Overexpression of gamma-glutamyl cyclotransferase 2;1 (CsGGCT2;1) reduces arsenic toxicity and accumulation in Camelina sativa (L.).

KEY MESSAGE: Overexpressing CsGGCT2;1 in Camelina enhances arsenic tolerance, reducing arsenic accumulation by 40-60%. Genetically modified Camelina can potentially thrive on contaminated lands and help safeguard food quality and sustainable food and biofuel production. Environmental arsenic contamination is a serious global issue that adversely affects human health and diminishes the quality of harvested produce. Glutathione (GSH) is known to bind and detoxify arsenic and other toxic metals. A steady level of GSH is maintained within cells via the γ-glutamyl cycle. The γ-glutamyl cyclotransferases (GGCTs) have previously been shown to be involved in GSH degradation and increased tolerance to toxic metals in plants. In this study, we characterized the GGCT2;1 homolog from Camelina sativa for its role in arsenic tolerance and accumulation. Overexpression of CsGGCT2;1 in Camelina under CaMV35S constitutive promoter resulted in strong tolerance to arsenite (AsIII). The overexpression (OE) lines had 2.6-3.5-fold higher shoots and sevenfold to tenfold enhanced root biomass on media supplemented with AsIII, relative to wild-type plants. The CsGGCT2;1 OE lines accumulated 40-60% less arsenic in root and shoot tissues compared to wild-type plants. Further, the OE lines had ~ twofold higher chlorophyll content and 35% lesser levels of malondialdehyde (MDA), an indicator of membrane damage via lipid peroxidation. There was a slight but non-significant increase in 5-oxoproline (5-OP), a product of GSH degradation, in OE lines. However, the transcript levels of Oxoprolinase 1 (OXP1) were upregulated, indicating the accelerated conversion of 5-OP to glutamate, which is further utilized for the resynthesis of GSH to maintain GSH homeostasis. Overall, this research suggests that genetically modified Camelina may have the potential for cultivation on contaminated marginal lands to reduce As accumulation; thereby could help in addressing food safety issues as well as future food and biofuel needs.

Nanoscale sulfur alleviates silver nanoparticle toxicity and improves seed and oil yield in Soybean (Glycine max).

Silver nanoparticles (AgNPs) are commonly used in many commercial products due to their antimicrobial properties, and their significant exposure in agricultural systems is anticipated. AgNPs accumulation in soil and subsequent uptake by plants can be harmful to plant growth and exposure to animals and humans through the food chain is a major concern. This study evaluated the potential protective role of nanosulfur (NS) and bulk sulfur (BS) at 200 and 400 mg/kg soil application in alleviating silver nanoparticle (AgNPs; 32 and 64 mg/kg) phytotoxicity to soybean [Glycine max (L) Merr.]. The treatments were added in the soil before soybean transplantation; growth, yield, nutrient, and silver accumulation were measured in the shoot, root, and seeds. Exposure to AgNPs significantly affected plant growth and yield, reducing nodule weight by 40%, fresh shoot weight by 66%, and seed yield by 68% when compared to controls. However, nanosulfur application in soil alleviated AgNPs toxicity, and importantly, this impact was nanoscale specific at the higher concentration because the benefits of corresponding bulk sulfur (BS) treatments were marginal. Specifically, nanosulfur at 400 mg/kg significantly increased seed yield (∼3-fold more than AgNP at 64 mg/kg) and shoot biomass (2.6-fold more than AgNP at 64 mg/kg) upon co-exposure with AgNPs, essentially alleviating AgNPs toxicity. Moreover, NS increased nodule mass by 3.5 times compared to AgNPs-treated plants, which was 170% greater than the Ag- and NS-free controls. Plants treated with NS with AgNPs co-exposure accumulated significantly less Ag in the shoots (∼80% reduction) and roots (∼95% reduction); no Ag contents were detected in seeds. These findings demonstrate the potential of sulfur, especially NS, as a sustainable soil amendment to reduce the accumulation and toxicity of AgNPs and as a valuable nano-enabled strategy to promote food safety and security.

