Pyramiding of γ-TMT and gly I transgenes in Brassica juncea enhances salinity and drought stress tolerance.

We previously generated Brassica juncea lines overexpressing either glyoxalase I (gly I) or γ-tocopherol methyltransferase (γ-TMT) involved in the glyoxalase system and tocopherol biosynthesis, respectively. These transgenic plants showed tolerance to multiple abiotic stresses. As tolerance is a complex trait that can be improved by pyramiding of several characteristics in a single genotype, we generated in this study B. juncea plants coexpressing gly I and γ-TMT by crossing the previously generated stable transgenic lines. The performance of the newly generated B. juncea lines coexpressing gly I and γ-TMT was compared with that of wild-type and the single transgenic lines under non-stressed and NaCl and mannitol stress conditions. Our results show a more robust antioxidant response of B. juncea plants coexpressing gly I and γ-TMT compared to the other lines in terms of higher chlorophyll retention, relative water content, antioxidant enzyme and proline levels, and photosynthetic efficiency and lower oxidative damage. The differences in response to the stress of the different lines were reflected in their yield parameters. Overall, we demonstrate that the pyramiding of multiple genes involved in antioxidant pathways could be a viable and useful approach for achieving higher abiotic stress tolerance in crop plants.

Novel insecticidal chitinase from the insect pathogen Xenorhabdus nematophila.

Xenorhabdus nematophila strain ATCC 19061 is an insect pathogen that produces various protein toxins which intoxicate and kill its larval host. In the present study, we have described the cloning, expression and characterization of a 76-kDa chitinase protein of X. nematophila. A 1.9 kb DNA sequence encoding the chitinase gene was PCR amplified and cloned. Further, the chitinase protein was expressed in Escherichia coli and purified by using affinity chromatography. Two highly conserved domains were identified GH18 and ChiA. The purified chitinase protein showed chitobiosidase activity, ß-N-acetylglucosaminidase and endochitinase activity, when enzyme activity was measured using respective substrates. The purified chitinase protein was found to be orally toxic to the larvae of a major crop pest, Helicoverpa armigera when fed to the larvae mixed with artificial diet. It also had adverse effect on the growth and development of the surviving larvae. Surviving larvae showed 9-fold reduction in weight, as a result the transformation of larvae into pupae was adversely affected. Our results demonstrated that the chitinase protein of X. nematophila has insecticidal property and can prove to be a potent candidate for pest control in plants.

Influence of eco-friendly phytotoxic metabolites from Lasiodiplodia pseudotheobromae C1136 on physiological, biochemical, and ultrastructural changes on tested weeds.

In this study, the active metabolites from both the wild strain of Lasiodiplodia pseudotheobromae C1136 and three genetically enhanced strains of C1136 were obtained through random mutagenesis. The effect of the active metabolites from these strains was evaluated in relation to physiological, biochemical, and ultrastructural changes on the leaves of two weeds (Amaranthus hybridus and Echinochloa crus-galli). The phytotoxic metabolites secreted by the genetically enhanced strains showed a decrease in the pigments (chl a, chl b, and carotenoids), carbohydrate content, and the amino acid profile. On the other hand, an increase in total phenols of the tested leaves was observed when compared with the untreated leaves. The scanning electron microscopy showed the presence of damages, necrosis, degradation, and ultrastructural changes on the tested leaf tissues of the weeds. Also, increased lipid peroxidation and electrolyte leakage were also observed on the tested weeds treated with phytotoxic metabolites secreted by the genetically enhanced strains. We also showed that the phytotoxins from the strains of C1136 are biocompatible and that it improved soil CO2 evolution, organic carbon content, and enzymatic activity (acidic and alkaline phosphatase, dehydrogenases, cellulase, catalase). The study validates the severe pathological effects of phytotoxic metabolites from the strains of C1136 on the leaves of the weeds presented in this study. The mode of action of the phytotoxic metabolites produced from this bioherbicidal isolates will go a long way in preventing environmental hazards.

Groundnut AhcAPX conferred abiotic stress tolerance in transgenic banana through modulation of the ascorbate-glutathione pathway.

