Identification of redox-regulated components of arsenate (As(V)) tolerance through thiourea supplementation in rice.

Arsenic (As) is a ubiquitously present environmental carcinogen that enters into the human food chain through rice grains. In our previous research, the application of thiourea (TU; a non-physiological thiol based ROS scavenger) has been demonstrated to enhance salt and UV stress tolerance as well as the crop yield under field conditions. These effects were associated with the ability of TU to maintain plant redox homeostasis. Since As stress also induces redox imbalance, the present research was initiated to evaluate the efficiency of TU in regulating As tolerance/accumulation in rice. The supplementation of TU (75 µM) to As(V) (25 µM) improved the root growth and also reduced the As concentration by 56% in the aerial parts, which could be attributed to significant downregulation of the Lsi2 transporter responsible for the translocation of As from root to shoot. The fact that these effects were not due to direct interaction between As and TU was confirmed from complexation studies using HPLC-(ICP-MS)-(ESI-MS). Short-term kinetic studies of GSH levels and the GSH/GSSG ratio confirmed the establishment of differential redox states in As and As + TU treated seedlings. The real-time RT-PCR based comparative expression profiling under As with/without TU treatment identified Sultr1;1 and Sultr1;2 as major redox-regulated sulfate transporters. Their specific induction in shoots coupled with enhanced root-to-shoot sulfate translocation (analyzed using (35)S-sulfate as a radiotracer) was observed under TU supplementation. Furthermore, the level of thiolic metabolites (PC2 in roots and GSH and PC3 in shoots) and activities of sulfur metabolism enzymes (ATP sulfurylase and cysteine synthase in roots and 5'-adenylylsulfate reductase in shoot) were also increased with As + TU as compared to As treatment. Thus, this study utilizes the interaction between As and TU to identify the critical redox regulated components of As tolerance in rice.

Biodegradation of tributyl phosphate using Klebsiella pneumoniae sp. S3.

Tributyl phosphate (TBP) has enormous applications in the field of extraction, fuel reprocessing, as defoamers and/or plasticizers. Excessive usage of this organophosphorus compound, poses an environmental threat. The present study deals with microbial degradation of TBP using Klebsiella pneumoniae S3 isolated from the soil. Diauxic growth curve pattern explains a preferential utilization of TBP. The strain S3 was able to biotransform TBP (1,000 mg L⁻¹) to dibutyl phosphate within 48 h and showed higher tolerance towards TBP up to 17.0 g L⁻¹. Toxicity of the parent as well as degraded product was assessed using comet assay. Generation of reactive oxygen species elaborates the oxidative stress imposed upon the bacterial strain by TBP. The antioxidant defense mechanism was studied using various biomarkers namely catalase, glutathione-S-transferase, and superoxide dismutase. The present study describes a faster and eco-friendly alternative for disposal of TBP.

Quantitative real-time expression profiling of aquaporins-isoforms and growth response of Brassica juncea under arsenite stress.

The present study analyzed the expression level of aquaporins of plasma membrane intrinsic protein (PIP) class in response to arsenite (AsIII) exposure of 100 µM from 0.5 h to 8 days in Brassica juncea. The expression levels of most of the PIPs were down-regulated during the course of AsIII exposure. This led to decrease in total water content of plants, which in turn hampered seedling growth. The level of reactive oxygen species (superoxide radicals and hydrogen peroxide), lipid peroxidation and root oxidizability increased significantly upon exposure to AsIII as compared to that of control leading to an increase in cell death. The study proposes that the down-regulation of PIPs happened presumably to regulate AsIII levels, which, however, occurred at the cost of reduced growth, disturbed water balance and induced oxidative stress.

Immobilization of the urease on eggshell membrane and its application in biosensor.

Eggshell membrane is a natural material, essentially made up of protein fibers having flexibility in the aqueous solution and possessing gas and water permeability. It is used as a biomembrane for immobilization of urease for the development of a potentiometric urea biosensor. Eggshell membrane was treated with polyethyleneimine (PEI) to impart polycation characteristics. Urease was immobilized on the PEI treated eggshell membrane through adsorption. SEM study was carried out to observe the changes in surface morphology after immobilization. FTIR study of membrane was carried out to observe the changes in IR spectra after immobilization of enzyme. Immobilized membrane was associated with ammonium ion selective electrode. Biosensor exhibited sigmoidal responses for the urea concentration range from 0.5 to 10mM. The response time of the biosensor was 120 s. A single membrane was reused for 270 reactions without loss of activity. The urease-eggshell membranes were stable for 2 months when stored in buffer even at room temperature.

