Effect of heat shock on the metabolism of glutathione in maize roots

High performance liquid chromatography analyses revealed that glutathione (GSH) and cysteine are two of the major low molecular weight thiol compounds in maize root extracts. Treatment of maize roots to heat shock temperatures of 40°C resulted in a decrease of cysteine levels and an increase of GSH levels. Pulse labeling of maize roots with [³⁵S]cysteine showed that the rate of incorporation of ³⁵S into GSH or glutathione disulfide (GSSG) in heat shocked tissues was twice that in nonheat shocked tissues. In addition, extracts from heat shocked maize, barley, and soybean tissues contained an unidentified low molecular weight compound that increased from 1.2- to 8-fold within 2 hours of heat shock treatment depending on the tissue and plant involved. Our results indicate that during heat shock there is an increase in the activity of the GSH synthetizing capacity in maize root cells. The elevated synthesis of GSH may be related to the cells capacity to cope with heat stress conditions.     

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.     

Effect of pretreatment with hydrogen sulfide donor sodium hydrosulfide on heat tolerance in relation to antioxidant system in maize (Zea mays) seedlings

Hydrogen sulfide (H₂S) is a signal molecule that is involved in plant growth, development and the acquisition of stress tolerance including heat tolerance, but the mechanism of H₂S-induced heat tolerance is not completely clear. In present study, the effect of sodium hydrosulfide (NaHS), a H₂S donor, treatment on heat tolerance of maize seedlings in relation to antioxidant system was investigated. The results showed that NaHS treatment improved survival percentage of maize seedlings under heat stress in a concentration-dependent manner, indicating that H₂S treatment could improve heat tolerance of maize seedlings. To further study mechanism of NaHS-induced heat tolerance, catalase (CAT), guaiacol peroxidase (GPX), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX) activities, and glutathione (GSH) and ascorbic acid (AsA) contents in maize seedlings were determined. The results showed that NaHS treatment increased the activities of CAT, GPX, SOD and GR, and GSH and AsA contents as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH +GSSG)] in maize seedlings under normal culture conditions compared with the control. Under heat stress, antioxidant enzymes activities, antioxidants contents and the ratio of the reduced antioxidants to total antioxidants in control and treated seedlings all decreased, but NaHS-treated seedlings maintained higher antioxidant enzymes activities and antioxidants levels as well as the ratio of reduced antioxidants to total antioxidants. All of above-mentioned results suggested that NaHS treatment could improve heat tolerance of maize seedlings, and the acquisition of this heat tolerance may be relation to enhanced antioxidant system activity.     

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.     

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     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Effects of exogenous hydrogen sulfide on the redox states of ascorbate and glutathione in maize leaves under salt stress

This study investigated the effects of exogenous hydrogen sulfide (H₂S) on the redox states of ascorbate (AsA) and glutathione (GSH) in maize leaves under NaCl (100 mM) stress. Salt stress increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), Γ-glutamylcysteine synthetase (Γ-ECS), and L-galactono-1,4-lactone dehydrogenase (GalLDH), malondialdehyde content and electrolyte leakage, and reduced the ratios of reduced and oxidised forms of AsA (AsA/DHA) and GSH (GSH/GSSG) compared with control. Pretreatment with NaHS (H₂S donor) further enhanced the activities of the above enzymes except MDHAR and ameliorated the decrease in the ratios of AsA/DHA and GSH/GSSG compared with the salt stress alone. Pretreatment with NaHS significantly reduced the malondialdehyde content and electrolyte leakage induced by the salt stress. Pretreatment with NaHS alone did not affect any of the above mentioned parameters compared with the control. Our results suggest that exogenous H2S could maintain the redox states of ascorbate and glutathione by up-regulating the ascorbate and glutathione metabolism and thus play an important role for acquisition of salt stress tolerance in maize.     

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.     

Metal uptake,antioxidant status and membrane potential in maize roots exposed to cadmium and nickel

Root growth of the seedlings of maize cultivars Premia and Blitz exposed to 2 μM cadmium (Cd), nickel (Ni) or both metals acting simultaneously (Cd + Ni) for 72 h was significantly reduced but not ceased. The effect was more pronounced in the seedlings of the cv. Blitz. The heavy metals (HMs) contents increased significantly in the roots. Simultaneous application of metals had an antagonistic effect on either Cd or Ni uptake in Premia but not in Blitz. In control roots the contents of ascorbic acid (AsA) and dehydroascorbic acid (DHA) were lower and gluthatione (GSH) content was higher in Premia than in Blitz. A decrease of AsA content was induced by all metal treatments in Premia but only by Cd + Ni in Blitz while an increase was induced by single metals in this cultivar. All metal treatments increased DHA contents in both cultivars. GSH content decreased significantly in Premia treated with Cd or Cd + Ni, and in Blitz treated with Ni. Unlike the contents of AsA, DHA and GSH, the increased metal concentrations in root cells did not affect the membrane potential (E _M). The changes in antioxidant contents depended on both, maize genotypes and HMs treatments. Nevertheless, the results indicated a role of antioxidative system in minimizing the effects of oxidative stress and protecting cell membranes in both maize cultivars.     

Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family

We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the version reported by Moore et al. [Moore, R. E., Davies, M. S., O'Connell, K. M., Harding, E. I., Wiegand, R. C., and Tiemeier, D. C. (1986) Nucleic Acids Res. 14:7227-7235] in eight reading frame shifts which result in partial amino acid sequence conservation with the rat GSH S-transferase sequences. The GST I and GST III sequences share approximately 45% amino acid sequence homology. Both the GST I and the GST III mRNAs contain different repeating motifs in front of the initiation codon ATG. Multiple poly(A) addition sites have been identified for these two classes of maize GSH S-transferase messages. Genomic Southern blotting results suggest that both GST I and GST III are present in single or low copies in the maize (GT112 RfRf) genome.     

Influence of glutathione chemical effectors in the response of maize to arsenic exposure

To support the key role of glutathione (GSH) in the mechanisms of tolerance and accumulation of arsenic in plants, this work examines the impact of several effectors of GSH synthesis or action in the response of maize (Zea mays L.) to arsenic. Maize was exposed in hydroponics to iso-toxic rates of 150 μM arsenate or 75 μM arsenite for 9 days and GSH effectors, flurazole (an herbicide safener), l-buthionine-sulfoximine (BSO, a known inhibitor of GSH biosynthesis), and dimercaptosuccinate (DMS) and dimercaptopropanesulfonate (DMPS) (two thiols able to displace GSH from arsenite-GSH complexes) were assayed. The main responses of plants to arsenic exposure consisted of a biomass reduction (fresh weight basis) of about 50%, an increase of non-protein thiol (NPTs) levels (especially in the GSH precursor γ-glutamylcysteine and the phytochelatins PC? and PC?) in roots, with little effect in shoots, and an accumulation of between 600 and 1000 ppm of As (dry weight basis) in roots with very little translocation to shoots. Growth inhibition caused by arsenic was partially or completely reversed in plants co-treated with flurazole and arsenate or arsenite, respectively, highly exacerbated in plants co-treated with BSO, and not modified in plants co-treated with DMS or DMPS. These responses correlated well with an increase of both NPTs levels in roots and glutathione transferase activity in roots and shoots due to flurazole treatment, the decrease of NPTs levels in roots caused by BSO and the lack of effect on NPT levels caused by both DMS and DMPS. Regarding to arsenic accumulation in roots, it was not modified by flurazole, highly reduced by BSO, and increased between 2.5- and 4.0-fold by DMS and DMPS. Therefore, tolerance and accumulation of arsenic by maize could be manipulated pharmacologically by chemical effectors of GSH.     

Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants

The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [³⁵S]Cys injected into endosperms of seedlings was partly metabolized to [³⁵S]sulfate. The scutella absorbed both [³⁵S]sulfate and [³⁵S]Cys and transformed 68 to 87% of the radioactivity into [³⁵S]GSH. [³⁵S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.     

Optimization of Callus Induction Conditions from Immature Embryos in Maize and Plant Regeneration

This research uses the immature embryos of inbred maize lines (GSH9901, Hi01, Hi02, and Chang 7-2) as receptor materials to establish the callus induction system. These inbred lines provide the receptor materials for the genetic regeneration of maize and the verification of the genetic functions of maize. The factor experiment and orthogonal experiments were used to investigate the impacts of different genotypes, immature embryo size, shield orientation, 2, 4-D concentration, proline concentration, and folic acid concentration on the induction rate of embryogenic callus tissue. A sensitivity experiment testing glyphosate (Bar) and an antibiotic (Cefotaxime sodium) were also conducted. The results indicate that the immature embryos of inbred maize line GSH9901 were the most effective for callus tissue induction, and the immature embryos with a length of 1.6-2.0 mm produce the best result. The upward shield face is more successful for the formation of induced callus. Using orthogonal analysis, we found that the optimal combination for the induction system was A₃ (2,4-D concentration 0.25 mg mL^-1 ), B₁C₃ (proline concentration 0.8 mg mL^-1 ), and D₂ (folate Concentration 0.5 mg mL^-1) and the induction rate reached 84%. We found that cold storage at 4 °C for 1 d is more conducive for the formation of embryogenic callus than the other treatments tested. The sensitivity experiment for callus tissue screening revealed the critical concentration of glyphosate to be 10 mg ml^-1 , and the critical concentration of antibiotic is 250 mg ml^-1 . Using this combination of glyphosate and antibiotic resulted in regenerated plants. This study established the optimal conditions for immature embryo callus tissue induction in maize.     

