The role of glutathione metabolism in tolerance of tobacco BY-2 suspension cells to microcystin-RR

This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 mu g/mL) for 0-6 days resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 days exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 days exposure. The GSH/GSSG ratio was much higher than control in 10 mu g/mL MC-RR treated group at day 4, but after 6 days exposure, the ratios in all treated groups were lower than that of the control group.

植物体内谷胱甘肽还原酶的功能研究

谷胱甘肽还原酶（GR，EC1.6.4.2）是一重要的抗氧化酶，许多生理学和遗传工程研究都证明GR酶在抗氧化中的重要作用。但改变GR酶怎样影响植物的抗氧化系统却不清楚。GR是抗坏血酸－谷胱甘肽循环途径中的重要组成部分，其功能必然与其密切相关。本文用RNAi技术获得具有较低GR酶活性的转基因烟草，系统测定了非胁迫条件和胁迫条件下抗坏血酸－谷胱甘肽循环的变化，得出以下主要结果： 1．选择一烟草叶绿体GR酶编码基因（X76293, gi: 431954）进行RNAi载体构建，构建好的双元载体转化根癌农杆菌LBA4404，然后侵染转化烟草叶圆片。获得的转基因烟草具有30－70％的GR酶活性。分子检测结果表明GR在RNA和蛋白水平上与GR酶活性的变化一致。文中我们第一次用2-D电泳对烟草中GR同工酶进行分析，并确定发生抑制的GR同工酶在细胞中的定位。2-D电泳后的Western杂交检测到烟草的10种GR同工酶，pI值分布在4.5-6.3，其中3种GR同工酶定位在叶绿体内，其蛋白量占据所有GR酶含量的大部分。RNAi发生在叶绿体内和叶绿体外，表明发生抑制的GR同工酶的基因序列具有很高的同源性。igr转基因烟草在表型上与野生型对照烟草无明显差异。 2．所有igr转基因植株和对照植株中的活性氧（O2-和H2O2）、MDA含量和光合作用都无明显差异，表明正常生长条件下GR酶活性的降低不会引起氧化胁迫。测定正常生长条件下igr转基因烟草中谷胱甘肽库的变化。结果表明与对照烟草相比，GR酶活性降低70％会引起转基因植株中GSH/GSSG比率明显降低，而GSH和GSSG的含量稍有增加；测定抗坏血酸－谷胱甘肽循环的变化，结果显示igr转基因烟草中DHAR和MDHAR的酶活性升高，表明非胁迫条件下较低的GR酶活性可能会诱导抗坏血酸－谷胱甘肽循环不能正常的运转。这一作用可能与改变的谷胱甘肽库有关。GR酶活性降低30％的转基因烟草中未检测到这些变化，表明70％的GR酶活对于非胁迫条件下igr转基因烟草可能是足够的。 3. MV处理结果显示，igr转基因烟草的离体叶圆片和活体植株在MV处理后都发生比对照烟草严重的光漂白作用。igr转基因烟草的活性氧和MDA含量明显高于对照烟草，igr转基因烟草的光合作用明显低于对照烟草。以上这些指标表明igr转基因烟草对MV处理更为敏感。MV处理条件下igr转基因烟草谷胱甘肽的含量明显高于对照烟草，但是GSH/GSSG的比率明显低于对照烟草，GR酶活性仍明显低于对照烟草，表明在MV胁迫条件下igr转基因烟草中较低的GR酶活性不能有效的将GSSG还原生成GSH。igr转基因烟草中较高的谷胱甘肽净含量说明其谷胱甘肽的合成能力提高，但这仍不能补偿胁迫条件下较低GR酶引起的GSH/GSSG比率降低。MV处理条件下igr转基因烟草和对照烟草相比ASC的含量大大降低，导致DHA/ASC明显升高。测定MDHAR和DHAR的结果表明，MV处理后igr转基因烟草的MDHAR酶活性明显降低，这表明较低的GR酶活性引起ASC再生循环受到抑制。MV处理后较低的GR酶还引起igr转基因烟草中APX的活性大大降低。以上这些结果表明MV处理条件下降低GR酶活性会削弱抗坏血酸－谷胱甘肽循环，从而引起活性氧的大量积累，造成严重的氧化伤害。 4．低温处理的结果和MV处理的结果稍有不同。在GR酶活性较高的i2转基因烟草中所有检测指标与对照烟草无明显差异。而GR酶活性较低的i21、i28和i42植株与对照烟草相比表现出明显差异。低温下生长的对照烟草叶绿素含量明显高于i21、i28和i42植株。i21、i28和i42中活性氧（O2-和H2O2）和MDA的含量都明显高于对照烟草，表明低温处理下i21、i28和i42受到更严重的胁迫伤害。与MV处理后的变化相似，低温处理后i21、i28和i42中较低的 GR酶活性导致GSH/GSSG大大降低，ASC再生循环受抑制，APX活性明显降低，从而使抗坏血酸－谷胱甘肽循环不能高效的清除活性氧，导致ROS和MDA的大量积累，造成严重的低温伤害。

