The 9-lipoxygenase GhLOX1 gene is associated with the hypersensitive reaction of cotton Gossypium hirsutum to Xanthomonas campestris pv malvacearum.

Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 from Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S-lipoxygenase activity (LOX) responsible for lipid peroxidation. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene (GhLOX1) and the sequencing of its promoter. GhLOX1 was found to be highly expressed during Xcm induced HR. Sequence analysis showed that GhLOX1 is a putative 9-LOX, and GhLOX1 promoter contains SA and JA responsive elements. Investigation on LOX signalisation on cotyledons infiltrated with salicylic acid (SA), or incubated with methyl-jasmonate (MeJA) revealed that both treatments induced LOX activity and GhLOX1 gene expression. HR-like symptoms were observed when LOX substrates were then injected in treated (MeJA and SA) cotyledons or when Xcm compatible race 20 was inoculated on MeJA treated cotyledons. Together these results support the fact that GhLOX1 encodes a 9 LOX whose activity would be involved in cell death during cotton HR.     

Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in Phloem Fluids of Cucumber during Systemic Acquired Resistance Is Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase Activity in Petioles and Stems

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.     

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.     

外源水杨酸对辣椒倍半萜环化酶基因表达及抗氧化酶系的作用

分析了外源水杨酸对辣椒叶片倍半萜环化酶基因表达及抗氧化酶系的作用 .结果表明 ,在 0 .5～4mmol·L-1的浓度范围内 ,SA处理均能不同程度地诱导辣椒叶片中倍半萜环化酶基因转录并表达酶活性 ,但是酶活性较低且在SA处理 36h后才出现 ;SA处理后 ,辣椒叶片SOD和POD酶活性较对照增高 ,CAT酶活性较对照降低 ,相应地 ,H2 O2 浓度升高 .H2 O2 含量的升高与SA对辣椒叶片抗氧化酶活性的综合影响有关     

Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction

Extracellularly secreted plant peroxidases (POXs) are considered to catalyze the generation of reactive oxygen species (ROS) coupled to oxidation of plant hormone indole-3-acetic acid (IAA) and defense-related compounds salicylic acid (SA), aromatic monoamines (AMAs) and chitooligosaccharides (COSs). This review article consists of two parts, which describe H(2)O(2)-dependent and H(2)O(2)-independent mechanisms for ROS generation, respectively. Recent studies have shown that plant POXs oxidize SA, AMAs and COSs in the presence of H(2)O(2) via a conventional POX cycle, yielding the corresponding radical species, such as SA free radicals. These radical species may react with oxygen, and superoxide (O(2)(.-)) is produced. Through the series of reactions 2 moles of O(2)(.-) can be formed from 1 moles of H(2)O(2), thus leading to oxidative burst. It has been revealed that the ROS induced by SA, AMAs and COSs triggers the increase in cytosolic Ca(2+) concentration. Actually POXs transduce the extracellular signals into the redox signals that eventually stimulate the intracellular Ca(2+) signaling required for induction of defense responses. On the other hand, IAA can react with oxygen and plant POXs in the absence of H(2)O(2), by forming the ternary complex enzyme-IAA-O(2), which readily dissociates into enzyme, IAA radicals and O(2)(.-). This article covers the recent reports showing that extracellularly produced hydroxy radicals derived from O(2)(.-) mediate the IAA-induced cell elongation. Here a novel model for IAA signaling pathway mediated by extracellular ROS produced by cell-wall POXs is proposed. In addition, possible controls of the IAA-POX reactions by a fungal alkaloid are discussed.     

Transport of Salicylic Acid in Tobacco Necrosis Virus-Infected Cucumber Plants

The transport of salicylic acid (SA) was studied in cucumber (Cucumis sativus L.) using 14C-labeled benzoic acid that was injected in the cotyledons at the time of inoculation. Primary inoculation with tobacco necrosis virus (TNV) on the cotyledons led to an induction of systemic resistance of the first primary leaf above the cotyledon against Colletotrichum lagenarium as early as 3 d after inoculation. [14C]SA was detected in the phloem or in the first leaf 2 d after TNV inoculation, whereas [14C]benzoic acid was not detected in the phloem during the first 3 d after TNV inoculation of the cotyledons, indicating phloem transport of [14C]SA from cotyledon. In leaf 1, the specific activity of [14C]SA decreased between 1.7 and 8.6 times compared with the cotyledons, indicating that, in addition to transport, leaf 1 also produced more SA. The amount of SA transported after TNV infection of the cotyledon was 9 to 160 times higher than in uninfected control plants. Thus, SA can be transported to leaf 1 before the development of systemic acquired resistance, and SA accumulation in leaf 1 results both from transport from the cotyledon and from synthesis in leaf 1.     

