GSH/GSSG对盐胁迫下番茄幼苗谷胱甘肽化修饰和抗氧化系统的影响

以营养液栽培‘中蔬四号’番茄(Solanum lycopersicum Mill.)为研究材料,研究不同时期的盐胁迫下叶面喷施5 mmol/L还原型谷胱甘肽(reduced glutathione,GSH)和1 mmol/Lγ-谷氨酰半胱氨酸合成酶抑制剂(inhibitor of gamma-glutamylcysteine synthetase,BSO)对番茄幼苗叶片GSH和抗坏血酸(ascorbic acid,As A)水平、抗氧化酶活性及谷胱甘肽化抗氧化酶活性的影响。结果表明:盐胁迫、盐胁迫+BSO两种处理下喷施外源GSH显著升高了番茄幼苗叶片中GSH和As A含量、GSH/GSSG和As A/DHA比率以及超氧化物歧化酶(superoxidase dismutase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)、抗坏血酸过氧化物酶(ascorbate peroxidase,APX)、脱氢抗坏血酸还原酶(dehydroascorbate reductase,DHAR)、单脱氢抗坏血酸还原酶(monodehydroascorbate reductase,MDHAR)、谷胱甘肽还原酶(glutathione reductase,GR)和谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)的活性(NaCl+GSH处理除15 d的MDHAR活性;NaCl+BSO+GSH处理除15 d的DHAR活性);添加GSSG诱导的谷胱甘肽化修饰使盐胁迫下番茄幼苗叶片的SOD、MDHAR(15 d)、DHAR(5和15 d)、GR(5和10 d)、GPX(5和15 d)活性显著增加,同时GSH处理能够诱导上述酶发生去谷胱甘肽化修饰,从而维持细胞内的氧化还原平衡。由此表明外源GSH能够通过蛋白质谷胱甘肽化修饰对植物进行氧化还原(Redox)调控,以维持番茄体内Redox平衡,从而缓解盐害对番茄幼苗叶片造成的氧化胁迫,减少NaCl对植株造成的伤害。本研究为明确GSH/GSSG与番茄耐盐性的关系及其潜在生理机制,有效缓解番茄盐害提供了一定的理论支持。     

GLDH超表达水稻愈伤组织的耐盐性研究

为了探讨内源抗坏血酸(ascorbic acid,AsA)水平对水稻愈伤组织耐盐性的影响,以AsA合成关键酶半乳糖内酯脱氢酶(L-galactono-1,4-lactone dehydrogenase, GLDH)基因超表达型水稻(GO)为材料,研究了盐胁迫(1% NaCl)下GO愈伤组织继代生长及相关抗性生理指标的动态变化。结果表明,盐胁迫下GO愈伤组织生长明显优于野生型(wild type,WT),表现为生长速率较快,愈伤颗粒较大,致密,呈浅黄色。盐胁迫下GO愈伤组织中还原型AsA与总AsA含量不断下降,DHA含量总体上也呈下降趋势,但其氧化还原势(AsA/DHA)在胁迫初期上升,后期快速下降。进一步研究发现盐胁迫下GO愈伤组织中MDA含量先上升后快速下降,但其含量显著低于WT,而POD活性在胁迫初期略有降低,在后期又快速升高,其活性总体上显著高于对照。因此上述研究表明GLDH超表达水稻愈伤组织的耐盐性增强与其AsA含量提高有关。     

Ectopic expression of GmPAP3 enhances salt tolerance in rice by alleviating oxidative damage

GmPAP3, a purple acid phosphatase from soybean (Glycine max), was previously shown to alleviate salt stress in BY‐2 cells and Arabidopsis thaliana by reducing oxidative damage. To make use of GmPAP3 for crop improvement, we investigated whether the protective function of GmPAP3 is persistent in rice. Compared with the untransformed wild type, the transgenic rice plants exhibited enhanced germination rate, longer shoots and roots, and higher survival rate under salt stress, when compared to the untransformed control. In addition, the transgenic plants also showed increased activity of superoxide dismutase (SOD) and catalase (CAT), proline content, water content and chlorophyll content, but decreased electrolyte leakage and malondialdehyde content in leaves. These results indicate that the expression of GmPAP3 could be used to enhance salt tolerance in rice.     

