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1.
Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO2 diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.  相似文献   

2.
Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, γ-glutamate cysteine ligase (GSH1), responsible for synthesis of γ-glutamylcysteine (γ-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by γ-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of γ-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by γ-EC in vitro strongly suggests export of γ-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, γ-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.  相似文献   

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The terminal step of glutathione (GSH) synthesis is the condensation of γ-glutamyl-cysteine (γ-EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ-EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ-EC and GSH was studied in excised leaves and leaf discs from untransformed poplar ( Populus tremula × P. alba ) and poplar overexpressing γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2). Poplars strongly overexpressing γ-ECS (ggs28) had enhanced levels of γ-EC and GSH compared to untransformed poplars. The relationship between γ-EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50-fold higher than γ-EC. On darkening, γ-EC accumulated markedly and GSH declined, so that the GSH:γ-EC ratio was close to 1. These dark-induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ-EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ-EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ-EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.  相似文献   

5.
Aims: To investigate the effects of pH stress coupled with cysteine addition on glutathione (GSH) production in the treatment of high cell density culture of Candida utilis. Methods and Results: We have previously observed that most Candida utilis cells remained viable after being subjected to pH at 1·2 for 3 h and that some intracellular GSH leaked into the medium. A cysteine addition strategy was applied in fed‐batch production of GSH. A single cysteine addition resulted in higher GSH yield than two separate additions without pH stress. An increase in intracellular GSH content triggered inhibition of γ‐glutamylcysteine synthetase (γ‐GCS). A strategy that combines cysteine addition with low‐pH stress was developed to relieve the inhibition of γ‐GCS. Conclusion: Without pH stress, single shot and double shot cysteine addition yielded a total GSH of 1423 and 1325 mg l?1. In comparison, a low‐pH stress counterpart resulted in a total GSH of 1542 and 1730 mg l?1, respectively. With low‐pH stress, we observed GSH secretion into the medium at 673 and 558 mg l?1 and an increase in the γ‐GCS activity by 1·2‐ and 1·5‐fold, respectively. The specific GSH production yield increased from 1·76% to 1·91% (w/w) for single shot, and 1·64% to 2·14% for double shots. Significance and Impact of the Study: Low‐pH shift was applied to alleviate the feedback inhibition of intracellular GSH on γ‐GCS activity by secreting GSH into the medium. This strategy is coupled with cysteine addition to enhance GSH production in Candida utilis.  相似文献   

6.
Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.  相似文献   

7.
叶绿体虽然是植物细胞内一种极其重要的细胞器,但其分裂的分子机制尚不很清楚。已经证明FtsZ蛋白作为真核细胞分裂装置的一个关键成分,参与叶绿体的分裂过程。烟草的FtsZ基因属于2个不同的家族,在对NtFtsZ1家族成员研究的基础上,用正义和反义表达技术研究了NtFtsZ2家族成员NtFtsZ2-1基因在转基因烟草中的功能。显微分析结果表明NtFtsZ2-1基因的表达水平异常增强或减弱都会严重干扰叶绿体的正常分裂过程,导致叶绿体在形态和数目上的异常(体积明显增大,数目显著减少),而单个叶肉细胞中叶绿体的总表面积在正反义转基因烟草和野生型烟草之间保持了相对稳定,没有发生明显的变化。同时还证明NtFtsZ2-1基因表达的变化对叶绿素含量和叶绿体的光合作用能力没有直接的影响。据此我们认为NtFtsZ2-1基因参与叶绿体的分裂和体积的扩大,其表达水平的波动会改变植物中叶绿体的数目和大小,而且在叶绿体的数目与体积之间可能存在一种补偿机制,保证叶绿体能最大限度地吸收光能,从而使光合作用得以正常进行。  相似文献   

8.
With the aim of increasing the methionine level in alfalfa (Medicago sativa L.) and thus improving its nutritional quality, we produced transgenic alfalfa plants that expressed the Arabidopsis cystathionine gamma-synthase (AtCGS), the enzyme that controls the synthesis of the first intermediate metabolite in the methionine pathway. The AtCGS cDNA was driven by the Arabidopsis rubisco small subunit promoter to obtain expression in leaves. Thirty transgenic plants were examined for the transgene protein expression, and four lines with a high expression level were selected for further work. In these lines, the contents of methionine, S-methylmethionine (SMM), and methionine incorporated into the water-soluble protein fraction increased up to 32-fold, 19-fold, and 2.2-fold, respectively, compared with that in wild-type plants. Notably, in these four transgenic lines, the levels of free cysteine (the sulphur donor for methionine synthesis), glutathione (the cysteine storage and transport form), and protein-bound cysteine increased up to 2.6-fold, 5.5-fold, and 2.3-fold, respectively, relative to that in wild-type plants. As the transgenic alfalfa plants over-expressing AtCGS had significantly higher levels of both soluble and protein-bound methionine and cysteine, they may represent a model and target system for improving the nutritional quality of forage crops.  相似文献   