Regulation of Antioxidant Enzymes by Salicylic Acid in Cucumber Leaves

Activities of the antioxidant enzymes involved in superoxide anion (O2-) and hydrogen peroxide (H2O2) metabolism were determined and the contents of O2 and 14202 were also measured. All concentrations of sahcylic acid (SA) tested (0.5, 1.0, 2.5 and 5.0 mmoL/L) significantly enhanced superoxide dismutase (SOD) and peroxidase (POD) activities not only in the first treated true leaf (leaf 1 ) but also in the second untreated true leaf (leaf 2) of Cucumis sativus L. When the leaves were treated with 1 mmol/L SA within 6 to 72 h, the activity of POD increased by 22 % to 67% in the treated leaf 1 and by 14% to 86% in the untreated leaf 2. However, no changes were observed during 3 h after treatment and at 96 h following treatment. Measurement of O2- and H202 showed that there was a significant decrease in 02' content and an increase in H202 content after SA treatment, but catalase (CAT) activity was only slighfiy inhibited and this suggested that the reason of the increase in H2O2 by SA treatment is not due to the inhibition of CAT but rather the increase in SOD activity. It was also found that SA at all concentrations tested could not induce new SOD isozyme but it induced 1 to 2 bands of new POD isozyme within one day after treatment. The results indicate that SA might involve in the regulation of antioxidant enzymes.     

The Effects of Polyacrylic Acid, Acetylsalicylic Acid and Salicylic Acid on Resistance of Cucumber to Colletotrichum lagenarium

Injection of cucumber cotyledons with polyacrylic acid (PA), acetylsalicylic acid (ASA) and salicylic acid (SA) induced resistance to inoculations with Colletrichum Iagenarium when inoculation followed injection by 96 h but not by 24 h. Size and number of lesions were greatly decreased. Resistance was greatest in injected cotyledons but was also pronounced in non-injected halves of cotyledons and occasionally in first or second leaves.
Incorporation of PA, ASA and SA into liquid shake cultures did not significantly affect growth of the pathogen.
Gel electrophoresis of soluble proteins extracted from cotyledons showed that induction of resistance was not accompanied by the appearance of novel soluble proteins.     

Salicylic and jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by Septoria nodorum Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species' (ROS) (superoxide radical O2*- and H2O2) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk. has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O2*- and H2O2 compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

接种马铃薯软腐病菌引起的烟草叶片爆发

用马铃薯软腐病菌处理烟草后,以化学法测定了叶片中O-2含量,发现在接种后1～14 h内只出现一次O-2爆发,不同于以往植物悬浮细胞研究中出现的两次爆发结果.接种软腐病菌后2 h和6 h烟草叶片O-2的电子自旋共振波谱图(electron spin resonance,ESR)显示了同样的一次O-2爆发结果.接种后烟草叶片的线粒体和叶绿体O-2爆发与叶片的同步,显示此两种细胞器参与烟草叶片在软腐病菌作用下的O-2发生.无光照叶绿体O-2的ESR谱线不出现,表明叶绿体O-2可能来源于光合电子传递链.马铃薯软腐病菌作用下烟草叶片的O-2爆发是多位点的.     

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves

Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g-1 fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-Gtase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7-27.0 [mu]g g-1 fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]-GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity.     

Influence of UV-B Radiation on Early Seedling Growth and Translocation of 65Zn from Cotyledons in Cotton

The influence of UV-B radiation from filtered or unfiltered fluorescent sunlamps on early seedling growth and translocation of ⁶⁵Zn from cotyledons to the shoot was examined in two cultivars of cotton, Acala and Gregg. Ten-day-old seedlings which had been irradiated in the greenhouse for 6 h continuously each day for 14 days with 0.81 or 1.61 W × m^-2 UV-B radiation under two unfiltered FS-40 sunlamps, showed pronounced phytotoxic damage. This was characterized at first by bronzing and glazing of the cotyledons and later by upward curling of the leaves and abscission. Leaf expansion, dry matter accumulation, and mobilization of ⁶⁵Zn from the cotyledons was severely impaired in the young developing shoot under unfiltered UV-B radiation. A significant stress response also was observed in seedlings exposed to 0.61 W × m^-2 UV-B radiation through a polystyrene filter and 0.73 W × m^-2 UV-B radiation through a cellulose-acetate filter. This stress response was characterized by the formation of a red pigment in the petioles of the cotyledons, reduced leaf expansion, and reduced transport of ⁶⁵Zn. Control seedlings exposed to 0.03 W × m^-2 UV-B radiation through a mylar filter were green, had maximum leaf size and dry-matter accumulation, and had the greatest percentage of ⁶⁵Zn translocated from the cotyledons.     

