Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

Background and Aims

α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone.

Methods

α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties.

Key Results

The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population.

Conclusions

The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.     

Developmental Aspects of Detoxifying Enzymes in Fish (Salmo Iridaeus)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism.

A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development.

In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney.

No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.     

Expression of glyoxalase, glutathione peroxidase and glutathione S-transferase isoenzymes in different bovine tissues

(1) The tissue-specific expression of various glutathione-dependent enzymes, including glutathione S-transferase (GST), glutathione peroxidase and glyoxalase I, has been studied in bovine adrenals, brain, heart, kidney, liver, lung and spleen. Of the organs studied, liver was found to possess the greatest GST and glyoxalase I activity, and spleen the greatest glutathione peroxidase activity. The adrenals contained large amounts of these glutathione-dependent enzymes, but significant differences were observed between the cortex and medulla. (2) GST and glyoxalase I activity were isolated by S-hexylglutathione affinity chromatography. Glyoxalase I was found in all the organs examined, but GST exhibited marked tissue-specific expression. (3) The alpha, mu and pi classes of GST (i.e., those that comprise respectively Ya/Yc, Yb/Yn and Yf subunits) were all identified in bovine tissues. However, the Ya and Yc subunits of the alpha class GST were not co-ordinately regulated nor were the Yb and Yn subunits of the mu class GST. (4) Bovine Ya subunits (25.5-25.7 kDa) were detected in the adrenal, liver and kidney, but not in brain, heart, lung or spleen. The Yc subunit (26.4 kDa) was expressed in all those organs which expressed the Ya subunit, but was also found in lung. The mu class Yb (27.0 kDa) and Yn (26.1 kDa) subunits were present in all organs; however, brain, lung and spleen contained significantly more Yn than Yb type subunits. The pi class Yf subunit (24.8 kDa) was detected in large amounts in the adrenals, brain, heart, lung and spleen, but not in kidney or liver. (5) Gradient affinity elution of S-hexylglutathione-Sepharose showed that the bovine proteins that bind to this matrix elute in the order Ya/Yc, Yf, Yb/Yn and glyoxalase I. (6) In conclusion, the present investigation has shown that bovine GST are much more complex than previously supposed; Asaoka (J. Biochem. 95 (1984) 685-696) reported the purification of mu class GST but neither alpha nor pi class GST were isolated.     

Green and blue light photoreceptors are involved in maintenance of dormancy in imbibed annual ryegrass (Lolium rigidum) seeds

Light plays an important role in two separate processes within the seeds of Lolium rigidum (annual ryegrass). Dormant seeds of L. rigidum remain dormant when imbibed in the light, but once seeds have lost dormancy through dark-stratification, light stimulates their germination. This study characterizes the light qualities and quantities which are effective in maintenance of dormancy. Dormant seeds were stratified under narrow- and broad-waveband light to identify the potential photoreceptors involved in dormancy maintenance, and to determine whether dark-induced dormancy loss is reversible by light. Blue and green light both mediated dormancy maintenance in a far-red-independent manner. Red light resulted in dormancy maintenance only when far-red wavelengths were excluded, suggesting a redundant function of phytochrome. At low fluence rates, white light was more effective than monochromatic light, suggesting the action of multiple photoreceptors in dormancy maintenance. By contrast, nondormant seeds did not germinate unless provided with red light. These results indicate that seed dormancy maintenance is potentially mediated through the actions of blue and green light photoreceptors. Seed dormancy could thus be added to the growing list of plant responses that may be mediated by green light in a cryptochrome-independent manner.     