Morphological and biochemical responses of Balanites aegyptiaca to drought stress and recovery are provenance‐dependent

Balanites aegyptiaca is a drought‐tolerant tree naturally distributed in Africa and has a high potential for biofuel production and livelihood. To understand the plant tolerance to drought stress, B. aegyptiaca plants collected from five provenances were subjected for 4 weeks to drought stress through different regimes of soil volumetric water content (VWC, i.e. 25% control, 15% as moderate and 5% as a severe drought stress) followed by 2‐week recovery. Morpho‐physiological responses as well as the changes in antioxidant defences under water stress and recovery were investigated. Drought stress significantly reduced plant biomass‐related parameters, stomatal conductance, quantum efficiency and increased leaf temperature. Each provenance showed specific patterns of stress response reactions that were detected in a cluster analysis. The large leaf area and a high level of lipid peroxidation in Cairo provenance increased its sensitivity to severe drought. For provenances El‐Kharga and Yemen, the highest tocopherol contents and the highest catalytic activities of ascorbate peroxidase (SOD), catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were recorded. These traits contributed to the high drought tolerance of these two provenances in comparison with the other provenances. All plants recovered from stress and showed specifically increased activity of glutathione‐S‐transferase (GST) as a repair mechanism. Results showed that the drought tolerance level in B. aegyptiaca is provenance‐dependent.     

Effect of Coronatine on AsA-GSH Cycle of Cotton Seedling under Low Temperature Stress

[Objective] In order to explore the stress-resistant mechanism of coronatine, an indoor experiment was conducted to study the effect of coronatine on ascorbate-glutathione circulatory system of the vegetative organs of cotton seedling under low temperature stress. [Method] Xinluzao 57 was used as the experimental material, when the seedlings reached the two-leaf stage, they were treated at 25 ℃ and 4 ℃ after spraying water, marked as CK and LT; the same treatment was conducted after spraying 0.01 μmol·L^－1 coronatine, marked as COR and (LT+COR). After 1 d of treatment, the roots, stems and leaves of cotton seedling were collected to determine the antioxidant contents and antioxidant enzyme activities. [Result] Compared with the control treatment (CK), the APX, MDHAR activities and DHA, GSH, glutathione contents decreased, the GPX activity, AsA and ascorbate contents increased, and no significant effect on DHAR and GR activities were observed in roots, stems and leaves of cotton seedling after spraying water at low temperature (LT), while in leaves, the GSSG content decreased, AsA-DHA ratio and GSH-GSSG ratio showed the maximum increase. Compared with the LT treatment, spraying COR at low temperature (LT+COR) showed increased APX, MDHAR, DHAR, GPX, GR activities and AsA, DHA, GSH, ascorbate contents; glutathione content in roots, stems and leaves of cotton seedling, and GSSG content, AsA-DHA ratio and GSH-GSSG ratio obviously changed in leaves. [Conclusion] Spraying COR at low temperature could regulate the AsA-GSH metabolism and alleviate the damage caused by low temperature to cotton seedling. COR had the strongest relieving effect on seedling leaves.     

The Effects of Foliar Application of Ascorbic Acid (Vitamin C) on Antioxidant Enzymes Activities, Lipid Peroxidation and Proline Accumulation of Canola (Brassica napus L.) under Conditions of Salt Stress

The effects of salt stress on protein (PROT) content, lipid peroxidation, proline accumulation, chlorophyll (Chl) content, and superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) activity were studied in the leaves and roots of canola (Brassica napus L. cv. Okapi). Four weeks after sowing (at the V₄ stage), plants were exposed to salt stress by the application of NaCl solution (200 mm ) for 6 days daily, After 6 days followed by foliar application of ascorbic acid (AsA) solution (25 mm ). The activity of all the antioxidant enzymes assayed (except SOD in the roots) was increased significantly in the plants under conditions of salt stress. The application of AsA decreased enzyme activity in the leaves, but it had no effect on enzyme activity in the roots. The total PROT content of the leaves and roots decreased under the conditions of high salinity. AsA treatment of plants under salt stress increased the total PROT content significantly in both leaves and roots. Measurement of the malondialdehyde content of leaves and roots showed that lipid peroxidation was increased by interaction with damaging reactive oxygen species during salt stress, and that application of AsA reduced lipid peroxidation only in the leaves. The Chl content was also affected by salt stress. There was significant difference between the controls and salt‐stress treatments in Chl content. The results of the present study indicate that usage of AsA reduces the harmful effects of salinity and increases resistance to salinity in canola plant.     

Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses

Summary Trehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 ^∘C and 35^∘C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops.     

一氧化氮和亚甲基蓝对盐胁迫抗虫棉根系的抗氧化能力的影响

 通过检测抗氧化酶活性和抗氧化物含量,研究一氧化氮和亚甲基蓝对盐胁迫抗虫棉根系的抗氧化能力的影响。在正常条件和盐胁迫条件下分别用一氧化氮和亚甲基蓝处理抗虫棉幼苗,检测抗坏血酸(ASA)含量、抗坏血酸过氧化物酶(APX)活性、谷胱甘肽还原酶(GR)活性、谷胱甘肽(GSH/GSSG)值、脱氢抗坏血酸还原酶(DHAR)活性、单脱氢抗坏血酸还原酶(MDAR)活性,并作对比。结果表明：正常生长条件下添加NO能促进棉花幼苗生长,而添加亚甲基蓝(MB)显著抑制抗虫棉幼苗的生长;添加NO显著缓解了盐胁迫对棉花幼苗生长的抑制,提高了根系的抗氧化能力,盐胁迫下添加NO的同时添加MB可不同程度地降低以上抗氧化物质含量和抗氧化酶活力,解除NO对盐胁迫的促长作用。     

Antioxidative Response of Quinoa Exposed to Iso‐Osmotic,Ionic and Non‐Ionic Salt Stress

Antioxidants play an important role in adapting plants to abiotic stress by detoxifying reactive oxygen species (ROS). Involvement of antioxidant enzymes in abiotic stress tolerance of highly stress‐tolerant quinoa was studied in a climatic chamber at 6 mOsm (milliosmolar) ionic (300 mm NaCl) and non‐ionic (600 mm mannitol) salts combined with increasing levels of potassium K1 and K2 (6, 12 mm ), respectively. Fifteen days of salt treatment (both ionic and non‐ionic) decreased plant growth (shoot and root fresh weight), stomatal conductance and chlorophyll content index. Furthermore, both forms of salt stress increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase up to 2.33‐, 3.98‐, 4.78‐ and 5.55‐folds, respectively, compared to no salt treatment, whereas membrane stability index decreased corresponding to increase in lipid peroxidation (malondialdehyde), with salt treatments compared to non‐stressed plants. However, no significant effect of potassium and salt treatments has been noticed on the maximal photochemical efficiency of PSII. The results suggested that enhanced antioxidant enzymes activity under salt stress could be one of the factors responsible for abiotic stress tolerance in quinoa.     

Effects of Exogenous Abscisic Acid on Antioxidant System in Weedy and Cultivated Rice with Different Chilling Sensitivity under Chilling Stress

Two chilling‐tolerant genotypes, that is, weedy rice WR03‐45 and cultivated rice Lijiangxintuanheigu and two chilling‐sensitive genotypes, that is, weedy rice WR03‐26 and cultivated rice Xiuzinuo were used in this study to investigate the effects of exogenous abscisic acid (ABA) on protection against chilling damage as well as on changes in physiological features. The results showed that under chilling stress the increased levels of superoxide radical (), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) in WR03‐45 and Lijiangxintuanheigu were lower than those in WR03‐26 and Xiuzinuo. Activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR)) and non‐enzymatic antioxidants (ascorbate acid (AsA) and reduced glutathione (GSH)) were enhanced in WR03‐45 and Lijiangxintuanheigu, whereas they were decreased significantly in WR03‐26 and Xiuzinuo. Application of exogenous ABA reduced the chilling damage in the four genotypes. The pre‐treatment with ABA decreased the levels of , H₂O₂ and MDA caused by chilling stress in the four genotypes through increasing the activities of SOD, CAT, APX, GR and the contents of AsA and GSH in the four genotypes under chilling stress. Moreover, pre‐treatment with Fluridone, the ABA biosynthesis inhibitor, prohibited the effects of ABA through enhancing the oxidative damages and suppressing the antioxidant defence systems under chilling stress. The results indicate the mechanism for rice with chilling tolerance is to enhance the capacity of antioxidant defence systems under chilling stress. Furthermore, ABA plays important roles in the tolerance of rice against chilling stress for it could induce the capacity of whole antioxidant defence systems including enzymatic and non‐enzymatic constitutions under chilling stress.     

