Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Salicylic acid-mediated hydrogen peroxide accumulation and protection against Cd toxicity in rice leaves

The role of H₂O₂ in salicylic acid (SA)-induced protection of rice leaves against subsequent Cd toxicity was investigated. SA pretreatment resulted in an increase in the contents of endogenous SA, as judged by the expression of OsWRKY45 (a SA responsive gene), and H₂O₂ in rice leaves. Diphenyleneiodonium (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented SA-increased H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂-generating enzyme in SA-pretreated rice leaves. DPI and IMD also inhibited SA-increased activities of superoxide dismutase (SOD), ascorbate peroixdase (APX), and glutathione reductase (GR) activities, but had no effect on SA-increased catalase (CAT) activity. Moreover, SA-induced protection against subsequent Cd toxicity could also be prevented by DPI and IMD. The inhibitory effect of DPI and IMD on SA-induced protection against subsequent Cd toxicity could be reversed by exogenous H₂O₂. All these results suggested that SA-induced protection against subsequent Cd toxicity is mediated through H₂O₂. This conclusion is supported further by the observations that exogenous H₂O₂ application resulted in an increase in SOD, APX, and GR activities, but not CAT activity and a protection against subsequent Cd toxicity of rice leaves.     

Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity

The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H₂O₂ accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H₂O₂ accumulation in AMF-inoculated roots was decreased by 42.44 % compared with the control under LT. H₂O₂ predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H₂O₂ production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H⁺-ATPase, P-Ca²⁺-ATPase, V-type H⁺-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H₂O₂ accumulation in the roots of cucumber.     

Hydrogen Peroxide-Mediated Growth of the Root System Occurs via Auxin Signaling Modification and Variations in the Expression of Cell-Cycle Genes in Rice Seedlings Exposed to Cadmium Stress

The link between root growth, H₂O₂, auxin signaling, and the cell cycle in cadmium (Cd)‐stressed rice (Oryza sativa L. cv. Zhonghua No. 11) was analyzed in this study. Exposure to Cd induced a significant accumulation of Cd, but caused a decrease in zinc (Zn) content which resulted from the decreased expression of OsHMA9 and OsZIP. Analysis using a Cd‐specific probe showed that Cd was mainly localized in the meristematic zone and vascular tissues. Formation and elongation of the root system were significantly promoted by 3‐amino‐1,2,4‐triazole (AT), but were markedly inhibited by N,N’‐dimethylthiourea (DMTU) under Cd stress. The effect of H₂O₂ on Cd‐stressed root growth was further confirmed by examining a gain‐of‐function rice mutant (carrying catalase1 and glutathione‐S‐transferase) in the presence or absence of diphenylene iodonium. DR5‐GUS staining revealed close associations between H₂O₂ and the concentration and distribution of auxin. H₂O₂ affected the expression of key genes, including OsYUCCA, OsPIN, OsARF, and OsIAA, in the auxin signaling pathway in Cd‐treated plants. These results suggest that H₂O₂ functions upstream of the auxin signaling pathway. Furthermore, H₂O₂ modified the expression of cell‐cycle genes in Cd‐treated roots. The effects of H₂O₂ on root system growth are therefore linked to auxin signal modification and to variations in the expression of cell‐cycle genes in Cd‐stressed rice. A working model for the effects of H₂O₂ on Cd‐stressed root system growth is thus proposed and discussed in this paper.     

Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in Medicago sativa by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H₂S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H₂S production as well as the activation of two H₂S-synthetic enzymes activities, including _L-cysteine desulfhydrase (LCD) and _D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H₂S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H₂S production triggered by Cd, but also alleviated Cd toxicity in a H₂S-dependent fashion. By contrast, the inhibition of H₂S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H₂S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.     

Cadmium toxicity and translocation in rice seedlings are reduced by hydrogen peroxide pretreatment

A hydroponic experiment was carried out to study the role of hydrogen peroxide (H₂O₂) in enhancing tolerance and reducing translocation of cadmium (Cd) in rice seedlings. Plant growth (length and biomass of shoot and root) was significantly repressed by Cd exposure. However, pretreatment with 100 μM H₂O₂ for 1d mitigated Cd stress by inducing enzyme activities for antioxidation (e.g., superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX)) and detoxification (e.g., glutathione S-transferase (GST)) as well as by elevating contents of reduced glutathione (GSH) and ascorbic acid (AsA). As a result, H₂O₂ and malondialdehyde (MDA) content decreased in plants and the seedling growth was less inhibited. On the other hand, H₂O₂ pretreatment decreased Cd concentration in shoots, thus lowered the ratio of Cd concentration in shoots and roots (S/R), indicating that H₂O₂ may affect Cd distribution in rice seedlings. The improved Cd tolerance is partly due to an enhanced antioxidative system that efficiently prevents the accumulation of H₂O₂ during Cd stress. Increased Cd sequestration in rice roots may contribute to the decline of Cd translocation.     

