Wall Peroxidase Research Articles

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.

Effect of N deficiency on leaf growth and cell wall peroxidase activity in contrasting maize genotypes

Two maize genotypes differing in leaf elongation rate (high-LER and low-LER) were used for the investigation of the effects of nitrogen deficiency on leaf growth and development and activity of enzyme cell wall peroxidase in the leaf growth zone. Plants were grown in a growth cabinet in perlite as a substrate and watered with complete N-NO3 solution (+N) and N-NO3 deficient solution (−N). Comparison between the investigated genotypes showed that final leaf length in both N treatments was related with LER, but not with the duration of leaf elongation. Faster leaf elongation rate in high-LER compared with low-LER genotype, was associated with longer growth zone, a bigger number of cells in it, and higher cell flux rate, although cell elongation rate was similar in both genotypes. These lines of evidence indirectly indicated that leaves of the faster growing genotype were characterized by higher meristematic activity. Nitrogen deficiency reduced the flux of cells and cell elongation rate, length of cell division zone and the number of cells in whole zone, significantly for both genotypes, although duration of cell elongation was increased and final epidermal cell length was unchanged. These results showed that N deficiency reduced both cell division and cell elongation, which in turn resulted in decreased leaf length and prolonged time for leaf development. Nitrogen deficiency significantly increased both bulk and segmental cell wall peroxidase activity in the growth zone of both investigated genotypes, thus showing an interaction between leaf growth cessation and enzyme activity.

Impact of Cadmium and Copper Excess on Cell Wall Peroxidases in Pea Stems

Impact of Cadmium and Copper Excess on Cell Wall Peroxidases in Pea Stems

Lignin dehydrogenative polymerization mechanism: a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in β-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO-C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO-C was capable of oxidizing high-molecular-weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO-C characteristics are important to produce polymer lignin. The results suggest that CWPO-C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.

Influence of partial root drying on morpho-anatomical and physiological characteristics of tomato fruits: Lycopersicon esculentum l

Partial root drying (PRD) is a new irrigation and plant growing technique which improves water use efficiency without significant yield reduction. The aim of the presented thesis was to investigate the effect of this technique on the growth of vegetative and generative tomato organs, and especially fruits, its influence on the activity of enzyme cell wall peroxidase morpho-anatomical characteristics of flower and fruit pedicels, influence on fruits yield and quality and ions distribution in tomato organs. Tomato line L-4 (Lycopersicon esculentum L) was grown in commercial compost with the root system divided equally between two plastic pots. During the vegetative and generative phases of development, one half of the root system of PRD was maintained in a dry state, while the other half was optimally watered. After 10 days an inversion was made in such a way that previously wet side was exposed to drought and dried side was watered. Obtained results showed that as a consequence of PRD treatment the growth of whole plants was reduced with fruit size and yield of PRD plants being similar to control, as well as morpho-anatomical characteristics of pedicels, ions and lycopen contents although sugar content was higher in PRD than in control plants. These results suggest that with PRD technique we can halve the demand for irrigation water and increase the nutritional value of the tomato fruit.

Exogenous jasmonic acid mimics herbivore‐induced systemic increase in cell wall bound peroxidase activity and reduction in leaf expansion

Summary Jasmonic acid (JA), a ubiquitous regulator of the wound response in plants, is part of a long distance defence signalling pathway and when applied exogenously induces several defence related responses in many species including the activation of proteinase inhibitor (PIN) proteins and defence‐related metabolites. There is evidence for an induced systemic resistance mechanism linking increased cell wall peroxidase activity with reductions in leaf expansion rates in Rumex obtusifolius, following limited grazing by the chrysomelid beetle Gastrophysa viridula. We hypothesised that herbivory induces strengthening of cell walls in distal, non‐damaged tissue through increases in peroxidase activity and that this mechanism was mediated systemically by JA. JA was applied to the fully expanded fourth leaf of R. obtusifolius and expansion of leaf 8 was measured over 19 days. Treatment with exogenous JA induced a temporary reduction in the expansion rate of leaf 8 beginning 6 days after treatment. This reduction continued until day 7 and reduced final leaf areas in treated plants by approximately 20%. Final epidermal cell areas in leaf 8 were reduced by approximately 25%, while epidermal cell numbers remained unchanged. Cell wall bound peroxidase activity was measured in leaf 8 over a 6‐day period following application of JA to leaf 4. Activity increased approximately 9‐fold 2 days after treatment, before returning to control activities on day 3. Treatment of leaf 4 with JA reduced the gregariousness of G. viridula larvae on the fully expanded leaf 8. We postulate that JA acts as a signalling molecule in a long‐distance pathway responsible for inducing resistance to future attack through increases in cell wall bound peroxidase activity leading to cell wall toughening, while incurring a potential ecological cost in the form of reductions in subsequent leaf expansion.

