Hydrogen peroxide is not involved in HrpN from Erwinia amylovora-induced hypersensitive cell death in maize leaves

Harpin elicits rapid and localized programmed cell death in plants, also known as the hypersensitive response (HR). Here we report that HrpN from Erwinia amylovora led to rapid cell death in maize leaves within 24 h and also induced the expression of systemic acquired resistance genes, such as ZmPR1 and ZmPR5. Surprisingly, the results of DAB staining showed that there was no H₂O₂ accumulation in maize leaves during the HR process, and semi-quantitative RT-PCR revealed that there was also no difference in the expression of the ZmRboh genes. These results suggest that HrpN-induced cell death may be independent of H₂O₂ accumulation in maize leaves.     

WD40‐REPEAT 5a functions in drought stress tolerance by regulating nitric oxide accumulation in Arabidopsis

Nitric oxide (NO) generation by NO synthase (NOS) in guard cells plays a vital role in stomatal closure for adaptive plant response to drought stress. However, the mechanism underlying the regulation of NOS activity in plants is unclear. Here, by screening yeast deletion mutants with decreased NO accumulation and NOS‐like activity when subjected to H₂O₂ stress, we identified TUP1 as a novel regulator of NOS‐like activity in yeast. Arabidopsis WD40‐REPEAT 5a (WDR5a), a homolog of yeast TUP1, complemented H₂O₂‐induced NO accumulation of a yeast mutant Δtup1, suggesting the conserved role of WDR5a in regulating NO accumulation and NOS‐like activity. This note was further confirmed by using an Arabidopsis RNAi line wdr5a‐1 and two T‐DNA insertion mutants of WDR5a with reduced WDR5a expression, in which both H₂O₂‐induced NO accumulation and stomatal closure were repressed. This was because H₂O₂‐induced NOS‐like activity was inhibited in the mutants compared with that of the wild type. Furthermore, these wdr5a mutants were more sensitive to drought stress as they had reduced stomatal closure and decreased expression of drought‐related genes. Together, our results revealed that WDR5a functions as a novel factor to modulate NOS‐like activity for changes of NO accumulation and stomatal closure in drought stress tolerance.     

H2O2-induced Leaf Cell Death and the Crosstalk of Reactive Nitric/Oxygen Species([F])

In plants, the chloroplast is the main reactive oxygen species (ROS) producing site under high light stress. Catalase (CAT), which decomposes hydrogen peroxide (H₂O₂), is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H₂O₂‐induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 (noe1), higher H₂O₂ levels induced the generation of nitric oxide (NO) and higher S‐nitrosothiol (SNO) levels, suggesting that NO acts as an important endogenous mediator in H₂O₂‐induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species (RNS). Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H₂O₂‐induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response (HR), leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions. [ Chengcai Chu (Corresponding author)]     

Nitric oxide activates superoxide dismutase and ascorbate peroxidase to repress the cell death induced by wounding

Wounding caused by rain, wind, and pathogen may lead plants to onset defense response. Previous studies indicated that mechanical wounding stimulates plants to generate nitric oxide (NO) and hydrogen peroxide (H₂O₂). In this study, the functions of NO and H₂O₂ after wounding in sweet potato (Ipomoea batatas cv. Tainung 57) was further analyzed. Mechanical wounding damaged cells and resulted in necrosis, but the presence of NO donors or NO scavenger might reduce or enhance the cell death caused by wounding, respectively. The amount of H₂O₂ induced by wounding was also decreased or increased when plants were incubated with NO donors or NO scavenger, individually. These results indicate that NO may regulate H₂O₂ generation to affect cell death. NO-induced proteins isolated from two-dimensional electrophoresis were identified to be Copper/Zinc superoxide dismutases (CuZnSODs). The activities of CuZnSODs and ascorbate peroxidase (APX) could be enhanced by NO. In addition, the expression of CuZnSOD and APX was induced by wounding via NO, and their expression was further stimulated by NO through the generation of cGMP. The influx of calcium ions and the activity of NADPH oxidase were also involved in the NO signal transduction pathway inducing APX expression. Collectively, the generation of H₂O₂ in wounded plants might trigger cell death. Meanwhile, the production of NO induced by wounding stimulated signal transducers including cGMP, calcium ions, and H₂O₂ to activate CuZnSOD and APX, which further decreased H₂O₂ level and reduced the cell death caused by wounding.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

MMP?REDOX/NO Interplay in Periodontitis and Its Inhibition with Satureja hortensis L. Essential Oil

Satureja hortensis L. is an aromatic plant with antibacterial and antibiofilm activities against periodontopathogens. Here, we attempted to find out whether the antioxidant properties of S. hortensis L. essential oil (EO) could be used to inhibit matrix metalloproteinase (MMP) activities and prevent the induction of cell death by a pro‐oxidant insult. First, a landscape analysis of MMP and REDOX/nitric oxide (NO)‐related genes was performed (MRN model), and array data from periodontitis patients were plotted over the newly developed model. Thereafter, the antigelatinolytic activity of S. hortensis L. EO and its preventive effect against hydrogen peroxide (H₂O₂)‐induced cell death were tested in vitro (HaCaT cells). Up‐regulation of MMP genes in the MRN network (except for MMP‐10, ‐15, ‐16, ‐20, ‐25, and ‐26) and differential expression of genes coding for antioxidant enzymes were found among others in periodontitis samples. MMP2 and MMP9 were central genes in the MRN network model. Moreover, treatments with 1 and 5 μl/ml of S. hortensis L. EO inhibited both MMP‐2 and MMP‐9 activities, and H₂O₂‐induced cell death in vitro. We concluded that S. hortensis L. EO could be a promising host‐modulating agent, since oxidative stress and excessive MMP expression/activity are typical hallmarks of periodontal pathogenesis.     

