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.     

Proline and betaine provide protection to antioxidant and methylglyoxal detoxification systems during cold stress in <Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze

The aim of this study was to monitor the influence of proline and betaine exposure on antioxidant and methylglyoxal (MG) detoxification system during cold stress in Camellia sinensis (L.) O. Kuntze. Cold stress enhanced MG and lipid peroxidation levels in tea bud (youngest topmost leaf). This increase was resisted upon the exposure of tea bud to proline and betaine. Exposure of tea bud with proline and betaine also help in maintaining thiol/disulfide ratio during cold stress. Proline exposure enhanced glutathione-S-transferase and glutathione reductase (GR) activity, while betaine exposure increased only GR activity during cold stress. Furthermore, effect of proline/betaine was studied on glyoxalase pathway enzymes that are involved in MG detoxification and comprise of two enzymes glyoxalase I and glyoxalase II. Both proline and betaine showed protective effect on glyoxalase I and activating effect on glyoxalase II during cold stress in tea bud. This investigation, therefore, suggest that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.     

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.     

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.     

Overexpression of Glyoxalase III gene in transgenic sugarcane confers enhanced performance under salinity stress

Journal of Plant Research - The glyoxalase pathway is a check point to monitor the elevation of methylglyoxal (MG) level in plants and is mediated by glyoxalase I (Gly I) and glyoxalase II (Gly II)...     

The critical role of S-lactoylglutathione formation during methylglyoxal detoxification in Escherichia coli

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.     

Catalysis and structural properties of Leishmania infantum glyoxalase II: trypanothione specificity and phylogeny

The glyoxalase pathway catalyzes the formation of d-lactate from methylglyoxal, a toxic byproduct of glycolysis. In trypanosomatids, trypanothione replaces glutathione in this pathway, making it a potential drug target, since its selective inhibition might increase methylglyoxal concentration in the parasites. Two glyoxalase II structures were solved. One with a bound spermidine molecule (1.8 A) and the other with d-lactate at the active site (1.9 A). The second structure was obtained by crystal soaking with the enzyme substrate (S)-d-lactoyltrypanothione. The overall structure of Leishmania infantum glyoxalase II is very similar to its human counterpart, with important differences at the substrate binding site. The crystal structure of L. infantum glyoxalase II is the first structure of this enzyme from trypanosomatids. The differential specificity of glyoxalase II toward glutathione and trypanothione moieties was revealed by differential substrate binding. Evolutionary analysis shows that trypanosomatid glyoxalases II diverged early from eukaryotic enzymes, being unrelated to prokaryotic proteins.     

2-Oxoaldehyde metabolism in microorganisms

The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes. The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate. The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase. The regulation of the yeast glyoxalase system was analyzed. The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones. The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied.     

Modification of the glyoxalase system in human HL60 promyelocytic leukaemia cells during differentiation to neutrophils in vitro

The glyoxalase system of human promyelocytic leukaemia HL60 cells was substantially modified during differentiation to neutrophils. The activity of glyoxalase I was decreased and the activity of glyoxalase II was markedly increased relative to the level in control HL60 promyelocytes. There was a decrease in the apparent maximum velocity, Vmax, of glyoxalase I, and an increase in the Vmax of glyoxalase II. The apparent Michaelis constants for both enzymes remained unchanged. The flux of intermediates metabolised via the glyoxalase system increased during differentiation, as judged by the formation of D-lactic acid, whereas the percentage of glucotriose metabolised via the glyoxalase system remained unchanged. The cellular concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, were markedly decreased during differentiation. The maturation of HL60 promyelocytes is associated with an increased ability to metabolise S-D-lactoylglutathione by glyoxalase II and a concomitant decrease in the mean intracellular concentrations of S-D-lactoylglutathione and methylglyoxal. The maintenance of a high concentration of S-D-lactoylglutathione in HL60 promyelocytes may be related to the status of the microtubular cytoskeleton, since S-D-lactoylglutathione potentiates the GTP-promoted assembly of microtubules.     

The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli

The glyoxalase I gene ( gloA ) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E . coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.     

Role of rpoS in the regulation of glyoxalase III in Escherichia coli

Methylglyoxal is an endogenous electrophile produced in Escherichia coli by the enzyme methylglyoxal synthase to limit the accumulation of phosphorylated sugars. In enteric bacteria methylglyoxal is detoxified by the glutathione-dependent glyoxalase I/II system, by glyoxalase III, and by aldehyde reductase and alcohol dehydrogenase. Here we demonstrate that glyoxalase III is a stationary-phase enzyme. Its activity reached a maximum at the entry into the stationary phase and remained high for at least 20 h. An rpoS- mutant displayed normal glyoxalase I and II activities but was unable to induce glyoxalase III in stationary phase. It thus appears that glyoxalase III is regulated by rpoS and might be important for survival of non-growing E. coli cultures.     

