Hydrogen peroxide affects ion channels in lily pollen grain protoplasts

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model – protoplasts obtained from lily pollen grains at the early germination stage – to reveal the effect of H₂O₂ on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS‐sensitive currents on the pollen grain plasma membrane: the hyperpolarisation‐activated calcium current, which is strongly enhanced by H₂O₂, and the outward potassium current, which is modestly enhanced by H₂O₂. We used low concentrations of H₂O₂ that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.     

Early burst of reactive oxygen species positively regulates resistance of eggplant against bacterial wilt

Reactive oxygen species (ROS) play a crucial role in the early response to plant biotic and abiotic stresses. In this study, bacterial wilt‐resistant and wilt‐susceptible eggplants were inoculated with Ralstonia solanacearum and the ROS content was analysed. The result revealed an increased accumulation of hydrogen peroxide (H₂O₂) and superoxide (O₂^?) in resistant and susceptible eggplant roots after R. solanacearum inoculation. H₂O₂ and O₂^? accumulation increased earlier in the inoculated resistant eggplant root than in the inoculated susceptible eggplant root. Real‐time polymerase chain reaction results revealed that respiratory burst oxidase homologue (Rboh) A, RbohB, RbohF and PR1 expression levels increased in inoculated resistant eggplant roots at an early stage (0–60 h postinoculation) and were at higher expression levels than those in susceptible eggplant roots. Ascorbate peroxidase, peroxidase and catalase activities were higher in inoculated resistant eggplant roots than in susceptible eggplant roots at the early stage. Hence, an early ROS burst positively regulates bacterial wilt resistance in eggplant.     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.     

Identification of a hydrogen peroxide signalling pathway in the control of light-dependent germination in Arabidopsis

Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H₂O₂ modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H₂O₂ in different light qualities. H₂O₂ either promoted germination or repressed germination depending on the light wavelengths, showing that H₂O₂ acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H₂O₂-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.     

1Alpha,25-dihydroxyvitamin D3 modulation of adipocyte reactive oxygen species production

Objective: We have previously shown 1α,25‐dihydroxyvitamin D₃ [1α,25‐(OH)₂D₃] to inhibit mitochondrial uncoupling protein 2 (UCP2) expression in adipocytes and that in vivo suppression of calcitriol levels with calcium‐rich diets increases UCP2 expression. Because UCP2 plays a significant role in the clearance of reactive oxygen species (ROS), we studied the effect of calcitriol on ROS production and ROS‐induced adipocyte proliferation. Research Methods and Procedures: ROS production in human and murine adipocytes was stimulated by high glucose (30 mM) or H₂O₂ (100 nM). Results: Both approaches resulted in increased ROS production by 27% to 100% (p < 0.05) and increased cell proliferation by 15% to 39% (p < 0.03). These effects were augmented by the addition of mitochondrial uncoupling inhibitor guanosine 5′‐diphosphate (GDP; 100 μM) or 1α,25‐(OH)₂D₃ (10 nM) and attenuated by UCP2 overexpression, suggesting that inhibition of mitochondrial uncoupling suppresses clearance of ROS and increases adipocyte proliferation. The addition of α ± tocopherol (1 μM) inhibited cell proliferation in adipocytes treated with either H₂O₂ or high glucose, indicating that ROS plays a major role in the regulation of cell proliferation in adipocytes. Moreover, stimulation of ROS with high glucose and H₂O₂ resulted in a 2‐ to 5‐fold increase in adipocyte intracellular calcium ([Ca²⁺]i; p < 0.001), and calcium channel antagonism (nifedipine, 10 μM) suppressed ROS induced calcium influx and cell proliferation, indicating that [Ca²⁺]i may also regulate ROS production and exert a mitogenic effect in adipocytes. Discussion: These data support a role of 1α,25‐(OH)₂D₃, UCP2, and [Ca²⁺]i in the regulation of adipocyte ROS production.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Cross-talk between salicylic acid and NaCl-generated reactive oxygen species and nitric oxide in tomato during acclimation to high salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) generated by salicylic acid (SA) are considered to be functional links of cross‐tolerance to various stressors. SA‐stimulated pre‐adaptation state was beneficial in the acclimation to subsequent salt stress in tomato (Solanum lycopersicum cv. Rio Fuego). At the whole‐plant level, SA‐induced massive H₂O₂ accumulation only at high concentrations (10^?3–10^?2M), which later caused the death of plants. The excess accumulation of H₂O₂ as compared with plants exposed to 100 mM NaCl was not associated with salt stress response after SA pre‐treatments. In the root tips, 10^?3–10^?2M SA triggered the production of reactive oxygen species (ROS) and NO with a concomitant decline in the cell viability. Sublethal concentrations of SA, however, decreased the effect of salt stress on ROS and NO production in the root apex. The attenuation of oxidative stress because of high salinity occurred not only in pre‐adapted plants but also at cell level. When protoplasts prepared from control leaves were exposed to SA in the presence of 100 mM NaCl, the production of NO and ROS was much lower and the viability of the cells was higher than in salt‐treated samples. This suggests that, the cross‐talk of signalling pathways induced by SA and high salinity may occur at the level of ROS and NO production. Abscisic acid (ABA), polyamines and 1‐aminocyclopropane‐1‐carboxylic acid, the compounds accumulating in pre‐treated plants, enhanced the diphenylene iodonium‐sensitive ROS and NO levels, but, in contrast to others, ABA and putrescine preserved the viability of protoplasts.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non‐phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H₂O₂ on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti‐angiogenesis. Our results showed that H₂O₂ dose‐dependently increased the generation of O₂ ^? (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H₂O₂ at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H₂O₂ at 4 nmol/cm² strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm²). Also, H₂O₂ (200～500 µM) could stimulate apoptosis in HUVECs. All the effects of H₂O₂ on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H₂O₂ to produce O₂ ^? and then to regulate angiogenesis. In summary, our results suggest that H₂O₂ as well as O₂ ^? mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Tailoring Selectivity of Electrochemical Hydrogen Peroxide Generation by Tunable Pyrrolic‐Nitrogen‐Carbon