Seasonal variability of detoxificant response and heavy metal accumulation in tissues of both sexes in <Emphasis Type="Italic">Tinca tinca</Emphasis> (L.) from Lake Trasimeno

Tinca tinca were sampled seasonally from Lake Trasimeno in order to investigate the influence of heavy metal accumulation and changing environmental parameters on the antioxidant responses in this species. Liver, gills and kidney of both sexes were analyzed for total glutathione, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I, glyoxalase II, while muscle was analyzed for arsenic, lead, cadmium and mercury. The MRLs (maximum residue limits) set by actual regulation were not exceeded for heavy metals, but a seasonal and sex-linked variability of antioxidant parameters was observed, and major variations occurred especially in summer and autumn. In this shallow lake, heavy metal accumulation did not affect the biochemical variations, which are rather due to the decreased water level inducing hyperoxia and hypoxia within 24 h, high temperature and loss of mating and refuge areas.     

Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells

Salt stress impairs reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems, and causes oxidative damage to plants. Up-regulation of the antioxidant and glyoxalase systems provides protection against NaCl-induced oxidative damage in plants. Thiol–disulfide contents, glutathione content and its associated enzyme activities involved in the antioxidant defense and glyoxalase systems, and protein carbonylation in tobacco Bright Yellow-2 cells grown in suspension culture were investigated to assess the protection offered by proline and glycinebetaine against salt stress. Salt stress increased protein carbonylation, contents of thiol, disulfide, reduced (GSH) and oxidized (GSSG) forms of glutathione, and the activity of glutathione-S-transferase and glyoxalase II enzymes, but decreased redox state of both thiol–disulfide and glutathione, and the activity of glutathione peroxidase and glyoxalase I enzymes involved in the ROS and MG detoxification systems. Exogenous application of proline or glycinebetaine resulted in a reduction of protein carbonylation, and in an increase in glutathione redox state and activity of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. Neither proline nor glycinebetaine, however, had any direct protective effect on NaCl-induced GSH-associated enzyme activities. The present study, therefore, suggests that both proline and glycinebetaine provide a protective action against NaCl-induced oxidative damage by reducing protein carbonylation, and enhancing antioxidant defense and MG detoxification systems.     

Salt- and glyphosate-induced increase in glyoxalase I activity in cell lines of groundnut (Arachis hypogaea)

Glyoxalase I (EC 4.4.1.5) activity has long been associated with rapid cell proliferation, but experimental evidence is forthcoming, linking its role to stress tolerance as well. Proliferative callus cultures of groundnut ( Arachis hypogaea L. cv. JL24) showed a 3.3-fold increase in glyoxalase I activity during the logarithmic growth phase, correlating well with the data on FW gain and mitotic index. Inhibition of cell division decreased glyoxalase I activity and vice versa, thus further corroborating its role as a cell division marker enzyme. Cell lines of A. hypogaea selected in the presence of high salt (NaCl) and herbicide (glyphosate) concentrations, yielded 4.2- to 4.5-fold and 3.9- to 4.6-fold elevated glyoxalase I activity, respectively, in a dose dependent manner reflective of the level of stress tolerance. The stress-induced increase in enzyme activity was also accompanied by an increase in the glutathione content. Exogenous supplementation of glutathione could partially alleviate the growth inhibition of callus cultures induced by methylglyoxal and d -isoascorbic acid, but failed to recover the loss in glyoxalase I activity due to d -isoascorbic acid. The adaptive significance of elevated glyoxalase I activity in maintaining glutathione homeostasis has been discussed in view of our understanding on the role of glutathione in the integration of cellular processes with plant growth and development under stress conditions.     

Methylglyoxal as a novel signal molecule induces the salt tolerance of wheat by regulating the glyoxalase system,the antioxidant system,and osmolytes