水杨酸在水稻抗冷性中的作用

近年来大量研究表明水杨酸（salicylic acid, SA）在植物抵抗生物胁迫与非生物胁迫中都发挥着重要作用。然而在一些单子叶植物如水稻中SA的作用迄今仍不是很清楚。为了更深入地了解SA在水稻抵御冷胁迫中的作用，本研究选用两个抗冷性不同的水稻品种：‘长白九’（Oryza sativa cv. ‘Changbaijiu’）和‘中鉴’（Oryza sativa cv. ‘Zhongjian’）作为实验材料，其中‘长白九’为抗冷性较强的品种，而‘中鉴’为冷敏感的品种。在水稻幼苗长至三叶期后，分别对其施以三种浓度（0.5 mM, 1.0 mM, 2.0 mM）的SA溶液预处理24 h，然后置于5 °C下进行冷处理24 h。形态学观察及各项指标的测定结果表明： 一、冷处理后，‘长白九’和‘中鉴’根与叶片中的SA含量都大幅提高，且结合态SA升高的幅度明显大于其自由态形式。 二、外施不同浓度的SA溶液于水稻根部，24 h后，大量SA尤其是结合态SA积累于根中，且其积累量与处理浓度成正相关;而叶片中积累的SA则较少。 三、形态学及生理指标的测定结果显示，SA预处理没有提高甚至降低了两个水稻品种幼苗的抗冷性。并且SA处理浓度越大，幼苗受到冷伤害程度的越高。 四、对水稻幼苗叶片与根中的抗氧化酶活性进行分析发现，常温下SA处理显著提高了‘长白九’和‘中鉴’根中过氧化氢酶（catalase, CAT）和谷胱甘肽还原酶（glutathione reductase, GR）的活性;而在低温下SA预处理反而降低了两种水稻叶片与根中部分抗氧化酶的活性，推测低温下抗氧化酶活性的下降可能与水稻幼苗抗冷性的降低有关。 五、尽管两个水稻品种具有不同的冷敏感性，然而外施水杨酸均加剧了其低温伤害。分析认为，外施水杨酸后，水稻根部大幅升高的内源SA水平可能加剧了活性氧的产生，破坏了植物细胞内部的氧化还原平衡，从而导致水稻幼苗受到的冷害加重。

Oxidative stress response after prolonged exposure of domestic rabbit to a lower dosage of extracted microcystins

Oxidative stress response after prolonged exposure to a low dose of microcystins (MCs) was studied in liver, kidney and brain of domestic rabbits. Rabbits were treated with extracted MCs (mainly MC-LR and MC-RR) at a dose of 2 MC-LReq. mu g/kg body weight or saline solution every 24 h for 7 or 14 days. During the exposure of MCs, increase of lipid peroxidation (LPO) levels were detected in all the organs studied, while antioxidant enzymes responded differently among different organs. The enzyme activities Of Superoxide dismutase (SOD). catalase (CAT) and glutathione reductase (GR) in liver decreased in the MCs treated animals. In brain, there were obvious changes in glutathione peroxidase (GPx) and GR, while only CAT was obviously influenced in kidney. Therefore, daily exposure at a lower dosage of MCs, which mimicked a natural route of MCs. could also induce obvious oxidative stress in diverse organs of domestic rabbits. The oxidative stress induced by MCs in brain was as serious as in liver and kidney, suggesting that brain may also be a target of MCs in mammals. And it seems that animals may have more time to metabolize the toxins or to form an adaptive response to reduce the adverse effects when exposed to the low dose of MCs. (C) 2008 Elsevier B.V. All rights reserved.