Heavy metal stress and sulfate uptake in maize roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.     

Can antioxidant’s reactive oxygen species (ROS) scavenging capacity contribute to aged seed recovery? Contrasting effect of melatonin,ascorbate and glutathione on germination ability of aged maize seeds

It is well known that antioxidants such as AA (reduced ascorbate), glutathione (GSH) (reduced glutathione) and melatonin can delay seed ageing. Can they recover aged seed? Artificial aged maize seeds were obtained and their reduced germination rate (GR) and high lipid peroxidation were recorded. Exogenous melatonin was applied on these aged seeds and enhanced GR was observed. However, treatment with other antioxidants such as AA, GSH or DMTU (dimethyl thiourea) did not significantly improve or even reduce the GR of aged seeds. In addition, melatonin improved germination ability of theses aged seeds can be significantly impaired by DDC (diethyldithiocarbamic acid, a specific inhibitor of superoxide dismutase or superoxide dismutase (SOD)) and ATZ (aminotriazol, a specific inhibitor of catalase or CAT). In a further study, we found that melatonin but not other antioxidants (AA, GSH and DMTU) significantly induced CAT and SOD activities of aged seeds after imbibition. Accordingly, melatonin significantly reduced lipid peroxidation in aged seeds than that of other antioxidants. Taken together, these data suggest that melatonin induced antioxidant enzyme but not its direct reactive oxygen species (ROS) scavenging capacity contributing to recovery of aged maize seeds.     

Role of Exogenous Glutathione in Alleviating Abiotic Stress in Maize (Zea mays L.)

Journal of Plant Growth Regulation - The role of exogenous GSH in improving abiotic stress tolerance in maize was investigated in this study. GSH-treated plants showed significantly higher...     

外源糖浸种缓解盐胁迫下玉米种子萌发

以玉米品种‘垦玉6号’为材料,在150 mmol·L^-1NaCl胁迫条件下,研究葡萄糖(Glc)和蔗糖(Suc)浸种对玉米种子萌发阶段耐盐性的影响.结果表明: 盐胁迫下,0.5 mmol·L^-1 Glc、Suc浸种可促进玉米种子萌发及幼苗早期生长,其中Glc浸种玉米胚芽和胚根长及相应干质量增加到盐处理的1.5、1.3、2.1、1.8倍;Suc浸种玉米分别增加到1.7、1.3、2.7、1.9倍;盐胁迫下Glc、Suc浸种可减少胚芽中硫代巴比妥酸反应物(TBARS)和过氧化氢(H2O2)含量,与盐处理相比分别降低24.9%、20.6%;Glc、Suc浸种可显著提高盐胁迫下玉米胚芽超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、谷胱甘肽过氧化物酶(GPX)、谷胱甘肽还原酶(GR)的活性,并诱导葡萄糖6 磷酸脱氢酶(G6PDH)活性的升高,其中Glc浸种玉米SOD、APX、GPX、GR、G6PDH活性较盐处理分别提高66.2%、62.9%、32.0%、38.5%、50.5%,Suc浸种玉米较盐处理分别提高67.5%、59.8%、30.0%、38.5%、50.4%;Glc、Suc浸种胚芽中抗坏血酸 (ASA)、谷胱甘肽(GSH)含量及ASA/DHA、GSH/GSSG显著提高,其中G6PDH活性与外源糖诱导的较强的抗氧化能力密切相关.Glc、Suc浸种还可提高盐胁迫下玉米胚芽中K⁺/Na⁺,分别为盐处理的2.3、2.4倍.外源 Glc、Suc浸种可通过提高玉米种子抗氧化能力及维持体内K⁺和Na⁺离子平衡缓解盐胁迫对玉米种子萌发的抑制效应.     

抗氧化系统在H2O2诱导的玉米幼苗耐热性形成中的作用

H2O2预处理可显著增强玉米幼苗的耐热性.H2O2预处理后,玉米幼苗抗氧化酶谷胱甘肽还原酶(GR)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)的活性及还原型抗氧化剂抗坏血酸(ASA)和谷胱甘肽(GSH)的水平显著提高,且H2O2预处理过的幼苗在高温处理期间及其后的恢复过程中均能保持相对较高的抗氧化酶活力和还原型/氧化型抗氧化剂比例.