Simultaneous exposure of wheat (Triticum aestivum L.) to CuO and S nanoparticles alleviates toxicity by reducing Cu accumulation and modulating antioxidant response.

Widespread use of metal-based nanoparticles (NPs) may result in the increased accumulation of metals in agricultural soil, which could affect crop productivity and contaminate the food-chain. The effect of sulfur nanoparticles (S NPs, 200 mg/L) co-exposure on the toxicity of CuO nanoparticles (CuO NPs, 25 and 50 mg/L) to wheat seedlings was investigated in a hydroponic system. CuO NPs exposure significantly inhibited the growth of wheat seedlings, causing 43.6% and 54.1% decreases in the fresh biomass of plants and 82.8% and 83.1% decrease in the total chlorophyll contents at 25 and 50 mg/L (CuONP25 and CuONP50), respectively, as compared to controls. CuO NPs exposure at both concentrations increased the malondialdehyde (MDA) content in shoot and root tissues by 66.4-67.9% and 47.7-48.8%, respectively. Further, CuO NPs exposure elevated the activities POD, SOD, and CAT by 2.19-2.27, 5.82-6.09, and 1.44-1.95 times in roots, and by 45.2-67.8%, 86.7-154.5%, and 22.5-56.1% in shoot, respectively, in comparison to control. The addition of S NPs alone increased wheat biomass by 11.0% and total chlorophyll contents by 4.4%, compared to controls. Further, simultaneous exposure to S NPs (200 mg/L) and CuO NPs (25 or 50 mg/L) alleviated the CuO NPs toxicity; wheat biomass was 47.8% and 37.7% higher in CuONP25 + SNP and CuONP50 + SNP treatments, respectively, as compared to CuO NPs alone treated plants. Co-exposed plants showed reduced levels of total reactive oxygen species (ROS), O2·- and H2O2. Additionally, S NPs exposure reduced Cu uptake and accumulation in both root and shoot tissue by 32.2-54.4% and 38.3-57.5%, respectively. In summary, S NPs alleviated CuO NPs toxicity to wheat seedlings, most likely by reducing Cu bioavailability and accumulation of Cu in plant tissues, and also altered S nutrition and the modulation of antioxidant response in plants. These results showed that S NPs application has the potential to alleviate CuO NP toxicity and increase wheat productivity affected by metals toxicity.

Nanotechnology-enabled biofortification strategies for micronutrients enrichment of food crops: Current understanding and future scope.

Nutrient deficiency in food crops severely compromises human health, particularly in under privileged communities. Globally, billions of people, particularly in developing nations, have limited access to nutritional supplements and fortified foods, subsequently suffering from micronutrient deficiency leading to a range of health issues. The green revolution enhanced crop production and provided food to billions of people but often falls short with respect to the nutritional quality of that food. Plants may assimilate nutrients from synthetic chemical fertilizers, but this approach generally has low nutrient delivery and use efficiency. Further, the overexposure of chemical fertilizers may increase the risk of neoplastic diseases, render food crops unfit for consumption and cause environmental degradation. Therefore, to address these challenges, more research is needed for sustainable crop yield and quality enhancement with minimum use of chemical fertilizers. Complex nutritional disorders and 'hidden hunger' can be addressed through biofortification of food crops. Nanotechnology may help to improve food quality via biofortification as plants may readily acquire nanoparticle-based nutrients. Nanofertilizers are target specific, possess controlled release, and can be retained for relatively long time periods, thus prevent leaching or run-off from soil. This review evaluates the recent literature on the development and use of nanofertilizers, their effects on the environment, and benefits to food quality. Further, the review highlights the potential of nanomaterials on plant genetics in biofortification, as well as issues of affordability, sustainability, and toxicity.

Graphitic Carbon Nitride (C3N4) Reduces Cadmium and Arsenic Phytotoxicity and Accumulation in Rice (Oryza sativa L.).

