Arsenate toxicity and stress responses in the freshwater ciliate Tetrahymena pyriformis

The arsenic metabolism in different biological organisms has been studied extensively. However, little is known about protozoa. Herein, we investigated the cell stress responses of the freshwater ciliate Tetrahymena pyriformis to arsenate toxicity. An acute toxicity assay revealed an 18-h EC(50) arsenate concentration of ca. 40 μM, which caused significant changes in the cell shape, growth and organism mobility. Whereas, under exposure to 30 μM arsenate, T. pyriformis could grow reasonably well, indicating a certain resistance of this organism. Arsenic speciation analysis revealed that 94-98% of the total arsenate in cells of T. pyriformis could be transformed to monomethylarsonic acid, dimethylarsinic acid and a small proportion of arsenite after 18 h of arsenate exposure, thus indicating the major detoxification pathway by arsenic oxidation/reduction and biomethylation. Finally, comparative proteomic analysis unveiled significant changes in the expression of multiple proteins involved in anti-oxidation, sugar and energy metabolism, proteolysis, and signal transduction. Our results revealed multiple pathways of arsenate detoxification in T. pyriformis, and indicated that protozoa may play important roles in the biogeochemical cycles of arsenic.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Microbial oxidation of arsenite in a subarctic environment: diversity of arsenite oxidase genes and identification of a psychrotolerant arsenite oxidiser

Background  

Arsenic is toxic to most living cells. The two soluble inorganic forms of arsenic are arsenite (+3) and arsenate (+5), with arsenite the more toxic. Prokaryotic metabolism of arsenic has been reported in both thermal and moderate environments and has been shown to be involved in the redox cycling of arsenic. No arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been reported in cold environments (i.e. < 10°C).     

Identification of genes and proteins involved in the pleiotropic response to arsenic stress in Caenibacter arsenoxydans, a metalloresistant beta-proteobacterium with an unsequenced genome

The effect of high concentrations of arsenic has been investigated in Caenibacter arsenoxydans, a beta-proteobacterium isolated from an arsenic contaminated environment and able to oxidize arsenite to arsenate. As the genome of this bacterium has not yet been sequenced, the use of a specific proteomic approach based on nano-high performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) studies and de novo sequencing to perform cross-species protein identifications was necessary. In addition, a random mutational analysis was performed. Twenty-two proteins and 16 genes were shown to be differentially accumulated and expressed, respectively, in cells grown in the presence of arsenite. Two genes involved in arsenite oxidation and one in arsenite efflux as well as two proteins responsible for arsenate reduction were identified. Moreover, numerous genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes were also up- or down-regulated, which demonstrates that bacterial response to arsenic is pleiotropic.     

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.     

Regulation of sugar metabolism in rice (Oryza sativa L.) seedlings under arsenate toxicity and its improvement by phosphate

The effect of arsenate with or without phosphate on the growth and sugar metabolism in rice seedlings cv. MTU 1010 was studied. Arsenate was found to be more toxic for root growth than shoot growth and water content of the seedlings gradually decreased with increasing concentrations. Arsenate exposure at 20 μM and 100 μM resulted in an increase in reducing sugar content and decrease in non-reducing sugar content. There was a small increase in starch content, the activity of starch phosphorylase was increased but α-amylase activity was found to be decreased. Arsenate toxicity also affected the activities of different carbohydrate metabolizing enzymes. The activities of sucrose degrading enzymes viz., acid invertase and sucrose synthase were increased whereas, the activity of sucrose synthesizing enzyme, viz. sucrose phosphate synthase declined. The combined application of arsenate with phosphate exhibited significant alterations of all the parameters tested under the purview of arsenate treatment alone which was congenial to better growth and efficient sugar metabolism in rice seedlings. Thus, the use of phosphorus enriched fertilizers may serve to ensure the production of healthy rice plants in arsenic contaminated soils.     

Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.     

Proteomic analysis of copper stress responses in the roots of two rice (Oryza sativa L.) varieties differing in Cu tolerance

Background and aims

Copper (Cu) is an essential micronutrient required for growth and development of plants. However, excess Cu is toxic to plants. To understand the mechanisms involved in copper stress response, a proteomic approach was used to investigate the differences in Cu stress-induced protein expression between a Cu-tolerant variety (B1139) and a Cu-sensitive one (B1195) of rice.

