Differential Responses of Antioxidant Enzymes and Lignin Metabolism in Susceptible and Resistant Sweetpotato Cultivars during Root-Knot Nematode Infection.

Root-knot nematodes (RKN) cause significant damage to sweetpotato plants and cause significant losses in yield and quality. Reactive oxygen species (ROS) play an important role in plant defenses, with levels of ROS-detoxifying antioxidant enzymes tightly regulated during pathogen infection. In this study, ROS metabolism was examined in three RKN-resistant and three RKN-susceptible sweetpotato cultivars. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were assessed, as was lignin-related metabolism. In RKN-infected roots, both resistant and susceptible cultivars increased SOD activity to produce higher levels of hydrogen peroxide (H2O2). However, H2O2 removal by CAT activity differed between cultivars, with susceptible cultivars having higher CAT activity and lower overall H2O2 levels. In addition, the expression of phenylpropanoid-related phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase genes, which encode enzymes involved in lignin metabolism, were higher in resistant cultivars, as were total phenolic and lignin contents. Enzyme activities and H2O2 levels were examined during the early (7 days) and late (28 days) phases of infection in representative susceptible and resistant cultivars, revealing contrasting changes in ROS levels and antioxidant responses in the different stages of infection. This study suggests that differences in antioxidant enzyme activities and ROS regulation in resistant and susceptible cultivars might explain reduced RKN infection in resistant cultivars, resulting in smaller RKN populations and overall higher resistance to infection and infestation by RKNs.

Flooding Tolerance in Sweet Potato (Ipomoea batatas (L.) Lam) Is Mediated by Reactive Oxygen Species and Nitric Oxide.

Flooding is harmful to almost all higher plants, including crop species. Most cultivars of the root crop sweet potato are able to tolerate environmental stresses such as drought, high temperature, and high salinity. They are, however, relatively sensitive to flooding stress, which greatly reduces yield and commercial value. Previous transcriptomic analysis of flood-sensitive and flood-resistant sweet potato cultivars identified genes that were likely to contribute to protection against flooding stress, including genes related to ethylene (ET), reactive oxygen species (ROS), and nitric oxide (NO) metabolism. Although each sweet potato cultivar can be classified as either tolerant or sensitive to flooding stress, the molecular mechanisms of flooding resistance in ET, ROS, and NO regulation-mediated responses have not yet been reported. Therefore, this study characterized the regulation of ET, ROS, and NO metabolism in two sweet potato cultivars-one flood-tolerant cultivar and one flood-sensitive cultivar-under early flooding treatment conditions. The expression of ERFVII genes, which are involved in low oxygen signaling, was upregulated in leaves during flooding stress treatments. In addition, levels of respiratory burst oxidase homologs and metallothionein-mediated ROS scavenging were greatly increased in the early stage of flooding in the flood-tolerant sweet potato cultivar compared with the flood-sensitive cultivar. The expression of genes involved in NO biosynthesis and scavenging was also upregulated in the tolerant cultivar. Finally, NO scavenging-related MDHAR expressions and enzymatic activity were higher in the flood-tolerant cultivar than in the flood-sensitive cultivar. These results indicate that, in sweet potato, genes involved in ET, ROS, and NO regulation play an important part in response mechanisms against flooding stress.

Characterization of components of a reducing system for SoxR in the cytoplasmic membrane of Escherichia coli.

A reducing system of SoxR, a regulator of redox-active molecules, was identified as rsxABCDGE gene products and RseC in Escherichia coli through genetic studies. We found that ApbE was an additional component of the reducer system. Bacterial two hybrid analysis revealed that these proteins indeed had multiple interactions among themselves. RseC and RsxB formed the core of the complex, interacting with more than five other components. RsxC, the only cytoplasmic component of the system, interacted with SoxR. It might be linked with the rest of the complex via RsxB. Membrane fractions containing the wild type complex but not the mutant complex reduced purified SoxR using NADH as an electron source. These results suggest that Rsx genes, RseC, and ApbE can form a complex using NAD(P)H to reduce SoxR.

Regulation of iron homeostasis by peroxide-sensitive CatR, a Fur-family regulator in Streptomyces coelicolor.

