Exogenous spermidine and spermine enhance cadmium tolerance of <Emphasis Type="Italic">Potamogeton malaianus</Emphasis>

In order to investigate the effects of spermidine (Spd) and spermine (Spm) on cadmium stress, the content of chlorophyll, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), soluble protein and proline, the rate of O₂^·− generation, and activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)) in Potamogeton malaianus Miq. were measured. Exogenous application of Spd or Spm significantly enhanced the level of proline, retarded the loss of chlorophyll, enhanced photosynthesis, decreased the rate of O₂^·− generation and H₂O₂ content, and prevented Cd-induced lipid peroxidation. Spd and Spm also effectively maintained the balance of antioxidant enzyme activities under Cd stress; however, GR activity was found to increase only slightly in response to polyamines (PAs). The antioxidant systems, which were modified by PAs, were able to moderate the radical-scavenging system and to lessen in this way the oxidative stress. These results suggest that both Spd and Spm can enhance Cd tolerance of P. malaianus.     

Effect of salicylic acid potentiates cadmium-induced oxidative damage in <Emphasis Type="Italic">Oryza sativa</Emphasis> L. leaves

In the present investigation, we studied the possible potentiating effect of salicylic acid (SA) under Cd toxicity in Oryza sativa L. leaves. Cd treatments for 24 h reduced the shoot length, dry biomass and total chlorophyll content followed by high Cd accumulation in shoots. About 16 h presoaking with SA resulted in partial protection against Cd, as observed by minor changes in length, biomass and total chlorophyll. SA priming resulted in low Cd accumulation. Enhanced thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H₂O₂) and superoxide anion (O₂ ⁻) content were seen when Cd was applied alone, while under SA priming the extent of TBARS, H₂O₂ and O₂ ⁻ were significantly low, suggesting SA-regulated protection against oxidative stress. The antioxidant enzymes like Catalase (CAT), guaiacol peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD) showed varied activities under Cd alone. CAT activity increased after Cd treatment, followed by a decline in GPX and GR activity. SOD also declined at the highest concentrations with an initial increase. Under SA-priming conditions, the efficiency of the antioxidant enzymes was significantly elevated. GPx and SOD activity showed significant increase in activity. The ascorbate activity increased after Cd treatment, followed by a decline in glutathione under SA-free condition. SA priming showed gradual increase in these non-enzymic antioxidants. Our results indicate that Cd-induced oxidative stress can be regulated by SA.     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Protective Effects of the Synthetic Cannabinoids CP55,940 and JWH-015 on Rat Brain Mitochondria upon Paraquat Exposure

The effects of cannabinoids in mitochondria after acute oxidative stress insult are not fully established. We investigated the ability of CP55,940 and JWH-015 to scavenge reactive oxygen species and their effect on mitochondria permeability transition (MPT) in either a mitochondria-free superoxide anion generation system, intact rat brain mitochondria or in sub-mitochondrial particles (SMP) treated with paraquat (PQ). Oxygen consumption, mitochondrial membrane potential (Δψ_m) and MPT were determined as parameters of mitochondrial function. It is found that both cannabinoids effectively attenuate mitochondrial damage against PQ-induced oxidative stress by scavenging anion superoxide radical (O₂ ^∙−) and hydrogen peroxide (H₂O₂), maintaining Δψ_m and by avoiding Ca²⁺-induced mitochondrial swelling. Understanding the mechanistic action of cannabinoids on mitochondria might provide new insights into more effective therapeutic approaches for oxidative stress related disorders.     

Influences of nitrogen load on the growth and photosynthetic responses of <Emphasis Type="Italic">Quercus serrata</Emphasis> seedlings to O<Subscript>3</Subscript>

The objectives of this study were to clarify the influences of nitrogen (N) load on the growth and photosynthetic responses of Quercus serrata seedlings to O₃ and to obtain basic data for evaluating the critical levels of O₃ for protecting Q. serrata forests in Japan. The effects of O₃ and/or N load on growth and photosynthetic activity of Q. serrata seedlings were investigated during the two growing seasons. Two-year-old seedlings were assigned to 12 experimental treatments, which were comprised of the combination of four gas treatments (charcoal-filtered air and three levels of O₃ at 1.0, 1.5 and 2.0 times ambient concentration) and three N treatments (0, 20 and 50 kg ha⁻¹ year⁻¹). During the second growing season, no significant interactive effects of O₃ and N load on the growth and net photosynthetic rate of the seedlings were detected. Threrfore, we concluded that N supply to the soil at ≤50 kg ha⁻¹ year⁻¹ does not significantly influence the growth and photosynthetic responses of Q. serrata seedlings to O₃. Based on the O₃ exposure-response relationships for the whole-plant growth of the seedlings, the critical level of O₃ for Q. serrata was estimated to be approximately 36 nmol mol⁻¹ as the average 15-h O₃ concentration during the one growing season.     