Identification and profiling of arsenic stress-induced microRNAs in Brassica juncea.

MicroRNAs (miRNAs) constitute a novel mechanism of gene regulation affecting plant development, growth, and stress response. To study the role of miRNAs in arsenic (As) stress, microarray profiling of miRNAs was performed in Brassica juncea using a custom Phalanx Plant OneArray containing 381 unique miRNA probes representing 618 miRNAs from 22 plant species. miRNA microarray hybridization of roots exposed to As for 1h and 4h revealed that a total of 69 miRNAs belonging to 18 plant miRNA families had significantly altered expression. The As-responsive miRNAs also exhibited a time- and organ-dependent change in their expression. Putative target prediction for the miRNAs suggested that they regulate various developmental processes (e.g. miR156, miR169, and miR172), sulphur uptake, transport, and assimilation (miR395, miR838, and miR854), and hormonal biosynthesis and/or function (e.g. miR319, miR167, miR164, and miR159). Notable changes were observed in the level of auxins [indole-3-acetic acid (IAA), indole-3- butyric acid, and naphthalene acetic acid], jasmonates [jasmonic acid (JA) and methyl jasmonate], and abscisic acid. The exogenous supply of JA and IAA improved growth of plants under As stress and altered expression of miR167, miR319, and miR854, suggesting interplay of hormones and miRNAs in the regulation of As response. In conclusion, the present work demonstrates the role of miRNAs and associated mechanisms in the plant's response towards As stress.

Evaluation of transgenic tobacco plants expressing a bacterial Co-Ni transporter for acquisition of cobalt.

Phytoremediation is a viable strategy for management of toxic wastes in a large area/volume with low concentrations of toxic elemental pollutants. With increased industrial use of cobalt and its alloys, it has become a major metal contaminant in soils and water bodies surrounding these industries and mining sites with adverse effects on the biota. A bacterial Co-Ni permease was cloned from Rhodopseudomonas palustris and introduced into Nicotiana tabacum to explore its potential for phytoremediation and was found to be specific for cobalt and nickel. The transgenic plants accumulated more cobalt and nickel as compared to control, whereas no significant difference in accumulation of other divalent ions was observed. The transgenic plants were evaluated for cobalt content and showed increased acquisition of cobalt (up to 5 times) as compared to control. The plants were also assessed for accumulation of nickel and found to accumulate up to 2 times more nickel than control. At the same initial concentration of cobalt and nickel, transgenic plant preferentially accumulated cobalt as compared to nickel. The present study is perhaps the first attempt to develop transgenic plants expressing heterologous Co transporter with an improved capacity to uptake cobalt.

Mechanisms of arsenic tolerance and detoxification in plants and their application in transgenic technology: a critical appraisal.

Arsenic (As) contamination of the environment has emerged as a serious problem. Consequently, there is an urge to understand plants' responses to As. The analysis of various hypertolerant and hyperaccumulator plants and comparison of their responses with non-tolerant and nonaccumulators have provided valuable information about the mechanisms of As tolerance and detoxification. Therefore, we understand why most of the pteridophytes are able to hyperacumulate As, why it is difficult to find hyperaccumulators among angiosperms and why rice is able to translocate As to its grains more efficiently than any other cereal crop. This information can be employed to generate As hyperaccumulators in angiosperms and to develop safe cultivars of rice for human consumption through biotechnological approaches. Although measurable success, in terms of application in the field, has so far not been achieved, transgenic research has yielded promising results, which shed light on the approaches to be taken up in future endeavor. In this review, we discuss the mechanisms of As tolerance and detoxification in plants and transgenic research conducted.

Immobilization of lipase on cotton cloth using the layer-by-layer self-assembly technique.

Lipase from Thermomyces lanuginosus was assembled into multiple layers on polyethylenimine treated cotton flannel cloth, utilising the enzymes property of forming bimolecular aggregates via layer-by-layer (LBL) immobilization technique. An increase in lipase activity with increasing enzyme layers confirmed lipase aggregation. A study to compare the activity of enzyme bound by classical LBL technique, containing alternate layers of polyethylenimine and lipase and the modified approach indicated above, showed that more enzyme was bound to cloth in the modified approach. A total of 13 U/cm(2) of enzyme were bound to cloth till the fifth layer whereas only 10.2 U/cm(2) were bound till the fifth bilayer in the classical approach. The successful assembly of lipase molecules has shown that this modified technique is a promising approach to immobilize enzymes that aggregate through hydrophobic interactions as nano-films on cloth.