Effects of chromium on hypocotyl elongation, wall components, and peroxidase activity of Phaseolus vulgaris seedlings

In this investigation, the effect of the heavy metal chromium (Cr) on hypocotyl elongation of Phaseolus vulgaris seedlings in light was studied. The seedlings were subjected to two concentrations of Cr (0.5 and 1.0 mM) in the form of K2Cr2O7. Hypocotyl elongation (growth) was measured in terms of length. Change in wall components (pectic polysaccharides and xyloglucan) and peroxidase activity (soluble and bound) were also studied. Both concentrations of Cr inhibited hypocotyl length and a clear concentration effect was observed. Maximum pectic polysaccharide content was observed at a high concentration of Cr and a minimum was observed in the distilled water control. A clear inverse correlation was observed, supporting the conclusion that pectic polysaccharides may be involved in cell wall loosening. Even xyloglucan (high and low) content showed a clear inverse correlation with hypocotyl length. It appears that Cr inhibits hypocotyl elongation and xyloglucan and pectic polysaccharide contents are not degraded to smaller oligosaccharides as they occur more in seedlings treated with Cr compared to seedlings treated with distilled water. Further soluble and bound peroxidase activity was assayed with four hydrogen donors— ferulic acid, caffeic acid, pyrocatechol, and pyrogallol during the initial phase of hypocotyl elongation. A clear inverse correlation between length and peroxidase activity was observed. All hydrogen donors showed a clear concentration effect, specificity, and variation, and maximum activity occurred with caffeic acid. The role of peroxidase in the defense mechanism in response to Cr toxicity is discussed.

Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth.

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.

Reduced leaf expansion as a cost of systemic induced resistance to herbivory

Summary We investigated whether increases in cell wall peroxidases link the benefits of induced resistance and the potential ecological costs of reduced leaf expansion. Rumex obtusifolius is a host plant of the chrysomelid beetle Gastrophysa viridula. The effect of feeding by G. viridula on plant physiology and activity of cell wall‐bound peroxidase activity in the plant was measured under controlled conditions. Gastrophysa viridula were applied to fully expanded leaf 4, and expansion of leaf 8 was measured over 22 days. Removal of leaf tissue by the herbivore delayed subsequent leaf emergence in treated plants and temporarily reduced the expansion rate of leaf 8, beginning 5 days after treatment. This reduction continued for 4 days when compared with non‐treated plants, and reduced final leaf area by approximately 37%. Final epidermal cell areas in leaf 8 were reduced by approximately 13%, while epidermal cell numbers remained unchanged. Cell wall‐bound peroxidase activity was measured in leaf 8 over a 7‐day period following attack. Activity increased within 24 h following the start of treatment and peaked at day 3. We postulate a herbivore‐induced systemic mechanism linking reductions in cell expansion and subsequent leaf growth rates as a result of an increase in cell wall‐bound peroxidase activity. Herbivore grazing commonly reduces subsequent host growth, and the assumption has been that this is largely due to the consequences of leaf area lost to grazing. This study suggests that reduced growth, and the associated ecological costs result from induced resistance, and are the consequence of a more subtle direct physiological effect in constraining leaf expansion.

Identification of a cell wall peroxidase in red calli of Prunus incisa Thunb.

