The involvement of hydrogen peroxide in plant responses to stresses

The role of reactive oxygen species, especially H₂O₂, in plant response to stresses has been the focus of much attention. Hydrogen peroxide has been postulated to play multiple functions in plant defence against pathogens. (1) H₂O₂ may possess direct microbicidal activity at the sites of pathogen invasion. (2) It is used for cell-wall reinforcing processes: lignification and oxidative cross-linking of hydroxyproline-rich proteins and other cell-wall polymers. (3) It was found to be necessary for phytoalexin synthesis. (4) H₂O₂ may trigger programmed plant cell death during the hypersensitive response that restricts the spread of infection. (5) H₂O₂ has been suggested to act as a signal in the induction of systemic acquired resistance and (6) it induces defence genes. Recently H₂O₂ has been proposed to be involved in the signal transduction pathways leading to acclimation and protection from abiotic stresses. The present review discusses new insights into the function of H₂O₂ in plant responses to biotic and abiotic stresses.     

The direct involvement of hydrogen peroxide in indoleacetic acid inactivation

Hydrogen peroxide appears to mask the chemical characteristics of indoleacetic acid. This was demonstrated by the Salkowski and Fluorescence tests. Stem elongation and root initiation were inhibited as a result of adding H₂O₂ to nutrient media containing IAA, however, upon the addition of purified catalase, most of the symptoms of IAA inactivation were reversed. It is suggested that in vivo IAA may be regulated partially by its conjugation with H₂O₂, and catalase may have a role in the IAA reactivation process. The accumulation of hydrogen peroxide in the cells as a result of catalase inhibition may lead to a temporary IAA inactivation, therefore effecting plant growth.     

The primary walls of cotton fibers contain an ensheathing pectin layer

Summary Cotton fiber walls (1–2 days post anthesis) are distinctly bilayered compared to those of nonfiber epidermal cells, with a more electron-opaque outer layer and a less electron-opaque, more finely fibrillar inner layer. When probed with antibodies and affinity probes to various saccharides, xyloglucans and cellulose are found exclusively in the inner layer and de-esterified pectins and extensin exclusively in the outer layer. Ovular epidermal cells that do not differentiate into fibers have no pectin sheath, but are labelled throughout with antixyloglucan and cellulase-gold probes. Middle lamellae between adjacent cells were clearly labelled with the antibodies to de-esterified pectins, however. Similarly, cell walls of leaf trichomes have a bilayered wall strongly enriched in pectin, whereas other epidermal cells are not bilayered and are pectin poor. These data indicate that one of the early markers of fiber and trichome cells from other epidermal cells involves the production of a pectin layer. The de-esterified pectins present in the ensheathing layer may allow for expansion and elongation of the fiber cells that does not occur in the other epidermal cells without such a sheath or may even be a consequence of the elongation process.     

Role of cytokinin in differentiation of secondary xylem fibers

The differentiation of secondary xylem fibers was studied in cultured hypocotyl segments of Helianthus annuus L. It is shown that cytokinin is both a limiting and controlling factor in the early stages of fiber differentiation. In the absence of kinetin or zeatin, there was no fiber differentiation. However, cytokinin could induce fiber differentiation only in the presence of indoleacetic and gibberellic acids. First fibers were observed in the tissue after 12 days in culture, and their number increased linearly during the following 2 weeks. At low cytokinin levels, there was a positive correlation between cytokinin concentration in the medium and the number of fibers formed in the explants. A similar correlation was also found at low gibberellic acid concentrations. At high concentration, zeatin was more effective than kinetin. It seems that later stages of fiber differentiation can occur in the absence of cytokinin. It is proposed that the mechanism which controls and determines the early stages of fiber differentiation is based on an interaction of three major hormonal signals: indoleacetic acid plus gibberellic acid from the leaves with zeatin from the root apices.     

Laccase-mediated system pretreatment to enhance the effect of hydrogen peroxide bleaching of cotton fabric

This study evaluates the bleaching efficiency of the hydrogen peroxide bleaching process combined with laccase-mediated system pretreatment (LMS-HPBP) in the treatment of scoured cotton fabric. By changing the factors of laccase-mediated system pretreatment and the hydrogen peroxide bleaching process and examining the subsequent whiteness value and retained tensile strength of the samples, we find three LMS-HPBP processes that are more environment friendly than the conventional hydrogen peroxide bleaching process (CHPBP): (i) bleaching with lower dosage of hydrogen peroxide; (ii) bleaching at reduced temperature; (iii) bleaching for shortened duration. Whiteness, retained tensile strength and K/S values of cotton fabric samples treated by i-iii processes were similar to or higher than those by CHPBP. X-ray diffraction (XRD) analysis also demonstrated that the three processes rendered fabric of both lower crystallinity and bigger crystallite size than those by CHPBP. In addition, the "green" short-flow process was developed to treat cotton fabric and the results obtained shows this method is feasible as a new energy-saving process.     

HCMV infection attenuates hydrogen peroxide induced endothelial apoptosis-- involvement of ERK pathway

Human cytomegalovirus (HCMV) exerts anti-apoptotic effect during early stage of infection, which provides HCMV time for propagation. We investigated pathways mediating the resistance to H(2)O(2)-induced cell death - a self-defense mechanism to remove infected cells. We found that human aortic endothelial cells (HAECs) infected with VHL/E strain of HCMV during first 3 days were resistant to H(2)O(2) (0-2 mM) induced apoptosis. This anti-apoptotic effect may be mediated by the upregulation of Bcl-2, an anti-apoptotic protein through the activation pro-survival pathway extracellular signal regulated kinase (ERK). Through this mechanism, HCMV is able to propagate and causes endothelial dysfunction, hence vascular disease.     

