N-Ethylmaleimide

Signaling molecule methylglyoxal ameliorates cadmium injury in wheat (Triticum aestivum L) by a coordinated induction of glutathione pool and glyoxalase system

A B S T R A C T
Methylglyoxal (MG) now is found to be an emerging signaling molecule. It can relieve the toxicity of cadmium (Cd), however its alleviating mechanism still remains unknown. In this study, compared with the Cd-stressed seedlings without MG treatment, MG treatment could stimulate the activities of glutathione reductase (GR) and gamma-glutamylcysteine synthetase (γ-ECS) in Cd-stressed wheat seedlings, which in turn induced an increase of reduced glutathione (GSH). Adversely, the activated enzymes related to GSH biosynthesis and increased GSH
were weakened by N-acetyl-L-cysteine (NAC, MG scavenger), 2,4-dihydroxy-benzylamine (DHBA) and 1,3-bis- chloroethyl-nitrosourea (BCNU, both are specific inhibitors of GR), buthionine sulfoximine (BSO, a specific in- hibitors of GSH biosynthesis), and N-ethylmaleimide (NEM, GSH scavenger), respectively. In addition, MG in- creased the activities of glyoxalase I (Gly I) and glyoxalase II (Gly II) in Cd-treated seedlings, followed by declining an increase in endogenous MG as comparision to Cd-stressed seedlings alone. On the contrary, the increased glyoxalase activity and decreased endogenous MG level were reversed by NAC and specific inhibitors of Gly I (isoascorbate, IAS; squaric acid, SA). Furthermore, MG alleviated an increase in hydrogen peroxide (H2O2) and malondialdehyde (MDA) in Cd-treated wheat seedlings. These results indicated that MG could al- leviate Cd toxicity and improve the growth of Cd-stressed wheat seedlings by a coordinated induction of glu- tathione pool and glyoxalase system.

1.Introduction
Cadmium (Cd) is a highly toxic and nonessential heavy metal for plants. Even at low concentrations, plant injury by Cd can be observed at molecular, subcellular, cellular, organic, and even the whole plant level (Liu et al., 2016; Rizwan et al., 2016; He et al., 2017; Huang et al., 2017). Exposure of plants to Cd stress, sophisticated changes can be detected at biochemical, physiological, and molecular levels. Cd stress can disturb stomata movement, transpiration, water uptake, nutrition homeostasis, respiration, and photosynthesis, finally leads to growth retardation and the loss of crop productivity (Liu et al., 2016; Rizwan et al., 2016; He et al., 2017; Huang et al., 2017). In addition, Cd is a potential threat to human health when it enters into the food chain. Cd has adversely effects on many systems such as urinary, respiratory, reproductive, and skeletal (Rizwan et al., 2016; He et al., 2017). Due to its stability, non-degradation, and toxicity, Cd pollution has attracted much attention in the fields of plant stress biology and environmental science (Liu et al., 2016; Rizwan et al., 2016; He et al., 2017; Huang et al., 2017). In general, Cd toxicity, similar to other abiotic stresses, usually leads to oxidative, methylglyoxal (MG) (one of the carbonyl stress), osmotic, ion, and nutrition stresses. Correspondingly, plants possess complexly and effectively adaptive mechanisms to Cd injury by stimulating the enhancement of reactive oxygen species (ROS) detoxification system (maintaining ROS homeostasis) and MG detoxification system (mainly glyoxalase system to remain MG at a low physiological level), osmor- egulation (accumulating osmolytes such as proline, glycine betaine, and soluble sugar, to maintain turgor pressure), and ion and nutrition homeostasis (regulating uptake and transport) (Liu et al., 2016; Rizwan et al., 2016; He et al., 2017; Huang et al., 2017).

