Involvement of oxidative stress and role of antioxidative defense system in growing rice seedlings exposed to toxic concentrations of aluminum

When seedlings of rice (Oryza sativa L.) cultivar Pant-12 were raised in sand cultures containing 80 and 160 μM Al³⁺ in the medium for 5–20 days, a regular increase in Al³⁺ uptake with a concomitant decrease in the length of roots as well as shoots was observed. Al³⁺ treatment of 160 μM resulted in increased generation of superoxide anion (O₂ ⁻) and hydrogen peroxide (H₂O₂), elevated amount of malondialdehyde, soluble protein and oxidized glutathione and decline in the concentrations of thiols (-SH) and ascorbic acid. Among antioxidative enzymes, activities of superoxide dismutase (SOD EC 1.15.1.1), guaiacol peroxidase (Guaiacol POX EC 1.11.1.7), ascorbate peroxidase (APX EC 1.11.1.11), monodehydroascorbate reductase (MDHAR EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (EC 1.6.4.2) increased significantly, whereas the activities of catalase (EC EC 1.11.1.6) and chloroplastic APX declined in 160 μM Al³⁺ stressed seedlings as compared to control seedlings. The results suggest that Al³⁺ toxicity is associated with induction of oxidative stress in rice plants and among antioxidative enzymes SOD, Guaiacol POX and cytosolic APX appear to serve as important components of an antioxidative defense mechanism under Al³⁺ toxicity. PAGE analysis confirmed the increased activity as well as appearance of new isoenzymes of APX in Al³⁺ stressed seedlings. Immunoblot analysis revealed that changes in the activities of APX are due to changes in the amounts of enzyme protein. Similar findings were obtained when the experiments were repeated using another popular rice cv. Malviya-36.     

Iron induction of lipid peroxidation and effects on antioxidative enzyme activities in rice leaves

Lipid peroxidation in relation to toxicity of detached rice leavescaused by excess iron (FeSO₄) was investigated. ExcessFeSO₄, which was observed to induce toxicity, enhanced the contentoflipid peroxidation but not the content of H₂O₂.Superoxidedismutase activity was reduced by excess FeSO₄. Ascorbate peroxidaseand glutathione reductase activities were increased by excess FeSO₄.Free radical scavengers, such as mannitol and reduced glutathione, inhibitedexcess iron-induced toxicity and at the same time inhibited excessiron-enhancedlipid peroxidation, suggesting that lipid peroxidation enhanced by excess ironis mediated through free radicals.     

Cadmium toxicity of rice leaves is mediated through lipid peroxidation

Oxidative stress, in relation to toxicity of detached rice leaves,caused by excess cadmium was investigated. Cd content inCdCl₂-treated detached rice leaves increased with increasingdurationof incubation in the light. Cd toxicity was followed by measuring the decreasein chlorophyll and protein. CdCl₂ was effective in inducing toxicityand increasing lipid peroxidation of detached rice leaves under both light anddark conditions. These effects were also observed in rice leaves treated withCdSO₄, indicating that the toxicity was indeed attributed to cadmiumions. Superoxide dismutase (SOD), ascorbate peroxidase (APOD), and glutathionereductase (GR) activities were reduced by excess CdCl₂ in the light.The changes in catalase and peroxidase activities were observed inCdCl₂-treated rice leaves after the occurrence of toxicity in thelight. Free radical scavengers reduced CdCl₂-induced toxicity and atthe same time reduced CdCl₂-induced lipid peroxidation and restoredCdCl₂-decreased activities of SOD, APOD, and GR in the light. Metalchelators (2,2-bipyridine and 1,10-phenanthroline) reducedCdCl₂ toxicity in rice leaves in the light. The reduction ofCdCl₂ toxicity by 2,2-bipyridine (BP) is closely associatedwith a decrease in lipid peroxidation and an increase in activities ofantioxidative enzymes. Furthermore, BP-reduced toxicity of detached riceleaves,induced by CdCl₂, was reversed by adding Fe²⁺ orCu²⁺, but not by Mn²⁺ or Mg²⁺.Reduction of CdCl₂ toxicity by BP is most likely mediated throughchelation of iron. It seems that toxicity induced by CdCl₂ mayrequire the participation of iron.     