CatR, a peroxide-sensing transcriptional repressor of Fur family, can de-repress the transcription of the catA gene encoding catalase upon peroxide stress in Streptomyces coelicolor. Since CatR-regulated genes other than catA and its own gene catR have not been identified in detail, the understanding of the role of CatR regulon is very limited. In this study, we performed transcriptomic analysis to identify genes influenced by both ΔcatR mutation and hydrogen peroxide treatment. Through ChIP-qPCR and other analyses, a new consensus sequence was found in CatR-responsive promoter region of catR gene and catA operon for direct regulation. In addition, vtlA (SCO2027) and SCO4983 were identified as new members of the CatR regulon. Expression levels of iron uptake genes were reduced by hydrogen peroxide and a DmdR1 binding sequence was identified in promoters of these genes. The increase in free iron by hydrogen peroxide was thought to suppress the iron import system by DmdR1. A putative exporter protein VtlA regulated by CatR appeared to reduce intracellular iron to prevent oxidative stress. The name vtlA (VIT1-like transporter) was proposed for iron homeostasis related gene SCO2027.

The Defense Response Involved in Sweetpotato Resistance to Root-Knot Nematode Meloidogyne incognita: Comparison of Root Transcriptomes of Resistant and Susceptible Sweetpotato Cultivars With Respect to Induced and Constitutive Defense Responses.

Sweetpotato (Ipomoea batatas [L.] Lam) is an economically important, nutrient- and pigment-rich root vegetable used as both food and feed. Root-knot nematode (RKN), Meloidogyne incognita, causes major yield losses in sweetpotato and other crops worldwide. The identification of genes and mechanisms responsible for resistance to RKN will facilitate the development of RKN resistant cultivars not only in sweetpotato but also in other crops. In this study, we performed RNA-seq analysis of RKN resistant cultivars (RCs; Danjami, Pungwonmi and Juhwangmi) and susceptible cultivars (SCs; Dahomi, Shinhwangmi and Yulmi) of sweetpotato infected with M. incognita to examine the induced and constitutive defense response-related transcriptional changes. During induced defense, genes related to defense and secondary metabolites were induced in SCs, whereas those related to receptor protein kinase signaling and protein phosphorylation were induced in RCs. In the uninfected control, genes involved in proteolysis and biotic stimuli showed differential expression levels between RCs and SCs during constitutive defense. Additionally, genes related to redox regulation, lipid and cell wall metabolism, protease inhibitor and proteases were putatively identified as RKN defense-related genes. The root transcriptome of SCs was also analyzed under uninfected conditions, and several potential candidate genes were identified. Overall, our data provide key insights into the transcriptional changes in sweetpotato genes that occur during induced and constitutive defense responses against RKN infection.

Transcriptomic changes in sweetpotato peroxidases in response to infection with the root-knot nematode Meloidogyne incognita.

A previous transcriptomic analysis of the roots of susceptible and resistant cultivars of sweetpotato (Ipomoea batatas) identified genes that were likely to contribute to protection against infection with the root-knot nematode Meloidogyne incognita. The current study examined the roles of peroxidase genes in sweetpotato defense responses during root-knot nematode infection, using the susceptible (cv. Yulmi) and resistant (cv. Juhwangmi) cultivars. Differentially expressed genes were assigned to gene ontology categories to predict their functional roles and associated biological processes. Comparison with Arabidopsis peroxidases identified a group of genes orthologous to Arabidopsis PEROXIDASE 52 (AtPrx52). An analysis of sweetpotato peroxidase genes determined their roles in protecting plants against root-knot nematode infection and enabled identification of important peroxidases. The interactions involved in sweetpotato resistance to nematode infection are discussed.

Zinc-dependent regulation of zinc import and export genes by Zur.

In most bacteria, zinc depletion is sensed by Zur, whereas the surplus is sensed by different regulators to achieve zinc homeostasis. Here we present evidence that zinc-bound Zur not only represses genes for zinc acquisition but also induces the zitB gene encoding a zinc exporter in Streptomyces coelicolor, a model actinobacteria. Zinc-dependent gene regulation by Zur occurs in two phases. At sub-femtomolar zinc concentrations (phase I), dimeric Zur binds to the Zur-box motif immediately upstream of the zitB promoter, resulting in low zitB expression. At the same time, Zur represses genes for zinc uptake. At micromolar zinc concentrations (phase II), oligomeric Zur binding with footprint expansion upward from the Zur box results in high zitB induction. Our findings reveal a mode of zinc-dependent gene activation that uses a single metalloregulator to control genes for both uptake and export over a wide range of zinc concentrations.

