Glutathione synthesis is regulated by nitric oxide in <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

Glutathione (GSH) is one of the main antioxidants in plants. Legumes have the specificity to produce a GSH homolog, homoglutathione (hGSH). We have investigated the regulation of GSH and hGSH synthesis by nitric oxide (NO) in Medicago truncatula roots. Analysis of the expression level of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) after treatment with sodium nitroprusside (SNP) and nitrosoglutathione (GSNO), two NO-donors, showed that γ-ecs and gshs genes are up regulated by NO treatment whereas hgshs expression is not. Differential accumulation of GSH was correlated to gene expression in SNP-treated roots. Our results provide the first evidence that GSH synthesis pathway is regulated by NO in plants and that there is a differential regulation between gshs and hgshs in M. truncatula. G. Innocenti and C. Pucciariello have contributed equally to the work.     

Nitric oxide is involved in the regulation of ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress

This study investigated the regulation of ascorbate and glutathione metabolism by nitric oxide in Agropyron cristatum leaves under water stress. The activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), and the contents of NO, reduced ascorbic acid (AsA), reduced glutathione (GSH), total ascorbate and total glutathione increased under water stress. These increases were suppressed by pretreatments with NO synthesis inhibitors N ^G-nitro-L-arginine methyl ester (L-NAME) and 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, application of L-NAME and cPTIO to plants sufficiently supplied with water did not affect the activities of above mentioned enzymes and the contents of NO and above mentioned antioxidants. Pretreatments with L-NAME and cPTIO increased the malondialdehyde (MDA) content and electrolyte leakage of plants under water stress. Our results suggested that water stress-induced NO is a signal that leads to the upregulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance.     

Herbicide tolerance in maize is related to increased levels of glutathione and glutathione-associated enzymes

Treatment of 10 days old maize seedlings with metribuzin and pretilachlor near the recommended field-dose resulted in differential reductions in shoot fresh and dry weights during the following 16 days. Metribuzin showed great and consistent reductions, however, the reduction induced by pretilachlor, mostly nullified by the end of the experiment. Moreover, there were differential accumulations of lipid peroxides, carbonyl groups and H₂O₂ in maize leaves; metribuzin caused the greatest accumulation. Meanwhile, levels of thiol forms and reduced glutathione (GSH) were much more induced by pretilachlor than metribuzin; the contrary was true regarding oxidized glutathione (GSSG). The ratio of GSH/GSSG was highest following pretilachlor treatment and least by metribuzin. On the other hand, activities of glutathione-S-transferases (GSTs, EC 2.5.1.18), γ-glutamyl-cysteine synthetase (γ-GCS, EC 6.3.2.2), glutathione synthetase (GS, EC 6.3.2.3), glutathione peroxidase (GPX, EC 1.15.1.1) and glutathione reductase (GR, EC 1.6.4.2) were more enhanced in maize leaves by pretilachlor than metribuzin. These findings suggest the occurrence of an oxidative stress differentially induced in maize by the herbicides, a state that was most pronounced with metribuzin. Pretilachlor was concluded to be the least phytotoxic to maize, while metribuzin was the most, this differential tolerance seemed to be related to the induction of GSH and GSH-associated enzymes.     

Phytochelatin synthesis in maize seedlings in response to excess zinc

Buthionine sulfoximine (BSO) specifically inhibits γ-glutamylcysteine synthetase and decreases a cellular level of glutathione (GSH) in maize seedling roots. Exogenous GSH restores Zn-phytochelatins synthesis in BSO-treated maize plants.     