Immobilization of microbial cells on inner epidermis of onion bulb scale for biosensor application.

Inner epidermis of onion bulb scales was used as a natural support for immobilization of microbial cells for biosensor application. A bacterium Sphingomonas sp. that hydrolyzes methyl parathion into a chromophoric product, p-nitrophenol (PNP), has been isolated and identified in our laboratory. PNP can be detected by electrochemical and colorimetric methods. Whole cells of Sphingomonas sp. were immobilized on inner epidermis of onion bulb scale by adsorption followed by cross-linking methods. Cells immobilized onion membrane was directly placed in the wells of microplate and associated with the optical transducer. Methyl parathion is an organophosphorus pesticide that has been widely used in the field of agriculture for insect pest control. This pesticide causes environmental pollution and ecological problem. A detection range 4-80 µM of methyl parathion was estimated from the linear range of calibration plot of enzymatic assay. A single membrane was reused for 52 reactions and was found to be stable for 32 days with 90% of its initial hydrolytic activity. The applicability of the cells immobilized onion membrane was also demonstrated with spiked samples.

Microbial biosensor for detection of methyl parathion using screen printed carbon electrode and cyclic voltammetry.

Whole cells of recombinant Escherichia coli were immobilized on the screen printed carbon electrode (SPCE) using glutaraldehyde. Recombinant E. coli was having high periplasmic expression of organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into two products, p-nitrophenol and dimethyl thiophosphoric acid. Cells immobilized SPCE was studied under SEM. Cells immobilized SPCE was associated with cyclic voltammetry and cyclic voltammograms were recorded before and after hydrolysis of methyl parathion. Detection was calibrated based on the relationship between the changes in the current observed at +0.1 V potential, because of redox behavior of the hydrolyzed product p-nitrophenol. As concentration of methyl parathion was increased the oxidation current also increased. Only 20 µl volume of the sample was required for analysis. Detection range of biosensor was calibrated between 2 and 80 µM of methyl parathion from the linear range of calibration plot. A single immobilized SPCE was reused for 32 reactions with retention of 80% of its initial enzyme activity.

Inheritance and molecular mapping of a gibberellin-insensitive dwarf mutant in groundnut (Arachis hypogaea L.).

A dark green dwarf mutant, TGM 167, was isolated from a gamma ray + sodium azide mutagenized population of cultivated groundnut breeding line, TFDRG 5. The mutant had a 45.8% reduction in height due to its shorter internodal length. Further, it was found to be insensitive towards exogenous GA(3) application, although it had nearly the same level of endogenous GA(3) as the parent. Genetic analysis revealed that the dwarfism is under the control of a single dominant gene. This dominant dwarfing gene was mapped with an SSR marker TC3H02 at a distance of 9.7 cM.

Survival of phosphate-solubilizing bacteria against DNA damaging agents.

Phosphate-solubilizing bacteria (PSBs) were isolated from different plant rhizosphere soils of various agroecological regions of India. These isolates showed synthesis of pyrroloquinoline quinone (PQQ), production of gluconic acid, and release of phosphorus from insoluble tricalcium phosphate. The bacterial isolates synthesizing PQQ also showed higher tolerance to ultraviolet C radiation and mitomycin C as compared to Escherichia coli but were less tolerant than Deinococcus radiodurans. Unlike E. coli, PSB isolates showed higher tolerance to DNA damage when grown in the absence of inorganic phosphate. Higher tolerance to ultraviolet C radiation and oxidative stress in these PSBs grown under PQQ synthesis inducible conditions, namely phosphate starvation, might suggest the possible additional role of this redox cofactor in the survival of these isolates under extreme abiotic stress conditions.

Genome-wide analysis of thiourea-modulated salinity stress-responsive transcripts in seeds of Brassica juncea: identification of signalling and effector components of stress tolerance.

