2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide and diphenyleneiodonium

The inhibition of root growth was investigated in wheat seedlings exposed to 3mM zinc (Zn). Zn treatment with or without 250 µM 2-phenyl-4,4,5,5,-tetrame-thylimidazoline-3-oxide-1-oxyl (PTIO) or 10 µM diphenylene iodonium (DPI) significantly inhibited growth, increased malondialdehyde content and lowered cell viability in roots. The most prominent changes of these three parameters at Zn+DPI treatment could be partly blocked by high PTIO concentration (1mM). The production of nitric oxide (NO) and hydrogen peroxide (H2O2) influenced each other under different treatments, with the highest NO level and the highest H2O2 accumulation in Zn+DPI-treated roots. Compared with Zn-stressed roots, catalase, soluble peroxidase (POD), ascorbate peroxidase and superoxide dismutase decreased in Zn+DPI-treated roots, suggesting that ROS generation from plasma membrane (PM) NADPH oxidase was associated with the regulation of antioxidant enzyme activities. Additionally, Zn-treated roots exhibited significant decreases in cell wall-bound POD, diamine oxidase and polyamine oxidase activities. Our results suggested that Zn-induced effects on root growth resulted from NO interaction with H2O2 and that Zn+DPI-induced strongest inhibition could be explained by the highest increase in the endogenous NO content and the reduction of extracellular ROS production.

Topics: Antioxidants; Cyclic N-Oxides; Enzyme Inhibitors; Free Radical Scavengers; Hydrogen Peroxide; Imidazoles; Lipid Peroxidation; Nitric Oxide; Onium Compounds; Plant Roots; Reactive Oxygen Species; Seedlings; Stress, Physiological; Triticum; Zinc

The effects of chitosan (beta-1,4 linked glucosamine, a fungal elicitor), on the patterns of stomatal movement and signaling components were studied. cPTIO (NO scavenger), sodium tungstate (nitrate reductase inhibitor) or L: -NAME (NO synthase inhibitor) restricted the chitosan induced stomatal closure, demonstrating that NO is an essential factor. Similarly, catalase (H(2)O(2) scavenger) or DPI [NAD(P)H oxidase inhibitor] and BAPTA-AM or BAPTA (calcium chelators) prevented chitosan induced stomatal closure, suggesting that reactive oxygen species (ROS) and calcium were involved during such response. Monitoring the NO and ROS production in guard cells by fluorescent probes (DAF-2DA and H(2)DCFDA) indicated that on exposure to chitosan, the levels of NO rose after only 10 min, while those of ROS increased already by 5 min. cPTIO or sodium tungstate or L: -NAME prevented the rise in NO levels but did not restrict the ROS production. In contrast, catalase or DPI restricted the chitosan-induced production of both ROS and NO in guard cells. The calcium chelators, BAPTA-AM or BAPTA, did not have a significant effect on the chitosan induced rise in NO or ROS. We propose that the production of NO is an important signaling component and participates downstream of ROS production. The effects of chitosan strike a marked similarity with those of ABA or MJ on guard cells and indicate the convergence of their signal transduction pathways leading to stomatal closure.

Topics: Abscisic Acid; Chelating Agents; Chitosan; Cyclic N-Oxides; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Free Radical Scavengers; Hydrogen Peroxide; Imidazoles; Kinetics; Microscopy, Confocal; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Onium Compounds; Oxidants; Pisum sativum; Plant Epidermis; Plant Growth Regulators; Plant Stomata; Reactive Oxygen Species; Time Factors; Tungsten Compounds

One of the key functions of nitric oxide (NO) in human is to dilate blood vessels. We tested glycerol trinitrate (GTN) and other well-known NO donors together with those bearing a >C=N-OH group for possible conversion to NO (or nitrites, respectively) by diaphorase (DP) and lipoamide dehydrogenase (LAD). Both, DP and LAD were unable to convert formamidoxime (FAM), acetone oxime (AC), acetohydroxamic acid (AHA) and Nomega-hydroxy-L-arginine (L-NOHA). On the other hand, we observed good conversion of GTN without the requirement of superoxide anion. However, superoxide anion participated to a varying extent in the conversion of other donors (formaldoxime (FAL), acetaldoxime (AO), nitroprusside (NP), S-nitrosoglutathione (SNOG), S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine (HA)). All DP- and LAD-mediated reactions were inhibited by diphenyleneiodonium chloride (DPI), (an inhibitor of flavine enzymes), in a concentration-dependent manner. For these inhibition reactions we determined Ki and IC50 values. In addition, we found that conversion of SNOG was significantly accelerated by glutathione reductase (GTR). Like with DP, 2-phenyl- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) was reduced also by LAD and thioredoxin reductase (TRR). In summary, we found that LAD significantly accelerates the conversion of a defined subset of NO donors to NO, especially GTN, and eliminates the NO scavenging effect of PTIO.

Topics: Biocatalysis; Clostridium kluyveri; Cyclic N-Oxides; Dihydrolipoamide Dehydrogenase; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Imidazoles; Kinetics; Nitric Oxide; Onium Compounds; Oxidation-Reduction

Hydrogen peroxide (H(2)O(2)) as a source of reactive oxygen species (ROS) significantly stimulated germination of switchgrass (Panicum virgatum L.) seeds with an optimal concentration of 20 mM at both 25 and 35 degrees C. For non-dormant switchgrass seeds exhibiting different levels of germination, treatment with H(2)O(2) resulted in rapid germination (<3 days) of all germinable seeds as compared to seeds placed on water. Exposure to 20 mM H(2)O(2) elicited simultaneous growth of the root and shoot system, resulting in more uniform seedling development. Seeds of big bluestem (Andropogon gerardii Vitman) and indiangrass [Sorghastrum nutans (L.) Nash] also responded positively to H(2)O(2) treatment, indicating the universality of the effect of H(2)O(2) on seed germination in warm-season prairie grasses. For switchgrass seeds, abscisic acid (ABA) and the NADPH-oxidase inhibitor, diphenyleneiodonium (DPI) at 20 microM retarded germination (radicle emergence), stunted root growth and partially inhibited NADPH-oxidase activity in seeds. H(2)O(2) reversed the inhibitory effects of DPI and ABA on germination and coleoptile elongation, but did not overcome DPI inhibition of root elongation. Treatment with H(2)O(2) appeared to enhance endogenous production of nitric oxide, and a scavenger of nitric oxide abolished the peroxide-responsive stimulation of switchgrass seed germination. The activities and levels of several proteins changed earlier in seeds imbibed on H(2)O(2) as compared to seeds maintained on water or on ABA. These data demonstrate that seed germination of warm-season grasses is significantly responsive to oxidative conditions and highlights the complex interplay between seed redox status, ABA, ROS and NO in this system.

Topics: Abscisic Acid; Cotyledon; Cyclic N-Oxides; Germination; Hydrogen Peroxide; Hydrolysis; Imidazoles; Models, Biological; NADPH Oxidases; Nitric Oxide; Onium Compounds; Peroxidase; Plant Proteins; Plant Roots; Poaceae; Reactive Oxygen Species; Seasons; Seeds