Role of nitric oxide in abscisic acid-induced subcellular antioxidant defense of maize leaves

The sources of nitric oxide (NO) production in response to abscisic acid (ABA) and the role of NO in ABA-induced hydrogen peroxide (H(2)O(2)) accumulation and subcellular antioxidant defense in leaves of maize (Zea mays L.) plants were investigated. ABA induced increases in generation of NO and activity of nitric oxide synthase (NOS) in maize leaves. Such increases were blocked by pretreatment with each of the two NOS inhibitors. Pretreatments with a NO scavenger or NR inhibitors inhibited ABA-induced increase in production of NO, but did not affect the ABA-induced increases in activity of NOS, indicating that ABA-induced NO production originated from sources of NOS and NR. ABA- and H(2)O(2)-induced increases in 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 pretreatments with the NO scavenger, inhibitors of NOS and NR, indicating that NO is involved in the ABA- and H(2)O(2)-induced subcellular antioxidant defense reactions. On the other hand, NO donor sodium nitroprusside (SNP) reduced accumulation of H(2)O(2) induced by ABA, and c-PTIO reversed the effect of SNP in decreasing the accumulation of H(2)O(2). SNP induced increases in activities of subcellular antioxidant enzymes, and the increases were substantially prevented from occurring by the pretreatment with c-PTIO. These results suggest that ABA induces production of H(2)O(2) and NO, which can up-regulate activities of the subcellular antioxidant enzymes, to prevent overproduction of H(2)O(2) in maize plants. There is a negative feedback loop between NO and H(2)O(2) in ABA signal transduction in maize plants.     

Role of nitric oxide dependence on nitric oxide synthase-like activity in the water stress signaling of maize seedling

Nitric oxide (NO) has been known as an important signal in plant antioxidative defense but its production and roles in water stress are less known. The present study investigated whether NO dependence on a NO synthase-lika (NOS) activity is involved in the signaling of drought-induced protective responses in maize seedlings. NOS activity, rate of NO release and drought responses were analyzed when NO donor sodium nitroprusside (SNP), NO scavenger c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramathylimidazoline-1-oxyl-3-oxide) and NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) were applied to both detached maize leaves and whole plants. Both NOS activity and the rate of NO release increased substantially under dehydration stress. The high NOS activity induced by c-PTIO as NO scavenger and NO accumulation Inhibited by NOS inhibitor L-NAME In dehydration-treated maize seedlings Indicated that most NO production under water deficit stress may be generated from NOS-like activity. After dehydration stress for 3 h, detached maize leaves pretreated with NO donor SNP maintained more water content than that of control leaves pretreated with water. This result was consistent with the decrease in the transpiration rate of SNP-treated leaves subjected to drought treatment for 3 h. Membrane permeability, a cell injury index, was lower in SNP-trested maize leaves under dehydration stress for 4 h when compared with the control leaves. Also, superoxide dismutsse (SOD) activity of SNP combined drought treatment maize leaves was higher than that of drought treatment alone, indicating that exogenous NO treatment alleviated the water loss and oxidative damage of maize leaves under water deficit stress. When c-PTIO as a specific NO scavenger was applied, the effects of applied SNP were overridden. Treatment with L-NAME on leaves also led to higher membrane permeability, higher transpiration rate and lower SOD activities than those of control leaves, indicating that NOS-like activity was involved in the antioxidative defense under water stress. These results suggested that NO dependence on NOS-like activity serves as a signaling component in the induction of protective responses and is associated with drought tolerance in maize seedlings.     

