Salicylic acid mediated by the oxidative burst is a key molecule in local and systemic responses of cotton challenged by an avirulent race of Xanthomonas campestris pv malvacearum

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O(2)(.-)), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H(2)O(2) infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2. 5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O(2)(.-)-generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H(2)O(2) is required for local and systemic accumulation of SA, which may locally control the generation of O(2)(.-). Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.     

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.

Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids.

To explore whether reactive oxygen species (ROS) play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a unique microdialysis or microcannula sampling technique was used in mice transfected with a mutant Cu,Zn-superoxide dismutase (SOD1) gene from humans with familial ALS, mice transfected with the normal human SOD1 gene, and normal mice. We demonstrate for the first time that the levels of hydrogen peroxide (H(2)O(2)) and the hydroxyl radical ((.)OH) are significantly higher, and the level of the superoxide anion (O(2)(.-)) is significantly lower in ALS mutant mice than in controls, supporting by in vivo evidence the hypothesis that the mutant enzyme catalyzes (.)OH formation by the sequence: O(2)(.-) --> H(2)O(2) --> (.)OH. This removes doubts regarding the relevance of elevated ROS in FALS raised by in vitro experiments. The levels of oxidation products are also significantly higher in the mutant mice than in controls, consistent with some previous reports. Only the superoxide concentration differs between two controls among all the measurements. Our findings correlate in vivo a gene mutation to both elevated H(2)O(2) and (.)OH and increased oxidation of cellular constituents. The elevated H(2)O(2) in mutant mice indicates impairment of its detoxification pathways, perhaps by changed interactions between SOD1 and H(2)O(2) detoxification enzymes.-Liu, D., Wen, J., Liu, J., Li, L. The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids.     

Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.     

Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.     

Physiological basis of different allelopathic reactions of cucumber and figleaf gourd plants to cinnamic acid

To provide an insight into the mechanism of interspecific interactions mediated by allelochemicals, cucumber and figleaf gourd seedlings were compared on their response to cinnamic acid, an autotoxin from root exudates of cucumber. Reactive oxygen species metabolism and plasma membrane H(+)-ATPase activity were examined in roots upon exposure to cinnamic acid. This exposure resulted in significant increases in activities of NADPH oxidase, superoxide dismutase, guaiacol peroxidase, and catalase, as well as in O(2)(.-) production and H(2)O(2) content, in cucumber roots but not in figleaf gourd roots. Notably, the cucumber roots produced significant amount of reactive oxygen species (ROS) immediately after cinnamic acid treatment, consequently increasing membrane peroxidation, decreasing membrane H(+)-ATPase activity, and losing root viability. By contrast, no such changes were observed in figleaf gourd roots. All these results indicated that there was an interspecies difference in the recognition of allelochemicals, which induced oxidative stress accompanied by root cell death in cucumber, an autotoxic plant, but not in figleaf gourd, a cucumber relative.     

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

SB202190 调节蚕豆保卫细胞中SA 诱导H2O2 产生

运用激光共聚焦扫描技术, 在p38 MAP激酶专一抑制剂SB202190处理下, 探索植物促分裂原活化蛋白激酶(mitogenactivated protein kinase, MAP激酶)介导蚕豆(Vicia faba)保卫细胞中H2O2为代表的活性氧(reactive oxygen species, ROS)信号机制, 发现: p38 MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca2+探针荧光强度增强, 与水杨酸 (salicylic acid, SA) 或脱落酸 (abscisic acid, ABA) 迅速加强2种探针荧光强度形成鲜明对比; 而该抑制剂分别与SA和ABA共同处理, 前者H2O2探针荧光强度没有增加, 而后者荧光强度仍然能够增加; 而进一步使用Ca2+螯合剂BAPTA和SB202190 ＋SA共同处理, H2O2探针荧光强度没有增加。这些结果初步表明: 无论胞质Ca2+浓度高低, SB202190调节蚕豆保卫细胞中SA诱导H2O2产生, 但是不调节植物逆境信使分子ABA 此类的反应。因此推测, 植物细胞中可能有类似动物和酵母细胞中的p38MAP激酶类, 并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知, 这是首次报道SB202190和SA共同调节植物保卫细胞中ROS信号过程。