During the process of plants respond and adapt to Cd stress, glutathione (GSH, γ-glutamyl- cysteinyl-glycine) plays a key role by directly and indirectly scavenging excessive ROS, regulating redox homeostasis, binding Cd directly, andsynthesizing phytochelatins and cysteine-rich proteins (Jozefczak et al., 2012; Hasanuzzaman et al., 2017a). In plants, GSH can be produced by de novo synthesis relying on both γ-glutamylcysteine synthetase (γ-ECS,a rate-limiting enzyme) and GSH synthetase, as well as the reduction ofoxidized glutathione (GSSG) by glutathione reductase (GR) (Jozefczak et al., 2012; Hasanuzzaman et al., 2017a). In pharmacological experi- ments, therefore, buthionine sulfoximine (BSO) and N-ethylmaleimide (NEM) are used as specific inhibitor of GSH biosynthesis and its sca- venger, respectively (Ma et al., 2007; Huang et al., 2008).Methylglyoxal (MG) is a highly reactive ketoaldehyde with α, β-carbonyl. It is one of the reactive carbonyl species (RCS). RCS, similar to ROS, commonly include MG, malondialdehyde (MDA), glyoxal (GO), 4-hydroxy-2-nonenal (HNE), and 2E-hexenal (HE) (Hossain et al., 2016; Li, 2016; Sankaranarayanan et al., 2017). MG is an inevitable side- product of photosynthesis and respiration (mainly glycolysis) in plants (Li, 2016). For a long time, MG is considered to be a cytotoxin, which can rapidly react with biomacromolecules (protein, DNA, and RNA) and biomembrane, producing advanced glycation end products (AGE). AGE can further disturb cell metabolism, and even leads to cell death (Hossain et al., 2016; Li, 2016; Sankaranarayanan et al., 2017).

Upon exposure to abiotic stress, plants can actively or passively accumulate1,3-bischloroethyl-nitrosourea (BCNU, a specific inhibitor of GR, Fitzgerald et al., 1991) + 700 μM MG (Cd+BCNU+MG), 150 μM Cd+ 700 μM NAC (Cd+NAC), 150 μM Cd + 500 μM DHBA, 150 μM Cd+ 500 μM BCNU; (2) 150 μM Cd + 500 μM N-ethylmaleimide (NEM, a GSH scavenger, Huang et al., 2008) + 700 μM MG (Cd+NEM+MG), 150 μM Cd + 500 μM buthionine sulfoximine (BSO, a specific inhibitor of GSH biosynthesis, Ma et al., 2007) + 700 μM MG (Cd+BSO+MG), 150 μM Cd + 500 μM NEM (Cd+NEM), 150 μM Cd + 500 μM BSO (Cd+BSO); (3) 150 μM Cd + 500 μM isoascorbate (ISA, a specific inhibitor of Gly I, Ramaswamy et al., 1984) + 700 μM MG (Cd+ISA+MG), 150 μM Cd + 500 μM squaric acid (SA, a specific inhibitor of Gly I, Ramaswamy et al., 1984) + 700 μM MG (Cd+SA+MG), 150 μM Cd + 500 μM ISA (Cd+ISA), 150 μM + 500 μM SA (Cd+SA). In experi-ments, the optimal concentrations of chemicals were chosen in the light of our previous study (Li et al., 2017b) and preliminary experiments. And then, the imbibed seeds were sequentially germinated and the seedlings were grown on t in a plant growth chamber with the same solution above, 100 μmol m−2 min−1, and 12 h photoperiod, as well asat 26 ℃ for 5 d. On the fifth day, the following parameters of seedlingswere measured, respectively.Recently, MG is found to be an emerging signaling molecule, which is involved in seed germination, pollen tube growth, reproduction, and stomata movement (Hossain et al., 2016; Li, 2016; Sankaranarayanan et al., 2017). Bless et al. (2017) reported that pretreatment with MG could promote seed germination and subsequent seedling growth of Brassica rapa L under zirconium stress. Our previous studies also showed that MG as signal molecule improved seed germination and subsequent seedling growth of wheat under NaCl stress. The acquisition of this salt tolerance was closely associated with MG and ROS detox- ification systems, as well as osmolytes (Li et al., 2017a). In addition, MG could alleviate Cd toxicity in wheat seedlings (Li et al., 2017b), how- ever the mechanism of which still remains unknown. Therefore, this study based on our previous research, using wheat seedlings as mate- rials, further illustrated the mechanism of MG-induced Cd tolerance, and the new viewpoint of MG as signal molecule was highlighted.