The inhibition of polyamine biosynthesis weakens the drought tolerance in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis>) associated with the alteration of extensive proteins

Changes of endogenous polyamine (PA) levels could be a key adaptive response to drought in plants. White clover pretreated with or without dicyclohexylamine (DCHA), an inhibitor of PA biosynthesis, was subjected to drought stress induced by 18% polyethylene glycol 6000 for 8 days in controlled growth chambers. Results showed that drought stress significantly increased endogenous PA content, whereas DCHA significantly decreased PA accumulation under drought stress. The attenuate PA biosynthesis was unfavorable for plant growth and drought tolerance, as reflected by significantly lower relative water content, relative growth rate, instantaneous water use efficiency, and cell membrane stability in leaves in response to drought. On the basis of proteomic analysis, the inhibition of PA synthesis decreased the accumulation of many key differentially expressed proteins including (1) ribosomal structure and biogenesis: elongation factor, ribosomal protein S10E, and 30S ribosomal protein; (2) amino acid transport and metabolism: cysteine synthase, delta-1-pyrroline-5-carboxylate synthetase, and glutamate decarboxylase; (3) carbohydrate metabolism and energy production: photosystem apoprotein, sucrose-phosphate synthase, phosphogluconate dehydrogenase, sucrose-phosphatase, NADH oxidoreductase, and ATP synthase; (4) antioxidant metabolism: catalase, peroxidase I, ascorbate peroxidase, and glutathione S-transferase; and (5) other biological processes: heat shock protein 70, heat shock protein 90, and calcium-dependent protein kinase associated with the decreased drought tolerance in white clover. These findings indicate that PAs play a critical role in the regulation of growth, ribosome, amino acid and energy metabolism, and antioxidant reactions in white clover under drought stress. Drought-induced increases in endogenous PAs could be one of key adaptive responses against drought stress in white clover.     

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 induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase cascade involved in antioxidant defense in maize leaves

* The role of nitric oxide (NO) and the relationship between NO, hydrogen peroxide (H(2)O(2)) and mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Both ABA and H(2)O(2) induced increases in the generation of NO in mesophyll cells of maize leaves, and H(2)O(2) was required for the ABA-induced generation of NO. Pretreatment with NO scavenger and nitric oxide synthase (NOS) inhibitor substantially reduced the ABA-induced production of NO, and partly blocked the activation of a 46 kDa MAPK and the expression and the activities of several antioxidant enzymes induced by ABA. Treatment with the NO donor sodium nitroprusside (SNP) also induced the activation of the MAPK, and enhanced the antioxidant defense systems. * Conversely, SNP treatment did not induce the production of H(2)O(2), and pretreatments with NO scavenger and NOS inhibitor did not affect ABA-induced H(2)O(2) production. * Our results suggest that ABA-induced H(2)O(2) production mediates NO generation, which, in turn, activates MAPK and results in the upregulation in the expression and the activities of antioxidant enzymes in ABA signaling.     

H_2S对干旱胁迫下白三叶幼苗抗氧化防御能力的影响

以‘拉丁诺’白三叶(Trifolium repens cv.‘Ladino’)为试验材料,研究外源H2S处理对PEG6 000(聚乙二醇)模拟干旱胁迫下白三叶叶片相对含水量(RWC)、膜脂过氧化、活性氧成分、抗氧化酶、抗坏血酸-谷胱甘肽循环代谢和非酶抗氧化物质的影响,以揭示H_2S调控白三叶抗旱性的生理机制。结果显示:(1)0.2 mmol/L的外源NaHS(H_2S供体)能显著提高干旱胁迫下白三叶的叶片相对含水量,维持显著较低的电解质渗透率(EL)和丙二醛(MDA)含量。(2)与直接干旱胁迫相比,干旱胁迫下外源添加NaHS处理的白三叶叶片内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著增强,抗坏血酸-谷胱甘肽循环代谢中关键酶抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱水抗坏血酸还原酶(MDHAR)和谷胱甘肽还原酶(GR)活性及其抗氧化中间产物抗坏血酸(AsA)、谷胱甘肽(GSH)含量也显著提高。(3)叶片类黄酮、总酚和原花青素的含量在一定的胁迫时间范围内亦显著增加,并伴随着活性氧成分O_2~(-·)产生速率和H_2O_2水平降低。研究认为,外源H2S能通过促进干旱胁迫下白三叶体内的多重抗氧化防御能力来提高其幼苗的抗旱性。     

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.     

