Superoxide-Dismutase Deficient Mutants in Common Beans (Phaseolus vulgaris L.): Genetic Control, Differential Expressions of Isozymes, and Sensitivity to Arsenic
28 Aug 2013-BioMed Research International (Hindawi Publishing Corporation)-Vol. 2013, pp 782450-782450
TL;DR: Gene expressions using qRT PCR confirmed higher expressions of Cu/Zn SOD transcripts in both mutants and the absence of Fe SOD in sodPv 1 and Mn S OD in SodPv 2 and ROS-imaging study revealed overaccumulation of both superoxides and H2O2 in leaves of double mutant.
Abstract: Two common bean (Phaseolus vulgaris L.) mutants, sodPv 1 and sodPv 2, exhibiting foliar superoxide dismutase (SOD) activity of only 25% and 40% of their mother control (MC) cv. VL 63 were isolated in EMS-mutagenized (0.15%, 8 h) M2 progeny. Native-PAGE analysis revealed occurrence of Mn SOD, Fe SOD, Cu/Zn SOD I and Cu/Zn SOD II isozymes in MC, while Fe SOD, and Mn SOD were not formed in sodPv 1 and sodPv 2 leaves, respectively. In-gel activity of individual isozymes differed significantly among the parents. SOD deficiency is inherited as recessive mutations, controlled by two different nonallelic loci. Gene expressions using qRT PCR confirmed higher expressions of Cu/Zn SOD transcripts in both mutants and the absence of Fe SOD in sodPv 1 and Mn SOD in sodPv 2. In 50 μM arsenic, Cu/Zn SODs genes were further upregulated but other isoforms downregulated in the two mutants, maintaining SOD activity in its control level. In an F2 double mutants of sodPv 1 × sodPv 2, no Fe SOD, and Mn SOD expressions were detectable, while both Cu/Zn SODs are down-regulated and arsenic-induced leaf necrosis appeared. In contrast to both mutants, ROS-imaging study revealed overaccumulation of both superoxides and H2O2 in leaves of double mutant.
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04 Mar 2015
TL;DR: Investigation of the functional relationship between hydrogen sulfide (H2S) and glutathione (GSH) in regulation of sulfate transporters and cysteine metabolisms revealed dual roles of H2S as stimulator of GSH-independent antioxidant defense and regulator of cysteines homeostasis through its metabolic diversion during As-exposure and blockage of G SH biosynthesis.
Abstract: Changes in expressions of up- and downstream thiol cascade were studied in leaves of Phaseolus vulgaris L. cv. VL-63 and its mutant, pvsod1 (deficient in superoxide dismutase activity) under 50 μM sodium arsenate (As), As + l-buthionine-sulfoximine (BSO) and As + BSO + Sodium hydrosulfide (NaHS)-treatments for 10 days. Main objective was to investigate the functional relationship between hydrogen sulfide (H2S) and glutathione (GSH) in regulation of sulfate transporters and cysteine metabolisms as up-stream thiol components and GSH, phytochelatins (PCs) and antioxidant defense response as downstream cascade under As-exposure. As treatment alone initiated coordinated inductions of sulfate transport, biosynthesis of cysteine, GSH, and PCs, and GSH-mediated antioxidant defense in the pvsod1 mutant. At As + BSO, GSH synthesis was blocked, resulting in significantly low GSH redox pool and steep decline in GSH-dependent antioxidant capacity of both the genotypes. However, unlike VL-63, cysteine-degradation pathway was induced in pvsod1 mutant, resulting in significant accumulation of endogenous H2S. The H2S-surge in the pvsod1 mutant stimulated ascorbate-dependent antioxidant defense and catalases and regulated O-acetylserine (thiol)lyase activity, preventing overaccumulation of H2O2 and free cysteine, respectively. No As-induced oxidative stress symptom was observed in the mutant. This trend was maintained at As + BSO + NaHS treatment, also. In contrast, failure to induce entire cascade from sulfate transport to downstream antioxidant defense led to onset of As-induced oxidative damage in VL-63 plant. Results revealed dual roles of H2S as (a) stimulator of GSH-independent antioxidant defense and (b) regulator of cysteine homeostasis through its metabolic diversion during As-exposure and blockage of GSH biosynthesis.
