to drought stress. Hence, these could be framed in stress adaptive mechanisms of horse gram. Being the first legume global PPI map, it would provide new insights into gene and protein regulatory networks for drought stress tolerance mechanisms in horse gram. Information compiled in the form of database (MauPIR) will provide the much needed high-confidence systems biology information for horse gram genes, proteins, and involved processes. This information would ease the effort and increase the efficacy for similar studies on other legumes. Public access is available at http://14.139.59.221/MauPIR/ .

Liposomes and phytosomes are kind of nanoparticles (NPs) which serve as an imperative tool for the delivery of various bioactive molecules. The molecules possessing lesser water solubility, bioavailability and retention time are encapsulated into both of these formulations. More specifically, liposomes can encapsulate both hydrophobic as well as hydrophilic molecules. While phytosomes contain only plant-based molecules having poor solubility in biological media like flavanones and terpenes. The various advantages of liposomes and phytosomes like biocompatibility, nontoxicity, ease to administer, decrease in dosage and increase in retention time make them potent vehicles for the drug delivery of various molecules. These nanoformulations find potential applications for delivery of various bioactive molecules, in tissue regeneration and as antimicrobials. This chapter highlights the importance of liposomes and phytosomes, methods of their preparation, mechanism of their action and applications.

The metallic nanoparticles (NPs) and their synthetic biology are an active area fascinating both the academics as well as scientific research applications in the field of nanotechnology. These nanostructures are a versatile class of materials such as metal NPs, metal oxide NPs, magnetic NPs and quantum dots for biomedical sciences and engineering due to their huge potential. A handful number of methods are adopted for the synthesis of one or the other kind of metal NPs which includes physical and chemical approaches. Each of the chemical and physical synthesis procedures deals with some limitations such as cost ineffectiveness, use of hazardous chemicals, formation of toxic end products and involvement of high-energy processes. To overcome these drawbacks, an alternative approach of environmentally benign and inexpensive biological synthesis mediated by plants or microbes has been essentially adopted. The variations in size, shape and surface chemistry of NPs affect the properties of metal NPs to a greater extent which in turn have an influence on their biological behaviour. Few metal NPs offer unique optical properties while others possess paramagnetic behaviour and quantum size effect which make them suitable in bio-imaging diagnostic techniques. Some metallic NPs play a role in tissue engineering and other therapeutic applications due to their ease of surface modifications, large surface area to volume ratio, unique electrical and anti-microbial activities. Instead of multi-disciplinary applications of metallic NPs, there is a great concern regarding the toxicity issues associated with the use of these NPs which need to be sorted out by looking out certain ways for favourable architecture involving the synthesis methods and parameters for designing the non-toxic NPs for improving the quality of life.

Nanoparticles (NPs) are being extensively used in the field of nanomedicines. Different types of NPs are administered into the body by various routes. NPs come in contact with cells inside the body. Cellular response of NPs is affected by size, shape, surface chemistry, and cellular uptake pathways of NPs. In addition to this, type of cells, various cell lines, and growth media are also found to affect the cellular response of NPs. NPs induce diverse cellular responses like apoptosis, necrosis, and reactive oxygen species (ROS) production. NPs also form a protein corona inside the biological media which may alter their identity and behaviour as compared to bare NPs. In this chapter, we have made an attempt to throw light on cellular uptake pathways of NPs, monitoring of endocytic pathways followed by NPs, factors affecting cellular responses of therapeutic NPs, and protein corona formation, characterisation and its implications on fate of NPs.

Biodegradable nanoparticles (BNPs) are seeking augmented attention for their ability to serve as a carrier for site-specific delivery of drugs, biomolecules, proteins and peptides in the body. They offer enhanced biocompatibility, better drug encapsulation and sustained release profile of a great number of drugs and biomolecules to be used in a variety of applications in nanomedicine. The use of BNPs for controlled drug delivery has shown significant therapeutic potential. Concurrently, targeted delivery technologies are becoming an increasingly important area of scientific investigation. The fate of BNPs is decided by virtue of their physiochemical properties in biological medium. Another important area which is gaining attention of researchers is the behaviour of BNPs in biological milieu. In the present chapter, efforts are made to give the readers a comprehensive outlook of the topic covering the following major points: (1) brief introduction to BNPs; (2) synthesis methodologies of BNPs; (3) characterisation of BNPs; (4) BNPs for drug delivery; (5) drug release mechanisms from BNPs; (6) targeted drug delivery using BNPs; (7) biological barriers encountered by BNPs; and (8) in vivo fate of BNPs.

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.