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

Chitooligosaccharide (COS) or oligochitosan has been shown to induce tobacco defense responses which are connected with nitric oxide (NO) and OIPK (oligochitosan-induced Ser/Thr protein kinase). The aim of this study was to reveal the relationship between NO production and OIPK pathway in the defense response of tobacco elicited by COS. NO generation was investigated by epidermal strip bioassay and fluorophore microscope using fluorophore diaminofluorescein diacetate (DAF-2DA). Tobacco epidermal cells treated with COS resulted in production of NO, which was first present in chloroplast, then in nucleus, finally in the whole cell; this NO production was sensitive to NO scavenger cPTIO and the mammalian NO synthase (NOS) inhibitor l-NAME, suggesting that NOS-like enzyme maybe involved in NO generation in tobacco epidermal cells. However, NOS and nitrate reductase (NR, EC 1.6.6.1) inhibitors reduced NO content in tobacco leaves by using NO Assay Kit, suggesting both NOS and NR were involved in NO production in tobacco leaves. Using a pharmacological approach and western blotting, we provide evidence that NO acts upstream of OIPK expression. NO scavenger, NOS inhibitor partly blocked the activation of OIPK and the activities of several defense-related enzymes induced by COS; treatment with NO donor sodium nitroprusside (SNP) induced the activation of OIPK and enhanced the defense systems. The results suggest that COS is able to induce NO generation, which results in up-regulation the activities of some defense-related enzymes through an OIPK-dependent or independent pathway.     

Relationships between polyamines, ethylene, osmoprotectants and antioxidant enzymes activities in wheat seedlings after short-term PEG- and NaCl-induced stresses

Contents of ethylene, osmoprotectants, levels and forms of polyamines (PAs) and activities of antioxidant enzymes in the leaves and roots were investigated for five wheat cultivar seedlings (differing in drought tolerance) exposed to osmotic stress (?1.5 MPa). Stress was induced by 2-day-long treatment of plants with polyethylene glycol 6000 (PEG) or NaCl added to hydroponic cultures. Nawra, Parabola and Manu cv. (drought tolerant) showed a marked increase in osmoprotectors (proline and soluble carbohydrates, mainly glucose, saccharose and maltose), free PAs (putrescine Put, spermidine Spd and spermine Spm) and Spd-conjugated levels, in both leaves and roots, after PEG-treatments. Radunia and Raweta (drought sensitive) exhibited smaller changes in the content of these substances. The analysis of enzymes involved in proline metabolism revealed the glutamate as a precursor of proline synthesis in PEG-induced stress conditions. The increase in the activity of antioxidative enzymes, especially catalase and peroxidases, was characteristic for tolerant wheat plants, but for sensitive ones, a decrease in superoxide dismutase and an increase in mainly glutathione reductase activities were observed. After NaCl-treatment smaller changes of all biochemical parameters were registered in comparison with PEG-induced stress. Exceptions were the higher values of ethylene content and a significant increase in saccharose, raffinose and maltose levels (only in stress sensitive plants). The proline synthesis pathway was stimulated from both glutamate and ornithine precursors. These results suggest that the accumulation of inorganic ions in NaCl-stressed plants may be involved in protective mechanisms as an additional osmoregultor. Thus, a weaker stressogenic effect as determined as water deficit by leaf relative water content and relative dry weight increase rate and differences in metabolite synthesis in comparison with PEG stress was observed. Proline seems to be the most important osmo-protector in osmotic stress initiated by both PEG and NaCl. The synthesis of sugars and PAs may be stimulated in a stronger stress conditions (PEG).     

Nitric oxide mediates brassinosteroid-induced ABA biosynthesis involved in oxidative stress tolerance in maize leaves