Alteration of oxidative and carbohydrate metabolism under abiotic stress in two rice (Oryza sativa L.) genotypes contrasting in chilling tolerance

Abiotic stress is a major limiting factor in crop production. Physiological comparisons between contrasting abiotic stress-tolerant genotypes will improve understanding of stress-tolerant mechanisms. Rice seedlings (S3 stage) of a chilling-tolerant (CT) genotype (CT6748-8-CA-17) and a chilling-sensitive (CS) genotype (INIAP12) were subjected to abiotic stresses including chilling (13/12 degrees C), salt (100mM NaCl), and osmotic (200mM mannitol). Measures of physiological response to the stresses included changes in stress-related sugars, oxidative products and protective enzymes, parameters that could be used as possible markers for selection of improved tolerant varieties. Metabolite analyses showed that the two genotypes responded differently to different stresses. Genotype survival under chilling-stress was as expected, however, CT was more sensitive to salt stress than the CS genotype. The CT genotype was able to maintain membrane integrity better than CS, perhaps by reduction of lipid peroxidation via increased levels of antioxidant enzymes during chilling stress. This genotype accumulated sugars in response to stress, but the accumulation was usually less than in the CS genotype. Chill-stressed CT accumulated galactose and raffinose whereas these saccharides declined in CS. On the other hand, the tolerance mechanism in the more salt- and water-deficit-tolerant CS may be associated with accumulation of osmoprotectants such as glucose, trehalose and mannitol.     

Amelioration of NaCl Stress by Arginine in Rice Seedlings: Changes in Endogenous Polyamines

Effects of NaCl (0.1 – 0.2 M) alone or in combination with 1 mM arginine on growth and endogenous polyamine (PA) content have been observed in two cultivars of rice differing in NaCl stress tolerance. The germination, seedlings fresh mass and water content decreased with increase in salinity in both the cultivars. This inhibition was partially alleviated by application of arginine. Cv. CSR-27 exhibited relatively better germination than cv. Bas-370 at different salinities. Total PA content increased in both the cultivars under NaCl stress alone and in combination with arginine. Putrescine to spermidine and spermine ratio was higher in NaCl-treated seedlings being more in cv. Bas-370 as compared to cv. CSR-27 and the ratio reversed to almost control level when arginine was applied along with NaCl.     

NaCl胁迫对西瓜嫁接苗叶片抗氧化酶活性及光合特性的影响

以耐盐性较强的葫芦品种‘超丰抗生王’为砧木,耐盐性较弱的西瓜品种‘秀丽’为接穗,采用营养液水培法,研究了NaCl胁迫对西瓜自根苗和嫁接苗保护酶活性、膜质过氧化及光合特性的影响。结果表明,NaCl胁迫下,嫁接苗和自根苗生物量显著下降,但嫁接苗下降幅度小于自根苗;NaCl胁迫抑制了西瓜自根苗和嫁接苗的气体交换参数,但是嫁接苗的净光合速率(Pn)、气孔导度(Gs)、胞间CO2浓度(Ci)及叶绿素含量显著高于自根苗;NaCl胁迫下西瓜嫁接苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著高于自根苗,丙二醛含量较自根苗低。上述结果表明,NaCl胁迫下嫁接苗通过维持较高的抗氧化酶活性来提高清除活性氧(ROS)的能力,从而降低氧化损伤,并保持较高光合速率,从而增强西瓜幼苗对盐胁迫的耐性。     

Chitosan enhances leaf membrane stability and antioxidant enzyme activities in apple seedlings under drought stress

Chitosan is a cationic marine polysaccharide with unique bioactive properties that make it an effective scavenger of reactive oxygen species. Chitosan application has been suggested as an aid for reducing oxidative injury caused by drought stress in crop plants. In order to confirm the antioxidant effects of exogenous chitosan, cell membrane stability and antioxidant enzyme activities were analyzed in leaves of apple seedlings placed under a period of drought stress. Pretreatment of apple seedling leaves with chitosan solution (20, 50, 100, 150 and 200 mg l⁻¹) prior to drought stress significantly decreased electrolyte leakage and the production of malondialdehyde in the leaves, while increasing antioxidant enzyme activities (superoxide dismutase, catalase), following imposition of drought stress conditions. An optimum response was obtained at a chitosan concentration of 100 mg l⁻¹. When apple seedlings were pretreated with 100 mg l⁻¹ of chitosan, cell membrane stability and antioxidant enzyme activities were enhanced for 21 days of drought treatment. Following restoration of moisture and a repeated drought stress, similar results were obtained on day 35. It is proposed that chitosan may act as an exogenous antioxidant that enhances resistance to oxidative stress during drought.     