11 citations
Cites background or methods or result from "Superoxide-Dismutase Deficient Muta..."
...org/PvGEA/) and reports on Phaseolus vulgaris (Liao et al. 2012; Talukdar and Talukdar 2013) and were presented in Table 1....
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...Pilot experiments indicated significant effect of arsenate, BSO and NaHS at these concentrations on plant biomass (Talukdar 2013; Talukdar and Talukdar 2013) and thus, were selected for the present study....
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...…of common bean (Phaseolus vulgaris L.) cultivar VL-63 and one pvsod1 mutant exhibiting low superoxide dismutase (SOD) activity (25 % of wild type) (Talukdar and Talukdar 2013) were surface sterilized with NaOCl (0.1 %, w/v) and continuously washed under running tap water followed by distilled…...
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...…by Primer ExpressTM V. 3.0 software (Applied Biosystems, USA) with the search of available sequence databases (http://www.phytozome.net/ commonbean.php; http://plantgrn.noble.org/PvGEA/) and reports on Phaseolus vulgaris (Liao et al. 2012; Talukdar and Talukdar 2013) and were presented in Table 1....
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...The result pointed out that cytosolic isoforms (Cu/Zn SODs) in both bean genotypes played pivotal roles in maintaining SOD activity during As-exposures, which confirmed earlier findings (Abercrombie et al. 2008; Talukdar and Talukdar 2013, 2014a)....
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01 Jan 2014
TL;DR: A mutant designated as pvcys exhibiting huge deficiency in foliar L-cysteine desulfhydrase and D-cySteine des sulfurhydrase activity was constitutively low in the mutant, even in response to external stress.
Abstract: A mutant designated as pvcys exhibiting huge deficiency in foliar L-cysteine desulfhydrase and D-cysteine desulfhydrase activity were isolated from an ethylmethane sulfonate-mutagenized M 2 population of a Phaseolus vulgaris L. genotype VL 63. The mutant showed growth inhibition and morpho-agronomic anomalies but exhibited high cysteine content and very low endogenous hydrogen sulfide concentration mainly due to crippling of cysteine degradation. Despite a normal glutathione and ascorbate redox pool, the mutant suffered oxidative stress due to over-accumulation of H 2 O 2 and consequent membrane damage by lipid peroxidation. Uniquely, this oxidative load was relieved in the mutant upon imposition to 20 and 40 µM sodium arsenate through consumption of excess cysteine to meet the growing demand for glutathione and subsequently, to confer tolerance to arsenate-induced oxidative stress. Both L-cysteine desulfhydrase and D-cysteine desulfhydrase activity was constitutively low in the mutant, even in response to external stress. The pvcys mutation was monogenic recessive in inheritance.
8 citations
Cites background from "Superoxide-Dismutase Deficient Muta..."
...…of GSH-mediated cellular defense during stress response has nicely been demonstrated in the model plant Arabidopsis thaliana, and in food legumes, like Phaseolus vulgaris, Pisum sativum, Lens culinaris, and Lathyrus sativus (Tsyganov et al. 2007; Talukdar 2012a, b; Talukdar and Talukdar 2013a,b)....
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01 Jan 2015
TL;DR: In this paper, the comprehensive knowledge generated in this area has been compiled and analyzed, and a comprehensive literature survey has been conducted to understand plant thiol metabolism from source (soil) to sink (grains).