The role of ABA in brassinosteroid (BR)-induced stress tolerance and the relationship between BR, nitric oxide (NO) and ABA under water stress induced by polyethylene glycol (PEG) were investigated in leaves of maize (Zea mays) plants. Water stress led to oxidative damage. Pre-treatment with the BR biosynthetic inhibitor brassinazole (Brz) aggravated the oxidative damage induced by PEG treatment, which was alleviated by the application of BR or ABA. Pre-treatment with the ABA biosynthetic inhibitor fluridone also aggravated the oxidative damage induced by PEG treatment; however, this was barely alleviated by the application of BR. BR treatment increased the content of ABA and up-regulated the expression of the ABA biosynthetic gene vp14 in maize leaves, which was blocked by pre-treatments with the NO scavenger cPTIO (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the nitric oxide synthase inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester. Moreover, BR treatment induced increases in the generation of NO in mesophyll cells of maize leaves, and treatment with the NO donor sodium nitroprusside (SNP) up-regulated the content of ABA and the expression of vp14 in maize leaves. Our results suggest that BR-induced NO production and NO-activated ABA biosynthesis are important mechanisms for BR-enhanced water stress tolerance in leaves of maize plants.     

Nitric oxide pretreatment enhances antioxidant defense and glyoxalase systems to confer PEG-induced oxidative stress in rapeseed

Nitric oxide (NO) is dynamic molecule implicated in diverse biological functions demonstrating its protective effect against damages provoked by abiotic stresses. The present study investigated that exogenous NO pretreatment (500?µM sodium nitroprusside, 24?h) prevented the adverse effect of drought stress [induced by 10% and 20% polyethylene glycol (PEG), 48?h] on rapeseed seedlings. Drought stress resulted in reduced relative water content with increased proline (Pro) level. Drought stress insisted high H₂O₂ generation and consequently increased membrane lipid peroxidation which are clear indications of oxidative damage. Drought stress disrupted the glyoxalase system too. Exogenous NO successfully alleviated oxidative damage effects on rapeseed seedlings through improving the levels of nonenzymatic antioxidant pool and upregulating antioxidant enzymes’ activities. Improvement of glyoxalase system (glyoxalase I and glyoxalase II activities) by exogenous NO was significant to improve plants’ tolerance. Nonetheless, regulation of Pro level and improvement of plant–water status were vital to confer drought stress tolerance.     

Metabolic and Physiological Changes Induced by Nitric Oxide and Its Impact on Drought Tolerance in Soybean

Nitric oxide (NO) is an important signaling molecule that plays a pivotal role in stress tolerance. To study the role of NO in drought tolerance and elucidate the underlying mechanisms, NO (0 and 100 μM) was applied to drought-treated soybean plants. Drought stress was imposed by PEG (5% (W/V) of PEG 6000. Nitric oxide improved growth of soybean plants under drought as evidenced by enhanced dry weight (30%). Nitric oxide caused a remarkable increase in activities of catalase and superoxide dismutase (SOD) and SOD expression (14.8-fold), which led to a significant decline in malondealdehyde content under drought conditions. Nitric oxide induced proline biosynthesis due to enhancing pyrroline-5- carboxylate synthetase (P5CS) expression (43.66-fold). The growth-promoting effect of NO application in soybean plants was concomitant with change in metabolic profile (phenolic acid and flavonoid compounds). Nitric oxide up-regulated of phenylalanine ammonia-lyase (PAL) expression in drought-treated plants and may influence on the phenylpropanoid production. Nitric oxide increased salicylic acid (SA) content in soybean plants under stress. So, NO and SA are jointly responsible for boosted tolerance to drought stress in soybean plants. The decrease in unsaturated fatty acid through NO application might reflect a reduction in oxidative damage. These results propose a multifaceted contribution of NO through regulation of physiological and metabolic processes in response to drought stress.

     

Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots

The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean (Phaseolus vulgaris) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN(3) (an NR inhibitor), but not affected by N(omega)-nitro-L-arginine (L-NNA) (an NOS inhibitor). Application of 2.5 mM Na(3)PO(4), an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na(3)PO(4). Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.     