BACKGROUND AND AIMS: Abiotic stresses including salinity are the major constraints to crop production. In this regard, the use of thiourea (TU) in imparting salinity-stress tolerance to Indian mustard (Brassica juncea) has been demonstrated earlier. To gain an insight into the mechanism of TU action, various molecular and biochemical studies were conducted. METHODS: Microarray analysis was performed in seeds subjected to distilled water (control), 1 m NaCl, 1 m NaCl + 6·5 mm TU and 6·5 mm TU alone for 1 h. Real-time PCR validation of selected genes and biochemical studies were conducted under similar treatments at 1 h and 6 h. KEY RESULTS: The microarray analysis revealed a differential expression profile of 33 genes in NaCl- and NaCl + TU-treated seeds, most of which are established markers of stress tolerance. The temporal regulation of eight selected genes by real-time PCR indicated their early and co-ordinated induction at 1 h in NaCl + TU only. Besides, NaCl + TU-treated seeds also maintained a higher level of abscisic acid, reduced to oxidized glutathione (GSH : GSSG) ratio and activities of catalase, phenylalanine ammonia lyase and glutathione-S-transferases, as compared with that of NaCl treatment. The addition of LaCl(3) (a specific calcium-channel blocker) restricted the responses of TU both at molecular and biochemical level suggesting the possible involvement of a cytosolic calcium burst in the TU-mediated response. The TU-alone treatment was comparable to that of the control; however, it reduced the expression of some transcription factors and heat-shock proteins presumably due to the stabilization of the corresponding proteins. CONCLUSIONS: The TU treatment co-ordinately regulates different signalling and effector mechanisms at an early stage to alleviate stress even under a high degree of salinity. This also indicates the potential of TU to be used as an effective bioregulator to impart salinity tolerance under field conditions.

An optical microbial biosensor for detection of methyl parathion using Sphingomonas sp. immobilized on microplate as a reusable biocomponent.

Organophosphorus pesticides such as methyl parathion have been widely used in the field of agriculture for insect pest control. These pesticides and their degradation products cause environmental pollution and ecological problem. With a view to monitor these pesticides biosensors are being developed. A bacterium Sphingomonas sp. from field soil has been isolated and identified in our laboratory that hydrolyzes the methyl parathion upto a chromophoric product, p-nitrophenol (PNP). PNP can be detected by electrochemical and colorimetric methods, which can be exploited to develop a biosensor for detection of the organophosphate pesticide. Whole cells of Sphingomonas bacteria were immobilized directly onto the surface of the wells of polystyrene microplates (96 wells) using glutaraldehyde as the cross-linker. SEM study confirmed the immobilization of Sphingomonas sp. Immobilized bacterial microplate was associated directly with the optical transducer, microplate reader. The microplate-based biosensor is having advantages as it has 96 reaction vessels and therefore it provides a convenient system for detecting multiple numbers of samples in a single platform. Detection range of the biosensor from the linear range was determined to be 4-80 µM methyl parathion. Cells-immobilized microplates were having reusability upto 75 reactions. Present study reports an innovative concept where the microplate can be used as immobilizing support for development of reusable microbial biocomponent.

Buckling-driven morphological transformation of droplets of a mixed colloidal suspension during evaporation-induced self-assembly by spray drying.

Morphological transformation during evaporation-induced self-assembly of a mixed colloidal suspension in micrometric droplets has been investigated. It has been demonstrated that a buckling-driven shape transition of drying droplets of mixed colloidal suspension takes place during evaporation-induced self-assembly. Further, it is also shown that the distortion modulations get significantly amplified with enhancement in volume fraction of anisotropic soft colloidal component of the mixed colloids. It has been argued that the reduction in elastic modulus of formed shell, at the boundary of a drying droplet, and the anisotropic nature of one of the colloidal components facilitate the deformation process. Hierarchical structures of these assembled colloidal grains have been probed using electron microscopy and scattering techniques.

Effect of variable sulfur supply on arsenic tolerance and antioxidant responses in Hydrilla verticillata (L.f.) Royle.

In the present study, Hydrilla verticillata plants were exposed to arsenate (AsV; 50 microM) and arsenite (AsIII; 5 microM) under variable S supply: deficient (2 microM S, -S), normal (1 mM S, +S) and excess (2 mM S, +HS). Arsenic accumulation (microg g(-1) dw) in +HS plants was about 2-fold higher upon exposure to both AsV (30) and AsIII (50) than that observed in +S (12 & 24) and -S (14 & 26) plants. Despite lower As accumulation, -S plants experienced the maximum oxidative stress owing to an inadequate response of enzymatic and molecular antioxidants and significant decline in total thiols and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). By contrast +HS plants had significant increase in total thiols and an improved redox status, did not demonstrate any negative impact to antioxidants except catalase and hence experienced the least increase in oxidative stress parameters. In conclusion, an increase in S supply to plants may improve their accumulation capacity for As through enhanced tolerance caused by a positive effect on thiol metabolism and antioxidant status of the plants.

Excess cell mass as an internal carbon source for biological denitrification.

