Cloning of a putative extracellular Cu/Zn superoxide dismutase and functional differences of superoxide dismutases in invasive and indigenous whiteflies

Superoxide dismutases (SODs) are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase (ecCuZnSOD) complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of Bt‐ecCuZnSOD was more than 10‐fold higher in the invasive Middle East Asia Minor 1 (MEAM1) than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature (4 °C), the expression of Bt‐ecCuZnSOD gene was significantly up‐regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level of B. tabaci intracellular CuZnSOD (Bt‐icCuZnSOD), Bt‐ecCuZnSOD and mitochondrial MnSOD (Bt‐mMnSOD) was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable (cotton) to unfavorable host plants (tobacco). On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt‐ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt‐mMnSOD was expressed equally in both species on cotton, Bt‐mMnSOD messenger RNA was up‐regulated in MEAM1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion of MEAM1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.     

Cu/Zn superoxide dismutase is differentially regulated in period gene-mutant mice

The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9 h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.     

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers

In animals, the small GTP-binding proteins, Rac and Rho, of theras superfamily participate in the signal rransduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.     

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS

A dominant mutation in the gene for copper-zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat-shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1(G93A) transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1(G93A) mice as disease progressed, while levels of expression of two other heat-shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1-containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.     

Structure of Cu/Zn superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6

The deep-sea yeast Cryptococcus liquefaciens strain N6 shows high tolerance towards heavy metals, and can grow in the presence of high concentrations of copper ions. Enzymatic analysis indicated that copper ions induced the Cu/Zn superoxide dismutase activity of strain N6 (Cl-SOD1). In this study, the 1.2 Å resolution crystal structure of Cl-SOD1 has revealed several significant residue substitutions compared to the other Cu/Zn SODs. In the electrostatic loop, notably, His135 and Pro136 replace the well-conserved linear residues, while Thr133 substitutes a highly conserved glycine. The electrostatic loop has been shown to be involved in the copper uptake process, and these substitutions have caused an inward dragging of the turn region of the loop. As the introduction of proline and abolishment of glycine decrease loop flexibility, this structural reorganization may have helped stabilize the loop conformation, possibly resulting in more efficient copper uptake and a more stabilized copper-bound form.     

Molecular characterization and oxidative stress response of an intracellular Cu/Zn superoxide dismutase (CuZnSOD) of the whitefly, Bemisia tabaci

Superoxide dismutases (SODs) are important for the survival of insects under environmental and biological stresses; however, little attention has been devoted to the functional characterization of SODs in whitefly. In this study, an intracellular copper/zinc superoxide dismutase of whitefly (Bemisia tabaci) (Bt-CuZnSOD) was cloned. Sequence analysis indicated that the full length cDNA of Bt-CuZnSOD is of 907 bp with a 471 bp open reading frame encoding 157 amino acids. The deduced amino acid sequence shares common consensus patterns with the CuZnSODs of various vertebrate and invertebrate animals. Phylogenetic analysis revealed that Bt-CuZnSOD is grouped together with intracellular CuZnSODs. Bt-CuZnSOD was then over-expressed in E. coli and purified using GST purification system. The enzymatic activity of purified Bt-CuZnSOD was assayed under various temperatures. When whiteflies were exposed to low (4°C) and high (40°C) temperatures, the in vivo activity of Bt-CuZnSOD was significantly increased. Furthermore, we measured the activities of several antioxidant enzymes, including SOD, catalase and peroxidase, in the whitefly after transferring the whitefly from cotton to tobacco (an unfavorable host plant). We found that the activity of SOD increased rapidly on tobacco plant. Taken together, these results suggest that the Bt-CuZnSOD plays a major role in protecting the whitefly against various stress conditions.     

The dimeric assembly of Photobacterium leiognathi and Salmonella typhimurium SodC1 Cu,Zn superoxide dismutases is affected differently by active site demetallation and pH: an analytical ultracentrifuge study

To establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PlSOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation.The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PlSOD is in reversible monomer-dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O _inf2 ^sup– , to O₂ and H₂O₂, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may he injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H₂O₂. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

Genetic variation for superoxide dismutase level in Drosophila melanogaster

We have studied genetic variation for levels of activity of the enzyme superoxide dismutase (SOD) in Drosophila melanogaster. We have constructed 34 lines homozygous for a given second and a given third chromosome derived from eight original lines; all lines were homozygous for the fast (F) allele of Sod. The variation in the relative levels of SOD CRM ranges from 1 to 1.6. The second chromosomes modify the SOD level, even though the structural Sod locus is in the third chromosome, and the specific effect of a given second chromosome depends on the particular third chromosome with which it is combined. This indicates that the variation in SOD content is controlled by polygenic modifiers present in the second (and in the third) chromosome. In addition to these trans-acting modifiers, we have isolated a cis-acting element (Sod ^CAl ) that reduces SOD CRM levels to 3.5% of a typical F/F homozygote. Sod ^CAl is either a mutation in a regulatory site closely linked to the structural locus or a change in the coding sequence affecting the rate of degradation of the enzyme.This research was supported by a Fellowship of the Swiss NSF to J.-D.G., and by Contract PA 200-14 Mod #4 with the U.S. Department of Energy.     

Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide-dismutase: II. Induction of specific CD8+ cytotoxic lymphocytes and sensitized CD4+ IFN-gamma-producing cell

Previously we reported that immunization with Escherichia coli DH5alpha-expressing Brucella abortus Cu/Zn superoxide dismutase [E. coli (pBSSOD)] induces a protective immune response in BALB/c mice. Here we studied the type of immune defense that the recombinant E. coli induces in mice using as our experimental model Brucella superoxide dismutase Cu/Zn presented by J744.A1 to sensitized lymphocytes as the target of specific lysis or as cytokine inductors. The results indicate that E. coli carrying the Cu/Zn gene was able to induce specific cytotoxic T cells, mainly from CD8(+) subpopulation and IFN-gamma-producing cells belonging in their vast majority to the CD4(+) subpopulation.     

Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity

Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 U_SOD mg_prot ⁻¹) and the highest volumetric yield (8.8 × 10⁵ U_SOD l⁻¹) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu²⁺ and Zn²⁺ supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 × 10⁶ U_SOD l⁻¹. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.