2.Materials and methods
Wheat seeds (Triticum aestivum L. cv. Yunmai 41) were sown on the cotton wool in culture vessels with the following solutions to imbibe and germinate after being sterilized according to previously described methods (Li et al., 2017b): (1) 0 (control), 150 μM CdCl2 (abbreviatedas Cd, the same as the following), 150 μM Cd + 700 μM MG (Cd+MG),150 μM Cd + 700 μM N-acetyl-L-cysteine (NAC, a MG scavenger, Dhar et al., 2010) + 700 μM MG (Cd+NAC+MG), 150 μM Cd + 500 μM2,4-dihydroxybenzylamine (DHBA, a specific inhibitor of GR, Fitzgerald et al., 1991) + 700 μM MG (Cd+DHBA+MG), 150 μM Cd + 500 μMand SA were assayed as per our previous methods (Li et al., 2017a, 2017b).Glutathione reductase (GR, EC 1.6.4.2) in wheat seedlings treated with Cd alone or in combination with MG, NAC, DHBA, and BCNU was extracted. Its activity was measured according to our previous methods (Li et al., 2013b) and expressed as μmol g−1 fresh weight (FW) min−1.A Gamma-glutamylcysteine synthetase (γ-ECS, EC 6.3.2.2) in wheat seedlings treated with Cd alone or in combination with MG, NAC,DHBA, and BCNU was extracted and its activity was assayed as per the methods of Shan et al. (2017). Wheat seedlings were ground in liquid nitrogen, and then extracted with 0.1 M Tris-HCl (pH8.0). The extract was centrifuged at 20,000×g for 15 min. The resulting supernatant was used for the assay of γ-ECS activity. The reaction mixture (1 mL) wascomposed of 0.1 M Tris-HCl (pH 8.0), 0.25 mM glutamate, 10 mM ATP,1 mM dithioerythritol, 2 mM cysteine, and 50 μL of supernatant. Re- action was started after adding supernatant and maintained at 25 °C for 1 h. Then one milliliter of phosphorus agent (3 mM H2SO4: distilledwater: 2.5% ammonium molybdate: 10% AsA = 1:2:1:1) was addedand mixed. The mixture was incubated at 45 °C for 30 min. The ab- sorbance at 660 nm was read.

The activity of γ-ECS was calculated using molar coefficient of 5.6 × 103 M−1 cm−1 and expressed as μmol g−1 FW min−1.GSH in wheat seedlings treated with Cd alone or in combination with MG, NAC, NEM, and BSO was extracted. Its content was de- termined according to the methods of Griffiths (Griffiths, 1980) and expressed as μmol g−1 FW.Gly I (EC 4.4.1.5) and Gly II (EC 3.1.2.6) in wheat seedlings treated with Cd alone or in combination with MG, NAC, IAS, and SA were extracted and their activities were determined as the proceduredescribed by Mostofa et al. (2015) and Hasanuzzaman et al. (2011). Gly I and Gly II activities were counted using the molar absorption coeffi- cient of 3.37 × 103 M−1 cm−1 (for S-D-lactoylglutathione) and 1.36 × 104 M−1 cm−1 (for 2-nitro-5-thiobenzoic acid), respectively, and ex- pressed as μmol g−1 FW min−1.MG in wheat seedlings treated with Cd alone or in combination with MG and NAC was extracted and its content was estimated according to the methods of Mostofa et al. (2015). The product (N-α-acetyl-S-(1- hydroxy-2-oxo- prop-1-yl) cysteine) that MG reacts with N-acetyl-L-cy- steine was recorded at 288 nm. MG content was calculated using the molar absorption coefficient of 249 M−1 cm−1 (for N-α-acetyl-S-(1-hydroxy −2-oxo-prop-1-yl) cysteine) and expressed as μmol g−1 FW.H2O2 and MDA in wheat seedlings treated with Cd alone or in combination with MG and NAC were extracted and their contents were determined based on the methods of Mostofa et al. (2014) and Li et al. (2013a), respectively. H2O2 and MDA contents were calculated using the molar absorption coefficient of 2.8 × 105 M−1 cm−1 (for H2O2-Ticompound) and 1.55× 105 M−1 cm−1 (for 3,5,5-Trimethylox-azolidine-2,4-dione), respectively, and expressed as nmol g−1 FW.The experiment was set up according to a completely randomized design at least three replications and two replications in each time. The data were processed statistically using software package SPSS version21.0 (SPSS, Chicago, USA) based on the analysis of variance (one-way ANOVA) and different letters indicate significant differences between treatments at P < 0.05. Figures were drawn by SigmaPlot 12.5 (Systat Software Inc., London, UK), data represented in Figures are means ± standard error (SE). 