Oltipraz protects the passive smoke induced changes in renal glyoxalase system of rats

The present study was performed to investigate the effect of oltipraz on passive smoke-induced alteration in renal glyoxalase system of rats. Adult Sprague-Dawley rats were exposed daily to passive cigarette smoke in a whole-body exposure chamber 6 h per day for 2, 4 and 12 weeks. The animals being sacrificed after 2 and 12 weeks were maintained on control diet, powdered 4% Teklad rat chow (Harlan Teklad, Madison, WI, USA). The 4 weeks group was divided into three subgroups, one receiving control diet, other two receiving control diet supplemented with two doses of oltipraz (either 167 or 500 ppm), starting 1 week prior to initiation of smoke exposure until the end of the experiment. The activity of glyoxalase I was higher in animals exposed for 4 and 12 weeks of passive smoke than those exposed for 2 weeks. There was no significant difference between 4 and 12 weeks. Glyoxalase II activity was lower in animals exposed to passive smoke for 4 weeks than those exposed for 2 weeks. However, the activity approached the basal level after 12 weeks of exposure. Furthermore, oltipraz treatment maintained the activity of both glyoxalase closer to the basal levels.     

Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a small intracellular thiol molecule which is considered as a strong non-enzymatic antioxidant. Glutathione regulates multiple metabolic functions; for example, it protects membranes by maintaining the reduced state of both α-tocopherol and zeaxanthin, it prevents the oxidative denaturation of proteins under stress conditions by protecting their thiol groups, and it serves as a substrate for both glutathione peroxidase and glutathione S-transferase. By acting as a precursor of phytochelatins, GSH helps in the chelating of toxic metals/metalloids which are then transported and sequestered in the vacuole. The glyoxalase pathway (consisting of glyoxalase I and glyoxalase II enzymes) for detoxification of methylglyoxal, a cytotoxic molecule, also requires GSH in the first reaction step. For these reasons, much attention has recently been directed to elucidation of the role of this molecule in conferring tolerance to abiotic stress. Recently, this molecule has drawn much attention because of its interaction with other signaling molecules and phytohormones. In this review, we have discussed the recent progress in GSH biosynthesis, metabolism and its role in abiotic stress tolerance.     

Nitric oxide modulates antioxidant defense and the methylglyoxal detoxification system and reduces salinity-induced damage of wheat seedlings

The present study investigates the possible regulatory role of exogenous nitric oxide (NO) in antioxidant defense and methylglyoxal (MG) detoxification systems of wheat seedlings exposed to salt stress (150 and 300 mM NaCl, 4 days). Seedlings were pre-treated for 24 h with 1 mM sodium nitroprusside, a NO donor, and then subjected to salt stress. The ascorbate (AsA) content decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) and the GSH/GSSG ratio increased with an increase in the level of salt stress. The glutathione S-transferase (GST) activity increased significantly with severe salt stress (300 mM). The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glutathione peroxidase (GPX) activities did not show significant changes in response to salt stress. The glutathione reductase (GR), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, especially at 300 mM NaCl, with a concomitant increase in the H₂O₂ and lipid peroxidation levels. Exogenous NO pre-treatment of the seedlings had little influence on the non-enzymatic and enzymatic components compared to the seedlings of the untreated control. Further investigation revealed that NO pre-treatment had a synergistic effect; that is, the pre-treatment increased the AsA and GSH content and the GSH/GSSG ratio, as well as the activities of MDHAR, DHAR, GR, GST, GPX, Gly I, and Gly II in most of the seedlings subjected to salt stress. These results suggest that the exogenous application of NO rendered the plants more tolerant to salinity-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene.

