Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings

Cysteine, γ-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, γ-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl₂ concentrations up to 200 micromolar. At 50 micromolar Cd²⁺, γ-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd²⁺, the concentration of γ-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd²⁺ concentrations, a significant increase in γ-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd²⁺. Labeling of isolated roots with [³⁵S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of γ-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd.     

外源NO对盐胁迫下黑麦草幼苗活性氧代谢、多胺含量和光合作用的影响

采用溶液培养法研究了外源一氧化氮(nitric oxide,NO)供体硝普钠(sodium nitroprusside,SNP)对100 mmol·L^-1 NaCl胁迫下黑麦草幼苗生长、活性氧代谢、多胺含量和光合作用的影响。结果表明,50 μmol·L^-1 SNP显著提高盐胁迫下黑麦草幼苗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性及谷胱甘肽(GSH)、精胺(Spm)、亚精胺(Spd)含量和(Spm+Spd)/Put比值,降低了腐胺(Put)、超氧阴离子(O^—·₂)、H₂O₂和丙二醛(MDA)含量,使幼苗叶片叶绿素和类胡萝卜素含量、净光合速率(P_n)和气孔导度(G_s)升高,胞间CO₂浓度(C_i)下降,相对生长量增加。叶绿素荧光动力学资料显示,SNP处理降低盐胁迫下黑麦草叶片的初始荧光(F₀),表明它对光合膜系统具有保护效应。SNP处理不仅提高了盐胁迫下叶片的最大荧光(F_m)、PSⅡ潜在光化学效率(F_v/F₀)和PSⅡ最大光化学效率(F_v/F_m),而且提高了PSⅡ实际光化学效率(Φ_PSⅡ)、光化学荧光猝灭系数(q_P)、表观光合电子传递速率(ETR)和光化学速率(PCR),降低了非光化学荧光猝灭系数(NPQ)和天线热耗散(D),而1 mmol·L^-1 NO清除剂PTIO和0.5 μmol·L^-1 NaNO₂处理(对照)则无此效应。由此表明,外源NO可能通过提高植株的抗氧化能力及光能的捕获与转换而增强盐胁迫下黑麦草叶片的光合能力,从而缓解盐胁迫对幼苗生长的抑制作用。     

Purification and properties of glutathione synthetase from spinach (Spinacia oleracea) leaves

Glutathione synthetase (γ-l-glutamyl-l-cysteine:glycine ligase [ADP-forming], EC 6.3.2.3) was partially-purified (100-fold) from spinach (Spinacia oleracea) leaves and its properties determined. At least part of the enzyme activity is localized in chloroplasts. The properties of the enzyme suggest that GSH synthesis would be facilitated at the pH and Mg²⁺ concentration in the stroma of illuminated chloroplasts, but glutathione synthetase does not appear to be ‘light-activated’ in isolated type A chloroplasts.     

Effects of a root-colonized dark septate endophyte on the glutathione metabolism in maize plants under cadmium stress

A high Cd-tolerant dark septate endophyte (DSE), Exophiala pisciphila, was inoculated into maize (Zea mays L.) roots under Cd stress. The Cd content, enzymes activity and thiol compound content relevant to glutathione (GSH) metabolism in maize leaves were analyzed. The Cd content in maize shoots increased with increasing Cd stress, but the DSE significantly reduced the Cd content at the 40?mg/kg Cd treatment. Cd stress increased the enzyme activity of glutathione reductase (GR), glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px) as well as the thiol compound contents of sulfur, thiols (-SH) and oxidized glutathione (GSSG). The content of reduced GSH and the GSH/GSSG ratio reached a peak at the 5?mg/kg Cd treatment but then decreased with increasing Cd stress. Furthermore, the DSE significantly enhanced the GR and GSH-Px activity and increased the contents of -SH and GSH under low Cd stress (5 and 10?mg/kg), but decreased the γ-glutamylcysteine synthetase and GST activity under high Cd stress (20 and 40?mg/kg). Highly positive correlations between the Cd content with enzymes activity and enzymes activity with thiol compound content were observed. Results indicated that DSE played a role in activating GSH metabolism in maize leaves under Cd stress.     