Decay of the Chloroplast Pool of Ascorbate Switches on the Oxidative Burst in UV‐B‐Irradiated Rice

A parallel was drawn between UV‐B irradiation and the oxidative burst in the chloroplast lamellae of rice plants (Oryza sativa L. cv. Safari). After irradiation with a total biological effective UV‐B of 20.825 kJ m^?2 between the 15th and 21st days following germination, it was found that superoxide, hydroxyl radicals and hydrogen peroxide increased significantly in stressed leaves. The levels of ascorbate and the kinetics of superoxide dismutase, glutathione reductase, dehydroascorbate reductase, ascorbate peroxidase and catalase became increasingly inhibited. The developing oxidative burst also diminished photosynthetic pigments and galactolipids accumulation, while the de‐epoxidation state of xanthophylls and lipids peroxidation increased. Following grana disorganisation, the accumulation of chloroplast polypeptides having 72/69, 33/32, 28/26, 22/20 and 18/16 kDa decreased, but the relative proportion of the 49/46 kDa polypeptides increased. The 69/65 kDa polypeptides also diminished on the 15th day. These alterations implicated the failure of both photosystems functioning, but the inhibition of the Hill reactions coupled to the water splitting complex was uppermost. In the 28th day, the chloroplast lamellae of leaves grown after UV‐B exposure revealed a general recovery from oxidative burst and a subsequent higher stacking of thylakoids. It is concluded that UV‐B irradiation becomes lethal when ascorbate oxidation in the xanthophyll cycle limits the antioxidant enzymes kinetics, triggering chloroplast proteolysis and acyl lipid peroxidation because of reactive oxygen species propagation.     

Overexpression of BpMADS4 from silver birch (Betula pendula Roth.) induces early-flowering in apple (Malus × domestica Borkh.)

To shorten the juvenile stage of apple (Malus × domestica Borkh.) the BpMADS4 gene from silver birch (Betula pendula Roth.) was constitutively overexpressed in 25 transgenic apple clones. All clones were characterized by PCR, RT‐PCR and Real Time PCR. Solitary flowers were produced on in vitro shoots of eight transgenic clones and most of them appeared to be morphologically normal. Twenty shoots of each clone were rooted and transferred to a glasshouse. Glasshouse plants of clones T1165, T1187 and T1190 developed flowers. Several plants of T1165 and T1187 started floral initiation within 3–4 months following transfer to the glasshouse. Primary flowers were solitary and in a terminal position on the main shoot. Lateral flower clusters, consisting of three to five individual flowers, were also found. Pollen vitality and tube germination of glasshouse‐grown flowers were investigated, and there were no significant differences compared to pollen of non‐transgenic control plants. Preliminary crosses using flowers of glasshouse plants resulted in small apple fruits. It would seem that this is the first report on in vitro flower induction in transgenic apple.     

5‐Aminolevulinic Acid Activates Antioxidative Defence System and Seedling Growth in Brassica napus L. under Water‐Deficit Stress

The present study assesses the effects of 5‐aminolevulinic acid (ALA, 0, 0.1, 1 and 10 mg l^?1) on the growth of oilseed rape (Brassica napus L. cv. ZS758) seedlings under water‐deficit stress induced by polyethylene glycol (PEG 6000, 0 and ?0.3 MPa). Water‐deficit stress imposed negative effects on seedling growth by reducing shoot biomass, cotyledon water potential, chlorophyll content and non‐enzymatic antioxidants (glutathione and ascorbic acid) levels. On the other hand, water‐deficit stress enhanced the malondialdehyde (MDA) content, reactive oxygen species (ROS) production, enzymatic antioxidants activities, reduced/oxidized glutathione ratio (GSH/GSSG) and reduced/oxidized ascorbic acid (ASA/DHA) ratio in seedlings. Application of ALA at lower dosages (0.1 and 1 mg l^?1) improved shoot weight and chlorophyll contents, and decreased MDA in rape seedlings, whereas moderately higher dosage of ALA (10 mg l^?1) hampered the growth. The study also indicated that 1 mg l^?1 ALA improved chlorophyll content, but reduced MDA content and ROS production significantly under water‐deficit stress. Lower dosages of ALA (0.1 and 1 mg l^?1) also enhanced GSH/GSSG and ASA/DHA as compared to the seedlings under water‐deficit stress. The antioxidant enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase and superoxide dismutase) enhanced their activities remarkably with 1 mg l^?1 ALA treatment under water‐deficit stress. It was also revealed that 1 mg l^?1 ALA treatment alone induced the expression of APX, CAT and GR substantially and under water‐deficit stress conditions ALA treatment could induce the expression of POD, CAT and GR to a certain degree. These results indicated that 0.1–1 mg l^?1 ALA could enhance the water‐deficit stress tolerance of oilseed seedlings through improving the biomass accumulation, maintaining a relative high ratio of GSH/GSSG and ASA/DHA, enhancing the activities of the specific antioxidant enzymes and inducing the expression of the specific antioxidant enzyme genes.     