Nitric oxide alleviates manganese toxicity by preventing oxidative stress in excised rice leaves

In the present study, we have investigated the effects of nitric oxide (NO) on alleviating manganese (Mn)-induced oxidative stress in rice leaves. Exogenous MnCl₂ treatment to excised rice leaves for 24 and 48 h resulted in increased production of H₂O₂ and lipid peroxides, decline in the levels of antioxidants, glutathione and ascorbic acid, and increased activities of antioxidative enzymes, superoxide dismutase, guaiacol peroxidase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Treatment of rice leaves with 100 μM sodium nitroprusside (SNP), a NO donor, was effective in reducing Mn-induced increased levels of H₂O₂, lipid peroxides and increased activities of antioxidative enzymes. The levels of reduced ascorbate and glutathione were considerably recovered due to SNP treatment. The effect of SNP was reversed by the addition of NO scavenger, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO) suggesting that ameliorating effect of SNP is due to release of NO. The results indicate that MnCl₂ induces oxidative stress in excised rice leaves, lowers the levels of reduced ascorbate and glutathione, and elevates activities of the key antioxidative enzymes. NO appears to provide a protection to the rice leaves against Mn-induced oxidative stress and that exogenous NO application could be advantageous in combating the deleterious effects of Mn-toxicity in rice plants.     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

Accumulation of maize γ-zein and γ-zein: KDEL to high levels in tobacco leaves and differential increase of BiP synthesis in transformants

Two gene constructs (pROK.TG1L and pROK.TG1LK) were utilized to achieve accumulation of maize γ-zein to high levels in tobacco (Nicotiana tabacum L.) leaves. Both the chimaeric genes contained the γ-zein-coding region preceded by the 5′untranslated leader from the coat protein mRNA of TMV, but one of them (pROK.TG1LK) was modified in its protein-coding region by the addition of the ER retention signal KDEL. The accumulation of γ-zein and γ-zein:KDEL in leaves was compared with heterologous protein accumulation in tobacco plants previously transformed with a γ-zein cDNA harbouring a native 5′UTR. Replacement of γ-zein 5′UTR with the TMV leader dramatically increased γ-zein production. Furthermore, γ-zein:KDEL-expressing plants, on average, accumulated twice as much foreign protein in their leaves as pROK.TG1L plants. The two-fold increase in the level of γ-zein:KDEL can probably be attributed to an improvement in the mechanism for ER retention of zeins in the transgenic cells. Transformants also showed increased production of BiP, though to a lesser extent in γ-zein:KDEL-expressing plants compared with pROK.TG1L plants. It is therefore likely that γ-zein:KDEL retention is made less dependent on the chaperone assistance of BiP by the presence of the KDEL signal on the γ-zein mutant. Received: 15 October 1999 / Accepted: 28 February 2000     

Light-dependent modulation of foliar glutathione synthesis and associated amino acid metabolism in poplar overexpressing γ-glutamylcysteine synthetase

Glutathione (GSH), γ-glutamylcysteine (γ-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula × P. alba) and poplars expressing Escherichia coli genes for γ-glutamylcysteine synthetase (γ-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing γ-ECS, foliar γ-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of γ-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and γ-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate γ-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, γ-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, γ-EC was negligible in the light but increased in the dark. Cysteine content was little affected by light in any of the poplar types. No light-dependent changes in the extractable activities of γ-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing γ-ECS at elevated CO₂ or low O₂ largely abolished the inverse light-dependent changes in γ-EC and GSH. Low O₂ did not affect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for γ-EC synthesis is augmented. Received: 17 December 1996 / Accepted: 28 January 1997     

Stress responses of wheat leaves to dehydration: Participation of endogenous NO and effect of sodium nitroprusside

Dynamics of endogenous NO in the leaves of 7-day-old seedlings of spring wheat (Triticum aestivum L., cv. Debyut) and the effect of exogenous NO donor (sodium nitroprusside, SNP) on the development of oxidative stress and activity of antioxidant enzymes in the leaves under water deficiency were investigated. Quick and phasic accumulation of NO in the leaves was observed under growing dehydration (0–3 h) and subsequent rehydration (0–3 h), which points to identical response of NO signal system to opposite changes in the water status of plants. A decrease in relative turgidity of tissues brought about accumulation of H₂O₂ and MDA therein. Protective effect of NO donor infiltrated in the leaves was associated with an elevation of ascorbate peroxidase and catalase activities and suppression of lipid peroxidation upon dehydration. Pretreatment with SNP (50–250 μM) induced the elevation of NO level in the leaves both before action of the stress agent and in the beginning (0–30 min) of dehydration. The obtained results suggest that brief increase in endogenous NO is necessary for triggering protective and adaptive responses in wheat plants during the development of water deficiency.     