Reactive carbonyl species methylglyoxal (MG) has always been regarded as a cytotoxic metabolite, but now is emerging to function as signal molecule in plants. However, whether MG can induce salt tolerance is elusive. In this study, treatment of wheat seeds with NaCl reduced seed germination, plant height, root length, fresh weight, and dry weight, indicating the inhibitive effects of NaCl on seed germination and seedling growth. The inhibitive effects of NaCl were alleviated by applying exogenous MG, but aggravated by the MG scavenger N-acetyl-L-cysteine (NAC), suggesting that MG could induce the salt tolerance of wheat. In addition, MG increased glyoxalase I and glyoxalase II activities and decreased endogenous MG content in wheat seedlings under NaCl stress, whereas coapplication of NAC weakened glyoxalase activity and enhanced the endogenous MG level. Also, MG activated superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase activities; increased glutathione and ascorbic acid levels; and decreased superoxide radical production and H₂O₂ and malondialdehyde contents under NaCl stress, while NAC reversed these physiological parameters. Furthermore, MG also induced the accumulation of proline, glycine betaine, and soluble sugar under NaCl stress, whereas this accumulation was weakened by NAC. This work reported for the first time that MG could induce the salt tolerance of wheat, and the acquisition of this salt tolerance was involved in the activation of the glyoxalase system and antioxidant system, as well as the accumulation of osmolytes.     

Biochemical characterization of the antioxidant system in the scallop Adamussium colbecki, a sentinel organism for monitoring the Antarctic environment

 The scallop Adamussium colbecki can be profitably used for monitoring Antarctic coastal environments but its utility would be increased if chemical analyses of pollutants were integrated with data on their biological effects. Since oxidative stress is a common pathway of toxicity induced by xenobiotics, a preliminary biochemical characterization was carried out on the antioxidant system of this species and baseline data collected for future assessment of the anthropogenic impact in this remote area. The digestive gland and gills were investigated for levels of glutathione and the activity of several glutathione-dependent and antioxidant enzymes: gluthathione reductase, EC 1.6.4.2; glyoxalase I, EC 4.4.1.5; glyoxalase II, EC 3.1.2.6; gluthathione S-transferases, EC 2.5.1.18; Se-dependent, EC 1.11.1.9 and Se-independent, EC 2.5.1.18 gluta-thione peroxidases; catalase, EC 1.11.1.6; and super-oxide dismutase, EC 1.15.1.1. The same enzymatic activities were measured for comparison in the Mediterranean molluscs Mytilus galloprovincialis and Pecten jacobaeus. Very high levels of glutathione S-transferases were found in the digestive gland of both species of scallop compared to mussels, suggesting the importance of different feeding behaviour among these molluscs. However, catalase activity, much higher in Adamussium colbecki than in the Mediterranean molluscs, may represent a biochemical adaptation to the Antarctic marine environment with high levels of dissolved oxygen. Enzymes from the Antarctic species appeared to be generally more active at low temperatures but, with a few exceptions, their activities increased at higher temperatures. Received: 20 March 1996/Accepted: 29 May 1996     

Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc-spiked soils

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.     

Relative distribution of glutathione transferase, glyoxalase I and glyoxalase II in helminths

Glutathione transferase, glyoxalase I and glyoxalase II activities were not evenly distributed among the major helminth groups. Intestinal cestodes and digeneans had higher glutathione transferase activity than parasitic nematodes. High glyoxalase II activity was found in cestodes and digeneans but no glyoxalase I was detectable. Glyoxalase I and II were both detected in nematodes. These results are discussed in relation to the enzymes' suggested role in protection against secondary lipid peroxidation products.     

Oxidative Stress Tolerance Mechanism in Rice under Salinity

The research was conducted to investigate comparative oxidative damage including probable protective roles of antioxidant and glyoxalase systems in rice (Oryza sativa L.) seedlings under salinity stress. Seedlings of two rice genotypes: Pokkali (tolerant) and BRRI dhan28 (sensitive) were subjected to 8 dSm⁻¹ salinity stress for seven days in a hydroponic system. We observed significant variation between Pokkali and BRRI dhan28 in phenotypic, biochemical and molecular level under salinity stress. Carotenoid content, ion homeostasis, antioxidant enzymes, ascorbate and glutathione redox system and proline accumulation may help Pokkali to develop defense system during salinity stress. However, the activity antioxidant enzymes particularly superoxide dismutase (SOD), catalase (CAT) and non-chloroplastic peroxidase (POD) were observed significantly higher in Pokkali compared to salt-sensitive BRRI dhan28. Higher glyoxalase (Gly-I) and glyoxalase (Gly-II) activity might have also accompanied Pokkali genotype to reduce potential cytotoxic MG through non-toxic hydroxy acids conversion. However, the efficient antioxidants and glyoxalase system together increased adaptability in Pokkali during salinity stress.     