腐植酸对干旱胁迫下谷子幼苗叶片抗坏血酸-谷胱甘肽循环的影响

为探讨腐植酸（HA）对干旱胁迫下谷子抗氧化系统的调节机制,以晋谷21号和张杂10号为材料,采用浸种法研究了不同浓度腐植酸（0、50、100、200、300mg/L）对干旱胁迫下谷子幼苗叶片抗坏血酸-谷胱甘肽（AsA-GSH）循环的影响。结果显示,100和200mg/L腐植酸处理显著提高了干旱胁迫下谷子幼苗叶片抗坏血酸过氧化物酶（APX）和脱氢抗坏血酸还原酶（DHAR）的活性,促进了抗氧化物质AsA和GSH的再生,使AsA/DHA和GSH/GSSH值增加,有效缓解了活性氧的积累。研究表明,腐植酸可通过提高AsA-GSH循环相关酶活性及抗氧化物质含量,缓解干旱胁迫对谷子幼苗造成的氧化损伤,从而提高谷子的抗旱性。     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation of indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for insect resistance

The rice leaffolder (RLF), Cnaphalocrocis medinalis is an important pest of rice that causes severe damage in many areas of the world. The plants were transformed with fully modified (plant codon optimized) synthetic Cry1C coding sequences as well as with the hpt and gus genes, coding for hygromycin phosphotransferase and β-glucuronidase, respectively. Cry1C sequences placed under the control of doubled 35S promoter plus the AMV leader sequence, and hpt and gus genes driven by cauliflower mosaic virus 35S promoter, were used in this study. Embryogenic calli after cocultivation with Agrobacterium were selected on the medium containing hygromycin B. A total of 67 hygromycin-resistant plants were regenerated. PCR and Southern blot analyses of primary transformants revealed the stable integration of Cry1C coding sequences into the rice genome with predominant single copy integration. R₁ progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. These transgenic lines were highly resistant to rice leaffolder (RLF), Cnaphalocrocis medinalis as revealed by insect bioassay.     

MeJA regulates enzymes involved in ascorbic acid and glutathione metabolism and improves chilling tolerance in loquat fruit

This study investigated the regulation of ascorbate acid (AsA) and glutathione metabolism and chilling tolerance by methyl jamonate (MeJA) in loquat fruit. The results showed that application of MeJA to loquat fruit inhibited the incidence of chilling injury manifested as internal browning (IB) and increased AsA and reduced glutathione (GSH) contents due to the inhibition of ascorbate oxidase activity and enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities. Meanwhile, MeJA also enhanced activities of ascorbate peroxidase, glutathione peroxidase and glutathione-S-transferase. Our results suggested that MeJA can regulate the ascorbate and glutathione metabolism and has important roles in alleviating oxidative damage and enhancing chilling tolerance in loquat fruit.     

Genetic transformation of pigeonpea with rice chitinase gene

With a long‐term plan to develop transgenic pigeonpea with resistance to fungal disease, the transfer of a rice chitinase gene to pigeonpea [Cajanus cajan (L.) Millsp.] is reported here. The rice chitinase gene harboured in the plasmid pCAMBIA 1302:RChit was delivered via the Agrobacterium‐mediated method to the cotyledonary node explants followed by subsequent regeneration of complete plants on selection media containing hygromycin. Putative transformed pigeonpea plants were recovered with stringent selection pressure and confirmed using molecular techniques. Stable integration and expression of the chitinase gene has been confirmed in the T0 and T1 transgenics through molecular analysis.