A stress inducible cytosolic ascorbate peroxidase gene (AhcAPX) was ectopically expressed in banana (cv. Grand naine) plants to strengthen their antioxidant capacity. High level of AhcAPX gene transcripts and enzyme suggested constitutive and functional expression of candidate gene in transgenic (TR) plants. The tolerance level of in vitro and in vivo grown TR banana plantlets were assessed against salt and drought stress. The TR banana plants conferred tolerance against the abiotic stresses by maintaining a high redox state of ascorbate and glutathione, which correlated with lower accumulation of H2O2, O2 ⋅- and higher level of antioxidant enzyme (SOD, APX, CAT, GR, DHAR and MDHAR) activities. The efficacy of AhcAPX over-expression was also investigated in terms of different physiochemical attributes of TR and untransformed control plants, such as, proline content, membrane stability, electrolyte leakage and chlorophyll retention. The TR plants showed higher photochemical efficiency of PSII (Fv/Fm), and stomatal attributes under photosynthesis generated reactive oxygen species (ROS) stress. The outcome of present investigation suggest that ectopic expression of AhcAPX gene in banana enhances the tolerance to drought and salt stress by annulling the damage caused by ROS.

Meeting the challenge of developing food crops with improved nutritional quality and food safety: leveraging proteomics and related omics techniques.

Eliminating malnutrition remains an imminent priority in our efforts to achieve food security and providing adequate calories, proteins, and micronutrients to the growing world population. Malnutrition may be attributed to socio-economic factors (poverty and limited accessibility to nutritional food), dietary preferences, inherent nutrient profiles of traditional food crops, and to a combination of all such factors. Modern advancements in "omics" technology have made it possible to reliably predict, diagnose, and suggest ways to remedy the low protein content and bioavailability of key micronutrients in food crops. In this review, we briefly describe how proteomics techniques can potentially be used for improving the nutrient profile of major crops, through high throughput multiplexed assays. Food safety is another important issue where proteomics and related platforms can offer solution for absolute quantitation of food allergens and mycotoxins present in the plant-based food. The purpose of the present review is to discuss the proteomic-based strategies in food crops to meet the challenges of overcoming malnutrition throughout the world.

Multiple abiotic stress tolerance in Vigna mungo is altered by overexpression of ALDRXV4 gene via reactive carbonyl detoxification.

Vigna mungo (blackgram) is an important leguminous pulse crop, which is grown for its protein rich edible seeds. Drought and salinity are the major abiotic stresses which adversely affect the growth and productivity of crop plants including blackgram. The ALDRXV4 belongs to the aldo-keto reductase superfamily of enzymes that catalyze the reduction of carbonyl metabolites in the cells and plays an important role in the osmoprotection and detoxification of the reactive carbonyl species. In the present study, we developed transgenic plants of V. mungo using Agrobacterium mediated transformation. The transgene integration was confirmed by Southern blot analysis whereas the expression was confirmed by RT-PCR, Western blot and enzyme activity. The T1 generation transgenic plants displayed improved tolerance to various environmental stresses, including drought, salt, methyl viologen and H2O2 induced oxidative stress. The increased aldose reductase activity, higher sorbitol content and less accumulation of the toxic metabolite, methylglyoxal in the transgenic lines under non-stress and stress (drought and salinity) conditions resulted in increased protection through maintenance of better photosynthetic efficiency, higher relative water content and less photooxidative damage. The accumulation of reactive oxygen species was remarkably decreased in the transgenic lines as compared with the wild type plants. This study of engineering multiple stress tolerance in blackgram, is the first report to date and this strategy for trait improvement is proposed to provide a novel germplasm for blackgram production on marginal lands.

A novel pilin subunit from Xenorhabdus nematophila, an insect pathogen, confers pest resistance in tobacco and tomato.

KEY MESSAGE: Overexpression of insecticidal pilin subunit from Xenorhabdus nematophila protects transgenic tobacco and tomato plants against Helicoverpa armigera. Xenorhabdus nematophila is a pathogenic bacterium producing toxins that kill the larval host. Previously, we characterized a pilin subunit of X. nematophila which was found to be a pore-forming toxin and cytotoxic to the larval hemocytes of Helicoverpa armigera by causing agglutination and lysis of the cells. In the present study, we report the efficacy of the insecticidal pilin subunit expressed in transgenic tobacco and tomato plants for control against H. armigera. A 537 bp mrxA gene encoding the 17 kDa insecticidal pilin subunit was transferred into the genome of tobacco and tomato, respectively, via Agrobacterium-mediated transformation. The stable integration of the 537 bp mrxA gene in the transgenic plants was confirmed by Southern blot analysis and expression of mrxA gene was confirmed by RT-PCR and Western blot analyses. The transgenic plants appeared healthy and phenotypically normal but proved toxic to the insects in insect bioassays, showing 100% insect mortality and reduced damage of the transgenic plants. Based on these observations, it is suggested that pilin subunit can be used as a potential candidate for control of H. armigera and may open new strategies for pest control in agricultural plants.

