Superoxide-Dismutase Deficient Mutants in Common Beans (Phaseolus vulgaris L.): Genetic Control, Differential Expressions of Isozymes, and Sensitivity to Arsenic

Redox State as a Central Regulator of Plant-Cell Stress Responses

Abstract: Plants are frequently exposed to different stressful factors, both of biotic or abiotic nature, which limit their growth and productivity. To survive under stress conditions, plants must activate stress-specific signalling pathways, which finally lead to morphological, physiological, and biochemical changes that allow to adapt to the adverse environment. Cellular redox homeostasis, determined by a complex interplay between pathways that produce and scavenge reactive oxygen species (ROS), plays a key role in the adaptive response. Each deviation in the cellular redox state, due to an imbalance of ROS production and/or scavenging, is indicative of environmental disturbance and works as a signal. Under stress conditions, different ROS are produced in many cell compartments. Plants have very proficient, versatile and flexible antioxidant machinery, which comprises enzymes and metabolites with distinct biochemical properties and distinct sub-cellular localization. The antioxidant systems play a key role in the control of redox homeostasis, determining either the extent or the specificity of ROS signals and the downstream redox-dependent responses. Redox signalling is responsive to a number of environmental cues, and the complex and dynamic pathways of redox regulation occur in different cell compartments. The redox-dependent modification of sensitive signalling proteins is proposed as a key mode of redox signal transmission. Each redox-dependent interaction is opportunely regulated by a restricted environment, whose change transfers the complex system of information and influences the plant response to external changes.

Retraction Note to: Coordinated response of sulfate transport, cysteine biosynthesis, and glutathione-mediated antioxidant defense in lentil (Lens culinaris Medik.) genotypes exposed to arsenic.

Abstract: Response of sulfate transporters, thiol metabolism, and antioxidant defense system was studied in roots of two lentil (Lens culinaris Medik.) genotypes grown in arsenic (10, 25, and 40 μM As(V))-supplemented nutrient solution, and significant changes compared to control (0 μM As(V)) were observed mainly at 25 and 40 μM. In L 414, high glutathione (GSH) redox (0.8-0.9) was maintained with elevated thiol synthesis, powered by transcriptional up-regulation of LcSultr1;1 and LcSultr1;2 sulfate transporters and significant induction of LcSAT1;1 and LcSAT1;2 (serine acetyltransferase), OAS-TL (O-acetylserine(thiol)-lyase), γ-ECS (γ-glutamylcysteine synthetase), and PCS (phytochelatin synthase) genes predominantly within 12-24 h of As exposure at 25 μM and within 6-12 h at 40 μM. This thiolic potency in L 414 roots was effectively complemented by up-regulation of gene expressions and consequent enhanced activities of superoxide dismutase, ascorbate peroxidase (APX), dehydroascorbate reductase, glutathione reductase (GR), and glutathione-S-transferase (GST) isoforms at 25 and 40 μMAs, efficiently scavenging excess reactive oxygen species to prevent onset of As-induced oxidative stress and consequent inhibition of root growth in L 414. In contrast, down-regulation of vital sulfate-uptake transporters as well as entire thiol-metabolizing system and considerably low APX, GST, and GR expressions in DPL 59 not only resulted in reduced GSH redox but also led to over-accumulation of H2O2. This triggered membrane lipid peroxidations as the marks of As-induced oxidative damage. Results indicated coordinated response of thiol-metabolism and antioxidant defense in conferring As-tolerance in lentil, and GSH is the key point in this cascade.

Phytoremediation potential of Salvinia molesta for arsenite contaminated water: role of antioxidant enzymes

Abstract: Antioxidant enzymes are important components in the defense against arsenic (As) stress in plants. Here, we tested the hypothesis that Salvinia molesta, an aquatic fern, counteracts the harmful arsenite (AsIII) effects by activating scavenging reactive oxygen species (ROS) enzymes. Thus, our objective was to investigate the role of the superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) in S. molesta tolerance to AsIII and indicate the use of this plant in remediation of contaminated water. Plants were grown in nutrient solution at pH 6.5 and exposed to 0, 5, 10, or 20 µM AsIII for 96 h (analyses of As absorption, mineral nutrient content, and relative growth rate) and for 24 h (analyses of oxidative stress indicators and enzymatic antioxidant defenses). In the floating leaves, there was a greater basal activity of the antioxidant enzymes and less accumulation of As than in submerged leaves. The submerged leaves, which function as roots in S. molesta, accumulated more As than floating leaves, and SOD and CAT activities were inhibited. Thus, there was a greater production of ROS and oxidative stress. Our results show that S. molesta presents enzymatic antioxidant defenses to alleviate AsIII toxicity and are more effectives in the floating leaves. These results are important to elucidate the AsIII tolerance mechanisms in S. molesta and the possibility of their use in contamined water phytoremediation. Additional studies exposing plants to more prolonged stress and using AsIII concentrations closer to those found in contaminated environments will confirm this claim.