Improved Performance of Transgenic Glycinebetaine-Accumulating Rice Plants under Drought Stress

Plasmid DNA (pChlCOD), containing the selectable hygromycin phosphotransferase hpt gene for hygromycin B resistance and the Arthrobacter globiformis codA gene for choline oxidase which catalyzes the direct conversion of choline to glycinebetaine, was delivered into rice plants using Agrobacterium-mediated gene transfer via scutellum-derived calli. Southern, Northern and Western blot analyses demonstrated that the foreign gene had been transferred, integrated into rice chromosomal DNA and expressed. Drought test indicated that glycinebetaine acts as an osmoprotectant and its production in transgenic rice plant helped the cells to maintain osmotic potential and increased root growth, and thus enhanced the ability of the plants to tolerate water deficit This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of short- and long-term salinity on the activities of antioxidative enzymes and lipid peroxidation in tomato roots

Changes in the antioxidative enzyme activities (SOD, CuZnSOD, GSH-Px, GST), as well as TBARS content in 5-week-old tomato (Lycopersicon esculentum Mill. cv “Perkoz”) roots were examined 1, 3 h (short-term stress) and 1–14 days (long-term stress) after a single application of 50 mM (mild stress) and 150 mM NaCl (severe stress). The severe stress caused an increase in GST, GSH-Px and SODs activities from the beginning of the experiment while mild stress induced enhancement of GST activity from the second day of experiment. The maximum increase in SODs after both NaCl solutions were applied and in GST activity after the higher NaCl dose on the second day of the experiment was observed. Moreover, after 1 h of NaCl treatment with both tested NaCl solutions, the highest induction of GSH-Px activity appeared. TBARS content was elevated from the first hour of salt stress and decreased only 14 days after 50 mM NaCl application which was accompanied by high induction of GSH-Px activity. In conclusion, enhanced activities of tested enzymes indicate their involvement in early and late defence systems under salinity stress. Moreover, the dynamics of the changes in the antioxidant enzymes suggests that the second day following NaCl application is a crucial moment of the experiment with regard to salt-mediated oxidative stress.     

Influence of a Brassinosteroid Analogue on Antioxidant Enzymes in Rice Grown in Culture Medium with NaCl

We studied the effects of a polyhydroxylated spirostanic brassinosteroid analogue (BB-16) on the activities of antioxidant enzymes in rice seedlings grown in vitro in culture medium supplemented with NaCl. Seedlings were grown in medium with 75 mM NaCl and 0.001 or 0.01 mg dm⁻³ BB-16 for 16 d or 3-d-old seedlings were exposed for 4 d to 0, 0.001 or 0.01 mg dm⁻³ BB-16 then further grown in medium with 75 mM NaCl without BB-16. Seedlings exposed to 0.01 mg dm⁻³ BB-16 for 16 d showed significant increase in the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) and a slight increase in ascorbate peroxidase (APX). On the other hand, 4-d exposure to BB-16 only increased SOD and CAT activities at concentration 0.001 mg dm⁻³. GR activity was not altered by this BB-16 treatment. These results indicated that BB-16, which is structurally modified in the lateral chain in relation to natural brassinosteroids, changes the activity of key antioxidant enzymes, which might confer tolerance to saline stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

NaCl-Induced Changes in Putrescine Content and Diamine Oxidase Activity in Roots of Rice Seedlings

The effects of NaCl on putrescine (Put) content and diamine oxidase (DAO) activity in roots of rice seedlings were examined. NaCl treatment lowered the content of Put and increased the activity of DAO in roots. Our current results indicate that Cl^– is not required for NaCl-induced decline in Put content and increase in DAO activity in roots. Put content in roots of rice seedlings exposed to NaCl is possibly regulated by DAO activity.     