Methods

Rice seedlings were exposed to 8 μM Cu for 3 days, with plants grown in the normal nutrient solution containing 0.32 μM Cu serving as the control. Proteins were extracted from the roots and separated by two-dimensional PAGE. Thirty four proteins were identified using MALDI-TOF mass spectrometry.

Results

Thirty-four protein spots were found to be differently expressed in the Cu-stressed roots in at least one variety of rice, including those involved in antioxidative defense, redox regulation, stress response, sulfur and glutathione (GSH) metabolism, carbohydrate metabolism, signal transduction, and some other proteins with various functions. Nine proteins, including putative cysteine synthase, probable serine acetyltransferase 3, L-ascorbate peroxidase 1, putative glutathione S-transferase 2, and thioredoxin-like 3-3, exhibited a greater increase in response to Cu stress in the Cu-tolerant variety B1139 compared with the Cu–sensitive variety B1195.

Conclusion

The majority of the proteins showing differential expression in response to Cu exposure are involved in the redox regulation, and sulfur and GSH metabolism, suggesting that these proteins, together with antioxidant enzymes, play an important role in the detoxification of excess Cu and maintaining cellular homeostasis.     

Proteomics of rice in response to heat stress and advances in genetic engineering for heat tolerance in rice

Rice is the most important food crop worldwide. Global warming inevitably affects the grain yields of rice. Recent proteomics studies in rice have provided evidence for better understanding the mechanisms of thermal adaptation. Heat stress response in rice is complicated, involving up- or down-regulation of numerous proteins related to different metabolic pathways. The heat-responsive proteins mainly include protection proteins, proteins involved in protein biosynthesis, protein degradation, energy and carbohydrate metabolism, and redox homeostasis. In addition, increased thermotolerance in transgenic rice was obtained by overexpression of rice genes and genes from other plants. On the other hand, heterologous expression of some rice proteins led to enhanced thermotolerance in bacteria and other easily transformed plants. In this paper, we review the proteomic characterization of rice in response to high temperature and achievements of genetic engineering for heat tolerance in rice.     

Arsenic uptake and accumulation in rice (Oryza sativa L.) irrigated with contaminated water

Long-term use of arsenic contaminated groundwater to irrigate crops, especially paddy rice (Oryza sativaL.) has resulted in elevated soil arsenic levels in Bangladesh. There is, therefore, concern regarding accumulation of arsenic in rice grown on these soils. A greenhouse pot experiment was conducted to evaluate the impact of arsenic-contaminated irrigation water on the growth and uptake of arsenic into rice grain, husk, straw and root. There were altogether 10 treatments which were a combination of five arsenate irrigation water concentrations (0–8 mg As l^–1) and two soil phosphate amendments. Use of arsenate containing irrigation water reduced plant height, decreased rice yield and affected development of root growth. Arsenic concentrations in all plant parts increased with increasing arsenate concentration in irrigation water. However, arsenic concentration in rice grain did not exceed the maximum permissible limit of 1.0 mg As kg^–1. Arsenic accumulation in rice straw at very high levels indicates that feeding cattle with such contaminated straw could be a direct threat for their health and also, indirectly, to human health via presumably contaminated bovine meat and milk. Phosphate application neither showed any significant difference in plant growth and development, nor in As concentrations in plant parts.     

Rice is more efficient in arsenite uptake and translocation than wheat and barley

Rice is efficient at arsenic (As) accumulation, thus posing a potential health risk to humans and animals. Arsenic bioavailability in submerged paddy soil is enhanced due to mobilisation of arsenite, but rice may also have an inherently greater ability to take up and translocate arsenite than other cereal crops. To test this hypothesis, rice, wheat and barley were exposed to 5 µM arsenate or arsenite for 24 h. Arsenic uptake and distribution, and As speciation in the xylem sap and nutrient solution were determined. Regardless of the As form supplied to plants, rice accumulated more As in the shoots than wheat or barley. Arsenite uptake by rice was double of that by wheat or barley, whereas arsenate uptake was similar between rice and wheat and approximately a third smaller in barley. The efficiency of As translocation from roots to shoots was greater when plants were supplied with arsenite than with arsenate, and in both treatments rice showed the highest translocation efficiency. Arsenite was the main species of As (86–97%) in the xylem sap from arsenite-treated plants of all three species. In the arsenate-treated plants, 84%, 45% and 63% of As in the xylem sap of rice, wheat and barley, respectively, was arsenite. Arsenite efflux to the external medium was also observed in all three plant species exposed to arsenate. The results show that rice is more efficient than wheat or barley in arsenite uptake and translocation, probably through the highly efficient pathway for silicon.     