Abstract: Growing plants have a constitutive demand for thiol (sulfur) to synthesize protein, sulfolipid, and other essential sulfur (S)-containing molecules for growth. The uptake and subsequent distribution of sulfate is regulated in response to demand and environmental factors. Sulfate transport consists of both constitutive and sulfur nutrition-dependent regulated transport. The acquisition of sulfur by plants has become an increasingly important concern for the agriculture due to the decreasing trends of S-emissions from industrial sources and the consequent limitation of inputs from deposition. The recognition of the importance of sulfate for plant growth and vigor and hence crop yield, as well as the nutritional importance of sulfur for human and animal diets, has increasingly been recognized. Cysteine synthesis in plants is a fundamental process for protein biosynthesis and all anabolic pathways that require reduced sulfur. Cysteine is the first committed molecule in plant metabolism that contains both sulfur and nitrogen, and, thus, the regulation of its biosynthesis is of utmost importance for the synthesis of a number of essential metabolites in plant pathways. Cysteine is incorporated into proteins and glutathione directly or serves as a sulfur donor for the synthesis of S-containing compounds such as methionine and its derivatives S-adenosylmethionine and S-methylmethionine and many secondary compounds. Furthermore, cysteine acts as a general catalyst in redox reactions through the nucleophilic properties of its sulfur atom, utilizing dithiol–disulfide interchange, as displayed in the thioredoxin and the glutaredoxin systems. Molecular characterization involving transcriptomics, proteomics, and metabolomics profiling in major crops like rice, barley, wheat, maize, and legumes along with model plant Arabidopsis thaliana revealed that sulfate uptake, distribution, and reductive assimilation are regulated in fine-tune depending on sulfur status and demand and that this cascade is integrated with plant photosynthesis, nutrient transports, antioxidant defense system, hormonal signaling, kinase cascades, carbohydrate metabolism, and during plants’ experiences with different biotic and abiotic stresses. This cascade can be manipulated in favor of enhanced plant growth and nutritional benefits—as, for example, effort has been initiated in food and feed legumes (chickpeas, narrow-leafed lupin, soybeans) and other plants with enhanced S-containing amino acids, threonine, glutathione, protein quality, protease inhibitors, and trace elements and with lysine, protein content, and compositions in cereal grains. This emerging prospect can be ushered by using latest cutting-edge functional genomics tools and better understanding of plant thiol-metabolism from source (soil) to sink (grains) in diverse arenas of “thiolomics.” In this chapter, the comprehensive knowledge generated in this area has been compiled and analyzed.
6 citations
28 Mar 2014
TL;DR: Biochemical analysis through cysteine synthesizing pathway in leaves revealed that activity of serine acetyl transferase was normal in both the mutant progenies but both were highly deficient in foliar O -acetylserine(thiol)-lyase (OAS-TL) activity.
Abstract: Lentil is a cool-season pulse crop, rich in protein but deficient in two sulphur-containing amino acids cysteine and methionine. Due to low genetic variability in existing germplasm, induced mutagenic technique has been adopted in lentil, and two mutant lines exhibiting poor growth and low dry weight were isolated in M 2 -mutagenized (0.10% and 0.15% EMS, 6 h) population of variety L 414. Further analysis revealed that plants from both mutant lines were highly deficient in seed cysteine (Cys) content, and thus, were tentatively designated as cysLc1 and cysLc2 mutants. Mutant plants were advanced to M 3 generation. Biochemical analysis through cysteine synthesizing pathway in leaves revealed that activity of serine acetyl transferase (SAT) was normal in both the mutant progenies but both were highly deficient in foliar O -acetylserine(thiol)-lyase (OAS-TL) activity. Transcriptomic analysis by qRT-PCR confirmed normal expression of SAT in both mutants but revealed differential expressions of two OAS-TL isoforms; OAS-TL 1 isoform was not detectable in cysLc1 mutant while expression of OAS-TL 2 isoform was totally repressed in leaves of cysLc2 mutant. Genetic studies and test of allelism pointed out that both the mutants were recessive and were complementing with each other to produce normal in F 1 and normal along with mutant plants in F 2 progeny. The progeny plants exhibiting normal phenotype showed normal mRNA transcripts of both OAS-TL isoforms. Being stable and self-fertile, the mutants will give vital clues in genetic basis of thiol-metabolic network of lentil crops.