Time-Dependent Oxidative Stress Responses of Crucian Carp (Carassius auratus) to Intraperitoneal Injection of Extracted Microcystins

This study was conducted to investigate time-dependent changes in oxidative enzymes in liver of crucian carp after intraperitoneally injection with extracted microcystins 600 and 150 mu g kg(-1) body weight. The results showed that activities of antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase generally exhibited a rapid increase in early phase (1-3 h post injection), but gradually decreased afterwards (12-48 h) compared with the control, with an evident time-dependent effect. These zigzag changes over time contributed a better understanding on oxidative stress caused by microcystins in fish.

Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus)

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely present in the environment, wildlife and human. We studied the cellular toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on oxidative stress and induction of apoptosis in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 1, 5, 15 and 30 mg L-1) for 24 h, and a dose-dependent decrease in cell viability was determined using trypan blue exclusion method. Significant induction of reactive oxygen species (ROS) accompanied by increases in activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found, while activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were decreased. Glutathione (GSH) content was reduced following treatment of PFOA and PFOS. A dose-dependent increase in the lipid peroxidation (LPO) level (measured as maleic dialdehyde, MDA) was observed only in the PFOA exposure groups, whereas LPO remained unchanged in the PFOS exposure groups. Furthermore, a significant activation of caspase-3, -8, -9 activities was evident in both PFOS and PFOA exposure groups. Typical DNA fragmentation (DNA laddering) was further characterized by agarose gel electrophoresis. The overall results demonstrated that PFOS and PFOA are able to produce oxidative stress and induce apoptosis with involvement of caspases in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Effects of hexachlorobenzene on antioxidant status of liver and brain of common carp (Cyprinus carpio)

Hexachlorobenzene (HCB)-induced oxidative damages have been published in rats while the effects have not yet been reported in fishes. Juvenile common carps (Cyprinus carpio) were exposed to waterborne HCB from 2 to 200 mu g l(-1) for 5, 10 or 20 days. Liver and brain were analyzed for various parameters of oxidative stress. There were no significant changes of glutathione (GSH) content and superoxide dismutase (SOD) activity in liver after 5 or 10 days exposure, whereas obvious drops were observed at higher concentrations after 20 days exposure. Significant decreases of GSH content and SOD activity in brain were found during all the exposure days. In brain, HCB also significantly elevated the contents of reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS, as an indicator of lipid peroxidation products), glutathione disulfide (GSSG), and activities of nitric oxide synthase (NOS), glutathione peroxidase (GPx), and glutathione reductase (GR), and inhibited activities of acetylcholinesterase (AchE) and glutathione S-transferase (GST). The results clearly demonstrated that environmentally possible level of HCB could result in oxidative stress in fish and brain was a sensitive target organ of HCB toxicity. (c) 2006 Elsevier Ltd. All rights reserved.