水稻CPR5基因参与氧化胁迫响应

为探讨水稻(Oryza sativa L.)中CPR5 基因的功能,以其cDNA 文库为基础进行序列比对,并将1 个同源性较高的序列命名为OsCPR5.生物信息学分析表明,OsCPR5 的开放阅读框长度为1551 bp,编码516 个氨基酸.软件预测该编码蛋白可能是1 个具有5 个跨膜区的膜蛋白和核定位蛋白.组织表达和亚细胞定位分析表明,OsCPR5 在根中的表达水平较高,且广泛分布在细胞膜、细胞质和细胞核上.逆境胁迫分析实验表明,植物激素脱落酸(Abscisic acid, ABA)、过氧化氢(Hydrogen peroxide,H₂O₂)、氯化钠(NaCl)、甲基紫精(Methyl viologen, MV)等环境胁迫可诱导OsCPR5 的上调表达,其中氧化胁迫相关的MV 和H₂O₂ 处理效果最为明显.拟南芥(Arabidopsis thaliana)转基因株系atcpr5/OsCPR5 在浓度为0.5 μmol L^-1、 1.0 μmol L^-1 的MV以及6 mmol L^-1、 8 mmol L^-1 的H₂O₂ 处理下,种子萌发率明显高于atcpr5 突变体.这揭示了OsCPR5 基因在植物抗氧化胁迫响应中具有一定的作用.     

CO2激光预处理对PEG6000胁迫下小麦幼苗根中抗氧化酶活性的影响

用CO2激光对小麦种子分别辐照0、1、3、5min,待其生长至12d时,用10%（W/V）PEG6000胁迫其幼苗,研究激光预处理对PEG6000水分胁迫下小麦幼苗根部脂质过氧化伤害的防护作用。结果表明,CO2激光预处理3min可使水分胁迫的小麦幼苗根部MDA、H2O2含量和O2.-产生速率显著降低（P〈0.05）,可显著提高（P〈0.05）小麦幼苗根部SOD、POD、CAT、APX活性和根长、根干重。激光预处理3min可抑制由水分胁迫引起的小麦幼苗根部脂质过氧化作用。     

Enhancement of the Tolerance to Oxidative Stress in Cucumber (Cucumis sativus L.) Seedlings by UV-B Irradiation: Possible Involvement of Phenolic Compounds and Antioxidative Enzymes

Cucumis sativus L.) seedlings were irradiated or not irradiated with UV-B for several days in environment-controlled growth chambers. The first leaves irradiated with UV-B were retarded in growth but simultaneously acquired a remarkably high tolerance to oxidative stress, as induced by paraquat treatment, compared with the non-irradiated leaves. This enhanced tolerance was observed within 1d after the start of UV-B irradiation and was maintained during the 12 d period of UV-B treatment. The effects of UV-B on several antioxidative enzymes were examined, and activities of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase, but not of glutathione reductase, were found to be enhanced. However, activation of these enzymes occurred only from 6 d after the start of irradiation. In contrast, accumulation of phenolic compounds was observed within 1d after the start of UV-B irradiation. HPLC analysis of phenolic compounds showed the distinct enhancement of a substance, which may have antioxidative properties in cucumber seedlings irradiated with UV-B. On the basis of these results, we conclude that not only antioxidative enzymes but also other factors in cucumber seedlings irradiated with UV-B, such as phenolic compounds, may participate in the enhanced tolerance to oxidative stress. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Involvement of Lipid Peroxidation in Water Stress-Promoted Senescence of Detached Rice Leaves

Role of lipid peroxidation and antioxidative enzymes (catalase, peroxidase, superoxide dismutase, ascorbate peroxidase and glutathione reductase) in water stress-promoted senescence of detached rice leaves was investigated. The senescence was followed by measuring the decrease in protein content. Increased lipid peroxidation was closely correlated with senescence in water stressed leaves. Decrease in superoxide dismutase activity was evident 8 h after beginning of water stress. However, decreased catalase, peroxidase, and ascorbate peroxidase activity was observed only when senescence was observed. Glutathione reductase was not affected by water stress. Free radical scavengers retarded water stress-enhanced senescence.     

Drought tolerance genes in rice

Quantitative trait loci (QTLs) for drought tolerance (DT) can be readily identified in available databases and in this paper, these QTLs were summarized in the form of a consensus map. An in silico strategy was then deployed to mine for candidate genes associated with DT QTLs using rice dbEST and rice genome databases. DT QTLs on rice chromosomes 1, 2, 4, 8, and 9 were selected to test the method. The result showed candidate genes associated with DT could be readily identified.     