Aim of the present work was to examine whether the SCOD (soluble chemical oxygen demand) released after the physical disruption of excess activated sludge can be used as an alternative carbon source for biological denitrification. In the first stage of research, we investigated the potential use of energy efficient hydrodynamic cavitation (HC) technique for the disruption of activated sludge. In a comparative study between ultrasonic cavitation (UC) and HC, it was observed that UC needs five times more energy than that of HC to release the same amount of SCOD. In the second stage of the experimental study, SCOD was successfully used as an alternative carbon source (alternative to sodium acetate) for biological denitrification. The critical weight ratio (SCOD/NO(3)-N) of seven ensured 100% removal of nitrate. Nitrate removal kinetics indicated that denitrification with SCOD as a carbon source gives higher specific denitrification rate (by approximately 200%) as compared to conventional carbon source (sodium acetate).

Investigation of uranium accumulation potential and biochemical responses of an aquatic weed Hydrilla verticillata (L.f.) Royle.

The uranium (U) accumulation potential and ensuing biochemical responses were studied in Hydrilla verticillata (L.f.) Royle upon exposure to U (0, 20 and 100 mg L(-1)). There was a concentration-duration dependent increase in U accumulation with the maximum being 78 mg g(-1) DW at 100 mg L(-1) U after 24 h. Plants experienced an initial phase of the maximum toxicity (within 30 min) followed by almost complete recovery after 24 h. The recovery was attributed to an integrated modulation in the level of both enzymatic and molecular antioxidants (viz., guaiacol peroxidase, ascorbate peroxidase, catalase, proline, total phenolics) and also the constituents of thiol metabolism (viz., cysteine and glutathione). Thus, plants were found to be able to accumulate significant amount of U in a short time and to tolerate it efficiently. Hence, they may find application in U phytoremediation considering there accumulation ability, fast growth due to weed-like habit and world-wide distribution.

Increasing sulfur supply enhances tolerance to arsenic and its accumulation in Hydrilla verticillata (Lf.) Royle.

The present study was aimed to analyze the effects of variable S supply on arsenic (As) accumulation potential of Hydrilla verticillata (Lf.) Royle. Plants were exposed to either arsenate (AsV; 50 microM) or arsenite (AsIII; 5 microM) for 4 h and 1 day while S supply was varied as deficient (2 microM, -S), normal (1 mM, +S) and excess (2 mM, +HS). The level of As accumulation (microg g(-1) dw) after 1 day was about 2-fold higher upon exposure to either AsV (30) or AsIII (50) in +HS plants than that being in +S (12 and 24) and -S (14 and 26) plants. The +HS plants showed a significant stimulation of the thiol metabolism upon As exposure. Besides, they did not experience significant toxicity, measured in terms of malondialdehyde accumulation; an indicator of oxidative stress. By contrast, -S plants suffered from oxidative stress probably due to negative impact to thiol metabolism. Variable S supply also modulated the activity of enzymes of glycine and serine biosynthesis indicating an interconnection between S and N metabolism. In conclusion, an improved supply of S to plants was found to augment their ability for As accumulation through stimulated thiol metabolism.

Comparative biochemical and transcriptional profiling of two contrasting varieties of Brassica juncea L. in response to arsenic exposure reveals mechanisms of stress perception and tolerance.

The mechanisms of perception of arsenic (As)-induced stress and ensuing tolerance in plants remain unresolved. To obtain an insight into these mechanisms, biochemical and transcriptional profiling of two contrasting genotypes of Brassica juncea was performed. After screening 14 varieties for As tolerance, one tolerant (TPM-1) and one sensitive (TM-4) variety were selected and exposed to arsenate [As(V)] and arsenite [As(III)] for 7 d and 15 d for biochemical analyses. The tolerant variety (TPM-1) demonstrated higher accumulation of As upon exposure to both 500 microM As(V) and 250 microM As(III) [49 microg g(-1) and 37 microg g(-1) dry weight (dw) after 15 d] as well as a better response of thiol metabolism as compared with the responses observed in the sensitive variety (TM-4). Transcriptional profiling of selected genes that are known to be responsive to sulphur depletion and/or metal(loid) stress was conducted in 15-d-old seedlings after 3 h and 6 h exposure to 250 microM As(III). The results showed an up-regulation of sulphate transporters and auxin and jasmonate biosynthesis pathway genes, whereas there was a down-regulation of ethylene biosynthesis and cytokinin-responsive genes in TPM-1 within 6 h of exposure to As(III). This suggested that perception of As-induced stress was presumably mediated through an integrated modulation in hormonal functioning that led to both short- and long-term adaptations to combat the stress. Such a coordinated response of hormones was not seen in the sensitive variety. In conclusion, an early perception of As-induced stress followed by coordinated responses of various pathways was responsible for As tolerance in TPM-1.