Ectopic expression of GroEL from Xenorhabdus nematophila in tomato enhances resistance against Helicoverpa armigera and salt and thermal stress.

The GroEL homolog XnGroEL protein of Xenorhabdus nematophila belongs to a highly conserved family of molecular chaperones/heat shock proteins (Hsps). XnGroEL was shown to possess oral insecticidal activity against a major crop pest Helicoverpa armigera. Under normal conditions, the Hsps/chaperones facilitate folding, assembly, and translocation of cellular proteins, while in stress conditions they protect proteins from denaturation. In this study, we describe generation of transgenic tomato plants overexpressing insecticidal XnGroEL protein and their tolerance to biotic and abiotic stresses. Presence of XnGroEL in the transgenic tomato lines conferred resistance against H. armigera showing 100% (p ≤ 0.001) mortality of neonates. In addition, XnGroEL provided thermotolerance and protection against high salt concentration to the tomato plants. Expression of XnGroEL minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane system of the plants under heat and salt stress. The enhanced tolerance to abiotic stresses correlated with increase in the anti-oxidative enzyme activity and reduced H2O2 accumulation in transgenic tomato plants. The variety of beneficial properties displayed by XnGroEL protein provides an opportunity for value addition and improvement of crop productivity.

Overexpression of CuZnSOD from Arachis hypogaea alleviates salinity and drought stress in tobacco.

KEY MESSAGE: Overexpression of CuZnSOD gene from Arachis hypogaea demonstrating its involvement in abiotic stress tolerance. Abiotic stress is accompanied by the formation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals, causing extensive cellular damage and inhibition of photosynthesis that limit the plant productivity. The level of ROS in cells needs to be tightly regulated and the toxic effects of ROS are countered by enzymatic as well as non-enzymatic antioxidant systems. The superoxide dismutase is the first enzyme involved in the detoxification of ROS and converts superoxide (O2(·-)) radicals to H2O2. A full-length cDNA clone encoding a CuZnSOD, named AhCuZnSOD, was isolated from the salt tolerant cell lines of Arachis hypogaea, stably thriving at 200 mM NaCl. The cell line showed higher transcript accumulation under multiple abiotic stresses, including drought, salinity, cold and oxidative stress treatment. The functional role of AhCuZnSOD in alleviation of abiotic stress was assessed by its overexpression in transgenic tobacco plants. The T1 transgenic plants showed improved tolerance to salinity and dehydration stress as indicated by higher seed germination and better chlorophyll content. The transgenic plants survived under longer periods of water deficiency and salinity stress and displayed improved recovery after rehydration compared to the wild type (WT) plants. The enhanced level of the transgene correlated with higher relative water content, less electrolyte damage, less malondialdehyde, higher antioxidant enzyme activity, H2O2 and O2(·-) accumulation under stress conditions compared to WT plants. Our results substantiate that increased levels of SOD activity brought about by overexpression of AhCuZnSOD gene may play an important role in ameliorating oxidative injury induced by various environmental stresses.

High efficiency transformation of banana [Musa acuminata L. cv. Matti (AA)] for enhanced tolerance to salt and drought stress through overexpression of a peanut salinity-induced pathogenesis-related class 10 protein.

Bananas and plantains (Musa spp. L.) are important subsistence crops and premium export commodity in several countries, and susceptible to a wide range of environmental and biotic stress conditions. Here, we report efficient, rapid, and reproducible Agrobacterium-mediated transformation and regeneration of an Indian niche cultivar of banana [M. acuminata cv. Matti (AA)]. Apical meristem-derived highly proliferative multiple shoot clump (MSC) explants were transformed with the Agrobacterium strain EHA105 harboring a binary vector pCAMBIA-1301 carrying hptII and uidA. Sequential agro-infiltration (10 min, 400 mmHg), infection (additional 35 min, Agrobacterium density A 600 = 0.8) and co-cultivation (18 h) regimen in 100 µM acetosyringone containing liquid medium were critical factors yielding high transformation efficiency (~81 %) corroborated by transient GUS expression assay. Stable transgenic events were recovered following two cycles of meristem initiation and selection on hygromycin containing medium. Histochemical GUS assay in several tissues of transgenic plants and molecular analyses confirmed stable integration and expression of transgene. The protocol described here allowed recovery of well-established putative transgenic plantlets in as little as 5 months. The transgenic banana plants could be readily acclimatized under greenhouse conditions, and were phenotypically similar to the wild-type untransformed control plants (WT). Transgenic plants overexpressing Salinity-Induced Pathogenesis-Related class 10 protein gene from Arachis hypogaea (AhSIPR10) in banana cv. Matti (AA) showed better photosynthetic efficiency and less membrane damage (P < 0.05) in the presence of NaCl and mannitol in comparison to WT plants suggesting the role of AhSIPR10 in better tolerance of salt stress and drought conditions.