The present study investigated the role of graphitic carbon nitride (C3N4) in alleviating cadmium (Cd)- and arsenic (As)-induced phytotoxicity to rice (Oryza sativa L.). A high-temperature pyrolysis was used to synthesize the C3N4, which was characterized by transmission electron microscopy, Fourier-transform infrared spectroscopy, and dynamic light scattering. Rice seedlings were exposed to C3N4 at 50 and 250 mg/L in half-strength Hoagland's solution amended with or without 10 mg/L Cd or As for 14 days. Both Cd and As alone resulted in 26-38% and 49-56% decreases in rice root and shoot biomass, respectively. Exposure to 250 mg/L C3N4 alone increased the root and shoot fresh biomass by 17.5% and 25.9%, respectively. Upon coexposure, Cd + C3N4 and As + C3N4 alleviated the heavy metal-induced phytotoxicity and increased the fresh weight by 26-38% and 49-56%, respectively. Further, the addition of C3N4 decreased Cd and As accumulation in the roots by 32% and 25%, respectively, whereas the metal contents in the shoots were 30% lower in the presence of C3N4. Both As and Cd also significantly altered the macronutrient (K, P, Ca, S, and Mg) and micronutrient (Cu, Fe, Zn, and Mn) contents in rice, but these alterations were not evident in plants coexposed to C3N4. Random amplified polymorphic DNA analysis suggests that Cd significantly altered the genomic DNA of rice roots, while no difference was found in shoots. The presence of C3N4 controlled Cd and As uptake in rice by regulating transport-related genes. For example, the relative expression of the Cd transporter OsIRT1 in roots was upregulated by approximately threefold with metal exposure, but C3N4 coamendment lowered the expression. Similar results were evident in the expression of the As transporter OsNIP1;1 in roots. Overall, these findings facilitate the understanding of the underlying mechanisms by which carbon-based nanomaterials alleviate contaminant-induced phyto- and genotoxicity and may provide a new strategy for the reduction of heavy metal contamination in agriculture.

Quantitative proteomic analysis reveals complex regulatory and metabolic response of Iris lactea Pall. var. chinensis to cadmium toxicity.

Cadmium pollution has become a serious environmental problem. Iris lactea var. chinensis showed strong Cd tolerance and accumulation ability, which has significant potential to be applied for the phytoremediation of Cd-contaminated soil. However, the lack of molecular information on the mechanism of I. lactea response to Cd limited the improvement of phytoremediation efficiency. In this study, label-free proteomics analysis of Cd response in I. lactea showed that there were 163 and 196 differentially expressed proteins (DEPs) in the shoots and roots, respectively. Bioinformatics analysis indicated the DEPs responding to Cd stress mainly involved in signal transduction, ion transport, redox etc., and participate in the pathway of amino acid biosynthesis, lignin biosynthesis, glycerolipid metabolism and glutathione metabolism. Besides, differential expression of seven DEPs was validated via gene expression analysis. Finally, we found that a Cd-induced mannose-specific lectin (IlMSL) from I. lactea enhanced the Cd sensitivity and increased Cd accumulation in yeast. The results of this study will enhance our understanding of the molecular mechanism of Cd tolerance and accumulation in I. lactea and ultimately provide valuable resources for using Cd tolerant genes for developing efficient strategies for phytoremediation of Cd-contaminated soils or limiting Cd accumulation in food crops.

Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase.