Polyamines modulate nitrate reductase activity in wheat leaves: involvement of nitric oxide

In the present work, the effect of polyamines (PAs) on nitrate reductase (NR) activity was studied in wheat leaves exposed to exogenously added PAs while assessing the nitric oxide (NO) involvement in the regulation of the enzyme activity. A biphasic response was observed along the time of treatment using 0.1 mM of putrescine (Put), spermidine (Spd) or spermine (Spm). At 3 h, Spd and Spm significantly reduced NR activity by 29 or 35%, respectively, whereas at 6 h, the activity of the enzyme decreased by an average of 25%. At 21 h, Put increased NR activity by 63%, while Spd and Spm elevated the enzyme activity by 114%. NR activity, that was reduced by 0.1 mM Spm at 3 and 6 h, returned almost to control values when c-PTIO (an NO scavenger) was used, confirming that NO was involved in the inhibition of NR activity. Nitric oxide was also mediating the PA-increase of the enzyme activity at longer incubation times, evidenced when the raise in NR activity produced by 0.1 mM Spm at the longest incubation time returned to the value of the control in the presence of cPTIO. Neither the protein expression nor the nitrate content were modified by PAs treatments. The involvement of PAs and NO in the regulation of NR activity is discussed.     

外源甲基乙二醛对干旱胁迫下板栗幼苗的影响

甲基乙二醛(MG)是一种在植物中具有多种功能的新型信号分子.为探究MG对板栗幼苗干旱胁迫的影响,以两年生'黄棚'板栗幼苗为试材,通过聚乙二醇(PEG)模拟干旱胁迫并进行MG及其清除剂N-乙酰半胱氨酸(NAC)处理,分析板栗幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(...     

Antioxidant response to drought in red and white clover

Antioxidant response to drought in red (Trifolium pratense L., cv. ??Start??) and white clover (Trifolium repens L, cv. ??Haifa?? and cv. ??Debut??) grown as soil cultures was evaluated in water-deprived and recovered plants. Drought provoked oxidative stress in leaves confirmed by the considerable changes in electrolyte leakage, malondialdehyde, hydrogen peroxides and proline contents. Immunoblot of ??-1-pyrroline-5-carboxylate synthetase (P5CS), which catalyzes the first two steps in proline biosynthesis, revealed strong induction of the enzyme in red clover plants submitted to drought. Water-deprived white clover plants exhibited distinct P5CS profiles. This was related to different drought tolerance of the studied T. repens cultivars. Isoenzyme analyses of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) demonstrated certain differences in antioxidant defence among the tested varieties. It was confirmed that MnSOD (in both T. repens and T pratense) and FeSOD (in T. repens) isoforms were the most affected by drought. The red clover cultivar ??Start?? exhibited the lowest FeSOD and POX activities which could contribute to its poor performance under water deprivation.     

NOS-like-mediated nitric oxide is involved in Pinus thunbergii response to the invasion of Bursaphelenchus xylophilus

The content of NO and H(2)O(2) as well as the activities of nitric oxide synthase (NOS)-like and nitrate reductase (NR) were monitored in the needles of Pinus thunbergii infected by Bursaphelenchus xylophilus. The results showed that the content of NO increased significantly only 8?h after the invasion of B. xylophilus, while H(2)O(2) increased 12?h after invasion. NO donor SNP could promote and NO scavenger cPTIO could prevent the production of NO and H(2)O(2). The content of NO changed earlier than that of H(2)O(2). In addition, the symptoms appeared 9, 5 and 12?days, respectively, after the inoculation with B. xylophilus, SNP pre-treatment and cPTIO pre-treatment followed by B. xylophilus infection. After B. xylophilus infection, the content of NO in P. thunbergii changed fiercely more earlier than the appearance of external symptoms, which indicated that the content of NO was related with the appearance and the development of the symptoms. The treatment with L-NNA (NOS inhibitor) inhibited the content of NO significantly, whereas, Na(2)WO(4) (NR inhibitor) had no effect. The further analysis of NOS revealed that NO changed in consistent with cNOS activity. To sum up, NO, as the upstream signal molecule of H(2)O(2), was involved in the pine early response to the invasion of B. xylophilus and influenced the accumulation of the content of H(2)O(2). Moreover, NOS-like rather than NR was responsible for the endogenous NO generation, which was modulated by cNOS during the interaction between P. thunbergii and B. xylophilus. Key message NO is involved in early response of P. thunbergii to the invasion of B. xylophilus and NOS is the key enzyme responsible for NO generation in P. thunbergii.