Red calli occur frequently in callus cultures of Prunus incisa Thunb. Calli that become red in color stop growing and turn brown while calli that remain green continue to grow. This study was carried out to compare the accumulation of antioxidant activity in red and green calli. The anthocyanin content, peroxidase isoforms and peroxidase activity were different in red and green calli. Red calli contained higher levels of anthocyanins, cell wall peroxidase activity and lower soluble peroxidase activity than green calli. A basic cell wall peroxidase (pI 10.0) was present only in red calli. Two acidic peroxidases (pI 6.0 and 6.8) had higher accumulation in green calli than in red calli. In the cell wall fraction of red calli, a peroxidase isoform with an apparent molecular mass of 30 kDa was found. MALDI-TOF mass spectrometry and internal amino acid sequence analysis indicate that this protein has a very high similarity with the cell wall peroxidase of Beta vulgaris L.

Hydrogen peroxide is required for poly(phenolic) domain formation during wound-induced suberization.

The requirement for hydrogen peroxide (H(2)O(2)) during suberization was demonstrated in wound-induced potato tubers by monitoring the extent of phenolic polymerization after the inhibition of H(2)O(2) production using diphenyleneiodonium (DPI). In DPI-treated tissues the extent of phenolic polymerization in suberized tissues, measured using DFRC (Derivatization Followed by Reductive Cleavage) and thioglycolic acid analyses, was greatly reduced relative to untreated controls. Concomitantly, a large quantity of new soluble phenolics accumulated in the DPI-treated tissue some of which were not present in the controls. We suggest that the inhibition of H(2)O(2) production prevented these phenolics from being oxidized by cell wall peroxidases. As a result, these phenolics were left unpolymerized and accumulated in the tissue. Several of the soluble phenolics were identified as hydroxycinnamic acid derivatives. From the data presented, it was concluded that H(2)O(2) is required for the polymerization step in the formation of the poly(phenolic) domain of suberized potato tubers.

Osmotic stress-induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings

The changes in activity of peroxidase (POD) extracted from the cell walls and the level of H 2 O 2 in rice seedling roots treated with mannitol and their correlation with root growth were investigated. Increasing concentrations of mannitol from 92 to 276 mM, which is iso-osmotic with 50 to 150 mM NaCI, progressively reduced root growth and increased POD activities extracted from the cell walls of rice roots. The reduction of growth was also correlated with an increase in H 2 O 2 level. Both diamine oxidase (DAO) and NADH peroxidase (NADH-POD) are known to be responsible for the generation of H 2 O 2 . Mannitol treatment increased DAO but not NADH-POD activities in roots of rice seedlings, suggesting that DAO contributes to the generation of H 2 O 2 in the cell walls of mannitol-treated roots. An increase in the level of H 2 O 2 and the activity of POD extracted from the cell walls of rice roots preceded root growth reduction caused by mannitol. An increase in DAO activity coincided with an increase in H 2 O 2 in roots caused by mannitol. Since DAO catalyses the oxidation of putrescine, the demonstration that mannitol increases the activity of DAO in roots is consistent with those that mannitol decreases the level of putrescine. In conclusion, cell-wall stiffening catalysed by POD is possibly involved in the regulation of root growth reduction caused by mannitol.

Evidence that hydroxyl radicals mediate auxin-induced extension growth.

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

Arbutin oxidation by pear ( Pyrus communis L.) peroxidases

The oxidation of arbutin (hydroquinone-β- d-glucopyranoside) by soluble and ionically bound to cell wall peroxidases (EC. 1.11.1.7) from pear ( Pyrus communis L.) shoots is described for the first time. Following the initiation of the reaction with hydrogen peroxide, arbutin was transformed to a product that was detected by UV spectrophotometry. The stoichiometry of the oxidized product formation versus hydrogen peroxide was nearly 2:1 suggesting that arbutin oxidation is a one-electron process. Higher oxidation rates were observed in a narrow pH zone and ionically bound peroxidases showed lower apparent K m for arbutin than soluble ones. Arbutin is considered to be an antioxidant and peroxidases from pear may influence the pro- or antioxidant properties of this compound.

Peroxidase involvement in the defense response of red raspberry to Didymella applanata (Niessl/Sacc.)