BACKGROUND: Camelina sativa has attracted much interest as alternative renewable resources for biodiesel, other oil-based industrial products and a source for edible oils. Its unique oil attributes attract research to engineering new varieties of improved oil quantity and quality. The overexpression of enzymes catalyzing the synthesis of the glycerol backbone and the sequential conjugation of fatty acids into this backbone is a promising approach for increasing the levels of triacylglycerol (TAG). In a previous study, we co-expressed the diacylglycerol acyltransferase (DGAT1) and glycerol-3-phosphate dehydrogenase (GPD1), involved in TAG metabolism, in Camelina seeds. Transgenic plants exhibited a higher-percentage seed oil content, a greater seed mass, and overall improved seed and oil yields relative to wild-type plants. To further increase seed oil content in Camelina, we utilized metabolite profiling, in conjunction with transcriptome profiling during seed development to examine potential rate-limiting step(s) in the production of building blocks for TAG biosynthesis. RESULTS: Transcriptomic analysis revealed approximately 2518 and 3136 transcripts differentially regulated at significant levels in DGAT1 and GPD1 transgenics, respectively. These transcripts were found to be involved in various functional categories, including alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. We quantified the relative contents of over 240 metabolites. Our results indicate major metabolic switches in transgenic seeds associated with significant changes in the levels of glycerolipids, amino acids, sugars, and organic acids, especially the TCA cycle and glycolysis intermediates. CONCLUSIONS: From the transcriptomic and metabolomic analysis of DGAT1, GPD1 and DGAT1 + GPD1 expressing lines of C. sativa, we conclude that TAG production is limited by (1) utilization of fixed carbon from the source tissues supported by the increase in glycolysis pathway metabolites and decreased transcripts levels of transcription factors controlling fatty acids synthesis; (2) TAG accumulation is limited by the activity of lipases/hydrolases that hydrolyze TAG pool supported by the increase in free fatty acids and monoacylglycerols. This comparative transcriptomics and metabolomics approach is useful in understanding the regulation of TAG biosynthesis, identifying bottlenecks, and the corresponding genes controlling these pathways identified as limitations, for generating Camelina varieties with improved seed and oil yields.

Exogenous glutathione increased lead uptake and accumulation in Iris lactea var. chinensis exposed to excess lead.

Long- and short-term hydroponic experiments were conducted to study the effect of different concentrations of exogenous glutathione (GSH) on Pb uptake, translocation, and gene expresses in Iris lactea var. chinensis exposed to excess lead (Pb). Exogenous GSH remarkedly promoted Pb uptake and translocation in long-term (14 d) experiment, and the GSH-dose-dependent increases in shoot and root Pb contents existed obviously when GSH concentrations were lower than 800 mg·L-1. The fresh weight in gradual rise in plants was observed with the increase of exogenous GSH concentration. In short-term (24 h) experiment, Pb contents in roots under Pb with L-buthionine sulfoximine (BSO, a known inhibitor of GSH biosynthesis) treatments were significantly lower than that under Pb exposure alone. The transcript levels of three genes (Ilγ-ECS, IlGS, and IlPCS) involved in GSH synthesis and metabolism, showed no significant change in expression pattern except that upregulation after 24 h of treatment with Pb and GSH in comparison with that of the single Pb treatment. Further, the level of IlGS transcript after exposure for 4 h was much higher than that of Ilγ-ECS and IlPCS transcripts. All these results obtained here suggest that exogenous GSH can increase Pb accumulation, detoxification, and translocation to the shoot.

Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression.

Plant seed oil-based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum-derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co-expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol-3-phosphate dehydrogenase (GPD1) genes under the control of seed-specific promoters. Plants co-expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild-type plants. Further, DGAT1- and GDP1-co-expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild-type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1- and GPD1-co-expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield.

Overexpression of rice glutaredoxins (OsGrxs) significantly reduces arsenite accumulation by maintaining glutathione pool and modulating aquaporins in yeast.

Arsenic (As) is an acute poison and class I carcinogen, can cause a serious health risk. Staple crops like rice are the primary source of As contamination in human food. Rice grown on As contaminated areas accumulates higher As in their edible parts. Based on our previous transcriptome data, two rice glutaredoxins (OsGrx_C7 and OsGrx_C2.1) were identified that showed up-regulated expression during As stress. Here, we report OsGrx_C7 and OsGrx_C2.1 from rice involved in the regulation of intracellular arsenite (AsIII). To elucidate the mechanism of OsGrx mediated As tolerance, both OsGrxs were cloned and expressed in Escherichia coli (Δars) and Saccharomyces cerevisiae mutant strains (Δycf1, Δacr3). The expression of OsGrxs increased As tolerance in E. coli (Δars) mutant strain (up to 4 mM AsV and up to 0.6 mM AsIII). During AsIII exposure, S. cerevisiae (Δacr3) harboring OsGrx_C7 and OsGrx_C2.1 have lower intracellular AsIII accumulation (up to 30.43% and 24.90%, respectively), compared to vector control. Arsenic accumulation in As-sensitive S. cerevisiae mutant (Δycf1) also reduced significantly on exposure to inorganic As. The expression of OsGrxs in yeast maintained intracellular GSH pool and increased extracellular GSH concentration. Purified OsGrxs displays in vitro GSH-disulfide oxidoreductase, glutathione reductase and arsenate reductase activities. Also, both OsGrxs are involved in AsIII extrusion by altering the Fps1 transcripts in yeast and protect the cell by maintaining cellular GSH pool. Thus, our results strongly suggest that OsGrxs play a crucial role in the maintenance of the intracellular GSH pool and redox status of the cell during both AsV and AsIII stress and might be involved in regulating intracellular AsIII levels by modulation of aquaporin expression and functions.

Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with Enhanced Glutathione Production by Overexpressing Bacterial γ-Glutamylcysteine Synthase.

Silver nanoparticles (Ag NPs) are widely used in consumer products, and their release has raised serious concerns about the risk of their exposure to the environment and to human health. However, biochemical mechanisms by which plants counteract NP toxicity are largely unknown. We have previously engineered Crambe abyssinica plants expressing the bacterial γ-glutamylecysteine synthase (γ-ECS) for enhancing glutathione (GSH) levels. In this study, we investigated if enhanced levels of GSH and its derivatives can protect plants from Ag NPs and AgNO3 (Ag(+) ions). Our results showed that transgenic lines, when exposed to Ag NPs and Ag(+) ions, were significantly more tolerant, attaining a 28%-46% higher biomass and 34-49% more chlorophyll content, as well as maintaining 35-46% higher transpiration rates as compared to those of wild type (WT) plants. Transgenic γ-ECS lines showed 2-6-fold Ag accumulation in shoot tissue and slightly lower or no difference in root tissue relative to levels in WT plants. The levels of malondialdehyde (MDA) in γ-ECS lines were also 27.3-32.5% lower than those in WT Crambe. These results indicate that GSH and related peptides protect plants from Ag nanotoxicity. To our knowledge, this is the first direct report of Ag NP detoxification by GSH in transgenic plants, and these results will be highly useful in developing strategies to counteract the phytotoxicty of metal-based nanoparticles in crop plants.

Sulfur mediated reduction of arsenic toxicity involves efficient thiol metabolism and the antioxidant defense system in rice.

Arsenic (As) contamination is a global issue, with South Asia and South East Asia being worst affected. Rice is major crop in these regions and can potentially pose serious health risks due to its known As accumulation potential. Sulfur (S) is an essential macronutrient and a vital element to combat As toxicity. The aim of this study was to investigate the role of S with regards to As toxicity in rice under different S regimes. To achieve this aim, plants were stressed with AsIII and AsV under three different S conditions (low sulfur (0.5mM), normal sulfur (3.5mM) and high sulfur (5.0mM)). High S treatment resulted in increased root As accumulation, likely due to As complexation through enhanced synthesis of thiolic ligands, such as non-protein thiols and phytochelatins, which restricted As translocation to the shoots. Enzymes of S assimilatory pathways and downstream thiolic metabolites were up-regulated with increased S supplementation; however, to maintain optimum concentrations of S, transcript levels of sulfate transporters were down-regulated at high S concentration. Oxidative stress generated due to As was counterbalanced in the high S treatment by reducing hydrogen peroxide concentration and enhancing antioxidant enzyme activities. The high S concentration resulted in reduced transcript levels of Lsi2 (a known transporter of As). This reduction in Lsi2 expression level is a probable reason for low shoot As accumulation, which has potential implications in reducing the risk of As in the food chain.

A γ-glutamyl cyclotransferase protects Arabidopsis plants from heavy metal toxicity by recycling glutamate to maintain glutathione homeostasis.

Plants detoxify toxic metals through a GSH-dependent pathway. GSH homeostasis is maintained by the γ-glutamyl cycle, which involves GSH synthesis and degradation and the recycling of component amino acids. The enzyme γ-glutamyl cyclotransferase (GGCT) is involved in Glu recycling, but the gene(s) encoding GGCT has not been identified in plants. Here, we report that an Arabidopsis thaliana protein with a cation transport regulator-like domain, hereafter referred to as GGCT2;1, functions as γ-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in Saccharomyces cerevisiae produced phenotypes that were consistent with decreased GSH content attributable to either GSH degradation or the diversion of γ-glutamyl peptides to produce 5-oxoproline (5-OP). 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic (As) via GSH degradation. Recombinant GGCT2;1 converted both GSH and γ-glutamyl Ala to 5-OP in vitro. GGCT2;1 transcripts were upregulated in As-treated Arabidopsis, and ggct2;1 knockout mutants were more tolerant to As and cadmium than the wild type. Overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Under As toxicity, the overexpression lines showed minimal changes in de novo Glu synthesis, while the ggct2;1 mutant increased nitrogen assimilation by severalfold, resulting in a very low As/N ratio in tissue. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress by recycling Glu.

A novel stress-associated protein 'AtSAP10' from Arabidopsis thaliana confers tolerance to nickel, manganese, zinc, and high temperature stress.

We describe here the functional characterization of a novel AtSAP10, a member of the Stress Associated Protein (SAP) gene family, from Arabidopsis thaliana ecotype Columbia. AtSAP10 contains an A20 and AN1 zinc-finger domain at the N- and C-terminal, respectively. Arabidopsis SAP10 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (Ni, Cd, Mn, Zn, and As), high and low temperatures, cold, and ABA. Overexpression of AtSAP10 in Arabidopsis conferred strong tolerance to heavy metals such as Ni, Mn, and Zn and to high temperature stress. AtSAP10 transgenic plants under these stress conditions grew green and healthy, attained several-fold more biomass, and had longer roots as compared to wild type plants. Further, while these transgenic plants accumulated significantly greater amounts of Ni and Mn in both shoots and root tissues, there was no significant difference in the accumulation of Zn. AtSAP10 promoter-GUS fusion studies revealed a root and floral organ-specific expression of AtSAP10. Overexpression of AtSAP10-GFP fusion protein showed the localization in both nucleus and cytoplasm. Taken together, these results showed that AtSAP10 is a potentially useful candidate gene for engineering tolerance to heavy metals and to abiotic stress in cultivated plants.

Expression profiling of Crambe abyssinica under arsenate stress identifies genes and gene networks involved in arsenic metabolism and detoxification.

BACKGROUND: Arsenic contamination is widespread throughout the world and this toxic metalloid is known to cause cancers of organs such as liver, kidney, skin, and lung in human. In spite of a recent surge in arsenic related studies, we are still far from a comprehensive understanding of arsenic uptake, detoxification, and sequestration in plants. Crambe abyssinica, commonly known as 'abyssinian mustard', is a non-food, high biomass oil seed crop that is naturally tolerant to heavy metals. Moreover, it accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae family. Thus, C. abyssinica has great potential to be utilized as an ideal inedible crop for phytoremediation of heavy metals and metalloids. However, the mechanism of arsenic metabolism in higher plants, including C. abyssinica, remains elusive. RESULTS: To identify the differentially expressed transcripts and the pathways involved in arsenic metabolism and detoxification, C. abyssinica plants were subjected to arsenate stress and a PCR-Select Suppression Subtraction Hybridization (SSH) approach was employed. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The transcripts corresponding to the subtracted cDNAs showed strong upregulation by arsenate stress as confirmed by the semi-quantitative RT-PCR. CONCLUSIONS: Our study revealed novel insights into the plant defense mechanisms and the regulation of genes and gene networks in response to arsenate toxicity. The differential expression of transcripts encoding glutathione-S-transferases, antioxidants, sulfur metabolism, heat-shock proteins, metal transporters, and enzymes in the ubiquitination pathway of protein degradation as well as several unknown novel proteins serve as molecular evidence for the physiological responses to arsenate stress in plants. Additionally, many of these cDNA clones showing strong upregulation due to arsenate stress could be used as valuable markers. Further characterization of these differentially expressed genes would be useful to develop novel strategies for efficient phytoremediation as well as for engineering arsenic tolerant crops with reduced arsenic translocation to the edible parts of plants.

Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography.

The HPLC method presented here for the quantification of metal-binding thiols is considerably shorter than most previously published methods. It is a sensitive and highly reproducible method that separates monobromobimane tagged monothiols (cysteine, glutathione, gamma-glutamylcysteine) along with polythiols (PC(2), PC(3), PC(4) and PC(5)) within 23min from a wide variety of samples. Total run time of the method is 35min. Detection limits for thiols is 33fmol for 10microlL injection. This method will be applicable to study the metal detoxification mechanisms for a wide variety of cell cultures and tissues of plants and trees including algae, Arabidopsis, crambe, rice, and red spruce.

The plant MT1 metallothioneins are stabilized by binding cadmiums and are required for cadmium tolerance and accumulation.

The small Arabidopsis genome contains nine metallothionein-like (MT) sequences with classic, cysteine-rich domains separated by spacer sequences, quite unlike the small conserved MT families found vertebrate genomes. Phylogenetic analysis revealed four ancient and divergent classes of plant MTs that predate the monocot-dicot divergence. A distinct cysteine spacing pattern suggested differential metal ion specificity for each class. The in vivo stability of representatives of the four classes of plant MT proteins and a mouse MT2 control expressed in E. coli were enhanced by cadmium (Cd). Particular MTs were also stabilized by arsenic (As), copper (Cu), and or zinc (Zn). To understand why plants have such a diversity of MT sequences, the Arabidopsis MT1 class, comprised of three genes, MT1a, MT1b, and MT1c, was characterized in more detail in plants. MT1 family transcripts were knocked down to less than 5-10% of wild-type levels in Arabidopsis by expression of a RNA interference (RNAi) construct. The MT1 knockdown plant lines were all hypersensitive to Cd and accumulated several fold lower levels of As, Cd, and Zn than wildtype, while Cu and Fe levels were unaffected. The ancient class of MT1 protein sequences may be preserved in plant genomes, because it has distinct metal-binding properties, confers tolerance to cadmium, and can assist with zinc homeostasis.

Arsenic and mercury tolerance and cadmium sensitivity in Arabidopsis plants expressing bacterial gamma-glutamylcysteine synthetase.

Cysteine sulfhydryl-rich peptide thiols are believed to play important roles in the detoxification of many heavy metals and metalloids such as arsenic, mercury, and cadmium in plants. The gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the synthesis of the dipeptidethiol gamma-glu-cys (gamma-EC), the first step in the biosynthesis of phytochelatins (PCs). Arabidopsis thaliana, engineered to express the bacterial gamma-ECS gene under control of a strong constitutive actin regulatory sequence (A2), expressed gamma-ECS at levels approaching 0.1% of total protein. In response to arsenic, mercury, and cadmium stresses, the levels of gamma-EC and its derivatives, glutathione (GSH) and PCs, were increased in the A2::ECS transgenic plants to three- to 20-fold higher concentrations than the increases that occurred in wild-type (WT). Compared to cadmium and mercury treatments, arsenic treatment most significantly increased levels of gamma-EC and PCs in both the A2::ECS transgenic and WT plants. The A2::ECS transgenic plants were highly resistant to arsenic and weakly resistant to mercury. Although exposure to cadmium produced three- to fivefold increases in levels of gamma-EC-related peptides in the A2::ECS lines, these plants were significantly more sensitive to Cd(II) than WT and trace levels of Cd(II) blocked resistance to arsenic and mercury. A few possible mechanisms for gamma-ECS-enhanced arsenic and mercury resistance and cadmium hypersensitivity are discussed.

Engineering a root-specific, repressor-operator gene complex.

Strong, tissue-specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a 'repressor-operator gene complex' (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root-specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using beta-glucuronidase (GUS) and mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root-specific tobacco RB7 promoter is demonstrated.

Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity.

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.