Simultaneous Activation of Iron- and Thiol-Based Sensor-Regulator Systems by Redox-Active Compounds.

Bacteria in natural habitats are exposed to myriad redox-active compounds (RACs), which include producers of reactive oxygen species (ROS) and reactive electrophile species (RES) that alkylate or oxidize thiols. RACs can induce oxidative stress in cells and activate response pathways by modulating the activity of sensitive regulators. However, the effect of a certain compound on the cell has been investigated primarily with respect to a specific regulatory pathway. Since a single compound can exert multiple chemical effects in the cell, its effect can be better understood by time-course monitoring of multiple sensitive regulatory pathways that the compound induces. We investigated the effect of representative RACs by monitoring the activity of three sensor-regulators in the model actinobacterium Streptomyces coelicolor; SoxR that senses reactive compounds directly through oxidation of its [2Fe-2S] cluster, CatR/PerR that senses peroxides through bound iron, and an anti-sigma factor RsrA that senses RES via disulfide formation. The time course and magnitude of induction of their target transcripts were monitored to predict the chemical activities of each compound in S. coelicolor. Phenazine methosulfate (PMS) was found to be an effective RAC that directly activated SoxR and an effective ROS-producer that induced CatR/PerR with little thiol-perturbing activity. p-Benzoquinone was an effective RAC that directly activated SoxR, with slower ROS-producing activity, and an effective RES that induced the RsrA-SigR system. Plumbagin was an effective RAC that activated SoxR, an effective ROS-producer, and a less agile but effective RES. Diamide was an RES that effectively formed disulfides and a weak RAC that activated SoxR. Monobromobimane was a moderately effective RES and a slow producer of ROS. Interestingly, benzoquinone induced the SigR system by forming adducts on cysteine thiols in RsrA, revealing a new pathway to modulate RsrA activity. Overall, this study showed that multiple chemical activities of a reactive compound can be conveniently monitored in vivo by examining the temporal response of multiple sensitive regulators in the cell to reveal novel activities of the chemicals.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1.

Factors affecting redox potential and differential sensitivity of SoxR to redox-active compounds.

SoxR is a [2Fe-2S]-containing sensor-regulator, which is activated through oxidation by redox-active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth-inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe-2S] binding site were proposed as key determinants of sensitivity. ScSoxR-like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR-like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxR(R) (127) (L) (/) (P) (131) (V) and ScSoxR(L126R/V130P) were estimated to be -192 ± 8 mV and -273 ± 10 mV, respectively, approaching that of ScSoxR (-185 mV) and EcSoxR (-290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe-2S], making it harder to get oxidized. This reveals a mechanism to modulate redox-potential in [Fe-S]-containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology.

Comparative study of SoxR activation by redox-active compounds.

SoxR from Escherichia coli and related enterobacteria is activated by a broad range of redox-active compounds through oxidation or nitrosylation of its [2Fe-2S] cluster. Activated SoxR then induces SoxS, which subsequently activates more than 100 genes in response. In contrast, non-enteric SoxRs directly activate their target genes in response to redox-active compounds that include endogenously produced metabolites. We compared the responsiveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, towards natural or synthetic redox-active compounds. EcSoxR responded to all compounds examined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh -440 mV) and menadione sodium bisulphite (Eh -45 mV) and to NO generators. PaSoxR was insensitive only to some NO generators. Whole-cell EPR analysis of SoxRs expressed in E. coli revealed that the [2Fe-2S](1+) of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid-point redox potential of purified ScSoxR was determined to be -185 ± 10 mV, higher by approximately 100 mV than those of EcSoxR and PaSoxR, supporting its limited response to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs towards various oxidants.

Conservation of thiol-oxidative stress responses regulated by SigR orthologues in actinomycetes.