5 citations
TL;DR: Based on RNA-seq data, tissue-specific expression revealed that DcCSD2 had higher expression in both xylem and phloem, and Dc CSD2 was differentially expressed in dark stress, which will serve as a basis for further functional insights into the DcSOD gene family.
Abstract: Superoxide dismutase (SOD) proteins are important antioxidant enzymes that help plants to grow, develop, and respond to a variety of abiotic stressors. SOD gene family has been identified in a number of plant species but not yet in Daucus carota. A total of 9 DcSOD genes, comprising 2 FeSODs, 2 MnSODs, and 5 Cu/ZnSODs, are identified in the complete genome of D. carota, which are dispersed in five out of nine chromosomes. Based on phylogenetic analysis, SOD proteins from D. carota were categorized into two main classes (Cu/ZnSODs and MnFeSODs). It was predicted that members of the same subgroups have the same subcellular location. The phylogenetic analysis was further validated by sequence motifs, exon–intron structure, and 3D protein structures, with each subgroup having a similar gene and protein structure. Cis-regulatory elements responsive to abiotic stresses were identified in the promoter region, which may contribute to their differential expression. Based on RNA-seq data, tissue-specific expression revealed that DcCSD2 had higher expression in both xylem and phloem. Moreover, DcCSD2 was differentially expressed in dark stress. All SOD genes were subjected to qPCR analysis after cold, heat, salt, or drought stress imposition. SODs are antioxidants and play a critical role in removing reactive oxygen species (ROS), including hydrogen peroxide (H2O2). DcSODs were docked with H2O2 to evaluate their binding. The findings of this study will serve as a basis for further functional insights into the DcSOD gene family.
5 citations
References
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TL;DR: 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 with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
225,085 citations
TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
139,407 citations
TL;DR: The mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions are described and the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.
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.
9,908 citations
"Superoxide-Dismutase Deficient Muta..." refers background in this paper
...As can induce oxidative stress through generation of reactive oxygen species (ROS) [4, 5], and moderate accumulation of ROS significantly affects nuclear gene expression [9]....
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...Finally, ROSmight change gene expression by targeting and modifying the activity of transcription factors [9]....
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TL;DR: 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..
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.
3,298 citations
"Superoxide-Dismutase Deficient Muta..." refers background in this paper
...and constitute the first line of defense against the toxicity of superoxide radicals [1, 2]....
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...The toxicity of superoxide radicals has been attributed to their interactionwith other cellular constituents, in particular with hydrogen peroxide [1]....
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TL;DR: The finding that the upstream sequences of Mn and peroxisomal Cu/Zn SODs have three common elements suggests a common regulatory pathway, which is borne out in the research literature.
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.
2,378 citations
"Superoxide-Dismutase Deficient Muta..." refers background or methods in this paper
...Based on the metal cofactor used by the enzyme, SODs are classified into three groups as iron SOD (Fe SOD), manganese SOD (Mn SOD), and copperzinc SODs (Cu/Zn SOD) [3]....
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...Cu/Zn SOD is normally quite stable, due in large part to copper and zinc binding and oxidation of an intramolecular disulfide [3]....
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...SOD isozymes are located in different cellular compartments [3]....
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...The Cu/Zn SODs are the most prolific SOD isozymes in chloroplast [3, 43, 44], and therefore their overexpression has immense significance in ROS metabolism and oxidative balance in leaves of present As-treated mother plants and mutant lines....
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...Information about structural and functional aspects of SOD isozymes could benefit agricultural crop production through a better understanding of the genetic programs by which plants optimize photosynthetic activity in their green tissues during diverse types of stress conditions [3, 22, 25]....
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