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.     

Cloning and characterization of glyoxalase I from soybean

Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No. X68819) resembling a GST being described as a glyoxalase I. To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures. When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or gamma-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean. When the clone X68819 was expressed in E. coli, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3. GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzymes are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%).     

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.     

Selenium Pretreatment Upregulates the Antioxidant Defense and Methylglyoxal Detoxification System and Confers Enhanced Tolerance to Drought Stress in Rapeseed Seedlings

In order to observe the possible regulatory role of selenium (Se) in relation to the changes in ascorbate (AsA) glutathione (GSH) levels and to the activities of antioxidant and glyoxalase pathway enzymes, rapeseed (Brassica napus) seedlings were grown in Petri dishes. A set of 10-day-old seedlings was pretreated with 25 μM Se (Sodium selenate) for 48 h. Two levels of drought stress (10% and 20% PEG) were imposed separately as well as on Se-pretreated seedlings, which were grown for another 48 h. Drought stress, at any level, caused a significant increase in GSH and glutathione disulfide (GSSG) content; however, the AsA content increased only under mild stress. The activity of ascorbate peroxidase (APX) was not affected by drought stress. The monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activity increased only under mild stress (10% PEG). The activity of dehydroascorbate reductase (DHAR), glutathione S-transferase (GST), glutathione peroxidase (GPX), and glyoxalase I (Gly I) activity significantly increased under any level of drought stress, while catalase (CAT) and glyoxalase II (Gly II) activity decreased. A sharp increase in hydrogen peroxide (H₂O₂) and lipid peroxidation (MDA content) was induced by drought stress. On the other hand, Se-pretreated seedlings exposed to drought stress showed a rise in AsA and GSH content, maintained a high GSH/GSSG ratio, and evidenced increased activities of APX, DHAR, MDHAR, GR, GST, GPX, CAT, Gly I, and Gly II as compared with the drought-stressed plants without Se. These seedlings showed a concomitant decrease in GSSG content, H₂O₂, and the level of lipid peroxidation. The results indicate that the exogenous application of Se increased the tolerance of the plants to drought-induced oxidative damage by enhancing their antioxidant defense and methylglyoxal detoxification systems.     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

Paclobutrazol mitigates salt stress in <Emphasis Type="Italic">indica</Emphasis> rice seedlings by enhancing glutathione metabolism and glyoxalase system

Stress-induced methylglyoxal (MG) functions as a toxic molecule, inhibiting plant physiological processes such as photosynthesis and antioxidant defense systems. In the present study, an attempt was made to investigate the MG detoxification through glutathione metabolism in indica rice [Oryza sativa L. ssp. indica cv. Pathumthani 1] under salt stress by exogenous foliar application of paclobutrazol (PBZ). Fourteen-day-old rice seedlings were pretreated with 15 mg L^?1 PBZ foliar spray. After 7 days, rice seedlings were subsequently exposed to 0 (control) or 150 mM NaCl (salt stress) for 12 days. Prolonged salt stress enhanced the production of MG molecules and the oxidation of proteins, leading to decreased activity of glyoxalase enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II). Consequently, the decreased glyoxalase activities were also associated with a decline in reduced glutathione (GSH) content and glutathione reductase (GR) activity. PBZ pretreatment of rice seedlings under salt stress significantly lowered MG production and protein oxidation, and increased the activities of both Gly I and Gly II. PBZ also increased GSH content and GR activity along with the up-regulation of glyoxalase enzymes, under salt stress. In summary, salinity induced a high level of MG and the associated oxidative damage, while PBZ application reduced the MG toxicity by up-regulating glyoxalase and glutathione defense system in rice seedlings.