Carboxin-Resistant Mutant of Ustilago maydis Is Impaired in Its Pathogenicity for Zea mays

We analyzed the pathogenicity of chitin synthetase (chs) disruptants of Ustilago maydis obtained with the carboxin-resistant or the hygromycin-resistant cassettes. We found that only chitin synthetase (chs) mutants obtained by gene disruption with the carboxin resistance cassette lost their virulence to maize (Zea mays) seedlings. Carboxin is a systemic fungicide that inhibits respiration by preventing the oxidation of succinate. We demonstrated that carboxin-resistant transformants were affected in the levels of succinate dehydrogenase and respiratory activities when compared with hygromycin-resistant disruptants. We propose that loss of virulence in the carboxin-resistant transformants is owing to loss of respiratory fitness, which probably represents an important component of virulence in this fungus. Received: 13 May 1999 / Accepted: 26 June 1999     

Nitric oxide counteracts cytotoxic processes mediated by reactive oxygen species in plant tissues

Many environmental conditions subject plants to oxidative stress, in which reactive oxygen species (ROS) are overproduced. These ROS act as transduction signals in plant defense responses, but also cause effects that result in cellular damage. Since nitric oxide (NO) is a bioactive molecule able to scavenge ROS, we analyzed its effect on some cytotoxic processes produced by ROS in potato (Solanum tuberosum L. cv. Pampeana) leaves. Two NO donors: (i) sodium nitroprusside and (ii) a mixed solution of ascorbic acid and NaNO₂, were able to prevent chlorophyll loss mediated by the methyl viologen herbicide diquat (a ROS generator), with effective concentrations falling between 10 and 100 μM of the donors. This protection was mimicked by thiourea and penicillamine, two antioxidant compounds. Residual products from NO generation and decomposition failed to prevent chlorophyll decline. A specific NO scavenger, the potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), arrested NO-mediated chlorophyll protection. In addition, some events mediated by ROS during infection of potato leaves with Phytophthora infestans (race 1, 4, 7, 8, 10, 11, mating type A2) were also examined. In this sense, NO proved to markedly decrease ion leakage and the number of lesions, indicative of cell death, produced upon infection in potato leaves. The NO-mediated decrease in ion leakage was also inhibited by carboxy-PTIO. Fragmentation of DNA diminished when P. infestans-infected potato leaves were treated with 100 μM SNP. These results suggest that, acting as an antioxidant, NO can strongly counteract many ROS-mediated cytotoxic processes in plants. Moreover, the evidence of NO functionality in the plant kingdom is strengthened by this work. Received: 18 December 1998 / Accepted: 19 January 1999     

Influence of GA3, sucrose and solid medium/bioreactor culture on in vitro flowering of Spathiphyllum and association of glutathione metabolism

Hormonal control of flower induction and inflorescence development in vitro was investigated in Spathiphyllum. The effects of gibberellic acid (GA₃) and sucrose on inflorescence development were studied in plantlets regenerated in tissue culture. GA₃ was mandatory for the shift from the vegetative to the reproductive stage. The effect of sucrose concentration on inflorescence bud development was studied in plantlets cultured in MS medium supplemented with 10 mg l⁻¹ GA₃. Sucrose concentration at 3 or 6% induced inflorescence development in, respectively, 83–85% of the plantlets. The effect of GA₃ and sucrose on inflorescence differentiation and development were also recorded in liquid culture using air-lift bioreactor. The best response was found in the same medium which was standardized as an optimum for solid culture, but the results were better than solid culture. In order to study the relationship between glutathione (GSH) and flowering, we also measured the oxidized and reduced GSH content in leaves throughout the culture period on 2 weeks interval. The GSH accumulation was more after 4 weeks until 6 weeks in GA₃ treated plantlets. Similarly, glutathione reductase which is involved in the recycling of reduced GSH providing a constant intracellular level of GSH, was also higher in GA₃ treated plantlets. The transient increase in GSH contents also correlated with the changes in measured γ-glutamylcysteine synthetase (γ-ECS) activity over the same period. The antioxidant enzyme activity in GA₃ treated plantlets also suggests that the plants suffered increased oxidative stress during the period of GA₃ treatment which subsequently increases GSH synthesis through activation of γ-ECS and this promotes flowering by increasing endogenous GSH.     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Expression of maize γ-zein and β-zein genes in transgenic Nicotiana tabacum and Lotus corniculatus