Lanthanum improves the cadmium tolerance of <Emphasis Type="Italic">Zea mays</Emphasis> seedlings by the regulation of ascorbate and glutathione metabolism

The effect of lanthanum on the metabolism of ascorbate (AsA) and glutathione (GSH) in the leaves of maize seedlings under cadmium stress was investigated. The findings showed that Cd remarkably increased electrolyte leakage (EL), the activities of ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase (MDHAR), glutathione reductase, L-galactono-1,4-lactone dehydrogenase, and γ-glutamylcysteine synthetase, and the content of reduced AsA, reduced GSH, total AsA, total GSH, malondialdehyde (MDA), and Cd, compared with control. However, Cd significantly decreased the dry biomass of roots and shoots. Treatment with La + Cd evidently increased the activities of above enzymes except MDHAR, the content of reduced AsA, reduced GSH, total AsA and total GSH, and the dry biomass of roots and shoots, compared with Cd stress alone. Meanwhile, treatment with La + Cd remarkably decreased EL and the content of Cd and MDA compared with Cd stress alone. Our results suggested that La could be used as a regulator to improve the Cd tolerance of maize for its role in the alleviation of Cd-induced oxidative damage by regulating the metabolism of AsA and GSH.     

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.     

A possible mechanism for the apocynin-induced nitric oxide accumulation in plants

Nitric oxide (NO) is a small, ubiquitous bioactive molecule, postulated as a broad spectrum anti-stress compound. The NADPH oxidase inhibitor apocynin induces the accumulation of endogenous NO in leaves of maize seedlings through a nitric oxide synthase (NOS)-like activity, and confers an augmented tolerance to UV-B-induced oxidative damage. Here we propose a mechanism for the apocynininduced NO increase in plants. NOS catalyzes the oxidation of arginine to citrulline and NO. It is suggested that apocynin inhibits arginase, the enzyme that hydrolyzes L-arginine to urea and L-ornithine, increasing the arginine availability for arginine-dependent NO synthesis. Superoxide (O₂⁻) is a strong NO scavenger due to its high reactivity with NO to give peroxynitrite (ONOO⁻). Superoxide is mainly produced by plant NADPH oxidase (pNOX). Inhibition of pNOX by apocynin at relatively high NO concentration, could reduces the formation of O₂⁻ and ONOO⁻, increasing the availability of a huge amount of NO. We consider apocynin as a very attractive compound for studying NO-regulated processes in plants since it can replace the use of NO donors and overcome the subsequent technical problems.Key words: apocynin, nitric oxide, NO, UV-B, oxidative stress, nitric oxide synthase, NOSNO is a small, highly diffusible atmospheric gas and a ubiquitous bioactive molecule, proposed as a broad spectrum anti-stress compound.¹ Because NO is a reactive gas with a short halflife in air, the vast majority of NO research in living organisms has involved application of NO donors. Floryszak and co workers² argue that although treating plant tissue with NO donors is a simple methodological approach, it has yielded some technical problems because the process of donor decomposition depends on numerous external factors. For instance, the mostly used NO donor, sodium nitropruside (SNP), is extremely photosensitive and its degradation is promoted also by oxygen and temperature.³ Nonreductive decomposition of S-nitrosothiols as S-nitrosoglutathione (GSNO) releases NO, but it is dependent on light, temperature and pH.⁴It was reported that the steady-state level of NO is increased by apocynin in human endothelial cells.^5,6 Apocynin (4-hydroxy-3-methoxyacetophenone,acetovanillone, CAS 498-02-2) is a methoxysubstituted catechol originally extracted from the roots of Picrorrhiza kurroa, a small perennial herb that grows in the Himalayas. Extracts of P. kurroa are used in traditional medicine for treating diseases associated with chronic inflammation.^7,8We have recently demonstrated that apocynin induces the dose-dependent accumulation of NO in leaves of maize seedlings through a nitric oxide synthase (NOS)-like activity. This NO production is bioactive and antioxidant since it confers an augmented tolerance to UV-B induced oxidative stress. Therefore, the use of apocynin as an alternative approach to study NO functionality in plants has been proposed.⁹ Here it is postulated that the apocynin-induced NO increase in plants is due to the confluence of at least two effects: the inhibition of arginase and NADPH oxidase.     