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.     

Pesticides induced oxidative stress in thymocytes

The role of oxidative stress in immune cell toxicity caused by the pesticides lindane, malathion and permethrin was investigated in thymic cells from C57BL/6 mice. Thymocytes treated with any of these pesticides (concentrations ranging between 50–150 μM) were found to generate both superoxide (^•O₂ ⁻) and H₂O₂. The production of ^•O₂ ⁻ was detected with hydroethidine-ethidium bromide assay. H₂O₂ production was monitored with a flow cytometric fluorescent (DCFH-DA) assay. All three pesticides stimulated ^•O₂ ⁻ release after 5 min exposure. Lindane and permethrin, but not malathion, continued to have significant (p ≤ 0.05) effects on ^•O₂ ⁻ generation following 15 min of exposure. The lindane + malathion mixture was found to cause more-than-additive increase in ^•O₂ ⁻ production compared to individual pesticide treatments (at both 5 and 15 min). However, the effect of the lindane + permethrin mixture was not significantly different than individual components of this mixture. The effects of these pesticides on levels of antioxidant enzymes were also investigated, and only mixtures were found to have significant (p ≤ 0.05) effects. Thus, lindane + malathion and lindane + permethrin mixtures increased total superoxide dismutase (SOD) specific activity, had no effect on catalase levels and inhibited GSH-peroxidase and GSH-reductase specific activities. Although the results of these studies do not explain the mechanism of action of these pesticides on the generation of ^•O₂ ⁻ and H₂O₂, it is worthy of note that mixtures of these chemicals have oxidative responses greater than those of single chemicals. An erratum to this article can be found at     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

Cadmium modulates NADPH oxidase activity and expression in sunflower leaves

The production of reactive oxygen species (ROS) and the ways by which ROS are generated are very important facts related to heavy metal toxicity in plants. In this work, superoxide anion (O₂ ^·−) generation diminished in cadmium treated sunflower (Helianthus annuus L.) leaf discs, and this reduction was time and Cd-concentration dependent. In line with these findings, we observed that NADPH-dependent oxidase activity was significantly inhibited by 0.1 and 0.5 mM Cd²⁺ treatments and the expression of the NADPH oxidase putative gene related to O₂ ^·− synthesis in sunflower leaves was 83 % inhibited by 0.1 mM CdCl₂ and almost completely depleted by 0.5 mM CdCl₂.     

Cholic acid changes defense response to oxidative stress in soybean induced by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The oxidative stress and antioxidant systems in soybean leaves and roots infected with plant pathogen Aspergillus niger were studied following treatment with different concentrations of cholic acid. Several oxidative stress parameters were analyzed: production of superoxide (O₂ ^·−) and hydroxyl radicals (^·OH), lipid peroxidation (LP), and superoxide dismutase (SOD; EC 1.15.1.1) activity, as well as the content of reduced glutathione (GSH). Results showed that inoculation with A. niger led to the increase of O₂ ^·− production and GSH quantities in leaves and ^·OH in roots. The highest activity of SOD occured in infected plants treated with cholic acid in concentrations of 40 and 60 mg L⁻¹ which ultimately led to a decrease in O₂ ^·− production. Inoculation with Aspergillus in combination with elevated cholic acid concentrations also increased ^·OH production which is correlated with increased LP. These results may support the idea of using cholic acid as an elicitor to trigger hypersensitive response in plant cells. Use of cholic acid may also actively contribute to soybean plants defense response against pathogen attack.     

Introduction to bioreactors of shake-flask inocula leads to development of oxidative stress in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Inoculation of bioreactors with shake-flask cultures present the organism with an immediate shift from an environment with little O₂ to one in which O₂ is typically at 100% saturation. The inoculation of such shake-flasks cultures into bioreactors sparged with 1 vvm air or 1 vvm air/O₂ mix i.e. 50% O₂ enrichment is an oxidatively stressful event, as judged by immediate increases in the intracellular concentrations of superoxide anion radical (O₂^·−) (from 4,600 to 11,600 RLU mg DCW⁻¹ and 5,500 to 23,000 RLU mg DCW⁻¹ respectively) and changes in the activities of the major antioxidant enzymes superoxide dismutase and catalase in all cultures. There are further effects on metabolic indices, particularly decreased nutrient consumption in oxygenated cultures (from 0.16 to 0.12 g starch g DCW h⁻¹) and decreased protein production, indicating that inoculation of the bioreactor exerts a global burden on the cellular metabolic networks.     