荞麦(Fagopyrum tataricum Gaertn.和F. esculentum Moench.)对紫外线B辐射的响应研究

近二十多年来，基于对臭氧层衰减、紫外线B（UV-B）增强的担心，研究者希望了解到紫外线辐射对不同作物的影响情况，增强UV-B辐射条件下是否对作物的生长发育、产量质量构成威胁。在本试验中，我们首先探讨了双子叶作物黄瓜（Cucumis sativus）和大豆（Glycine max）对不同紫外波段的生物效应[分别为B-UVA（315-400 nm），N-UVA（315-340 nm），B-UVB（275-400 nm）和N-UVB（290-340 nm），UV-（>400nm）作对照]。我们观察到所有的UV波段处理都使黄瓜和大豆的生长受到抑制，并且细胞受到不同程度的氧化伤害；UV波段处理的作用效果与不同波段的紫外有效生物辐射剂量有关。处理差异在UV-B波段内部和UV-A波段内部同样存在。植物生长UV辐射公式（BSWF）能很好的预测本试验UV-B波段内的平均植物效应，但不能预测UV-A波段的植物效应。短波UV-A的生物作用强于长波UV-A。光合色素的变化与UV波谱差异和种间差异有关。在高的紫外/可见光背景下，UV-A处理同UV-B同样导致光合色素的降低，但黄瓜类胡萝卜素/叶绿素比例升高。与其他研究者的试验结果比较后，我们认为紫外线B辐射的生物效应一致性很高，但紫外线A波段的生物学效应存在较大争议。因此我们在本试验的基础上仅进行荞麦[苦荞(Fagopyrum tataricum Gaertn.)和甜荞(Fagopyrum esculentum Moench.)]对紫外线B波段的响应研究。 我们对苦荞品种-圆籽荞进行了连续两个生长季节的大田半控制试验以观察UV-B辐射对苦荞生长、发育、产量及叶片色素的影响；试验小区进行降低UV-B、近充足UV-B和增强UV-B辐射处理。我们的试验表明在不同强度UV-B辐射下苦荞的生长、地上部生物量积累及最终产量都有所下降，但苦荞的发育加快；当前条件下的日光紫外线B辐射对植物生长和产量也造成负面影响。植物光合色素被日光及增强UV-B辐射降低；UV化合物及卢丁含量在中低剂量的UV-B辐射强度下显著升高，但在高剂量的增强UV-B辐射下短期升高后迅速下降。我们的试验表明苦荞是一个对UV-B高度敏感的作物。苦荞对UV-B的敏感性与UV-B剂量、外界环境因素及生长季节有关。 单个苦荞品种的试验结果使我们认识到外界UV-B辐射已经对苦荞生长发育构成逆境条件，未来全球气候变化条件下增强紫外线B辐射可能使其处于更不利的生长环境中。因此我们有进行了多个种群进行UV-B响应观察并筛选耐性种群。我们对15个苦荞种群进行增强UV-B辐射处理（6.30 kJ m2 UV-BBE，模拟当地25%的臭氧衰减），我们观察苦荞UV-B辐射效应存在显著的种内差异，UV-B辐射对多数种群具有抑制作用，但对一些种群还有刺激作用。我们采用主成分分析方法与作物UV-B响应指数（RI）来评价苦荞作物UV-B辐射耐性。我们发现作物的UV-B耐性不仅与其原产地背景UV-B强度有关，而且与作物相对生长效率、次生代谢产物含量（如卢丁）及其他因素有关。我们观察到苦荞伸展叶总叶绿素变化与UV-B耐性成正相关；室内苦荞幼苗的UV-B辐射致死试验表明：苦荞种群死亡率与其UV-B耐性成负相关。 此外，我们对甜荞的UV-B辐射响应也进行了初步研究。选取美姑甜荞、巧家甜荞和云龙甜荞进行5个梯度的增强UV-B辐射室外模拟试验。我们观察到UV-B辐射显著降低了甜荞的生长、生物量及产量；并严重影响了甜荞的生殖生长，降低了花序数、种子数和结实率；并且UV-B辐射对甜荞的抑制作用存在显著的剂量效应。三种甜荞品种存在显著的种内差异，其中美姑品种UV-B耐性最强，且膜脂受UV-B辐射氧化伤害最小，这与该品种UV-B辐射下较高的GR酶活性、APX酶活性和PPO酶活性、以及含量更高的抗坏血酸有关。甜荞的次生代谢也受到增强UV-B辐射的影响，其香豆酰类化合物在UV-B辐射下升高显著，而槲皮素含量也在高剂量UV-B辐射下有所增加；卢丁含量依赖UV-B辐射剂量而变化，中低剂量UV-B辐射下其卢丁含量逐渐升高，但在高剂量辐射下逐渐下降。 通过对生长在高海拔地区的荞麦作物(苦荞和甜荞)进行的室外研究，我们认识到作物不同品种存在很大的耐性差异，这就为UV-B耐性育种创造了有利条件。进一步加大荞麦种质资源筛选力度并深入认识荞麦抗性机理，在此基础上通过杂交或其他基因融合手段培育抗性品种，对高剂量UV-B辐射地区的荞麦产量的提高将起到重要推动作用，并使荞麦生产能有效应对未来全球气候变化条件下UV-B辐射可能升高的威胁。 During last few decades, due to concern of ozone layer depletion and enhancement of ultraviolet B radiation（UV-B, 280-315 nm), the agronomist want to know the responses of different crop species to UV-B. In the first experiment of our study, the effect of different UV band [B-UVA（315-400 nm), N-UVA（315-340 nm), B-UVB（275-400 nm), N-UVB（290-340 nm）and UV-（>400nm, as control)] on the cucumber（Cucumis sativus）and soybean（Glycine max）were investigated in growth room. Spectra-dependent differences in growth and oxidation indices existed within UV-A bands as well as UV-B bands. The general biological effects of different band were UV- ＜ B-UVA＜ N-UVA＜N-UVB＜B-UVB. The plant growth biologically spectra weighting function（BSWF）matched well with average plant response in UV-B region, but not in UV-A region. Shorter UV-A wavelength imposed more negative impact than longer UV-A wavelength did in both species. The effect on photosynthetic pigment was related to different UV bands and different species. The photosynthetic pigment content was decreased by UV-A spectra as well as UV-B spectra. In comparison with the results of previous studies, we found that the wavelength-dependent biological effect of ultraviolet B radiation has high consistency, but the biological effect of ultraviolet-A radiation was inconsistent. We narrow our following study on the effect of ultraviolet B radiation on the buckwheat（tartary buckwheat and common buckwheat). The tartary buckwheat（Fagopyrum tataricum Gaertn.）cultivars Yuanziqiao was grown in the sheltered field plots for two consecutive seasons under reduced, near-ambient and two supplemental levels of UV-B radiation. The crop growth, photosynthetic pigments, total biomass, final seed yield and thousand-grain weight were decreased by near-ambient and enhanced UV-B radiation, while crop development was promoted by enhanced UV-B radiation. Leaf rutin concentration and UV-B absorbing compound was generally increased by UV-B with the exception of 8.50 kJ m-2 day-1 supplemental levels. Our results showed that tartary buckwheat is a potentially UV-B sensitive species. Study on one cultivars showed that ambient solar radiation had present a stress to tartary buckwheat. This makes it