Plant Growth and Leaf Antioxidant Metabolism of Four Elite Grass Pea (Lathyrus sativus) Genotypes, Differing in Arsenic Tolerance

Abstract: Response of four improved grass pea (Lathyrus sativus L.) genotypes to arsenic was tested in pot experiment using 30 mg As l−1 up to 60 days of growth after commencement of treatment (DAC). Arsenic exposure significantly reduced growth and seed yield potential of vars B1 and BioL-212, but no such effect was observed in bold-seeded mutant and pod indehiscent mutant lines. Results revealed normal leaf photosynthesis and antioxidant metabolism at 20 DAC in both varieties. However, high superoxide dismutase activity coupled with low ascorbate redox and declining ascorbate peroxidase level led to abnormal rise in H2O2 content at reproductive stages (40 and 60 DAC), consequently, resulting in significantly enhanced arsenic-induced oxidative damage and physiological impediment in both varieties. By contrast, H2O2 level in both the mutants was remarkably modulated at reproductive stage (40 DAC) in a highly controlled manner by balanced action of antioxidant defense. This favored normal photosynthesis and ensured good grain yield even under prolonged arsenic exposure by effectively preventing oxidative damage to membrane in both the mutants. Native PAGE analysis revealed that enzyme isoforms were involved in the regulation of foliar H2O2 metabolism, the level of which was found extremely critical in determining arsenic tolerance of grass pea genotypes.

Exogenous thiourea modulates antioxidant defence and glyoxalase systems in lentil genotypes under arsenic stress

Abstract: Arsenic (As) is a wide-spread toxic and carcinogenic metalloid, affecting crop productivity worldwide. Lentil, an edible grain legume, is increasingly exposed to soil arsenic contamination. However, our understandings regarding mechanistic details and mitigation strategies against arsenic toxicity in edible legume are extremely poor. Main purpose of the present study was to investigate the As-effects and its mitigation by thiourea (TU), a sulfhydryl bioregulator, in lentil. Four widely grown lentil genotypes were grown in nutrient media, supplemented with 30 μM sodium arsenate (As), As + 6.5 mM TU and As + 13 mM TU, keeping an untreated control for 10 d. As severely affected plant dry weight by accumulating in shoots and roots. However, TU application sequestered As in crop roots and prevented up-ward translocation of As. TU coordinately modulated glyoxalase system I and II (Gly I and II) and ascorbate (AsA)-glutathione (GSH) redox, and antioxidant defense enzymes in both leaves and roots of four genotypes. Elevation of Gly system prevented toxic methyl glyoxal overaccumulation whereas stimulated AsA-GSH cycle enzymes and Glutathione s-transferase and catalase effectively scavenged H 2 O 2 and prevented reactive oxygen species (ROS) -mediated onset of oxidative damage in four genotypes, as was evident from ROS-imaging study. Results suggested exogenous TU stimulated the Gly and antioxidant defense in fine tune against As-induced oxidative damage in lentil genotypes.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Abstract: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.

Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method

Abstract: The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction

Abstract: Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

Superoxide Radical and Superoxide Dismutases

Abstract: O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Role of superoxide dismutases (SODs) in controlling oxidative stress in plants

Abstract: Reactive O2 species (ROS) are produced in both unstressed and stressed cells. Plants have welldeveloped defence systems against ROS, involving both limiting the formation of ROS as well as instituting its removal. Under unstressed conditions, the formation and removal of O2 are in balance. However, the defence system, when presented with increased ROS formation under stress conditions, can be overwhelmed. Within a cell, the superoxide dismutases (SODs) constitute the first line of defence against ROS. Specialization of function among the SODs may be due to a combination of the influence of subcellular location of the enzyme and upstream sequences in the genomic sequence. The commonality of elements in the upstream sequences of Fe, Mn and CuuZn SODs suggests a relatively recent origin for those regulatory regions. The differences in the upstream regions of the three FeSOD genes suggest differing regulatory control which is borne out in the research literature. The finding that the upstream sequences of Mn and peroxisomal CuuZn SODs have three common elements suggests a common regulatory pathway. The tools are available to dissect further the molecular basis for antioxidant defence responses in plant cells. SODs are clearly among the most important of those defences, when coupled with the necessary downstream events for full detoxification of ROS.