Enzymatic synthesis of glutathione using engineered Saccharomyces cerevisiae

Two single gene cassettes, each containing one of the individual gene (γ-glutamylcysteine synthetase gene GSH1 or glutathione synthetase gene GSH2), were constructed under the control of alcohol dehydrogenase (ADH1) promoter and their respective native terminators. The recombinant plasmids constructed with Kan ^r or Hyg ^r as the selective markers and were transformed into Saccharomyces cerevisiae separately and jointly. Three engineered strains, GSH1-enhanced strain S.TS013/GSH1, GSH2-enhanced strain S.TS013/GSH2 and GSH1+GSH2 double-enhanced strain S.TS013/GSH1+GSH2, were constructed. Glutathione production using the recombinant strains to improve was then determined. By the cell dosage proportions of two engineered strains (S.TS013/GSH1, S.TS013/GSH2) and a two-stage reaction, GSH productivity increased by 84 and 59 % over that of the host strain and the S.TS013/GSH1+GSH2 strain, respectively.     

Starch synthetases from Vitis vinifera and zea mays

ADPglucose: α-1,4-glucan α-4-glucosyltransferases (starch synthetases) from leaves of Vitis vinifera and leaves and kernels of Zea mays were chromatographed on DEAE-cellulose columns. One form of the enzyme was present in grape leaves having activity both in the presence and absence of primer. Two forms were present in both leaves and kernels of maize. The second peak of activity in maize leaves and the first peak in maize kernels synthesized a polyglucan in the absence of primer. A peak of branching enzyme (Q-enzyme) occurred between the two starch synthetase peaks with both tissues. When fractions containing starch synthetase and branching enzyme were added to the first leaf starch synthetase peak, up to 100-fold activation of the unprimed reaction occurred. Branching enzyme did not stimulate the unprimed activity of the first kernel peak and no branching enzyme could be detected in this peak. The unprimed product was a branched polyglucan with mainly α-1,4-links.     

Nitric oxide improves chilling tolerance of maize by affecting apoplastic antioxidative enzymes in leaves

The effects of nitric oxide (NO) on chilling tolerance (freezing injury, ice nucleation activity, contents of hydrogen peroxide and superoxide anion, and lipid peroxidation level) and the activities of apoplastic antioxidant enzymes (peroxidase and superoxide dismutase) were investigated in the leaves of maize (Zea mays) exposed to short-term chilling. NO treatment was carried out through spraying of sodium nitroprusside (SNP), which is a donor of NO, in concentrations of 0.0, 0.1 and 1 μM on the leaves of 10-day plants. The plants then were transferred into the chilling condition (10/7 °C) 2 days before the harvesting of leaves (14th and 21th days). Application of 0.1 μM NO had more effect on the alleviation by decreasing the freezing injury in maize at least for 11 days after the application. Both concentrations of NO generally increased ice nucleation activity of apoplastic proteins extracted from leaves. The SNP applications decreased the contents of reactive oxygen species such as hydrogen peroxide and superoxide anion and the level of lipid peroxidation, while further increasing the activities of the apoplastic antioxidant enzymes studied. The results show that exogenous NO treatment provides important contributions to increasing the chilling tolerance of maize by regulating the biochemical mechanisms of chilling response, including apoplastic antioxidant enzymes. It can be seen that the NO treatment can play positive roles in alleviating chilling-induced damage in maize. Therefore, it is suggested that NO treatments may contribute to research studies related to diminishing chilling-induced damage in agricultural applications.     

γ-Glutamylcysteinylserine — A New Homologue of Glutathione in Plants of the Family Poaceae*

In addition to glutathione (γ-GluCysGly), many species of the family Poaceae have another tripeptide which has the amino acid sequence γ-GluCysSer. This thiol was isolated from etiolated leaves of wheat (Triticum aestivum L. cv. Star). Its structure was elucidated by quantitative amino acid analysis after total hydrolysis and by partial hydrolysis with carboxypeptidase A and γ-glutamyltranspeptidase. The content of γ-GluCysSer in the leaves of T. aestivum is increased by incubation with sulfate and is severely diminished by incubation with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase. Oxidized γ-GluCysSer is reduced by yeast glutathione reductase with a rate somewhat lower than for glutathione, but the new tripeptide is not a substrate of glutathione-S-transferase from equine liver. Besides homoglutathione (γ-GluCysßAla), a tripeptide found in plants of the order Fabales, the tripeptide γ-GluCysSer is the second homologue of glutathione detected in plants.