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A hexagonal mesoporous molecular sieve-like structure of MCM41 and SBA15 with a large surface area was used to immobilize protein L-ribose isomerase (L-RI) through covalent linkages. The amino group of APTES functionalized nanosilica support MCM41 and SBA15 interacted with glutaraldehyde to promote bidentate linkage and on other side with amino group of enzyme. The use of mesoporous silica matrix for immobilization was observed to conserve the distinctive properties of the protein. The various operational conditions optimized for covalent conjugation of protein with the silica support were found to be dependent on enzyme support ratio, immobilization temperature and time. The immobilization yield of L-RI on MCM41 and SBA15 was achieved to be 60 % (600 mg enzyme /g matrix) and 45 % (450 mg enzyme/g matrix), respectively under the optimized conditions. The immobilized biocatalyst was characterized by various analytical techniques like HR-TEM, EDS, FTIR, TGA and BET. Effects of different experimental conditions were optimized to study enzyme kinetics, pH, temperature, bioconversion, reusability, metal ion effect and storage stability. The biocatalytic efficiency (k

The biocatalysts are broadly explored in the biological transformation processes. The enzyme cascade catalysis involves various catalytic activities in a sequential process to produce the desired product including the formation of reaction intermediates. Enzyme immobilization is a method in which enzymes are confined within a support or matrix either physically or chemically to enhance their relative stability and catalytic activity in the enzyme cascade catalysis. In view of this, L-arabinose isomerase (L-AI) and L-ribose isomerase (L-RI) were immobilized on zeolite based metal framework as a micro-composite construct (DEMC@L-AI+L-RI) using linker, and metal ions. Such immobilization could be of great significance and provide several advantages like mesoporous surface for enzyme adsorption, desirable functionality in the production of products in enzyme cascade reaction, high storage stability and enhanced recyclability. The developed DEMC@L-AI+L-RI was characterized using SEM, FTIR, CLSM and TGA. The immobilization yield was 32% and loading of enzyme was 22% on the surface of micro-composite. The DEMC@L-AI+L-RI showed relatively stable catalytic activity at pH 5-6 and temperature 40 °C. The catalytic efficiency (k

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.

A large number of computational tools have been documented in recent years for identification of target-specific valid single-guide (sg) RNAs (18–20 nucleotide long sequence) that is an important component for the efficient utilization of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats—CRISPR-associated Protein) system. Despite optimization of Cas9, other major concerns are on-target efficiency and off-target activity that depend upon the sequence(s) of target-specific sgRNA(s). However, a very little attention has been paid for identification of the best-hit sgRNA for precise targeting as well as minimizing the off-target effects. The aim of this present work is to offer comparative insight into existing CRISPR software tools with their unique features (including targeted genome) and utilities. These available web tools were found to be designed based upon only a few limited mathematical models. Among all these available web tools, three (Benchling, Desktop and CRISPR-P) have been curated as exclusively available for plant genome-editing purpose. These three software tools have been comprehensively described and analyzed with single same target enquiry from two randomly selected genes (IDM2 and IDM3 from Arabidopsis thaliana). Interestingly, all these selected tools generated different results (sgRNAs) even for the same query. In fact, the sequence of sgRNA is considered an important parameter to determine the efficiency and specificity of sgRNAs for precise genome editing. Thus, there is an urgent requirement to pay attention for a validated sgRNA-designing tool for precise DNA editing in plants. In conclusion, this work will encourage building up a consensus for developing a universal valid sgRNA designing for different organisms including plants.

The present study aims at optimisation of enhanced solute impregnation through ultrasonication (US) at 37 kHz using central composite design of response surface methodology (RSM). Black carrots were blanched at 98 °C/3 min followed by immersion in salt-spices based solutions for 8 h, treated with bath sonication at specific intervals (2, 46 h) for 5, 10 and 15 min. Responses for optimum water loss (WL), solute gain (SG), anthocyanins retention, texture and highest antioxidant activity were optimized in effect of NaCl%, spices mix% and US time. The suggested models were of good fit while lack of fit as non-significant (p 0.0500). Best combination was compared for anthocyanins retention on pressure-cooking (120 °C/15 psi) with fresh, blanched, salt-spiced without US subjected to hot-air convective drying (55 °C/5 h). Ultrasonication-assisted salt-spiced convective dried (USPCD) carrots retained highest anthocyanin content (41%) post cooking which correlated with increased glass transition (T

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

Tomato processing industry generates huge waste like tomato skin, seed, and pulp which creates environmental issues. Since tomato pomace contains bioactive compounds and pigments, present study was conducted to investigate the effect of tomato pomace addition on physicochemical characteristics and shelf-life stability of the developed bread and muffin. Refined flour was partially substituted with 35 and 40% tomato pomace in bread and muffin, respectively. Tomato pomace addition in bread and muffin was observed to significantly (p

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

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.

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 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.

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.

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.

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...

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.

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.

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.

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.

With the advent of LC-MS, identification and characterization of compounds from plant origin has become very easy now a day. In this effort, several plant-based compounds have been identified possessing therapeutic propensities. This category includes cruciferous vegetables, garlic, ginger, onions, soybeans, and turmeric.

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.

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

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.

Nanoencapsulation of antioxidant molecules on protein nanoparticles (NPs) could be an advanced approach for providing stable, better food nutraceuticals and anticancer drugs. The bioavailability and stability of catechin (CAT) and epicatechin (ECAT) were very poor. In the present study, the CAT and ECAT were loaded on bovine serum albumin (BSA) NPs following desolvation method. The transmission electron microscope (TEM) and atomic force microscope (AFM) recorded size of CAT-BSA NPs and ECAT-BSA NPs were 45 ± 5 nm and 48 ± 5 nm respectively. The encapsulation efficiency of CAT and ECAT on BSA NPs was found to be 60.5 and 54.5 % respectively. CAT-BSA NPs and ECAT-BSA NPs show slow and sustained in vitro release. The CAT-BSA NPs and ECAT-BSA NPs were stable in solution at various temperatures 37°C, 47°C and 57°C. DPPH assay revealed that CAT and ECAT maintained their functional activity even after encapsulation on BSA NPs. Furthermore, the efficacy of CAT-BSA NPs and ECAT-BSA NPs determined against A549 cell lines was found to be improved. CAT and ECAT aptly encapsulated in BSA NPs, showed satisfactory sustained release, maintained antioxidant potential and found improved efficacy. This has thus suggested their more effective use in food and nutraceuticals as well as in medical field., EXCLI Journal ; Vol. 13, 2014

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

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

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