轮枝菌诱导棉花细胞的氧化迸发

大丽轮枝菌（Verticilium dahliae kleb.)是引起棉花黄萎病的病原真菌。使用从大丽轮枝菌V44（高毒）和V64（低毒）的菌丝体制备的各市县导物（I44和I64）作用悬浮培养的豫棉6号（感性）和豫棉8号（耐性）细胞系,用过氧化物酶法测定诱导后30min内的反应性氧变化,发现仅有不亲和性较高的体系,即弱毒力的大丽轮枝菌（V64）和耐性的豫棉8号所组成的体系（I64-Y8）表现最高的反应性氧迸发,3min-6min时增加61.8%。使用显著低于杀菌浓度的剂量的水杨酸（SA）和H2O2作用上述大丽轮枝菌,发现水杨酸和H2O2都能影响微生物,经1mmol/L水杨酸和0.2mmol/LH2O2作用后的微生物,其所产生的诱导物对植物细胞反应性氧的诱导作用要高于未被作用的微生物的诱导物,两种化学性质的影响的共同点是使反应性氧迸发的峰值时间提前,峰值增加,水杨酸提前12min,H2O2的影响还突出表现在使反应性氧迸发的曲线锐化,即峰使时间范围的平均每分钟增加率表现显著增加,9min-12min时达到109%。     

二氧化硫对小麦的氧化胁迫及其某些信号分子的调节

通过在密闭的培养箱中一次性通入不同体积浓度的SO₂气体,研究了小麦幼苗超氧自由基O2-含量和3种抗氧化酶活性的变化,探讨了信号分子水杨酸、乙烯和过氧化氢对SO₂氧化胁迫的调节作用.结果表明,当通入10和40 μl·L^-1 SO₂时,小麦叶片中O2-含量递增,过氧化物酶(POD)和过氧化氢酶(CAT)活性增强,但超氧化物歧化酶(SOD)活性降低.当SO₂浓度达到50 μl·L^-1时,POD和CAT活性也开始降低,此时叶片尖端出现坏死,叶片绿色部位滋生大量真菌.用1 mmol·L^-1水杨酸(SA)(pH6.5)浸泡小麦干种子6 h,或用10 mmol·L^-1 H₂O₂浸泡幼苗,O2-含量低于对照植株,而3种酶的活性高于对照植株,均可有效地减轻SO₂的氧化胁迫.在SO₂熏蒸下,乙烯显著抑制3种酶的活力,提高O2-的形成速率.SA与乙烯同时使用时,SA几乎完全消除了乙烯的负面作用.     

Impact of salicylic acid on the antioxidant enzyme system and hydrogen peroxide production in Cucumis sativus under chilling stress

Salicylic acid (SA) is a naturally produced compound and has been implicated to play important roles in defense of plants against diverse biotic and abiotic stresses. To understand how SA functions in the tolerance of cucumber (Cucumis sativus) to chilling stress, endogenous SA levels in two different cultivars with opposite chilling responsiveness were quantified. Membrane integrity, including malondialdehyde (MDA) content and leakage of electrolyte, was also examined in SA-pretreated cucumber plants under chilling conditions. In addition, activities of the two antioxidant enzymes peroxidase (POD) and catalase (CAT) were quantified, and hydrogen peroxide (H2O2) production was investigated histochemically in SA-treated leaves under chilling temperature. Chilling stress resulted in greater induction of SA levels in the chilling-tolerant cultivar Changchun mici in both leaves and seeds compared to the chilling-sensitive one Beijing jietou, while the former one contained higher levels of SA than the latter one in the seeds under normal conditions. Pretreatment with SA diminished the increased electrolyte leakage and MDA content caused by chilling in the leaves of both cultivars, while much less MDA and electrolyte leakage were produced in Changchun mici compared to Beijing jietou. Moreover, exogenous application of SA increased significantly the POD and CAT activities and soluble protein content. Most importantly, exogenous SA treatment could eliminate the accumulation of H202 in leaves and cotyledons of both cultivars caused by chilling stress. The data clearly demonstrated that the chilling-tolerant cultivar displays a higher SA level than the chilling-sensitive one, and that exogenous SA can enhance the chilling tolerance ability, which might be achieved through modulating the antioxidant system in cucumber.     

Systemic Responses in Arabidopsis thaliana Infected and Challenged with Pseudomonas syringae pv syringae

Attack of plants by necrotizing pathogens leads to acquired resistance to the same or other pathogens in tissues adjacent to or remotely located from the site of initial attack. We have used Arabidopsis thaliana inoculated with the incompatible pathogen Pseudomonas syringae pv syringae on the lower leaves to test the induction of systemic reactions. When plants were challenged with Pseudomonas syringae pv syringae in the upper leaves, bacterial titers remained stable in those preinfected on the lower leaves. However, there was a distinct decrease in symptoms that correlated with a local and systemic increase in salicylic acid (SA) and in chitinase activity. Peroxidase activity only increased at the site of infection. No changes in catalase activity were observed, either at the local or at the systemic level. No inhibition of catalase could be detected in tissue in which the endogenous levels of SA were elevated either naturally (after infection) or artificially (after feeding SA to the roots). The activity of catalase in homogenates of A. thaliana leaves could not be inhibited in vitro by SA. SA accumulation was induced by H2O2 in leaves, suggesting a link between H2O2 from the oxidative burst commonly observed during the hypersensitive reaction and the induction of a putative signaling molecule leading to system acquired resistance.