CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.     

Production of nitric oxide under Ultraviolet-B irradiation is mediated by hydrogen peroxide through activation of nitric oxide synthase

Excised leaves of kidney bean (Phaseolus vulgaris) were used to investigate the mechanism of NO generation under UV-B stress. We showed that two signaling molecules, NO and H₂O₂, were produced in the irradiated leaves. NO release was blocked by LNNA, an inhibitor of NOS. Application of CAT (EC 1.11.1.6) not only effectively eliminated H₂O₂ in the leaves, but also inhibited the activity of NOS and the emission of NO. In contrast, treatment with exogenous H₂O₂ increased both of those events. Therefore, we suggest that, under UV-B stress, NO production is mediated by H₂O₂ through greater NOS activity.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells

Heat shock(HS, 40℃, 10 min) induces hypericin production, nitric oxide(NO) generation, and hydrogen peroxide(H2O2) accumulation of Hypericum perforatum suspension cells.Catalase(CAT) and NO spe-cific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO) suppress not only the HS-induced H2O2 generation and NO burst, but also the HS-triggered hypericin produc-tion.Hypericin contents of the cells treated with both NO and H2O2 are significantly higher than those of the cells treated with NO alone, although H2O2 per se has no effects on hypericin production of the cells, which suggests the synergistic action between H2O2 and NO on hypericin production.NO treatment enhances H2O2 levels of H.perforatum cells, while external application of H2O2 induces NO generation of cells.Thus, the results reveal a mutually amplifying action between H2O2 and NO in H.perforatum cells.CAT treatment inhibits both HS-induced H2O2 accumulation and NO generation, while cPTIO can also suppress H2O2 levels of the heat shocked cells.The results imply that H2O2 and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells.Membrane NAD(P)H oxidase inhibitor diphenylene iodonium(DPI) and nitric oxide synthase(NOS) inhibitor S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea(PBITU) not only inhibit the mutually amplifying action between H2O2 and NO but also abolish the synergistic effects of H2O2 and NO on hypericin production, showing that the synergism of H2O2 and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification.Taken together, data of the present work demonstrate that both H2O2 and NO are essential for HS-induced hypericin production of H.perforatum suspension cells.Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.     

The role of nitric oxide in the cardiac effects of hydrogen peroxide

Oxidative stress mediated by hydrogen peroxide (H₂O₂) increases coronary flow (CF) in Langendorff-perfused rat hearts. We investigated the possible role of nitric oxide (NO) in H₂O₂-induced vasolidation. A dose-response study was conducted to find a concentration of H₂O₂ which increased CF without influencing left ventricular developed (LVDP) or end-diastolic (LVEDP) pressures. 80 (n = 10),100 (n = 7), 120 (n = 7),140 (n = 7),160 (n = 7), and 180 (n = 10) M H₂O₂ was infused for 10 min, followed by recovery for 50 min. 80 M H₂O₂ increased CF to a maximum of 143 ± 4 (mean ± S.E.M) percent of initial value after 15 min observation (p < 0.001 compared to buffer only), with no effect on LVDP or LVEDP. Another series of hearts were perfused with N-nitro-L-Arginine methylester (L-NAME, 1 M), methylene blue (MB, 50 M), or haemoglobin (Hb, 10 M), without (n = 7 in each) or with (n = 10 in each) 80 M H₂O₂ for 10 min. L-NAME, MB, and Hb alone increased CF, but attenuated the H₂O₂-induced increase of CF. LVDP was depressed when L-NAME, MB, or Hb were given in conjunction with 80 M H₂O₂. In summary, H₂O₂ concentration-dependently increased LVEDP and depressed LVDP. The H₂O₂-induced increase of CF was independent of concentration. Inhibition of NO synthesis, action, or soluble guanylate cyclase attenuated the H₂O₂-induced increase of CF, and depressed LVDP when given together with H₂O₂. H₂O₂ induces a NO-dependent vasodilation, and inhibition of NO is detrimental to left ventricular function after H₂O₂-mediated oxidative stress.     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

Foliar abscisic acid content underlies genotypic variation in stomatal responsiveness after growth at high relative air humidity

Background and Aims

Stomata formed at high relative air humidity (RH) respond less to abscisic acid (ABA), an effect that varies widely between cultivars. This study tested the hypotheses that this genotypic variation in stomatal responsiveness originates from differential impairment in intermediates of the ABA signalling pathway during closure and differences in leaf ABA concentration during growth.