Peroxidase activity of red raspberry canes was dependent on the cultivar and influenced the subsequent lignification. After inoculation with Didymella applanata, responsible for the spur blight cane disease, the activity of soluble cytoplasmic enzyme increased in the moderately resistant ‘Latham’ and susceptible ‘Malling Promise’, similarly for syringaldazine and guaiacol as hydrogen donors. Systemic induction found in ‘Latham’ was recognized as a symptom of defence mechanism responsible for fungal restriction. Locally enhanced peroxidase activity in the ‘M.Promise’ tissues was related to the local lignification and/or may be associated with the loss of cell integrity caused by pathogen penetration. Pathogen-induced changes of cell wall peroxidases were similar in both cultivars mentioned above. No influence of the infection was found in the high susceptible Zeva cultivar. Using native-PAGE analysis and horizontal starch electrophoresis of soluble fraction five constitutive acidic isoperoxidases were detected in ‘Latham’ and three in ‘M. Promise’. The infection process was accompanied by the appearance of two new anodic isoforms.

Induction of soluble and cell wall peroxidases by aphid infestation in barley.

Peroxidase enzymes have been found in soluble, ionically bound, and covalently bound forms and have been implicated in several physiological processes in plants. This paper investigates the effect of aphid infestation on soluble and bound-cell wall peroxidase activity and bound-cell wall isoform changes of barley plants. Peroxidase activity was measured in control plants and plants infested with the aphid Schizaphis graminum (Rondani). The activity of soluble peroxidases increased with time of infestation, older plants being more affected than younger ones. The increase in bound-cell wall peroxidase activity as a function of age was higher in infested than in control plants, being higher in ionically bound than in covalently bound peroxidases. When the aphids were removed from plants, the activities of both types of peroxidases decreased to control levels. Isoelectrofocusing analyses of the ionically bound peroxidases showed changes in the isoform pattern. A new isoform was induced by infestation. The activities of all covalently bound isoforms increased after infestation. The physiological implications of these changes are discussed.

Cell wall peroxidase against ferulic acid, lignin, and NaCl-reduced root growth of rice seedlings

Summary The changes in cell wall peroxidase activity against ferulic acid (FPOD) and lignin level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mmol L −1 progressively decreases root growth. The reduction of root growth by NaCl is closely correlated with the increase in FPOD activity extracted from the cell wall. In contrast, lignin level was reduced by NaCl. Since proline and ammonium accumulations are associated with root growth inhibition caused by NaCl, we determined the effect of proline or NH 4 Cl on root growth and FPOD in roots. Exogenous application of NH 4 Cl or proline markedly inhibited root growth and increased FPOD activity in rice seedlings in the absence of NaCl. An increase in FPOD activity in roots preceded inhibition of root growth caused either by NaCl, NH 4 Cl, or proline. Our results suggest that cell-wall stiffening catalyzed by FPOD may participate in the regulation of root growth reduction of rice seedlings caused by NaCl.

Cell wall peroxidase activity, hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H2O2 level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H2O2 level. Exogenous H2O2 was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH4Cl or proline on root growth, cell-wall POD activity and H2O2level in roots. External application of NH4Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H2O2 level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H2O2 level preceded inhibition of root growth caused by NaCl, NH4Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H2O2 generation in the cell wall of NaCl- or proline-treated roots. NH4Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H2O2 generation in cell wall of NH4Cl-treated roots.

Tomato root peroxidase isoenzymes: kinetic studies of the coniferyl alcohol peroxidase activity, immunological properties and role in response to salt stress

More than eleven soluble and ionically-bound cell wall peroxidase (EC 1.11.1.7; donor: hydrogen-peroxidase oxido-reductase) isoenzymes were found in tomato (Lycopersicon esculentum (L.) Mill. cv. Pera) roots. Isoperoxidases with isoelectric points 9.6, 8.2, 7.5, 6.5, and 3.6, were partially purified by ammonium sulfate precipitation, gel filtration on Sephacryl-S200 and ion exchange chromatography on DEAE Sephacel and/or SP-Sephadex C50 columns. Isoenzymes of pI 9.6, 3.6, and 6.5 showed the highest catalytic efficiencies with coniferyl alcohol, a true monolignol, used as substrate marker of peroxidases presumably involved in lignification. In addition, they showed cross-reactivity in Western blots of crude extracts when antibodies anti pI 3.6 isoenzyme were used for the immunodetection, despite their different pI values. It was demonstrated that the levels of pI 3.6 isoenzyme increased with the time of growth but did not respond to salt stress, unlike the isoenzyme with pI 9.6, which did. Thus, the expression of peroxidase isoenzymes involved in lignification may be differentially controlled by either developemental or stress signals.