Numerous thiol-reactive compounds cause oxidative stress where cells counteract by activation of survival strategies regulated by thiol-based sensors. In Streptomyces coelicolor, a model actinomycete, a sigma/antisigma pair SigR/RsrA controls the response to thiol-oxidative stress. To unravel its full physiological functions, chromatin immuno-precipitation combined with sequence and transcript analyses were employed to identify 108 SigR target genes in S. coelicolor and to predict orthologous regulons across actinomycetes. In addition to reported genes for thiol homeostasis, protein degradation and ribosome modulation, 64 additional operons were identified suggesting new functions of this global regulator. We demonstrate that SigR maintains the level and activity of the housekeeping sigma factor HrdB during thiol-oxidative stress, a novel strategy for stress responses. We also found that SigR defends cells against UV and thiol-reactive damages, in which repair UvrA takes a part. Using a refined SigR-binding sequence model, SigR orthologues and their targets were predicted in 42 actinomycetes. This revealed a conserved core set of SigR targets to function for thiol homeostasis, protein quality control, possible modulation of transcription and translation, flavin-mediated redox reactions, and Fe-S delivery. The composition of the SigR regulon reveals a robust conserved physiological mechanism to deal with thiol-oxidative stress from bacteria to human.

Transcriptional activation of the aldehyde reductase YqhD by YqhC and its implication in glyoxal metabolism of Escherichia coli K-12.

The reactive alpha-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) are generated in vivo from sugars through oxidative stress. GO and MG are believed to be removed from cells by glutathione-dependent glyoxalases and other aldehyde reductases. We isolated a number of GO-resistant (GO(r)) mutants from Escherichia coli strain MG1655 on LB plates containing 10 mM GO. By tagging the mutations with the transposon TnphoA-132 and determining their cotransductional linkages, we were able to identify a locus to which most of the GO(r) mutations were mapped. DNA sequencing of the locus revealed that it contains the yqhC gene, which is predicted to encode an AraC-type transcriptional regulator of unknown function. The GO(r) mutations we identified result in missense changes in yqhC and were concentrated in the predicted regulatory domain of the protein, thereby constitutively activating the product of the adjacent gene yqhD. The transcriptional activation of yqhD by wild-type YqhC and its mutant forms was established by an assay with a beta-galactosidase reporter fusion, as well as with real-time quantitative reverse transcription-PCR. We demonstrated that YqhC binds to the promoter region of yqhD and that this binding is abolished by a mutation in the potential target site, which is similar to the consensus sequence of its homolog SoxS. YqhD facilitates the removal of GO through its NADPH-dependent enzymatic reduction activity by converting it to ethadiol via glycolaldehyde, as detected by nuclear magnetic resonance, as well as by spectroscopic measurements. Therefore, we propose that YqhC is a transcriptional activator of YqhD, which acts as an aldehyde reductase with specificity for certain aldehydes, including GO.

SoxRS-mediated lipopolysaccharide modification enhances resistance against multiple drugs in Escherichia coli.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria that serves as a barrier against harmful molecules, including antibiotics. The waaYZ locus that encodes the LPS core biosynthetic function in Escherichia coli was found to be induced strongly by superoxide generators but not by H(2)O(2), ethanol, or heat shock. This induction was dependent on SoxRS, a superoxide and nitric oxide sensing system, through a soxbox in the waaY promoter that binds SoxS. A DeltawaaYZ mutant became more sensitive to some superoxide generators, and the activation of SoxR by these drugs became more sensitized in the mutant. Through phenotypic microarray analysis, we found that the mutant became sensitive to a wide variety of chemicals not restricted to oxidizing agents. We found that the mutant is under envelope stress and is altered in LPS composition, as monitored by the level of sigma(E) activation and changes in the electrophoretic mobility of LPS, respectively. waaY expression was also regulated by MarA (multiple-antibiotic resistance regulator), which shares a binding site (soxbox) with SoxS, and was induced by salicylate, a nonoxidative compound. These results demonstrate a novel way of protecting gram-negative bacteria against various compounds by modifying LPS, possibly through phosphorylation. Since either oxidant or nonoxidant compounds elicit resistance toward themselves and other toxic drugs, this mechanism could serve as an efficient way for pathogenic bacteria to enhance survival during antibiotic treatment within an oxidant-rich host immune environment.

A reducing system of the superoxide sensor SoxR in Escherichia coli.

The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide. When SoxR is activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression. How the oxidized SoxR returns to and is maintained in its reduced state has been under question. To identity genes that constitute the SoxR-reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants. Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE-rseABC operon. In-frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype. The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR. Electron paramagnetic resonance analysis of SoxR and measurement of re-reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR.