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Picrorhiza kurrooa Royle ex Benth., an important high altitude plant produces picrosides, the monoterpenoids of high therapeutic value. Production of geraniol in the iridoid branch of the picroside biosynthetic pathway is a key step in the biosynthesis of picrosides. Geraniol synthase, catalyzes the conversion of geranyl pyrophosphate into geraniol. In view of this, here we report the identification and sequence analysis of PkGES promoter. The PkGES promoter sequence was isolated following a directional genome walking approach. Presence of similar cis elements such as GT1 consensus elements, LTREs, DREs, WRKY binding elements, PREATPRODH motif, BIHD1OS motif, SEBFCONSSTPR10A motif and MeJA responsive elements in the promoter sequence of PkGES documented its multiple stress responsive characteristic signifying its role in adaptation. Further, PkGES showed differential expression pattern in leaves, rhizome and roots. To elucidate whether the differential expression pattern of PkGES correlates with its epigenetic regulation, DNA methylation analysis of promoter as well as coding region of PkGES was conducted. The DNA methylation levels in the promoter sequence of PkGES gene were found to be dominated at CG followed by CHH and CHG sequence contexts. Epigenetic regulation (DNA methylation) of promoter as well as coding region of PkGES suggested its role in the regulation of PkGES transcripts in diverse tissues. The present study has revealed the presence of several stress inducible cis elements in PkGES promoter sequence and its tissue specific epigenetic regulation in P. kurrooa.

The CRISPR–Cas9 is emerging genome editing tool and very easy and straightforward in operation that has been tested and explored for introduction of new traits in plant systems. Recently, a number of reports have documented utilization of this technology for providing tolerance against viral diseases mediated by begomoviruses. Begomoviruses infect dicot and are transmitted by white flies and cause devastating losses to yield of important agricultural crops including tomato, cassava and cotton. An overview of genomic structure of begomoviruses has been presented to understand the potential strategy for designing of effective sgRNAs to combat the viral replication for generating resistance against infection. This review provides the introduction, recent developments, and applications of the CRISPR–Cas9 system in plants and proposes a holistic methodology for generating cotton plant an example having resistance against begomoviruses. The genome editing using CRISPR–Cas9 system against complex of begomoviruses collectively termed as cotton leaf curl virus, which a major contributor to reduction of the cotton yield especially in Northern India and Pakistan is also discussed thoroughly. In conclusion, this potential strategy could be a sustainable approach for development of tolerant crops against diseases mediated by DNA viruses.

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.

With the advancement of biotechnological tools and techniques such as next generation sequencing, RNAomics, epigenomics, gene silencing, plant, microbe transformation, proteomics and metabolomics, the understanding of metabolic pathways and their manipulation for the desired characters became feasible. Metabolic engineering has been successful in the production of golden rice, bioprocess for artemisinin production, flavonoids in plant and microbes as well as generated biotic and abiotic stress tolerance in several crop plants. In view of the significance of metabolic engineering, this article includes recent techniques developed and their use in manipulation of glyoxalase metabolism for multiple abiotic stress tolerance in plants. The importance of engineering of flavonoids pathway for high value antioxidants production as well as improving the biotic and abiotic stress tolerance has been documented. Importance and success of metabolic engineering has been realized by its promising hope for sustainable technologies of bioactives production for mankind’s health as well as in the generation of improved crop varieties.

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323bp ORF encoding 441 amino acids protein with molecular weight 47.2kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1-21 amino acids, pre-peptide 22-143 amino acids, peptidase S8 domain 144-434 amino acids, and pro-peptide 435-441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed ∼50kDa and ∼40kDa bands, respectively. The pre-propeptide ∼11kDa removed from Alp1 resulted in mature protein of ∼35kDa with 1738Umg-1 specific activity. The recombinant protease was optimally active at 40°C and pH 9, and stable over 10-70°C and 6-12pH. The activity at low-temperature and alkaline pH was supported by high R/(R+K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry.

The necessity to develop new strategies for the control of Fusarium wilt of tomato signifies the identification of pathogencity genes and ascertaining their role to use them as molecular tools for fungicide development or to develop transgenics. Semi-quantitative gene expression studies have identified two pathogenicity genes, FOW2 and chsV, reported as ZnII)2Cys6-type transcription regulator and class V chitin synthase, respectively, as potential ones for being secreted all the time. The roles of these genes in the pathogenicity of Fusarium oxysporum and F. solani have been established by RNA interference (RNAi)-induced silencing (knockdown). The silencing vector encoding hairpin RNA of each of the gene fragment was constructed in a two-step PCR-based cloning, and introduced into the fungal genomic DNA. Silencing of either of the genes resulted in less virulent fungal phenotypes with altered physiological characteristics like sporulation and growth on solid media and a reduction in mRNA expression. The results therefore demonstrate the applicability of these pathogenicity genes as useful molecular targets for exploitation in Fusarium wilt control in tomato.

Flavonoids are main class of secondary metabolites and occur in different tissues and organs in diverse plant species. The higher accumulation of flavonoids in a wide variety of fruits and vegetables increases their economical value because flavonoids are good for human health. It has been established that flavonoids help the plants to protect against adverse environmental constraints and have not played a significant role in plant growth and development. A number of recent reports provided strong evidences in support of significant role of flavonoids in growth and development. The objective of this chapter is to provide an overview of the flavonoid biosynthetic pathway and review the significant contribution of flavonoids in growth and development of plants. This study provides an in-depth understanding of the role of flavonoids and is useful for further manipulation of flavonoids for growth and development of wide types of plant species.

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H2O2, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.

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.

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.

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.

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/ .

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.

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.

We report on gene silencing of glutathione synthetase (GSHS) that reduces reduced glutathione (GSH) content in somatic embryos of Camellia sinensis L. Using degenerate primers with cDNA of Camellia sinensis, a 457 bp GSHS gene fragment was cloned through polymerase chain reaction. This fragment was used in making ihpRNA. For this it was cloned in sense at AscI and SwaI and in anti-sense at Bam HI and XbaI restriction sites of pFGC5941 that has chalcone synthase (Chs) intron between SwaI and BamHI restriction sites. Resultant RNAi construct was used for C. sinensis somatic embryos transformation through Agrobacterium. After 11, 13 and 15 d of transformation, embryo GSHS transcript levels and GSH content decreased to a great extent which documented the feasibility of RNAi based gene silencing in C. sinensis.

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.

A cDNA encoding glutamine synthetase, one of the enzymes of the GS/GOGAT pathway, was cloned from Camellia sinensis (CsGS). The isolated cDNA consists of 1,071 nucleotides encoding a polypeptide of 356 amino acids with an estimated isoelectric point of 6.13. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of 39.2 kDa. The purified protein was confirmed by blotting with anti-His antibodies. Catalytic parameters of the protein were determined using glutamate and ATP as substrates. The observed Km was 9 mM and Vmax was 93 U/mg protein with glutamate as substrate, while with ATP Km and Vmax values were 6 mM and 70 U/mg protein, respectively. Purified enzyme showed pH optima at 8. Cations were found to be showing enhancing effect on the activity of GS enzyme and Mg2+ ion exhibited maximum enhancing effect among the various ions used in this study. This enzyme activity increased by 25% in presence of DTT and decreased by 18% when incubated with PMSF. Transcript analysis in tea bud, youngest leaf, showed that CsGS gene expression is stimulated in response to abscisic acid (ABA), salicylic acid (SA), and hydrogen peroxide (H2O2), while gibberellic acid (GA3) has no influence on its expression levels.

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.