An Enhancing Effect of Exogenous Mannitol on the Antioxidant Enzyme Activities in Roots of Wheat Under Salt Stress

The role of mannitol as an osmoprotectant, a radical scavenger, a stabilizer of protein and membrane structure, and protector of photosynthesis under abiotic stress has already been well described. In this article we show that mannitol applied exogenously to salt-stressed wheat, which normally cannot synthesize mannitol, improved their salt tolerance by enhancing activities of antioxidant enzymes. Wheat seedlings (3 days old) grown in 100 mM mannitol (corresponding to −0.224 MPa) for 24 h were subjected to 100 mM NaCl treatment for 5 days. The effect of exogenously applied mannitol on the salt tolerance of plants in view of growth, lipid peroxidation levels, and activities of antioxidant enzymes in the roots of salt-sensitive wheat (Triticum aestivum L. cv. Kızıltan-91) plants with or without mannitol was studied. Although root growth decreased under salt stress, this effect could be alleviated by mannitol pretreatment. Peroxidase (POX) and ascorbate peroxidase (APX) activities increased, whereas superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities decreased in Kızıltan-91 under salt stress. However, activities of antioxidant enzymes such as SOD, POX, CAT, APX, and GR increased with mannitol pretreatment under salt stress. Although root tissue extracts of salt-stressed wheat plants exhibited only nine different SOD isozyme bands of which two were identified as Cu/Zn-SOD and Mn-SOD, mannitol treatment caused the appearance of 11 different SOD activity bands. On the other hand, five different POX isozyme bands were determined in all treatments. Enhanced peroxidation of lipid membranes under salt stress conditions was reduced by pretreatment with mannitol. We suggest that exogenous application of mannitol could alleviate salt-induced oxidative damage by enhancing antioxidant enzyme activities in the roots of salt-sensitive Kızıltan-91.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

干旱胁迫对2种光合类型C4荒漠植物叶片光合特征酶和抗氧化酶活性的影响

以荒漠C₄草本植物蔷薇猪毛菜(NADP苹果酸酶型,NADP-ME）和粗枝猪毛菜（NAD苹果酸酶型,NAD-ME）为研究对象,采用盆栽控水试验设置正常供水和轻度、中度、重度干旱处理（土壤含水量分别为田间持水量80%、60%、45%和35%）,通过测定不同程度干旱胁迫下叶片含水量、C₄光合特征酶和抗氧化酶活性等指标,探讨不同类型C₄荒漠植物光合特征酶和抗氧化系统对干旱逆境的适应机制。结果显示：（1）2种植物叶片含水量均随干旱胁迫的加剧不同程度降低。（2）叶片磷酸烯醇式丙酮酸羧化酶（PEPC）活性在中度干旱胁迫下显著增加而在重度干旱胁迫下急剧下降;蔷薇猪毛菜NAD-ME活性和粗枝猪毛菜NADP-ME活性都很低,且它们基本不受干旱胁迫的影响;随干旱胁迫的加剧,蔷薇猪毛菜NADP-ME活性呈下降趋势,而粗枝猪毛菜NAD-ME活性先显著增加而在重度干旱胁迫下显著降低。（3）随着干旱胁迫的加剧,叶片超氧化物歧化酶（SOD）活性呈下降趋势,过氧化物酶（POD）活性在不同程度干旱胁迫下均有不同程度增加;过氧化氢酶（CAT）活性在中度干旱胁迫下均有不同程度的增加,但在重度干旱胁迫下蔷薇猪毛菜CAT活性降低,而粗枝猪毛菜CAT活性显著增加;丙二醛（MDA）含量随干旱胁迫的加剧均有不同程度的增加。研究认为,一定程度干旱胁迫下,2种荒漠植物的PEPC活性均有增加;不同光合类型C₄植物叶片脱羧酶（NADP-ME和NAD-ME）对干旱胁迫的响应有明显的差异。POD和CAT是这两种C₄植物适应干旱胁迫的主要抗氧化酶,但蔷薇猪毛菜CAT在重度干旱胁迫下没有起到积极保护作用。