在耐热性诱导中豌豆叶片过氧化氢和水杨酸含量与质膜ATP酶活性的变化及相互关系

在高温锻炼(37℃,2h)过程中,豌豆(Pisum sativum L.)叶片过氧化氢(H_2O_2)和游离态水杨酸(SA)含量与质膜ATP酶(H~ -ATPase)活性都有一个高峰,H_2O_2的迸发早于游离态SA的积累,而质膜H~ -ATPase活性高峰的出现则迟于SA高峰;活性氧清除剂、抗氧化剂、质膜NADPH氧化酶抑制剂和H_2O_2的淬灭剂预处理均可有效地阻止高温下H_2O_2和SA的积累以及质膜H~ -ATPase活性的增加。根据以上结果推测,H_2O_2、质膜H~ -ATPase和SA均参与耐热性诱导相关的信号传递,前者作用于SA的上游,而后者在SA下游起作用。     

Effect of exogenous H2O2 on antioxidant enzymes of Brassica juncea L. seedlings in relation to 24-epibrassinolide under chilling stress

Hydrogen peroxide is most stable molecule among reactive oxygen species, which play a vital role in growth and development of plant as signaling molecule at low concentration in response to various abiotic and biotic stresses. Exogenous application of H2O2 is known to induce chilling tolerance in plants. Brassinosteroids are plant steroid hormones known for their anti-stress properties. In this study, effect of exogenous H2O2 on antioxidant defense system of Brassica juncea L. seedlings was investigated in 24-epibrassinolide (24-EBL) treated and untreated seedlings under chilling stress. The surface sterilized seeds of B. juncea L. were germinated in petriplates containing different concentrations of H2O2 alone and in combination with 10(-8) M 24-EBL. Chilling treatment (4 degrees C) was given to 10-days old seedlings grown in different treatments for 6 h daily up to 3 days. 24 h recovery period was given to chilling treated seedlings by placing at 25 degrees C + 2 degrees C and harvested for antioxidant enzymes on 14th day after sowing (DAS). Treatment of 24-EBL in combination with H2O2 (15 and 20 mM) helped in reducing the toxicity of seed and seedlings due to H2O2 exposure on their germination rate, shoot and root length respectively. 24-EBL treatment at seed and seedling stage helped in alleviating the toxic effect of H2O2 through antioxidant defense system by increasing the activities of various enzymes involved in antioxidant defense system such as catalase (CAT, E.C. 1.11.1.6), ascorbate peroxidase (APOX, E.C. 1.11.1.11), and superoxide dismutase (SOD, E.C. 1.15.1.1). In conclusion, exogenous pretreatment of H2O2 to seeds of B. juncea L. adapted the seedlings to tolerate chilling stress, which was further ameliorated in combination of H2O2 with 24-EBL.     

Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize

Protein phosphorylation plays a central role in mediating abscisic acid (ABA) signaling transduction in plant cells, whereas many of the sensory proteins involving in ABA signaling pathway remain unclear. Here, using a modified in vitro kinase assay, our results showed that ABA and H2O2 induced a rapid activation of total protein kinases and calcium dependent protein kinases in the leaves of maize seedlings. However, ABA-induced activation of protein kinases was inhibited by reactive oxygen species (ROS) inhibitors or scavengers. Protein kinase inhibitors decelerated not only the ABA and H2O2 -induced kinase activity but also ABA or H2O2-induced antioxidant enzyme activity. Protein phosphorylation caused by ABA and H2O2 preceded ABA or H2O2 -induced antioxidant defense obviously. Using in-gel kinase assays, our results showed that several protein kinases with molecular masses of 66kDa, 52kDa, 49kDa and 35kDa respectively might mediate ABA and H2O2-induced antioxidant defense. And the 66kDa and 49kDa protein kinases may act downstream of ROS, and the 52kDa and 35kDa protein kinases may act between ABA and ROS in ABA-induced antioxidant defensive signaling.     

The oleate-stimulated phospholipase D, PLDdelta, and phosphatidic acid decrease H2O2-induced cell death in Arabidopsis

Hydrolysis of common membrane phospholipids occurs in response to various environmental stresses, but the control and cellular function of this hydrolysis are not fully understood. Hydrogen peroxide (H2O2) is a pivotal signaling molecule involved in various stress responses. Here, we show that the plasma membrane-bound phospholipase D, PLDdelta, is activated in response to H2O2 and that the resulting phosphatidic acid (PA) functions to decrease H2O2-promoted programmed cell death. The Arabidopsis genome has 12 PLD genes, and knockout of PLDdelta abolishes specifically the oleate-stimulated PLD activity. H2O2 treatment of Arabidopsis cells activates PLD enzyme activity, and ablation of PLDdelta abolishes that activation. PLDdelta-null cells display increased sensitivity to H2O2-induced cell death. The addition of PA to PLDdelta-null cells mitigates the H2O2 effect, whereas suppression of the H2O2-induced PA formation in wild-type cells increases the effect. PLDdelta-ablated plants exhibit increased susceptibility to stress. These results demonstrate that activation of oleate-stimulated PLDdelta constitutes an important step in the plant response to H2O2 and increasing plant stress tolerance.     