Anaerobic glycerol fermentation by Escherichia coli strains expressing genes from the Klebsiella pneumoniae dha regulon showed that cell growth and 1,3-propanediol (1,3-PD) production are significantly inhibited when 5 g/L or higher of glycerol is initially present. One reason for this inhibition may be methylglyoxal (MG) accumulation. Assays of both intracellular and extracellular MG levels indicated an accumulation of MG in anaerobic glycerol fermentation of transgenic E. coli. Pseudomonas putida glyoxalase I was expressed in the transgenic E. coli to enhance MG detoxification. The activity of glyoxalase I in the transgenic E. coli with the P. putida glyoxalase I under anaerobic conditions was 12-fold higher than that in the control cells. Compared to the control cells, the transgenic cells with the P. putida glyoxalase I displayed a reduction of 35-43% in intracellular MG and a decrease of 30% in extracellular MG. These decreases were statistically significant (P>94). Furthermore, the expression of the P. putida glyoxalase I in the transgenic E. coli markedly improved cell growth and resulted in a 50% increase in 1,3-PD production.     

Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione

Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.     

Genome-wide analysis of rice and <Emphasis Type="BoldItalic">Arabidopsis</Emphasis> identifies two glyoxalase genes that are highly expressed in abiotic stresses

Glyoxalase pathway, ubiquitously found in all organisms from prokaryotes to eukaryotes, consists of glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, which detoxify a cytotoxic molecule, methylglyoxal (MG). Increase in MG has been correlated with various diseases in humans and different abiotic stresses in plants. We have previously shown that overproduction of GLY I and/or GLY II enzymes in transgenic plants provide tolerance towards salinity and heavy metal stresses. We have identified nineteen potential GLY I and four GLY II proteins in rice and twenty two GLY I and nine GLY II proteins in Arabidopsis. An analysis of complete set of genes coding for the glyoxalase proteins in these two genomes is presented, including classification and chromosomal distribution. Expression profiling of these genes has been performed in response to multiple abiotic stresses, in different tissues and during various stages of vegetative and reproductive development using publicly available databases (massively parallel signature sequencing and microarray). AtGLYI8, OsGLYI3, and OsGLYI10 expresses constitutively high in seeds while AtGLYI4, AtGLYI7, OsGLYI6, and OsGLYI11 are highly stress inducible. To complement this analyses, qRT-PCR is performed in two contrasting rice genotypes, i.e., IR64 and Pokkali where OsGLYI6 and OsGLYI11 are found to be highly stress inducible.     

Stress-inducible overexpression of <Emphasis Type="Italic">glyoxalase I</Emphasis> is preferable to its constitutive overexpression for abiotic stress tolerance in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis>

The glyoxalase system catalyzes the conversion of cytotoxic methylglyoxal to d-lactate via the intermediate S-d-lactoylglutathione. It comprises two enzymes, Glyoxalase I (Gly I) and Glyoxalase II (Gly II), and reduced glutathione which acts as a cofactor by anchoring the substrates in the active sites of the two enzymes. The overexpression of both Gly I and Gly II, either alone or in combination, has earlier been reported to confer tolerance to multiple abiotic stresses. In the present study, we sought to evaluate the consequences of constitutive and stress-induced overexpression of Gly I on the performance and productivity of plants. Towards this end, several Gly I transgenic Brassica juncea lines (designated as R and S lines) were generated in which the glyoxalase I (gly I) gene was expressed under the control of either a stress-inducible rd29A promoter or a constitutive CaMV 35S promoter. Both the R and S lines showed enhanced tolerance to salinity, heavy metal, and drought stress when compared to untransformed control plants. However, the S lines showed yield penalty under non-stress conditions while no such negative effect was observed in the R lines. Our results indicate that the overexpression of the gly I gene under the control of stress-inducible rd29A promoter is a better option for improving salt, drought and heavy metal stress tolerance in transgenic plants.     

The glutathione-dependent glyoxalase pathway in the yeast Saccharomyces cerevisiae

Glyoxalase I (EC 4.4.1.5), which catalyzes the reaction methylglyoxal + GSH leads to S-lactoylglutathione, is a ubiquitous enzyme for which no clear physiological function has been shown. In the yeast Saccharomyces cerevisiae, methylglyoxal may derive from the spontaneous decay of intracellular glyceraldehyde-3-P, which may accumulate during growth on glycerol as the carbon source. The half-life time for the triose phosphate was found to be 4.6 h under physiological conditions (pH 6.2, 0.05 M phosphate at 30 degrees C). Glyoxalase I is induced by growth on glycerol or by the addition of methylglyoxal to the growth medium. The enzyme is also subject to carbon catabolite repression. A mutant strain, fully defective in glyoxalase I and bearing only one nuclear mutation, was obtained. The strain, which is killed by exposure to glycerol, excretes methylglyoxal into the medium. Growth of the mutant on glucose as carbon source appears to be similar to that of the wild type strain. This investigation has clearly demonstrated a physiological role of glyoxalase I in a eucaryotic cell.