Drought Induces Oxidative Stress and Enhances the Activities of Antioxidant Enzymes in Growing Rice Seedlings

When rice seedlings grown for 10 and 20 days were subjected to in vitro drought stress of −0.5 and −2.0 MPa for 24 h, an increase in the concentration of superoxide anion (O₂^.−), increased level of lipid peroxidation and a decrease in the concentration of total soluble protein and thiols was observed in stressed seedlings compared to controls. The concentration of H₂O₂ as well as ascorbic acid declined with imposition of drought stress, however glutathione (GSH) concentration declined only under severe drought stress. The activities of total superoxide dismutases (SODs) as well as ascorbate peroxidase (APX) showed consistent increases with increasing levels of drought stress, however catalase activity declined. Mild drought stressed plants had higher guaiacol peroxidase (GPX) and chloroplastic ascorbate peroxidase (c-APX) activity than control grown plants but the activity declined at the higher level of drought stress. The activities of enzymes involved in regeneration of ascorbate i.e. monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were higher in drought stressed plants compared to controls. Results suggest that drought stress induces oxidative stress in rice plants and that besides SOD, the enzymes of ascorbate-glutathione cycle, which have not been studied in detail earlier under stressful conditions, appear to function as important component of antioxidative defense system under drought stress.     

High-light-induced superoxide anion radical formation in cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach as detected by EPR spectroscopy

The generation of superoxide anion radical (O₂ ^·−) in the cytochrome b ₆ f complex (Cyt b ₆ f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O₂ ^·− was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN₃ and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O₂ ^·−, which is produced under high-light illumination of the Cyt b ₆ f from spinach, might be generated from a reaction involing ¹O₂, and the Rieske Fe-S protein could serve as the electron donor in the O₂ ^·− production. The mechanism of photoprotection of the Cyt b ₆ f complex by antioxidants is discussed.     

Expression of antioxidant defense genes in mung bean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) roots under water-logging is associated with hypoxia tolerance

This study was conducted to examine the extent of oxidative stress and the role of antioxidant enzymes on hypoxia tolerance in highly tolerant wild species Vigna luteola, and mung bean (Vigna radiata) cvs. T 44 (tolerant) and Pusa Baisakhi (susceptible). Two days of water-logging caused about 40–50% decline in superoxide radical (O₂ ^·−) and hydrogen peroxide (H₂O₂) contents in all the genotypes, however, further water-logging to 8 days caused significant increase in O₂ ^·− and H₂O₂ contents, and the values were 80–90% of the control values. In control and revived plants O₂ ^·− and H₂O₂ contents were higher in Pusa Baisakhi, while under water-logging stress T 44 and V. luteola showed greater increases in the O₂ ^·− and H₂O₂ contents. Hypoxia induced increase in superoxide dismutase, ascorbate peroxidase, and glutathione reductase activities were higher in T 44 and V. luteola compared with Pusa Baisakhi; and the increases in T 44 and V. luteola continued up to 8th day of water-logging, while in case of Pusa Baisakhi, the maximum increase was observed only on the 2nd day of water-logging. Gene expression studies showed enhanced expression of cytosolic-Cu/Zn-superoxide dismutase (SOD) and cytosolic-ascorbate peroxidase (APX) in the roots of waterlogged V. luteola and T 44, while little expression was observed in control or treated plants of Pusa Baisakhi. PCR band products were cloned and sequenced, and partial cDNAs of Cu/Zn-SOD and APX, respectively, were obtained. Results suggest that increase in the activity of antioxidant enzymes is to scavenge reactive oxygen species produced both during and after relief from water-logging stress.     

Photosynthesis of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> subjected to drought stress is enhanced by choline chloride through alleviating lipid peroxidation and increasing proline accumulation

Rehmannia glutinosa seedlings were pretreated with choline chloride (CC) in concentrations of 0, 0.7, 2.1 and 3.5 mM, and then subjected to drought and rewatering treatment to study the effects of CC on the generation of reactive oxygen species (O₂⁻, H₂O₂), lipid peroxidation, proline accumulation, water status and photosynthesis. The results showed that pretreatment with CC alleviated the inhibition of SOD and APX activity caused by drought stress, and therefore, the rate of O₂⁻ production and H₂O₂ concentration were reduced and lipid peroxidation decreased in pretreated plants. CC pretreatment also accelerated accumulation of proline, maintained higher Ψw and RWC, deferred leaf water loss during drought stress and retarded the drop in proline concentration after rewatering. Consequently, drought-induced decreases in F_m/F₀, F_v/F_m, Φ_PS2, q_P, and A and increase in q_NP were inhibited and the recovery of photosynthesis after rewatering was quicker in pretreated plants. Although differences in F_v/F_m, Φ_PS2 and q_P between treatments were not significant, there was a general trend that the effects of CC increased with the rise of its concentrations. The data suggested that 2.1 mM of CC be suitable for alleviating lipid peroxidation, promoting proline accumulation, retarding leaf water loss and improving photosynthesis of R. glutinosa seedlings under drought stress.     