An Ustilago maydis gene involved in H2O2 detoxification is required for virulence

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.     

NADPH oxidase-dependent hydrogen peroxide production, induced by salinity stress, may be involved in the regulation of total calcium in roots of wheat

Hydrogen peroxide (H(2)O(2)) is often generated by cells and tissues under environmental stress. In this work, we provide evidence that plasma membrane (PM) NADPH oxidase-dependent H(2)O(2) production might act as an intermediate step in the NaCl-induced elevation of calcium (Ca) in roots of wheat. Remarkable increases in the content of total Ca were observed not only in roots exposed to NaCl but also in roots of seedlings exposed to exogenous H(2)O(2). In roots, H(2)O(2) production increased upon exposure to salt stress. PM vesicles were isolated from roots, and NADPH oxidase activity was determined by measuring superoxide anion (O(2)(-)) production. NADPH oxidase-dependent O(2)(-) production was 11.6nmolmg(-1)proteinmin(-1) in control vesicles, but 19.6nmol after NaCl treatment (24h), indicating that salt stress resulted in the activation of the PM NADPH oxidase. Furthermore, the NaCl-induced increase in total Ca was partially abolished by the addition of 150U/mL catalase (CAT), a H(2)O(2) scavenger, and also by 10microM diphenylane iodonium (DPI), a NADPH oxidase inhibitor. This data suggest that NADPH oxidase-dependent H(2)O(2) production might be involved in the modulation of the Ca content in wheat roots. In conclusion, our results show that salinity stress increases the total Ca content of wheat roots, which is partly due to PM NADPH oxidase-dependent H(2)O(2) generation.     

Activation of Host Defense Mechanisms by Elevated Production of H2O2 in Transgenic Plants

Active oxygen species have been postulated to perform multiple functions in plant defense, but their exact role in plant resistance to diseases is not fully understood. We have recently demonstrated H2O2-mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In this study we provide further evidence that the H2O2-mediated disease resistance in potato is effective against a broad range of plant pathogens. We have investigated mechanisms underlying the H2O2-mediated disease resistance in transgenic potato plants. The constitutively elevated levels of H2O2 induced the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the level of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H2O2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a major contributing factor to the H2O2-mediated disease resistance in transgenic plants.     

Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene

Paradoxically, in eukaryotic cells, hydrogen peroxide (H(2)O(2)) accumulates in response to oxygen deprivation (hypoxia). The source of H(2)O(2) under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H(2)O(2) is mainly produced by activation of NAD(P)H-oxidases or by disruption of the mitochondrial electron transport chain (m-ETC). This study showed that hypoxia, and inhibitors of respiration like potassium cyanide (KCN) and sodium nitroprusside (SNP), trigger the production of H(2)O(2) in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H(2)O(2) levels induced by KCN, suggesting that, under respiratory stress, H(2)O(2) is mainly produced by disruption of the m-ETC. On the other hand, γ-aminobutyric acid (GABA), a metabolite that in plants alleviates oxidative stress by activating antioxidant enzymes, reduced significantly the levels of H(2)O(2) induced by KCN and, surprisingly, repressed the expression of genes encoding antioxidant enzymes such as ASCORBATE PEROXIDASE (VvAPX), GLUTATHIONE PEROXIDASE (VvGLPX), SUPEROXIDE DISMUTASE (VvSOD), and one of the CATALASE isoforms (VvCAT1), while VvCAT2 was upregulated. In contrast to GABA, hypoxia, H(2)O(2), and ethylene increased dramatically the expression of genes encoding antioxidant enzymes and enzymes of the alternative respiratory pathway such as ALTERNATIVE NADH-DEHYDROGENASES (VvaNDs) and ALTERNATIVE OXIDASES (VvAOXs). Hence, it is concluded that H(2)O(2) production is stimulated by respiratory stress in grapevine buds, that H(2)O(2) and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H(2)O(2) levels by up-regulating the expression of VvCAT2.     