Low-temperature bleaching of cotton induced by glucose oxidase enzymes and hydrogen peroxide activators

Glucose oxidase enzymes were used to produce hydrogen peroxide from glucose and oxygen in aqueous solutions. Different working conditions, that is, temperature, aeration with liquefied air, presence of cotton fibre and time of enzyme activity, were tested in order to obtain a solution with the highest possible concentration of hydrogen peroxide. The hydrogen peroxide produced was transformed into different peracids which could bleach the cotton fabric under mild conditions, at a pH between 7 and 8 and at a temperature of around 60°C. The conversion or activation of hydrogen peroxide was conducted with the bleach activators TAED, NOBS and TBBC. The concentrations of hydrogen peroxide and peracids in the solutions were measured with sodium thiosulphate titrations.

The results indicated that the formation of hydrogen peroxide with glucose oxidase was effective under optimal conditions, which are 50°C, pH 4.6 and aeration. Convenient activators for the conversion of hydrogen peroxide into peracids were TAED and TBBC, which enabled attainment of a relatively high degree of whiteness at pH 7.5 and temperature 50°C. Using the activator NOBS under these conditions did not provide enough peracid to markedly improve whiteness.     

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H₂O₂ concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H₂O₂ concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H₂O₂, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H₂O₂ and ABA-induced activities of APX and GR. ABA-induced accumulation of H₂O₂ was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H₂O₂ is involved in ABA-induced APX and GR activities in rice roots.     

The involvement of hydroxyl radical derived from hydrogen peroxide in lignin degradation by the white rot fungus Phanerochaete chrysosporium

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.     

The cellular production of hydrogen peroxide

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H₂O₂ indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H₂O₂ generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H₂O₂. Succinate, the most effective substrate, yields H₂O₂ at a rate of 0.5nmol/min per mg of protein in state 4. H₂O₂ generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H₂O₂ modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H₂O₂ at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H₂O₂ rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H₂O₂ (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H₂O₂/mol of NADP⁺ during the oxidation of NADPH. H₂O₂ is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H₂O₂/mol of NAD⁺). 8. Estimation of the rates of production of H₂O₂ under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H₂O₂/min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H₂O₂ on cellular metabolism.     

Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of ferryl intermediate

Hydrogen peroxide, one of the potent oxidants in muscle tissues, can induce very rapid oxidation of oxymyoglobin (MbO2) to metmyoglobin (metMb) with an apparent rate constant of 7.5 X 10(4) h-1 M-1 (i.e., 20.8 s-1 M-1) over the wide pH range of 5.5-10.2 in 0.1 M buffer at 25 degrees C. Its molecular mechanism, however, is quite different from that of the autoxidation of MbO2 to metMb. Kinetic analysis has revealed that the hydrogen peroxide oxidation proceeds through the formation of ferryl-Mb(IV) from deoxy-Mb(II), which is in equilibrium with MbO2, by a two-equivalent oxidation with H2O2. Once the ferryl species is formed, it reacts rapidly with another deoxy-Mb(II) in a bimolecular fashion so as to yield 2 mol of metMb(III). Under physiological conditions, the rate-determining step was the oxidation of the deoxy species by H2O2, its rate constant being estimated to be on the order of 3.6 X 10(3) s-1 M-1 at 25 degrees C. These findings leads us to the view that a good supply of dioxygen provides rather an important defense against the oxidation of myoglobin with hydrogen peroxide in cardiac and skeletal muscle tissues.     

Direct involvement of hydrogen peroxide in curvature of wheat coleoptile in blue-light-treated and dark-grown coleoptiles

Blue-light-induced photomorphogenesis is the sum total of a sequence of phenomena involving absorption of light by specific receptors, generation of a signal, processing transmembrane transport of signal, and the activation of a cascade of reactions in the cell interior. Though four blue-light receptors cryptochrome1, cryptochrome2, phototropin1, and phototropin2 have been identified, the signal transduction events associated with blue-light receptor activation are not understood. In this report, we demonstrate the generation and spatiotemporal distribution of H(2)O(2) in wheat coleoptile in response to blue light. Interception of the free-radical generation pathways dithiothreitol and propyl gallate rendered wheat coleoptile tips phototropically non-responsive. Unilateral application of H(2)O(2) onto the sub-apical region of a growing coleoptile brought about curvature in dark. Blue light also caused lipid peroxidation and augmented membrane rigidity of coleoptile cell membranes. We conclude that H(2)O(2) can act as a translocating second messenger that could bring about coleoptile curvature, and the signaling events may trigger Ca(2+) signaling cascades, changes in gene expression, and protein modifications.     

The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus our results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.     

Formation of hydrogen peroxide by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Summary Isolated cell-wall suspensions from horseradish in the presence of 5×10^-4 M MnCl₂ catalyze the production of hydrogen peroxide at the expense of either NADPH or NADH. This reaction is inhibited by scavengers of the superoxide free radical ion such as ascorbate or dihydroxyphenols or by superoxide dismutase, and stimulated by monophenols such as p-coumaric acid. On comparison with isolated (commercial) horseradish peroxidase it becomes evident that (a) cell-wall-bound peroxidase(s) is (are) responsible for the production of hydrogenperoxide, involving the superoxide free radical ion as an intermediate of the complex reaction chain.Abbreviation SOD superoxide dismutase