Methods

Stomatal anatomical features and stomatal responsiveness to desiccation, feeding with ABA, three transduction elements of its signalling pathway (H₂O₂, NO, Ca²⁺) and elicitors of these elements were determined in four rose cultivars grown at moderate (60 %) and high (90 %) RH. Leaf ABA concentration was assessed throughout the photoperiod and following mild desiccation (10 % leaf weight loss).

Key Results

Stomatal responsiveness to desiccation and ABA feeding was little affected by high RH in two cultivars, whereas it was considerably attenuated in two other cultivars (thus termed sensitive). Leaf ABA concentration was lower in plants grown at high RH, an effect that was more pronounced in the sensitive cultivars. Mild desiccation triggered an increase in leaf ABA concentration and equalized differences between leaves grown at moderate and high RH. High RH impaired stomatal responses to all transduction elements, but cultivar differences were not observed.

Conclusions

High RH resulted in decreased leaf ABA concentration during growth as a result of lack of water deficit, since desiccation induced ABA accumulation. Sensitive cultivars underwent a larger decrease in leaf ABA concentration rather than having a higher ABA concentration threshold for inducing stomatal functioning. However, cultivar differences in stomatal closure following ABA feeding were not apparent in response to H₂O₂ and downstream elements, indicating that signalling events prior to H₂O₂ generation are involved in the observed genotypic variation.     

Cross-talk between calcium-calmodulin and nitric oxide in abscisic acid signaling in leaves of maize plants

Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide （H2O2）, calcium （Ca^2＋）-calmodulin （CAM）, and nitric oxide （NO） in abscisic acid （ABA）-induced antioxidant defense were investigated in leaves of maize （Zea mays L.） plants. Treatments with ABA, H2O2, and CaCl2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. However, such increases were blocked by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca^2＋-induced increase in the production of NO. On the other hand, treatments with ABA and the NO donor sodium nitroprusside （SNP） also led to increases in the concentration of cytosolic Ca^2＋ in protoplasts of mesophyll cells and in the expression of calmodulin 1 （CaM1） gene and the contents of CaM in leaves of maize plants, and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor. Moreover, SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 （SOD4）, cytosolic ascorbate peroxidase （cAPX）, and glutathione reductase 1 （GR1） and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Our results suggest that Ca^2＋-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.     

Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli

Hydrogen peroxide (H₂O₂, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H₂O₂ presents several questions. First, the lethal H₂O₂ concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H₂O₂-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H₂O₂ toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H₂O₂ antimicrobial action, others instead report alleviation. To investigate this “NO paradox,” we treated Escherichia coli with various concentrations of H₂O₂-alone or H₂O₂+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H₂O₂ treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H₂O₂ toxicity by blocking H₂O₂ scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H₂O₂-alone or H₂O₂+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H₂O₂-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H₂O₂ concentrations is transiently alleviated by NO, reproducing the “NO paradox.” We conclude that NO potentiates H₂O₂ toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H₂O₂ concentrations, while transiently suppressing mode-two killing by immediately lethal high H₂O₂ concentrations.     

Nitric oxide reduces hydrogen peroxide accumulation involved in water stress-induced subcellular anti-oxidant defense in maize plants

Nitric oxide （NO） is a bioactive molecule involved in many biological events, and has been reported as pro-oxidant as well as anti-oxidant in plants. In the present study, the sources of NO production under water stress, the role of NO in water stress-induced hydrogen peroxide （H2O2） accumulation and subcellular activities of anti-oxidant enzymes in leaves of maize （Zea mays L.） plants were investigated. Water stress induced defense increases in the generation of NO in maize mesphyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. Water stress-induced defense increases in the production of NO were blocked by pretreatments with inhibitors of NOS and nitrate reductase （NR）, suggesting that NO is produced from NOS and NR in leaves of maize plants exposed to water stress. Water stress also induced increases in the activities of the chloroplastic and cytosolic anti-oxidant enzymes superoxide dismutase （SOD）, ascorbate peroxidase （APX）, and glutathione reductase （GR）, and the increases in the activities of anti-oxidant enzymes were reduced by pretreatments with inhibitors of NOS and NR. Exogenous NO increases the activities of water stress-induced subcellular anti-oxidant enzymes, which decreases accumulation of H2O2. Our results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO. The potential ability of NO to scavenge H2O2 is, at least in part, due to the induction of a subcellular anti-oxidant defense.     