3.Results In order to study the effect of MG alone or in combination with NAC (MG scavenger), DHBA, and BCNU (the last two are specific inhibitors of GR) on the growth of wheat seedlings and the activities of GR and γ-ECS under Cd stress, the seedling growth parameters and GR and γ-ECSactivities were determined. The results showed that Cd stress led to growth retardation of wheat seedlings, as reflected in a decrease in Cd- stressed seedling height and root length, compared with the control seedlings without Cd stress (Fig. 1A). In addition, growth retardation of seedlings by Cd was ameliorated by exogenous MG, while MG sca- venger NAC and specific inhibitors of GR, DHBA and BCNU, sig- nificantly weakened the alleviating effect of MG (Fig. 1A). These results indicated that Cd was toxic to the growth of wheat seedlings, however this toxicity could be mitigated by MG but deteriorated by NAC, DHBA, and BCNU.Similarly, Cd stress increased the activities of GR and γ-ECS in both aboveground parts and roots of wheat seedlings, and this increase wasfurther enhanced by addition of exogenous MG (Figs. 1B, 2). The en- hanced GR activity by MG in both overground parts and roots was crippled by NAC, DHBA, and BCNU, respectively (Fig. 1B). In addition, the enhanced γ-ECS activity by MG in both acrial parts and roots was eliminated by NAC, DHBA (except that of roots), and BCNU, respec-tively (Fig. 2). These data suggested that the alleviating effect of MG on Cd toxicity might be achieved by a coordinated induction of GR and γ-ECS in wheat seedlings.To further explore the effect of MG alone or in combination with NAC (MG scavenger), NEM (GSH scavenger), and BOS (specific in- hibitor of GSH biosynthesis) on the growth of Cd-stressed wheat seed- lings and GSH content, the seedling growth parameters and GSH con- tent were assayed. As shown in Fig. 3, MG mitigated the inhibitive effect of Cd on the seedling height and root length of wheat seedlings. The effect of combination of MG and NAC, NEM or BSO on the seedling height and root length under Cd stress exhibited different effects. For root length, significant difference was observed in wheat seedlings treated with Cd+NAC+MG, Cd+NEM+MG, and Cd+BSO+MG, compared with the Cd+MG group. For seedling height, significant difference was not recorded in seedlings treated with above combina- tions (Fig. 2A). In addition, in comparison to the Cd-stressed seedlings alone, NEM and BSO markedly eliminated the increased seedling height and root length by MG under Cd stress (Fig. 2A).GR and γ-ECS activities were induced by MG in both abovegroundparts and roots of the Cd-stressed wheat seedlings (Figs. 1B, 2). Simi- larly, MG elevated the content of GSH in both acrial parts and roots of the Cd-stressed wheat seedlings (Fig. 3). This elevation was impaired by NAC, NEM, and BSO, while completely eliminated by the combination of Cd and NEM or BSO (Fig. 3), analogue to the change in the activities of GR and γ-ECS (Figs. 1B, 2). These experimental results implied thatMG could improve GSH content in the Cd-stressed wheat seedlings, thisimprovement might be a common outcome of activation of GR and γ- ECS by MG.In addition to GSH pool, to illustrate the effect of MG alone or in combination