Protection against oxidative stress by vitamin D in cone cells

Photoreceptor degeneration (PD) refers to a group of heterogeneous outer retinal dystrophies characterized by the death of photoreceptors. Both oxidative stress and inflammation are involved in the pathogenesis of PD. We investigate whether vitamin D has a potential for the treatment of PD by evaluating the anti‐oxidative stress and anti‐inflammatory properties of the active form of vitamin D₃, 1,α, 25‐dihydroxyvitamin D₃, in a mouse cone cell line, 661W. Mouse cone cells were treated with H₂O₂ or a mixture of H₂O₂ and vitamin D; cell viability was determined. The production of reactive oxygen species (ROS) in treated and untreated cells was measured. The expression of key anti‐oxidative stress and inflammatory genes in treated and untreated cells was determined. Treatment with vitamin D significantly increased cell viability and decreased ROS production in 661W cells under oxidative stress induced by H₂O₂. H₂O₂ treatment in 661W cells can significantly down‐regulate the expression of antioxidant genes and up‐regulate the expression of neurotoxic cytokines. Vitamin D treatment significantly reversed these effects and restored the expression of antioxidant genes. Vitamin D treatment also can block H₂O₂ induced oxidative damages. The data suggested that vitamin D may offer a therapeutic potential for patients with PD.     

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.     

Nobiletin protects human retinal pigment epithelial cells from hydrogen peroxide–induced oxidative damage

Nobiletin (3′,4′,5,6,7,8‐hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, has been reported to have antioxidant effect. However, the effect of nobiletin on human retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H₂O₂) is still unclear. Therefore, we investigated the protective effect of nobiletin against H₂O₂‐induced cell death in RPE cells. Our results demonstrated that nobiletin significantly increased cell viability from oxidative stress. Nobiletin inhibited H₂O₂‐induced ROS production and caspase‐3/7 activity in ARPE‐19 cells. Furthermore, nobiletin significantly increased Akt phosphorylation in ARPE‐19 cells exposed to H₂O₂. Meanwhile, LY294002, an inhibitor of PI3K/Akt, abolished the protective effect of nobiletin against H₂O₂‐induced decreased cell viability and increased caspase‐3/7 activity in ARPE‐19 cells. In summary, these data show that nobiletin protects RPE cells against oxidative stress through activation of the Akt‐signaling pathway. Thus, nobiletin should be an oxidant that attenuates the development of age‐related macular degeneration.     

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.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

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.     

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose‐ and time‐dependent reduction in viability, and the agonist‐treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca²⁺ levels, resulting in Ca²⁺ uptake by the mitochondria and subsequent H₂O₂ accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP‐induced increase of intracellular ATP, essential for the activation of caspase‐like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca²⁺ concentration but plays a negligible role in eATP‐stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.     

OsHK3 is a crucial regulator of abscisic acid signaling involved in antioxidant defense in rice

In this study, the role of the rice(Oryza sativa L.)histidine kinase Os HK3 in abscisic acid(ABA)-induced antioxidant defense was investigated. Treatments with ABA, H2O2,and polyethylene glycol(PEG) induced the expression of Os HK3 in rice leaves, and H2O2 is required for ABA-induced increase in the expression of Os HK3 under water stress. Subcellular localization analysis showed that Os HK3 is located in the cytoplasm and the plasma membrane. The transient expression analysis and the transient RNA interference test in rice protoplasts showed that Os HK3 is required for ABA-induced upregulation in the expression of antioxidant enzymes genes and the activities of antioxidant enzymes. Further analysis showed that Os HK3 functions upstream of the calcium/calmodulin-dependent protein kinase Os DMI3 and the mitogen-activated protein kinase Os MPK1 to regulate the activities of antioxidant enzymes in ABA signaling. Moreover, Os HK3was also shown to regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase genes, Osrboh B and Osrboh E, and the production of H2O2 in ABA signaling. Our data indicate that Os HK3 play an important role in the regulation of ABA-induced antioxidant defense and in the feedback regulation of H2O2 production in ABA signaling.     

Nitric oxide and hydrogen peroxide are important signals mediating the allelopathic response of Arabidopsis to p‐hydroxybenzoic acid

Both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are important signals that mediate plant response to environmental stimulation. Their role in plants' allelopathic interactions has also been reported, but the underlying mechanism remains little understood. p‐Hydroxybenzoic acid (pHBA) has been proposed to be an allelopathic chemical. Here, we found that pHBA at 0.4 mM efficiently suppressed Arabidopsis growth. Meanwhile, pHBA rapidly induced the accumulation of NO and H₂O₂, where such effect could be reversed by NO or H₂O₂ metabolism inhibitors or scavengers. Also, pHBA‐induced NO and H₂O₂ could be compromised in NO synthesis mutants noa1, nia1 and nia2, or H₂O₂ metabolism mutant rbohD/F, but suppressing NO accumulation with a NO synthesis inhibitor or using NO synthesis‐related mutants did not reduce pHBA‐induced H₂O₂ accumulation. Furthermore, we found that the effect of pHBA on allelopathic inhibition of growth was aggravated in NO/H₂O₂ metabolism‐related mutants or reducing NO/H₂O₂ by different inhibitors, whereas the addition of an NO/H₂O₂ donor could partly relieve the inhibitory effect of pHBA on the growth of wild type. However, adding only an NO donor, but not low concentration of H₂O₂ as the donor, could relieve the inhibitory effect of pHBA on root growth in NO metabolism mutants. On the basis of these results, we propose that both NO and H₂O₂ are important signals that mediate Arabidopsis response to the allelopathic chemical pHBA, where during this process H₂O₂ may work upstream of the NO signal.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.