Nitric oxide inhibits blue light-specific stomatal opening via abscisic acid signaling pathways in Vicia guard cells

Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NO's effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H(+) pumping and H(+)-ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) restored all these inhibitory effects. ABA and hydrogen peroxide (H(2)O(2)) inhibited all of these blue light-specific responses in a manner similar to NO. c-PTIO partially restored the ABA-induced inhibition of all of these opening responses but did not restore inhibition of the responses by H(2)O(2). ABA, H(2)O(2) and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H(+) pumping in guard cells nor H(+) transport by H(+)-ATPase in the isolated membranes. From these results, we conclude that both NO and H(2)O(2) inhibit blue light-induced activation of H(+)-ATPase by inhibiting the component(s) between phototropins and H(+)-ATPase in guard cells and stimulate stomatal closure by ABA.     

Arbuscular mycorrhizal fungal inoculation increases phenolic synthesis in clover roots via hydrogen peroxide,salicylic acid and nitric oxide signaling pathways

Arbuscular mycorrhizal fungi can increase the host resistance to pathogens via promoted phenolic synthesis, however, the signaling pathway responsible for it still remains unclear. In this study, in order to reveal the signaling molecules involved in this process, we inoculated Trifolium repense L. with an arbuscular mycorrhizal fungus (AMF), Glomus mosseae, and monitored the contents of phenolics and signaling molecules (hydrogen peroxide (H₂O₂), salicylic acid (SA), and nitric oxide (NO)) in roots, measured the activities of l-phenylalanine ammonia-lyase (PAL) and nitric oxide synthase (NOS), and the expression of pal and chs genes. Results demonstrated that AMF colonization promoted the phenolic synthesis, in parallel with the increase in related enzyme activity and gene expression. Meanwhile, the accumulation of all three signaling molecules was also up-regulated by AMF. This study suggested that AMF increased the phenolic synthesis in roots probably via signaling pathways of H₂O₂, SA and NO in a signaling cascade.     

Consequences of MnSOD interactions with nitric oxide: nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^- ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^-  species initiate the formation of peroxynitrite (ONOO^- ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^-  decomposition and ONOO^- -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^-  is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Guard-cell signalling for hydrogen peroxide and abscisic acid

Guard cells can integrate and process multiple complex signals from the environment and respond by opening and closing stomata in order to adapt to the environmental signal. Over the past several years, considerable research progress has been made in our understanding of the role of reactive oxygen species (ROS) as essential signal molecules that mediate abscisic acid (ABA)-induced stomatal closure. In this review, we discuss hydrogen peroxide (H2O2) generation and signalling, H2O2-induced gene expression, crosstalk and the specificity between ABA and H2O2 signalling, and the cellular mechanism for ROS sensing in guard cells. This review focuses especially on the points of connection between ABA and H2O2 signalling in guard cells. The fundamental progress in understanding the role of ABA and ROS in guard cells will continue to provide a rational basis for biotechnological improvements in the development of drought-tolerant crop plants with improved water-use efficiency.     

Possible accumulation of non-active molecules of catalase and superoxide dismutase in S. cerevisiae cells under hydrogen peroxide induced stress

The effect of hydrogen peroxide on the activities of catalase and superoxide dismutase (SOD) in S. cerevisiae has been studied under different experimental conditions: various H₂O₂ concentrations, time exposures, yeast cell densities and media for stress induction. The yeast treatment with 0.25–0.50 mM H₂O₂ led to an increase in catalase activity by 2–3-fold. At the same time, hydrogen peroxide caused an elevation by 1.6-fold or no increase in SOD activity dependently on conditions used. This effect was cancelled by cycloheximide, an inhibitor of protein synthesis in eukaryotes. Weak elevation of catalase and SOD activities in cells treated with 0.25–0.50 mM H₂O₂ found in this study does not correspond to high level of synthesis of the respective enzyme molecules observed earlier by others. It is well known that exposure of microorganisms to low sublethal concentrations of hydrogen peroxide leads to the acquisition of cellular resistance to a subsequent lethal oxidative stress. Hence, it makes possible to suggest that S. cerevisiae cells treated with low sublethal doses of hydrogen peroxide accumulate non-active stress-protectant molecules of catalase and SOD to survive further lethal oxidant concentrations.     

