Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH₄ ⁺ and Na⁺ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.     

Regulation of gamma-glutamylcysteine synthetase expression in response to oxidative stress.

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gamma-glutamylcysteine synthetase (gamma-GCS) is the rate limiting enzyme for the GSH synthesis. Gamma-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of gamma-GCS gene, in the present study, we show evidences that gamma-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-alpha mediated by nuclear factor-kappaB (NF-kappaB), and impairment of the expression of gamma-GCS by TNF-alpha in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-kappaB subunits.     

Changes in ammonium ion content and glutamine synthetase activity in rice leaves caused by excess cadmium are a consequence of oxidative damage

Ammonium ion accumulation and the decrease in glutamine synthetase (GS)activity induced by CdCl₂ were investigated in relation to lipidperoxidation in detached rice leaves. CdCl₂ was effective inincreasing ammonium ion content, decreasing GS activity and increasing lipidperoxidation. Free radical scavengers (glutathione, thiourea, sodium benzoate)and an iron chelator (2,2-bipyridine) were able to inhibit the decreasein GS activity and ammonium ion accumulation caused by CdCl₂ and atthe same time inhibit CdCl₂-induced lipid peroxidation. Paraquat,which is known to produce oxygen radicals, decreased GS activity, increasedammonium ion content, and increased lipid peroxidation. GS1 appears to be thepredominant isoform present. Excess Cd caused a decrease in GS1 but not in GS2in detached rice leaves. An increase in lipid peroxidation preceded ammoniumionaccumulation and the decrease in GS1 activity. These results suggest that thedecrease in GS activity and the accumulation of ammonium ions in detached riceleaves are a consequence of oxidative damage caused by excess Cd.     

Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.)

OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast.     

Protein phosphatase Z modulates oxidative stress response in fungi

The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.     

Glutathione metabolism in Urtica dioica in response to cadmium based oxidative stress

To investigate the antioxidative response of glutathione metabolism in Urtica dioica L. to a cadmium induced oxidative stress, activities of glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px), content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation (LPO), and also accumulation of Fe, Zn, Mn, Cu besides Cd were determined in the roots, stems, and leaves of plants exposed to 0 (control), 0.045, and 0.09 mM CdCl₂ for 58 h. Whereas the Cd content continuously increased in all organs, the Fe, Zn, Mn, and Cu content decreased in dependence on the applied Cd concentration and incubation time. The Cd treatment resulted in increased GR and GST activities in all organs, however, GSH-Px activity was dependent on Cd concentration and plant organ. The GSH/GSSG ratio maintained above the control level in the stems at both Cd concentrations. The LPO was generally close to the control values in the roots and stems but it increased in the leaves especially at 0.09 mM Cd.     

水稻根系响应镉胁迫的蛋白质差异表达

为探讨水稻根系对镉胁迫的分子生理响应,以抗镉水稻PI312777和镉敏感水稻IR24为材料,设置Cd~(2+)浓度为0、50和100μmol/L的水培试验,处理7 d后分析了水稻根系的蛋白质差异表达。结果表明,在镉胁迫下水稻PI312777和IR24根系有18个蛋白质发生了差异表达,其中的12个得到MALDI-TOF/MS鉴定。这些鉴定的蛋白功能可分四类:(1)与活性氧(ROS)胁迫相关的过氧化物酶(POD)、蛋氨酸腺苷转移酶(MAT)、类萌发素蛋白前体;(2)与谷胱甘肽(GSH)合成相关的S-腺苷甲硫氨酸合成酶(SAMS)、谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH);(3)与逆境胁迫相关的ABA胁迫诱导蛋白含HVA22域蛋白、ABA-胁迫-成熟诱导蛋白5(ASR5);(4)与细胞分裂调控相关的GTP结合核蛋白Ran-2。镉胁迫下SAMS和GTP结合核蛋白Ran-2在两种水稻根系均发生上调表达;MAT、POD、类萌发素蛋白前体和GS发生下调表达;依赖NADP-GDH、GDH和磷酸甘油酸变位酶在IR24根部均发生下调表达,在PI312777根部仅在100μmol/L Cd~(2+)处理发生下调表达;含HVA22域蛋白在PI312777根部上调表达,在IR24根部发生下调表达;ASR5在PI312777根部上调表达,在IR24根部的表达无显著差异;100μmol/L Cd~(2+)胁迫下60S酸性核糖体蛋白P0在水稻PI312777根部表达下调,在IR24根部表达上调。可见,镉胁迫使水稻根部ROS增加,形成氧化胁迫反应,造成毒害作用,而水稻根通过调节SAMS和GS提高GSH合成降低镉毒害。ASR5和HVA22蛋白等逆境胁迫蛋白的表达差异则是水稻品种间抗性差异的重要原因之一。     