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

Cadmium toxicity is reduced by nitric oxide in rice leaves

We evaluate the protective effect of nitric oxide (NO) against Cadmium (Cd) toxicity in rice leaves. Cd toxicity of rice leaves was determined by the decrease of chlorophyll and protein contents. CdCl₂ treatment resulted in (1) increase in Cd content, (2) induction of Cd toxicity, (3) increase in H₂O₂ and malondialdehyde (MDA) contents, (4) decrease in reduced form glutathione (GSH) and ascorbic acid (ASC) contents, and (5) increase in the specific activities of antioxidant enzymes (superoxide dismutase, glutathione reductase, ascorbate peroxidase, catalase, and peroxidase). NO donors [N-tert-butyl-α-phenylnitrone, 3-morpholinosydonimine, sodium nitroprusside (SNP), and ASC + NaNO₂] were effective in reducing CdCl₂-induced toxicity and CdCl₂-increased MDA content. SNP prevented CdCl₂-induced increase in the contents of H₂O₂ and MDA, decrease in the contents of GSH and ASC, and increase in the specific activities of antioxidant enzymes. SNP also prevented CdCl₂-induced accumulation of NH₄ ⁺, decrease in the activity of glutamine synthetase (GS), and increase in the specific activity of phenylalanine ammonia-lyase (PAL). The protective effect of SNP on CdCl₂-induced toxicity, CdCl₂-increased H₂O₂, NH₄ ⁺, and MDA contents, CdCl₂-decreased GSH and ASC, CdCl₂-increased specific activities of antioxidant enzymes and PAL, and CdCl₂-decreased activity of GS were reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that protective effect by SNP is attributable to NO released. Reduction of CdCl₂-induced toxicity by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H₂O₂.     

Alterations in hepatic metabolism of sulfur-containing amino acids induced by ethanol in rats

Summary.  Alterations in hepatic metabolism of S-amino acids were monitored over one week in male rats treated with a single dose of ethanol (3 g/kg, ip). Methionine and S-adenosylhomocysteine concentrations were increased rapidly, but S-adenosylmethionine, cysteine, and glutathione (GSH) decreased following ethanol administration. Activities of methionine adenosyltransferase, cystathionine γ-lyase and cystathionine β-synthase were all inhibited. γ-Glutamylcysteine synthetase activity was increased from t = 8 hr, but GSH level did not return to control for 24 hr. Hepatic hypotaurine and taurine levels were elevated immediately, but reduced below control in 18 hr. Changes in serum and urinary taurine levels were consistent with results observed in liver. Cysteine dioxygenase activity was increased rapidly, but declined from t = 24 hr. The results show that a single dose of ethanol induces profound changes in hepatic S-amino acid metabolism, some of which persist for several days. Ethanol not only inhibits the cysteine synthesis but suppresses the cysteine availability further by enhancing its irreversible catabolism to taurine, which would play a significant role in the depletion of hepatic GSH. Received April 26, 2002 Accepted June 12, 2002 Published online October 14, 2002 Authors' address: Young C. Kim, Ph.D., Professor of Toxicology, College of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Ku, Seoul, Korea, Fax: +82-2-872-1795, E-mail: youckim@snu.ac.kr Abbreviations: CβS, cystathionine β-synthase; CDC, cysteine sulfinate decarboxylase; CDO, cysteine dioxygenase; CγL, cystathionine γ-lyase; GCS, γ-Glutamylcysteine synthetase; GSH, glutathione; MAT, methionine adenosyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.