9.
Insect glutathione S‐transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up‐regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had Vmax (reaction rate of the enzyme saturated with the substrate) and Km (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and Vmax and Km values of 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L for 1‐chloro‐2,4‐dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro‐diphenyl‐trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.  相似文献   

10.
Recently many researchers have proposed a protective role for morphine against tumor growth and metastasis, especially through induction of apoptosis in tumoral cells. These findings may lead to underestimation of cytotoxic effects of opioid drugs which are usually expected only at high doses. The present study was conducted to clarify whether repeated morphine administration, which is commonly used for relief from chronic pain, would interfere with liver antioxidant defence and hepatocytes vitality. Morphine was injected repeatedly at doses that have been reported to relieve cancer pain and reduce tumor spread in mice (5 and 10 mg/kg/day for nine consecutive days). The changes in hepatic glutathione concentration, its synthesis pathway and enzymatic antioxidant defense revealed the pro-oxidant effects of chronic morphine treatment on the liver. None of these changes were observed in those mice that were co-treated with naltrexone (opioid antagonist) and same doses of morphine. However induction of liver conjugating enzymes following morphine treatment was not receptor mediated. Moreover, chronic morphine treatment induced hepatocytes apoptosis. Interestingly, the apoptotic changes were antagonized by co-administration of either naltrexone or thiol antioxidant. In conclusion, although hepatotoxic effects of morphine at high doses have been reported previously, our findings propose that repeated morphine administration even at lower doses would induce oxidative stress in the liver, which may contribute to induction of apoptosis in hepatocytes. Since many of the observed adverse effects were mediated by opioid receptors, our results suggest that other opioid analgesics should also be used more cautiously.  相似文献   

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【目的】本文旨在挖掘野生型家蚕Bombyx mori和Ras1CA过表达转基因家蚕后部丝腺中的转录本差异,分析和验证细胞周期通路中的差异表达基因,从而探讨Ras1CA过表达转基因家蚕提高蚕丝产量的分子机制。【方法】利用转录组学比较野生型和Ras1CA过表达转基因家蚕后部丝腺中的基因转录本差异,经实时荧光定量PCR(q RT-PCR)验证。【结果】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺组织中有2 636个差异表达基因,其中细胞周期信号通路中有42个差异表达基因,包括细胞周期依赖性激酶(CDK)、细胞周期素(Cyclin)以及转录因子等。通过q RT-PCR检测cdk1、cyclin D1、cyclin D2、cdc7、cdh1、dp-1,2等6个基因在野生型和Ras1CA过表达转基因家蚕后部丝腺组织中的相对表达量,发现转基因家蚕中的表达量均显著高于野生型(P<0.05),其中cyclin D2的表达差异极显著(P<0.01)。q RT-PCR结果与转录本差异一致,表明Ras1CA过表达后,能够促进细胞周期通路基因的表达。【结论】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺中有大量差异表达基因,且Ras1CA能够在转录水平上调控细胞周期通路,影响后部丝腺组织的细胞分裂和器官生长,从而促进蚕丝蛋白的合成。  相似文献   

13.
We compared three transgenic poplar lines over‐expressing the bacterial γ‐glutamylcysteine synthetase (GSH1) targeted to plastids. Lines Lggs6 and Lggs12 have two copies, while line Lggs20 has three copies of the transgene. The three lines differ in their expression levels of the transgene and in the accumulation of γ‐glutamylcysteine (γ‐EC) and glutathione (GSH) in leaves, roots and phloem exudates. The lowest transgene expression level was observed in line Lggs6 which showed an increased growth, an enhanced rate of photosynthesis and a decreased excitation pressure (1‐qP). The latter typically represents a lower reduction state of the plastoquinone pool, and thereby facilitates electron flow along the electron transport chain. Line Lggs12 showed the highest transgene expression level, highest γ‐EC accumulation in leaves and highest GSH enrichment in phloem exudates and roots. This line also exhibited a reduced growth, and after a prolonged growth of 4.5 months, symptoms of leaf injury. Decreased maximum quantum yield (Fv/Fm) indicated down‐regulation of photosystem II reaction centre (PSII RC), which correlates with decreased PSII RC protein D1 (PsbA) and diminished light‐harvesting complex (Lhcb1). Potential effects of changes in chloroplastic and cytosolic GSH contents on photosynthesis, growth and the whole‐plant sulphur nutrition are discussed for each line.  相似文献   

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