Polyamines content and physiological and biochemical responses to ladder concentration of nickel stress in Hydrocharis dubia (Bl.) Backer leaves

Influence of ladder concentration of nickel (Ni) on the leaves of Hydrocharis dubia were studied after 3 days treatment. The accumulation of Ni, the content of polyamines, proline, malondialdehyde (MDA) and soluble protein, as well as the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in the leaves were investigated. The result indicated that the toxicity of Ni manifested in respective aspect of physiological and biochemical characters. Significant increase of Ni concentration in the leaf tissue was observed, which was concentration dependent. Visible symptoms of Ni toxicity: chlorosis and necrosis occurred following the 3rd day. Meantime, treatment with Ni resulted in the increase in the generation rate of O₂^•− in the leaves. SOD and CAT activities decreased significantly in response to Ni treatment, it was possibly the reason of accumulation of O₂^•−. However, a several-fold decrease in POD activities was found. Our results indicated that because of prolonged increases in O₂^•− level, oxidative damage, measured as the level of lipid peroxidation, occured in the leaves of Ni treated fronds. The changes of the content of polyamines (PAs) were also investigated in the leaves of Hydrocharis dubia. Ni treatment significantly increased the putrescine (Put) level and lowered spermidine (Spd) and spermine (Spm) levels, thereby significantly reducing the ratio of free (Spd + Spm)/Put in leaves, which has been considered as the signal under stress. Although the trend that PS-conjugated PAs and PIS-bound PAs changed the same as free PAs, they changed in more less extent.     

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Effects of Ca(NO<Subscript>3</Subscript>)<Subscript>2</Subscript> stress on oxidative damage,antioxidant enzymes activities and polyamine contents in roots of grafted and non-grafted tomato plants

The effects of Ca(NO₃)₂ stress on biomass production, oxidative damage, antioxidant enzymes activities and polyamine contents in roots of grafted and non-grafted tomato plants were investigated. Results showed that when exposed to 80 mM Ca(NO₃)₂ stress, the biomass production reduction in non-grafted plants was more significant than that of grafted plants. Under Ca(NO₃)₂ stress, superoxide anion radical (O₂•⁻) producing rate, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents of non-grafted plants roots were significantly higher than those of grafted plants, however, nitrate (NO₃ ⁻), ammonium (NH₄ ⁺) and proline contents, superoxide dismutase (SOD, EC1.15.1.1), peroxidase (POD, EC1.11.1.7), catalase (CAT, EC1.11.1.6) and arginine decarboxylase (ADC, EC 4.1.1.19) activities of grafted plants roots were significantly higher than those of non-grafted plants. Regardless of stress, free, conjugated and bound polyamine contents in roots of grafted plants were significantly higher than those of non-grafted plants. The possible roles of antioxidant enzymes, prolines and polyamines in adaptive mechanism of tomato roots to Ca(NO₃)₂ stress were discussed. Gu-Wen Zhang and Zheng-Lu Liu contributed equally to this work.     

Gain of function of stomatal movements in rooting <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. plants: regulation by H<Subscript>2</Subscript>O<Subscript>2</Subscript> is independent of ABA before the protruding of roots

Reactive oxygen species (ROS), namely superoxide radical (O₂ ⁻) and hydrogen peroxide (H₂O₂) are generated when plant tissues endure a variety of environmental stresses, including light stress. The extremely short life times of ROS makes the study of their production in planta very difficult. The use of ROS-specific tracer dyes, 3-3′ diaminobenzidine and nitroblue tetrazolium, together with high-resolution imaging provides the opportunity to identify sites of photooxidative stress response by ROS accumulation. This technique was applied to grapevine during the first 7 days after transfer from in vitro to ex vitro under an irradiance 4-fold higher than in vitro. ROS accumulation was detected in the first days of analysis, which gradually decreased to levels comparable to greenhouse leaves. O₂ ⁻ was uniformly distributed while H₂O₂ accumulated preferentially in veins, wounds and stomatal guard and surrounding cells. To evaluate the role of H₂O₂ in stomatal functioning and its crosstalk with abscisic acid (ABA) we focused on the percentage of coloured structures, stomatal aperture and ABA concentration. We propose that the high H₂O₂ level triggered by increased light is responsible for the activation of a signalling pathway over stomatal cells, in a process apparently irrespective of ABA regulation prior to root protrusion. This could explain the gain of function of a low yet consistent percentage of stomatal cells, essential for plant survival during the ontogenic period in analysis.