The electrochemical reduction of O₂ via a two‐electron reaction pathway to H₂O₂ provides a possibility for replacing the current anthraquinone process, enabling sustainable and decentralized H₂O₂ production. Here, a nitrogen‐rich few‐layered graphene (N‐FLG) with a tunable nitrogen configuration is developed for electrochemical H₂O₂ generation. A positive correlation between the content of pyrrolic‐N and the H₂O₂ selectivity is experimentally observed. The critical role of pyrrolic‐N is elucidated by the variable intermediate adsorption profiles as well as the dependent negative shifts of the pyrrolic‐N peak on X‐ray adsorption near edge structure spectra. By virtue of the optimized N doping configuration and the unique porous structure, the as‐fabricated N‐FLG electrocatalyst exhibits high selectivity toward electrochemical H₂O₂ synthesis as well as superior long‐term stability. To achieve high‐value products on both the anode and cathode with optimized energy efficiency, a practical device coupling electrochemical H₂O₂ generation and furfural oxidation is assembled, simultaneously enabling a high yield rate of H₂O₂ at the cathode (9.66 mol h^?1 g_cat^?1) and 2‐furoic acid at the anode (2.076 mol m^?2 h^?1) under a small cell voltage of 1.8 V.     

Zinc‐induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido‐reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²⁺. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H₂DCFDA and EPR measurements. The extent of zinc‐induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H₂O₂ production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, ^?CH₃ and ^?OH, were found in the cell walls of zinc‐treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell‐wall‐bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc‐promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²⁺ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.     

Synthesis,characterization and luminescent properties of europium complexes with 2,4,6‐tris‐(2‐pyridyl)‐s‐triazine as highly efficient sensitizers

Using 2,4,6‐tris‐(2‐pyridyl)‐s‐triazine (TPTZ) as a neutral ligand, and p‐hydroxybenzoic acid, terephthalic acid and nitrate as anion ligands, five novel europium complexes have been synthesized. These complexes were characterized using elemental analysis, rare earth coordination titrations, UV/vis absorption spectroscopy and infrared spectroscopy. Luminescence spectra, luminescence lifetime and quantum efficiency were investigated and the mechanism discussed in depth. The results show that the complexes have excellent emission intensities, long emission lifetimes and high quantum efficiencies. The superior luminescent properties of the complexes may be because the triplet energy level of the ligands matches well with the lowest excitation state energy level of Eu³⁺. Moreover, changing the ratio of the ligands and metal ions leads to different luminescent properties. Among the complexes, Eu₂(TPTZ)₂(C₈H₄O₄)(NO₃)₄(C₂H₅OH)·H₂O shows the strongest luminescence intensity, longest emission lifetime and highest quantum efficiency. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (¹O₂) formed during high light stress in higher plants. Although quenching of ¹O₂ by prenylquinols has been previously studied, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol‐9 (PQH₂‐9) in chemical quenching of ¹O₂ was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH₂‐9 and plastochromanol‐8 biosynthesis. In this work, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of ¹O₂ was associated with consumption of PQH₂‐9 and formation of its various oxidized forms. Oxidation of PQH₂‐9 by ¹O₂ leads to plastoquinone‐9 (PQ‐9), which is subsequently oxidized to hydroxyplastoquinone‐9 [PQ(OH)‐9]. We provide here evidence that oxidation of PQ(OH)‐9 by ¹O₂ results in the formation of trihydroxyplastoquinone‐9 [PQ(OH)₃‐9]. It is concluded here that PQH₂‐9 serves as an efficient ¹O₂ chemical quencher in Arabidopsis, and PQ(OH)₃‐9 can be considered as a natural product of ¹O₂ reaction with PQ(OH)‐9. The understanding of the mechanisms underlying ¹O₂ chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

外源H2O2对盐胁迫下小白菜种子萌发和幼苗生理特性的影响

以小白菜"甜脆青"为试材,研究不同浓度（5、10、25、50和100 mmol/L）过氧化氢（H₂O₂）浸种处理对100 mmol/L NaCl胁迫下小白菜（Brassica chinensis L.）种子萌发、幼苗生长及生理特性的影响。结果表明：100 mmol/L NaCl胁迫明显抑制小白菜种子的萌发状况和幼苗生长,发芽势、发芽指数、活力指数及幼苗根和芽长度和鲜重均明显降低,根和芽中CAT的活性及K⁺含量明显受到抑制,渗透调节物质、活性氧和MDA含量显著增加。不同浓度H₂O₂浸种处理提高了NaCl胁迫下小白菜种子发芽势、发芽指数和活力指数,促进小白菜根和芽的生长,增强了NaCl胁迫下根和芽中SOD、CAT和APX的活性及K⁺含量,降低O₂^-·产生速率及H₂O₂和MDA含量,进一步促进脯氨酸和可溶性糖含量的增加,降低体内Na⁺含量。其中以10 mmol/L H₂O₂处理缓解盐胁迫效果最好,明显缓解NaCl胁迫对小白菜种子萌发和幼苗生长的抑制。     

Production of Reactive Oxygen Intermediates (O2˙−, H2O2, 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 (O₂^˙−) 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 O₂^˙−, H₂O₂, 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 O₂^˙− 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 H₂O₂. (4) Inhibition of endogenous ^˙OH formation by O₂^˙− 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.