Interrelationship between calmodulin (CaM) and H2O2 in abscisic acid-induced antioxidant defense in the seedlings of Panax ginseng

Calmodulin (CaM), the predominant Ca(2+) receptors, is one of the best-characterized Ca(2+) sensors in all eukaryotes. In this study the role of CaM and the possible interrelationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA) induced antioxidant defense were investigated in the seedling of Panax ginseng. Treatment of ABA (100 μM) and H(2)O(2) (10 mM) increased the expression of Panax ginseng calmodulin gene (PgCaM) and significantly enhanced the expression of the antioxidant marker genes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and the activities of chloroplastic and cytosolic antioxidant enzymes. Pretreatments with two CaM antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W7) and inhibitor or scavenger, diphenyleneiodonium chloride, and dimethylthiourea of reactive oxygen species almost completely suppressed the up-regulation of antioxidant and PgCaM gene. Moreover, H(2)O(2) production and CaM content was almost completely inhibited by pretreatments with two CaM antagonists. In addition, the expressions of PgCaM gene under different biotic stress were analyzed at different time intervals. Thus it may suggests that CaM are involved in ABA-induced increased expression of PgCaM which triggers H(2)O(2) production through activating trans-plasma membrane NADPH oxidase, resulting in up-regulation of defense related antioxidant gene and also plays a pivotal role in defense response against pathogens.     

Participation of H2O2 in Enhancement of Cold Chilling by Salicylic Acid in Banana Seedlings

The possible physiological mechanism of enhancement of cold tolerance by salicylic acid (SA) in banana seedlings (Musa acuminata cv. Williams 8188) was explored. Measurements of leakage electrolyte after 2 d of recovery at 30/22 ℃ (day/night) following 3 d of cold stress at 7 ℃ showed that pretreatment with hydroponic solution containing SA 0.3－0.9 mmol/L as foliar spray under normal growth conditions (30/22 ℃) could significantly enhance cold tolerance of banana plants. The highest enhancing effect of SA occurred at 0.5 mmol/L and it showed the lowest leakage rate of electrolyte or smaller leaf wilting area after 2 d of recovery at normal temperature from 3 d of 7 ℃ or 5 ℃ cold stress. Higher concentrations (≥2.5 mmol/L) of SA, however, caused more electrolyte leakage, indicating that they aggravated chilling damage. Enhanced cold tolerance by SA could be related to H2O2 metabolism. Compared with water-treated seedlings (control), SA 0.5 mmol/L treatment inhibited activities of catalase (CAT) and ascorbate peroxidase (APX), increased peroxidase (POX) activity, but did not affect the activity of superoxide dismutase (SOD) under normal growth conditions, and these changes might lead to an accumulation of H2O2, whereas SA pretreatment enhanced the activities of CAT and APX, and reduced the increase in productions of H2O2 and thiobarbituric acid-reaction substances (TBARS) during subsequent 7 ℃ cold stress and recovery periods. Exogenous H2O2 treatments (1.5－2.5 mmol/L) also increased cold tolerance of banana seedlings. Furthermore, pretreatment of banana seedlings with dimethylthiourea (a trap for H2O2) significantly inhibited cold tolerance induced by SA. These results suggested that endogenous H2O2 may be required for SA-enhanced cold tolerance. The significance of the interaction of SA, H2O2 and H2O2-metabolizing enzymes during cold stress has been discussed.     

Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signaling in Arabidopsis thaliana

SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H2O2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H2O2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H2O2 in response to salt stress, suggesting that it is altered signaling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H2O2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H2O2.     

外源H2O2对水稻幼苗抗寒性的调节作用

在常温下用不同浓度的外源H2O2(0～20 mmol·L-1)预处理水稻幼苗,再进行12 h 6℃低温胁迫,根据幼苗相对含水量和质膜相对透性筛选最佳外源H2O2处理浓度,并分析最佳外源H2O2浓度下幼苗的渗透调节物质和活性氧相关指标的变化.结果表明:(1)0～8 mmol·L-1 H2O2预处理可以增加水稻幼苗的相对含水量,降低其质膜相对透性,并以4 mmol·L-1 H2O2的效果最佳.(2)低温胁迫后,与对照组相比,4 mmol·L-1外源H2O2预处理降低了水稻幼苗萎蔫程度,并使其总呼吸速率、交替途径容量都有增加,同时还抑制了丙二醛的含量,增加了可溶性糖、可溶性蛋白质和脯氨酸的含量.(3)外源H2O2预处理对水稻幼苗的内源H2O2含量以及O(-)/(·)2产生速率没有显著影响.研究发现,外源H2O2可以通过提高呼吸速率、降低脂质过氧化程度、增加碳氮代谢来有效增强水稻幼苗的抗寒性,它可能以一种独立于内源活性氧系统之外的方式发挥作用.