Structural characterization and functional validation of aldose reductase from the resurrection plant Xerophyta viscosa.

Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K M (1.2 mM) and k cat (14.5 s(-1)) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha-beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl2), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.

Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine.

In concern with frequent recurrence of anthrax in endemic areas and inadvertent use of its spores as biological weapon, the development of an effective anthrax vaccine suitable for both human and veterinary needs is highly desirable. A simple oral delivery through expression in plant system could offer promising alternative to the current methods that rely on injectable vaccines extracted from bacterial sources. In the present study, we have expressed protective antigen (PA) gene in Indian mustard by Agrobacterium-mediated transformation and in tobacco by plastid transformation. Putative transgenic lines were verified for the presence of transgene and its expression by molecular analysis. PA expressed in transgenic lines was biologically active as evidenced by macrophage lysis assay. Intraperitoneal (i.p.) and oral immunization with plant PA in murine model indicated high serum PA specific IgG and IgA antibody titers. PA specific mucosal immune response was noted in orally immunized groups. Further, antibodies indicated lethal toxin neutralizing potential in-vitro and conferred protection against in-vivo toxin challenge. Oral immunization experiments demonstrated generation of immunoprotective response in mice. Thus, our study examines the feasibility of oral PA vaccine expressed in an edible plant system against anthrax.

Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

A novel insecticidal GroEL protein from Xenorhabdus nematophila confers insect resistance in tobacco.

Xenorhabdus nematophila is an entomopathogenic bacteria. It secretes a GroEL homolog, XnGroEL protein, toxic to its larval prey. GroEL belongs to the family of molecular chaperones and is required for proper folding of cellular proteins. Oral ingestion of insecticidal XnGroEL protein is toxic to Helicoverpa armigera, leading to cessation of growth and development of the larvae. In the present study, the insecticidal efficacy of XnGroEL against H. armigera has been evaluated in transgenic tobacco plant expressing the protein. A 1.7-kb gene encoding the 58-kDa XnGroEL protein was incorporated into the tobacco genome via Agrobacterium-mediated transformation. The stable integration of the transgene was confirmed by Southern blot analysis and its expression by RT-PCR and western blot analyses in transgenic plants. The transgenic lines showed healthy growth and were phenotypically normal. Insect bioassays revealed significant reduction of 100 % in the survival of larvae (p < 0.001) and 55-77 % reduction in plant damage (p < 0.05 and p < 0.001) compared to the untransformed and vector control plants. The results demonstrate that XnGroEL is a novel potential candidate for imparting insect resistance against H. armigera in plants.

Effects of wild-type and α-tocopherol-enriched transgenic Brassica juncea on the components of xenobiotic metabolism, antioxidant status, and oxidative stress in the liver of mice.

Alpha (α)-tocopherol is the most biologically active and preferentially retained form of vitamin E in the human body and is known for its antioxidant and gene regulatory functions. Its increased intake is implicated in protection against diseases that involve an oxidative stress component. We have evaluated the chemopreventive potential of a diet supplemented with natural α-tocopherol-enriched transgenic (TR) Brassica juncea seeds. The modulation of phase I and phase II xenobiotic metabolism and of antioxidative enzymes was compared in the livers of mice fed on a control diet or on a diet supplemented with 2, 4, and 6 % (w/w) of wild-type (WT) or TR seeds. A dose-dependent increase in the specific activities of these enzymes was observed in those animals fed on diet supplemented with TR seeds. In comparison, an increase in the specific activities of antioxidative enzymes was substantial only at higher doses of WT seeds. Consequently, oxidative stress measured in terms of lipid peroxidation and lactate dehydrogenase activity was found to be lower in the case of mice fed with the supplemented diet. However, the chemopreventive potential of TR seeds was more pronounced than that of WT seeds. This study demonstrates the feasibility of fortifying diets with natural α-tocopherol for chemopreventive benefits by means of transgenic manipulation of a commonly used oilseed crop.

Modulation of antioxidant machinery in α-tocopherol-enriched transgenic Brassica juncea plants tolerant to abiotic stress conditions.