Here, Syzygium cumini leaf extract (LE) and seed extract (SE) were explored for the synthesis of gold nanoparticles (GNP). LE and SE as well as their polar (water) fractions showed potential for GNP synthesis. Comparative synthesis kinetics and morphological characterisation studies revealed the synthesis of smaller sized GNP by LE than SE. Only polar (water) fractions showed potential for GNP synthesis, which are smaller in size compared to their respective extracts. SE contained more polyphenols and biochemical constituents than LE and therefore, showed higher synthesis rate and bigger sized GNP. Atomic force microscope and scanning electron microscope analysis indicated that both extracts and their fractions catalysed the synthesis of spherical GNP. The average size of GNP synthesised by LE, leaf water fraction (LWF), SE and seed water fraction (SWF) were 24, 23, 35 and 32 nm, respectively. Fourier transform infrared analysis identified the biomolecules involved in the synthesis and stability of GNP. T...

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.

Small RNAs (sRNAs) are 18-30 nt non-coding regulatory elements found in diverse organisms, which were initially identified as small double-stranded RNAs in Caenorhabditis elegans. With the development of new and improved technologies, sRNAs have also been identified and characterized in plant systems. Among them, micro RNAs (miRNAs) and small interfering RNAs (siRNAs) are found to be very important riboregulators in plants. Various types of sRNAs differ in their mode of biogenesis and in their function of gene regulation. sRNAs are involved in gene regulation at both transcriptional and post-transcriptional levels. They are known to regulate growth and development of plants. Furthermore, sRNAs especially plant miRNAs have been found to be involved in various stress responses, such as oxidative, mineral nutrient deficiency, dehydration, and even mechanical stimulus. Therefore, in the present review, we focus on the current understanding of biogenesis and regulatory mechanisms of plant sRNAs and their responses to various abiotic stresses.

MicroRNAs (miRNAs) are a class of naturally occurring and small non-coding RNA molecules of about 21-25 nucleotides in length. Their main function is to downregulate gene expression in different manners like translational repression, mRNA cleavage and epigenetic modification. To predict new miRNAs in plants different computational approaches have been developed. In the present study, an EST based approach has been used to identify novel miRNAs in horsegram. Identification of miRNAs was initiated by mining the EST database available at NCBI. Total of 989 ESTs were obtained for the identification of miRNAs. These ESTs were subjected to CAP3 assembly to remove the redundancy. This resulted in an output of 72 contigs and 606 singletons as non redundant datasets. The miRNAs were then predicted by using miRNA-finder. A total of eight potential miRNAs were predicted and named as hor-miR1 to hor-miR8. None of identified miRNAs showed significant homology with the previously reported in plants and therefore should be considered novel. These miRNAs were inputted to miRU2 program to predict their targets. The target mRNAs for these miRNAs mainly belong to zinc finger, chromosome condensation, protein kinase, abscisic acid-responsive, calcineurin-like phosphoesterase, disease resistance and transcriptional factor family proteins. These targets appeared to be involved in plant growth and development and environmental stress responses.

BACKGROUND: Plant mediated synthesis of metallic nanoparticles has been studied and reported, however, to date, the biomolecules involved in the synthesis of metallic nanoparticles have not been characterized. This study was therefore undertaken to characterize the biomolecules of Syzygium cumini involved in the synthesis of silver nanoparticles. RESULTS: Synthesis kinetics and morphological characterization of silver nanoparticles (SNP) synthesized using leaf extract (LE) and seed extract (SE) as well as their polar (water) fractions from Syzygium cumini were compared. The polyphenols content and high performance liquid chromatography (HPLC) profile of different fractions revealed good correlation between size and synthesis rate of SNP. SE contains more polyphenols and biochemical constituents than LE and therefore, showed higher synthesis rate and bigger sized SNP. To analyse the nature of biomolecules involved in the synthesis of SNP, LE and SE were fractionated on a polarity basis by solvent–solvent partitioning. Only the water fractions of LE and SE showed potential for SNP synthesis. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis of SNP indicated that all fractions catalyze the synthesis of spherical nanoparticles. The average size of SNP synthesized by LE, leaf water fraction, SE and seed water fraction were 30, 29, 92, and 73 nm respectively. CONCLUSION: Results suggest that only highly polar soluble constituents are responsible for SNP synthesis. The size of SNP was found to be directly correlated with the amount of polyphenols as well as surfactants present in the reaction solution. Thus, the amount of polyphenols could be one of the crucial parameters determining the size and distribution of SNP. Copyright © 2010 Society of Chemical Industry

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.

RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5' overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5' overhang and the partnering between RDM12 and RDR2.

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.

Nanobiotechnology deals with the synthesis of nanostructures using living organisms. Among the use of living organisms for nanoparticle synthesis, plants have found application particularly in metal nanoparticle synthesis. Use of plants for synthesis of nanoparticles could be advantageous over other environmentally benign biological processes as this eliminates the elaborate process of maintaining cell cultures. Biosynthetic processes for nanoparticles would be more useful if nanoparticles were produced extracellularly using plants or their extracts and in a controlled manner according to their size, dispersity and shape. Plant use can also be suitably scaled up for large-scale synthesis of nanoparticles. In view of this, we have reviewed here the use of plants or their extracts in the synthesis of silver and gold nanoparticles for various human applications. Copyright © 2008 Society of Chemical Industry

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.