一氧化氮供体对过氧化氢引起的心肌细胞损伤的保护作用

关于一氧化氮(NO)对心肌细胞是否具有保护作用目前尚存在争议,为探讨NO对过氧化氢(H2O2)引起的心肌细胞损伤是否具有保护作用及其可能的机制,实验将体外培养的新生大鼠心肌细胞分为3组(1)阴性对照组(Normal组);(2)H2O2组H2O2(0.1mmol/L)与心肌细胞共育4h;(3)S-亚硝基-N-乙酰青霉胺(SNAP)+H2O2组NO供体SNAP(0.5mmol/L)处理心肌细胞10min后,加入H2O2与心肌细胞共育4 h.用流式细胞术检测心肌细胞凋亡率,心肌细胞损伤程度以心肌细胞存活率和乳酸脱氢酶(lactate dehydrogenase,LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(MDA)含量.通过激光共聚焦显微术检测在不同处理条件下心肌细胞胞内钙的变化.结果表明,正常心肌细胞LDH活性和细胞存活率分别为631.4±75.6 U/L和93.1±6.2%,细胞凋亡率为0;H2O2处理细胞后可使细胞LDH活性显著增高(1580.5±186.7 U/L,P＜0.01),细胞存活率明显下降(58.3±7.6%,P＜0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为26.4±5.7%;SOD活性较正常细胞19.67±0.85 NU/ml显著下降,为14.73±1.68 NU/m(P＜0.01),MDA含量较正常细胞6.95±0.83μmol/L显著增高,为15.35±3.49μmol/L(P＜0.01).SNAP预处理细胞可显著提高心肌细胞存活率(79.7±9.3%,P＜0.01),降低LDH活性和细胞凋亡率(分别为957.8±110.9 U/L和9.1±3.3%,P＜0.01);并提高细胞抗氧化能力,表现为较H2O2处理组的SOD活性增高(21.36±3.11 NU/ml,P＜0.01),MDA含量下降(9.12±1.47 μmol/L,P＜0.01).激光共聚焦显微镜检测结果表明,H2O2可升高细胞内钙,而SNAP则可降低细胞内钙,SNAP预处理细胞后可取消H2O2升高细胞内钙的作用.上述结果提示,NO供体SNAP可对抗H2O2对心肌细胞的损伤,其机制与提高心肌细胞抗氧化损伤能力和对抗H2O2引起的细胞内钙超载有关.     

NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用

 NO和H₂O₂是参与植物抗非生物胁迫反应的重要信号分子, 为了确定NO和H₂O₂在大豆(Glycine max)根尖和根边缘细胞(root border cells, RBCs)耐铝反应中的作用及其相互关系, 以‘浙春3号’大豆为材料, 研究了铝毒胁迫下大豆根尖内源NO和H₂O₂的变化, 以及外源NO和H₂O₂诱导大豆根尖和RBCs的耐铝反应。结果表明, 50 μmol·L^–1 Al处理48 h显著抑制大豆根的伸长, 提高Al在根尖的积累, 同时显著增加根尖内源NO和H₂O₂含量。施加0.25 mmol·L^–1外源NO供体亚硝基铁氰化钠(Na₂[Fe(CN)₅NO]·2H₂O, sodium nitroprusside, SNP)和0.1 mmol·L^–1H₂O₂, 能有效地缓解Al对大豆根伸长的抑制、根尖Al积累和RBCs 的死亡, 该缓解作用可以被0.05 mmol·L^–1 NO清除剂2-(4- 羧基苯)-4,4,5,5- 四甲基咪唑-1- 氧-3- 氧化物, 钾盐(C₁₄H₁₆N₂O₄·K, carboxy-PTIO, cPTIO)和150 U·mL^–1 H₂O₂清除酶(catalase, CAT)逆转。并且外源NO能够显著促进根尖H₂O₂的积累, 而外源H₂O₂对根尖NO的含量无显著影响。这表明NO和H₂O₂是诱导大豆根尖及RBCs耐铝反应的两种信号分子, NO可能通过调控H₂O₂的形成, 进而诱导大豆根尖及RBCs的耐铝反应。