Molecular characterization of the glutamine synthetase gene in the Pacific oyster Crassostrea gigas: expression study in response to xenobiotic exposure and developmental stage

In this study, we characterized the full-length cDNA and genomic sequence of the gene encoding cytosolic glutamine synthetase (CgGSII) in the Pacific oyster, Crassostrea gigas. A phylogenetic analysis of GS sequences showed that CgGS clustered with the invertebrate group as expected. We analyzed the expression of mRNA CgGSII using RT-PCR to follow the expression of this gene in gills and digestive gland of oysters exposed, under experimental conditions, to hypoxia and to several contaminants (hydrocarbons and two pesticide treatments, glyphosate and a mixture of atrazine, diuron and isoproturon). We also investigated the expression of CgGSII in different developmental stages of C. gigas. Our results show that CgGSII expression was highly regulated in xenobiotic-exposed oysters compared to the control for all the treatments. Likewise, CgGSII expression was highly regulated according to the developmental stage of C. gigas. Finally, use of CgGSII as a possible marker to monitor xenobiotic exposure in disturbed ecosystems is discussed.     

Halving the selenophosphate synthetase gene dose confers hypersensitivity to oxidative stress in Drosophila melanogaster

Several lines of evidence indicate that selenoproteins mainly act as cellular antioxidants. Here, we test this idea comparing the sensitivity to oxidative stress (paraquat and hydrogen peroxide) between wild type and heterozygous flies for the selenophosphate synthetase selD(ptuf) mutation. Whereas under normal laboratory conditions no difference in life span is observed, a significant decrease is seen in heterozygous flies treated with oxidant agents. In contrast, overexpression of the selD gene in motoneurons did not extend longevity. Our results strongly suggest that selD haploinsufficiency makes heterozygous flies more sensitive to oxidative stress and add further evidence to the role of selenoproteins as cellular antioxidants.     

Cloning of the glutamine synthetase gene fromspirulina platensis

An 8 Kilobase-pair (Kbp) HindIII fragment containing the coding sequence forSpirulina platensis glutamine synthetase [EC 6.3.1.1.] has been identified utilizing a probe derived fromAnabaena 7120 and cloned in the vector pAT153.     

Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress and telomere length

Early embryonic exposure to maternal glucocorticoids can broadly impact physiology and behaviour across phylogenetically diverse taxa. The transfer of maternal glucocorticoids to offspring may be an inevitable cost associated with poor environmental conditions, or serve as a maternal effect that alters offspring phenotype in preparation for a stressful environment. Regardless, maternal glucocorticoids are likely to have both costs and benefits that are paid and collected over different developmental time periods. We manipulated yolk corticosterone (cort) in domestic chickens (Gallus domesticus) to examine the potential impacts of embryonic exposure to maternal stress on the juvenile stress response and cellular ageing. Here, we report that juveniles exposed to experimentally increased cort in ovo had a protracted decline in cort during the recovery phase of the stress response. All birds, regardless of treatment group, shifted to oxidative stress during an acute stress response. In addition, embryonic exposure to cort resulted in higher levels of reactive oxygen metabolites and an over-representation of short telomeres compared with the control birds. In many species, individuals with higher levels of oxidative stress and shorter telomeres have the poorest survival prospects. Given this, long-term costs of glucocorticoid-induced phenotypes may include accelerated ageing and increased mortality.     