Arsenic stress affects the expression profile of genes of 14-3-3 proteins in the shoot of mycorrhiza colonized rice

The intimate association between the arbuscular mycorrhizal fungi and host plants helps the latter in phosphate acquisition in exchange of carbohydrates and in enhanced stress tolerance. Similarly, the ubiquitous 14-3-3 protein family is known to be a major regulator of plant metabolism and stress responses. However, the involvement of mycorrhiza and plant 14-3-3 proteins interaction in plant response to environmental stimuli, such as arsenic (As) stress, is yet unknown. In this study, we analysed the impact of the As stress on the expression profile of 14-3-3 genes in the shoot of mycorrhiza colonized rice (Oryza sativa) plants. Ten day old rice seedlings were kept for 45 days for mycorrhizal colonisation (10 g inoculum per 120 g soilrite) and were then subjected to 12.5 µM arsenate [As(V)] exposure for 1 and 3 days, in hydroponics. Arsenate stress resulted in significant change in expression of 14-3-3 protein genes in non-colonized and mycorrhiza colonized rice plants which indicated As mediated effects on 14-3-3 proteins as well as interactive impact of mycorrhiza colonization. Indeed, mycorrhiza colonization itself induced up-regulation of all 14-3-3 genes in the absence of As stress. The results thus indicate that 14-3-3 proteins might be involved in As stress signalling and the mycorrhiza induced As stress response of the rice plants.     

A CDC25 homologue from rice functions as an arsenate reductase

Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)(5)R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1.     

Proteomic identification of differentially expressed proteins in the anoxic rice coleoptile

Rice is the staple food for more than fifty percent of the world's population, and is therefore an important crop. However, its production is hindered by several biotic and abiotic stresses. Although rice is the only crop that can germinate even in the complete absence of oxygen (i.e. anoxia), flooding (low oxygen) is one of the major causes of reduced rice production. Rice germination under anoxia is characterized by the elongation of the coleoptile, but leaf growth is hampered. In this work, a comparative proteomic approach was used to detect and identify differentially expressed proteins in the anoxic rice coleoptile compared to the aerobic coleoptile. Thirty-one spots were successfully identified by MALDI-TOF MS analysis. The majority of the identified proteins were related to stress responses and redox metabolism. The expression levels of twenty-three proteins and their respective mRNAs were analyzed in a time course experiment.     

Proteome analysis of maize roots reveals that oxidative stress is a main contributing factor to plant arsenic toxicity

To gain insight into plant responses to arsenic, the effect of arsenic exposure on maize (Zea mays L.) root proteome has been examined. Maize seedlings were fed hydroponically with 300 microM sodium arsenate or 250 microM sodium arsenite for 24 h, and changes in differentially displayed proteins were studied by two-dimensional electrophoresis and digital image analysis. About 10% of total detected maize root proteins (67 out of 700) were up- or down-regulated by arsenic, among which 20 were selected as being quite reproducibly affected by the metalloid. These were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and 11 of them could be identified by comparing their peptide mass fingerprints against protein- and expressed sequence tag-databases. The set of identified maize root proteins highly responsive to arsenic exposure included a major and functionally homogeneous group of seven enzymes involved in cellular homeostasis for redox perturbation (e.g., three superoxide dismutases, two glutathione peroxidases, one peroxiredoxin, and one p-benzoquinone reductase) besides four additional, functionally heterogeneous, proteins (e.g., ATP synthase, succinyl-CoA synthetase, cytochrome P450 and guanine nucleotide-binding protein beta subunit). These findings strongly suggest that the induction of oxidative stress is a main process underlying arsenic toxicity in plants.     

Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed

Background  

High Salinity is a major environmental stress influencing growth and development of rice. Comparative proteomic analysis of hybrid rice shoot proteins from Shanyou 10 seedlings, a salt-tolerant hybrid variety, and Liangyoupeijiu seedlings, a salt-sensitive hybrid variety, was performed to identify new components involved in salt-stress signaling.