The antioxidant machinery in plants consists of several components with unique or overlapping functions that combat the deleterious production of reactive oxygen species (ROS) induced by stress conditions. Tocopherols are a group of powerful antioxidants having additional roles in signaling and gene expression, with α-tocopherol being the most potent form. In the present study, we used wild-type (WT) and α-tocopherol-enriched transgenic (TR) Brassica juncea plants grown under salt, heavy metal, and osmotic stress to compare their relative tolerance to these stresses and to assess the effects of increased α-tocopherol content on the other antioxidative enzymes and molecules. The oxidative damage caused by induced stress was lower in TR plants compared to WT plants as assessed by their higher relative water content and lower electrolyte leakage, malondialdehyde content as well as H(2)O(2) accumulation. Lesser superoxide and H(2)O(2) accumulation was also observed by histochemical staining in TR seedlings exposed to stress. Though no significant differences were evident under normal growth conditions, TR plants showed higher activities and transcript levels of antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase than WT plants under similar stress conditions. A decrease in ascorbate and glutathione content with marginally higher reductive ratios of these compounds was also observed in TR plants under the stress conditions. Our findings implicate the role of higher α-tocopherol levels in conferring better tolerance against salt, heavy metal, and osmotic stresses and also establish the existence of interplay between this lipid-soluble antioxidant and other water-soluble components of plant antioxidant defense.

The Xerophyta viscosa aldose reductase (ALDRXV4) confers enhanced drought and salinity tolerance to transgenic tobacco plants by scavenging methylglyoxal and reducing the membrane damage.

We report the efficacy of an aldose reductase (ALDRXV4) enzyme from Xerophyta viscosa Baker in enhancing the prospects of plant's survival under abiotic stress. Transgenic tobacco plants overexpressing ALDRXV4 cDNA showed alleviation of NaCl and mannitol-induced abiotic stress. The transgenic plants survived longer periods of water deficiency and salinity stress and exhibited improved recovery after rehydration as compared to the wild type plants. The increased synthesis of aldose reductase in transgenic plants correlated with reduced methylglyoxal and malondialdehyde accumulation and an elevated level of sorbitol under stress conditions. In addition, the transgenic lines showed better photosynthetic efficiency, less electrolyte damage, greater water retention, higher proline accumulation, and favorable ionic balance under stress conditions. Together, these findings suggest the potential of engineering aldose reductase levels for better performance of crop plants growing under drought and salt stress conditions.

Overexpression of gamma-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: physiological and chlorophyll a fluorescence measurements.

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the gamma-tocopherol methyl transferase (gamma-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of alpha-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the gamma-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial "O" level to the "P" (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that alpha-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.

Differential response of arsenic stress in two varieties of Brassica juncea L.

Present study showed the toxicity caused by Arsenite (As(III)) and its detoxification responses in two varieties (Varuna and Pusa Bold) of Brassica juncea. Comparisons were made in leaves and roots of both the varieties, which showed that the accumulation pattern in both the varieties were dose and duration dependent, being more in roots for two days and in leaves for four days. Increase/decrease of antioxidant enzymes activities (SOD, CAT, GPX) showed not much changes at the given concentrations except that the enzyme activities showed significant increase at the lower concentrations. Semi quantitative RT-PCR analysis of PCS showed more expression of its transcript in P. Bold as compared to Varuna variety due to As(III) stress. The analysis of isoenzyme pattern in leaves of P. Bold showed five and two major bands of SOD and GPX, respectively. As(III) treatment leads to the activation of MAPK activity indicating role of this important cascade in transducing As(III) mediated signals. The data presented indicates the differential responses in both the varieties and also that the increased tolerance in P. Bold may be due to the defensive role of antioxidant enzymes, induction of MAPK and up regulation of PCS transcript which is responsible for the production of metal binding peptides.

Heavy metal induced DNA changes in aquatic macrophytes: Random amplified polymorphic DNA analysis and identification of sequence characterized amplified region marker.

Plants have been used as good bio-indicators and genetic toxicity of environmental pollution in recent years. In this study, aquatic plants Hydrilla verticillata and Ceratophyllum demersum treated with 10 micromol/L Cd, 5 micromol/L Hg, and 20 micromol/L Cu for 96 h, showed changes in chlorophyll, protein content, and in DNA profiles. The changes in DNA profiles included variation in band intensity, presence or absence of certain bands and even appearance of new bands. Genomic template stability test performed for the qualitative measurement of changes in randomly amplified polymorphic DNA (RAPD) profiles, showed significant effect at the given concentration of metals. Cloning and sequencing of bands suggested that these markers although may not be homologous to any known gene but its conversion as a sequence characterized amplified region (SCAR) marke is useful in detecting the effects of genotoxin agents.