necessary to observe the UV-B response of many cultivars and screen tolerant cultivars. Fifteen populations of tartary buckwheat were experienced enhanced UV-B radiation simulating 25% depletion of the stratospheric ozone layer in Kunming region, and plant responses in growth, morphology and productivity were observed. Principal components analysis（PCA）was used to evaluate overall sensitivity of plant response to UV-B as well as response index. The different populations exhibited significant differences in responses to UV-B. The photosynthetic pigments of young seedlings were also affected significantly under field condition. On the other hand, the healthy seedlings of different populations were exposed to the high level of UV-B radiation in growth chambers to determine the plant lethality rate. The plant tolerance evaluated by multivariate analysis was positively related to total plant chlorophyll change, but negatively related to lethality rate. In other hand, the UV-B responses of the other important cultivated buckwheat species, common buckwheat（Fagopyrum esculentum Moench.), were also studied preliminarily. Three widespread cultivated variety（Meigu, Qiaojia and Yunlong cultivars）were provided with five level of enhanced UV-B radiation outdoors. We observed that the crop growth, development and production were significantly decreased, and reproductive production, like anthotaxy number, seed number and seed setting ratio, was also decreased. Dose-dependent inhibition effect caused by enhanced UV-B radiation also existed in common buckwheat. Significant intraspecific difference existed in those three cultivars. The Meigu cultivars with dwarfed growth and lower production have highest UV-B tolerance as well as lowest damage in cell membrane, this could be associated with profound enhancements of glutathione reductase（GR）activity, ascorbate peroxidase activity and polyphenol oxidase activity as well as higher ascorbic acid concentration. The secondary metabolism was also affected by UV-B radiation, with profound elevation of coumarin compound and moderate increase of quercetin concentration. Rutin concentration was peaked in 5kJ m-2 UV-B. The contrasting effect of UV-B radiation on different populations indicated that there existed abundant genetic resources for selecting tolerant populations of common and tartary buckwheat. Much effort needed be pose on screening of buckwheat germplasm and clarification of mechanism of buckwheat tolerance to UV-B. On this base the tolerant cultivars could be bred by hybridization and other gene transfusion method, this would help increase buckwheat yield in high ambient UV-B region and counteract the effect of possible enhanced UV-B radiation in future.

Examination of Antioxidative System's Responses in the Different Phases of Drought Stress and During Recovery in Desert Plant Reaumuria soongorica (Pall.) Maxim

The aim of this study was to test the protective roles of superoxide dismutases (SODs), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) against oxidative damage and their activities in different phases of the dry down process in Reaumuria soongorica (Pall.) Maxim. leaves. Drought stress was imposed during 100 consecutive days and rewatering after 16, 72, and 100 days. The concentration of hydrogen peroxide (H2O2), malondialdehyde, and SODs activities were elevated significantly with progressing drought stress. POD and CAT activities increased markedly in the early phase of drought and decreased significantly with further drought stress continuation, and POD activity was unable to recover after rewatering. Ascorbate, reduced glutathione, APX, and GR activities declined in the initial stages of drought process, elevated significantly with further increasing water deficit progression and recovered after rewatering. These results indicate that: (1) iron SODs-removing superoxide anion is very effective during the whole drought stress; (2) CAT scavenges H2O2 in the early phase of drought and enzymes of ascorbate-glutathione cycle scavenge H2O2 in further increasing drought stress; and (3) POD does not contribute to protect against oxidative damage caused by H2O2 under drought stress.