Scanning ion‐selective electrode technique and X‐ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt‐sensitive cucumber and salt‐tolerant pumpkin under NaCl stress

Grafting onto salt‐tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na⁺ in salt‐tolerant pumpkin and salt‐sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion‐selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na⁺, and a correspondingly increased H⁺ influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na⁺/H⁺ exchange in the root was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or vanadate [a plasma membrane (PM) H⁺‐ATPase inhibitor], indicating that Na⁺ exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na⁺/H⁺ antiporter across the PM, and the Na⁺/H⁺ antiporter system in salt stressed pumpkin roots was sufficient to exclude Na⁺. X‐ray microanalysis showed higher Na⁺ in the cortex, but lower Na⁺ in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na⁺, limit the radial transport of Na⁺ to the stele and thus restrict the transport of Na⁺ to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots.     

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.     

l‐cysteine desulfhydrase‐related H2S production is involved in OsSE5‐promoted ammonium tolerance in roots of Oryza sativa

Previous studies revealed that rice heme oxygenase PHOTOPERIOD SENSITIVITY 5 (OsSE5) is involved in the regulation of tolerance to excess ammonium by enhancing antioxidant defence. In this study, the relationship between OsSE5 and hydrogen sulfide (H₂S), a well‐known signalling molecule, was investigated. Results showed that NH₄Cl triggered the induction of l ‐cysteine desulfhydrase (l ‐DES)‐related H₂S production in rice seedling roots. A H₂S donor not only alleviated the excess ammonium‐triggered inhibition of root growth but also reduced endogenous ammonium, both of which were aggravated by hypotaurine (HT, a H₂S scavenger) or dl ‐propargylglycine (PAG, a l ‐DES inhibitor). Nitrogen metabolism‐related enzymes were activated by H₂S, thus resulting in the induction of amino acid synthesis and total nitrogen content. Interestingly, the activity of l ‐DES, as well as the enzymes involved in nitrogen metabolism, was significantly increased in the OsSE5‐overexpression line (35S:OsSE5), whereas it impaired in the OsSE5‐knockdown mutant (OsSE5‐RNAi). The application of the HT/PAG or H₂S donor could differentially block or rescue NH₄Cl‐hyposensitivity or hypersensitivity phenotypes in 35S:OsSE5‐1 or OsSE5‐RNAi‐1 plants, with a concomitant modulation of nitrogen assimilation. Taken together, these results illustrated that H₂S function as an indispensable positive regulator participated in OsSE5‐promoted ammonium tolerance, in which nitrogen metabolism was facilitated.     

Arbuscular mycorrhizas alter root system architecture of Citrus tangerine through regulating metabolism of endogenous polyamines

Cadmium (Cd) is readily taken up by the roots of rice seedlings, leading to growth reduction. H₂O₂ is a constituent of oxidative metabolism and is itself a reactive oxygen species. In this study, the participation of H₂O₂ in CdCl₂-inhibited growth of rice roots was investigated. CdCl₂ treatment increased H₂O₂ production in rice roots. CdCl₂ treatment had no effect on the activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase, but inhibited the activity of catalase (CAT) in rice roots. CdCl₂-inhibited root growth and -increased H₂O₂ content were lessened in the presence of diphenyleneiodonium chloride, an inhibitor of H₂O₂ generating NADPH oxidase. However, this stimulation of root growth in CdCl₂-treated seedlings is small (about 5%). Calcium (Ca) is important in many physiological processes in plants. Attempts were also made to determine whether the action of Ca on CdCl₂-inhibited growth of rice roots is associated with H₂O₂. CaCl₂ application reduced the production of H₂O₂, the decrease in CAT activity, and the inhibition of root growth caused by CdCl₂. The effects of CaCl₂ application could be reversed by exogenous H₂O₂. Our results indicate that the Cd causes a decline in CAT and to a lower extent a stimulation of NADPH oxidase in rice roots, with the subsequent generation of H₂O₂, an agent responsible for growth inhibition.     

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl₂ (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H₂O₂ and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl₂ as those non-HS. The content of H₂O₂ increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H₂O₂ under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H₂O₂ production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H₂O₂ during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

NaCl-induced heme oxygenase in roots of rice seedlings is mediated through hydrogen peroxide

Recent findings have suggested that H₂O₂ is an important signaling molecule for regulating plant responses to abiotic stress. H₂O₂ plays a critical role in NaCl stress. Heme oxygenase (HO) is known to play a protective role against oxidative stress. In this study, we examined the possible involvement of H₂O₂ in regulating NaCl-promoted HO activity in rice roots. Treatment with NaCl increased HO activity and H₂O₂ content in rice roots. As well, NaCl could induce OsHO1 mRNA expression. NaCl (150 mM) and NaNO₃ (150 mM) were equally effective in inducing HO activity. However, mannitol at the concentration (276 mM) iso-osmotic with 150 mM NaCl had no effect on HO activity. NaCl-promoted HO activity and OsHO1 expression in rice roots was reduced by NADPH oxidase inhibitors i.e. dipehnyleneiodonium and imidazole. Moreover, exogenous application of H₂O₂ enhanced the activity of HO and the mRNA level of OsHO1. Our data suggest that H₂O₂ production plays a positive role in NaCl- induced HO activity by enhancing its mRNA level in rice roots.