Expression of tea cytosolic glutamine synthetase is tissue specific and induced by cadmium and salt stress

Glutamine synthetase (GS) showed highest expression and activity in bud (youngest topmost leaf) of Camellia sinensis, lower in older leaves, while lowest activity in stem and roots. GS expression and activity was increased by ammonium and nitrate and also by cadmium and salt stress but decreased by copper, aluminum, drought, cold and heat stress.     

Increased vitamin E content in the lung after ozone exposure: a possible mobilization in response to oxidative stress

Vitamin E (vE) is a biological free radical scavenger capable of providing antioxidant protection depending upon its tissue content. In previous studies, we observed that vE increased significantly in rat lungs after oxidant exposure, and we postulated that vE may be mobilized to the lung from other body sites under oxidative stress. To test this hypothesis, we fed Long-Evans rats either a vE-supplemented or a vE-deficient diet, injected them intraperitoneally with 14C-labeled vE, and then exposed half of each group to 0.5 ppm ozone (O3) for 5 days. After exposure, we determined vE content and label retention in lungs, liver, kidney, heart, brain, plasma, and white adipose tissue. Tissue vE content of all tissues generally reflected the dietary level, but labeled vE retention in all tissues was inversely related to tissue content, possibly reflecting a saturation of existing vE receptor sites in supplemented rats. Following O3 exposure, lung vE content increased significantly in supplemented rats and decreased in deficient rats, but the decrease was not statistically significant, and vE content remained unchanged in all other tissues of both dietary groups. Retention of 14C-labeled vE increased in all tissues of O3-exposed rats of both dietary groups, except in vE-deficient adipose tissue and vE-supplemented brain, where it decreased, and plasma, where it did not change. The marked increases in lung vE content and labeled vE retention of O3-exposed vE-supplemented rats support our hypothesis that vE may be mobilized to the lung in response to oxidative stress, providing that the vitamin is sufficiently available in other body sites.     

Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate

A glutamine synthetase gene ( GS15) coding for soybean cytosolic glutamine synthetase (GS1) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC(3)) and a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies of GS15 (one 35S-GS15 line, one LBC (3) -GS15 line, and two rolD-GS15 lines) were tested for the expression of GS15, levels of GS1, GS activity, N accumulation, N(2) fixation, and plant growth at different levels of nitrate. Enhanced levels of GS1 were detected in leaves of three transformed lines (the 35S-GS15 and rolD-GS15 transformants), in nodules of three lines (the LBC (3) -GS15 and rolD-GS15 transformants), and in roots of all four transformants. Despite increased levels of GS1 in leaves and nodules, there were no differences in GS activity in these tissues or in whole-plant N content, N(2) fixation, or biomass accumulation among all the transgenic lines and the wild-type control. However, the rolD-GS15 transformants, which displayed the highest levels of GS1 in the roots of all the transformants, had significantly higher GS activity in roots than the wild type. In one of the rolD-GS15 transformed lines (Line 8), increased root GS activity resulted in a lower N content and biomass accumulation, supporting the findings of earlier studies with Lotus japonicus (Limami et al. 1999 ). However, N content and biomass accumulation was not negatively affected in the other rolD-GS15 transformant (Line 9) and, in fact, these parameters were positively affected in the 0.1 mM treatment. These findings indicate that overexpression of GS15 in various tissues of pea does not consistently result in increases in GS activity. The current study also indicates that the increase in root GS activity is not always consistent with decreases in plant N and biomass accumulation and that further investigation of the relationship between root GS activity and growth responses is warranted.     

Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves

Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants.     

Overexpression of cytosolic glutamine synthetase. Relation to nitrogen,light, and photorespiration

In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO(2) photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO(2) compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.