with NAC (MG scavenger), IAS, and SA (the last two are specific inhibitors of Gly I) on the growth of wheat seedlings and glyoxalase activity, the seedling height, root length, and Gly I and Gly II activities were measured. The results showed that wheat seedling growth, as indicated in seedling height and root length, had been in- hibited by Cd stress (Fig. 4A). The growth inhibition of seedlings was mitigated in Cd+MG, Cd+NAC+MG, Cd+IAS+MG, and Cd+SA+MG groups, especially Cd+MG (Fig. 4A). Under Cd stress, the alleviating effect of MG on growth was absolutely removed by IAS and SA, respectively, no significant difference compared with the seedlings treated with Cd (Fig. 4A). These data demonstrated that MG could al- leviate the toxicity of Cd in wheat seedlings, and this alleviation was eliminated by Gly I inhibitors, IAS and SA. In the same way, Cd stress could activate the activities of Gly I and Gly II in both aboveground parts and roots (Figs. 4B, 5). This activation was further enhanced by MG, while weakened by MG scavenger NAC (Figs. 4B, 5). In addition, Gly I inhibitors, IAS and SA, eliminated the increased activity of Gly I by MG in both acrial parts and roots, sig- nificantly lower than that of the control (Fig. 4B). For Gly II, the effect of IAS and SA on its activity in both overground parts and roots was not significant difference compared with the seedlings treated with Cd alone (Fig. 5). These results showed that MG activated the glyoxalase activity in wheat seedlings under Cd stress, which probably connected with the acquisition of Cd tolerance.To further uncover the effect of Cd stress on oxidative and MG stresses in wheat seedlings and the alleviating effect of MG, the contents of H2O2, MDA, and endogenous MG in wheat seedlings were detected. The results as indicated in Figs. 6 and 7, in comparison to the control, Cd stress induced the oxidative stress (as shown in an increase in H2O2 and MDA, both increased by approximately 2.2-fold) and MG stress (as reflected in a 2.5-fold increase in MG) in both leaves and roots. In ad- dition, the Cd-induced oxidative and MG stresses were ameliorated by exogenous MG treatment in both acrial parts and roots (Figs. 6 and 7). Adversely, the ameliorating effect of MG on both oxidative and MG stresses were weakened by NAC, MG scavenger (Fig. 7). These experi- mental results indicated that Cd could induce oxidative and MG stresses in wheat seedlings, and these stresses were alleviated by exogenous MG, which might be one of the physiological basis for MG-induced Cd tolerance. 4.Discussion Heavy metal Cd stress usually leads to plant damage at anatomical, morphological, physiological, biochemical, and molecular level. Cd can disturb photosynthesis, respiration, water and nutrient uptake, nitrogen(N) and sulfur (S) metabolism, and antioxidant machinery in plants, thus resulting in chlorosis, browning, growth retardation, and even ultimate death of plants (Yannarelli et al., 2007; Liu et al., 2016; Rizwan et al., 2016; He et al., 2017; Huang et al., 2017). Among these injuries, growth retardation is a common morphological symptom (Liu et al., 2016; Huang et al., 2017). Curiously, Lin et al. (2007) reported that Cd at low concentrations in soil could stimulate the growth of wheat seedlings, as indicated by an increase in fresh weight (FW) and seedling height. This stimulatory effect may be associated with the enhancement of cell division and proliferation. Similar phenomena, that is, an increase in dry weight (DW) was observed by Stolt et al. (2003) in hydroponic experiments, but higher Cd concentrations sig- nificantly inhibited seedling growth. Our previous (Li et al., 2017b) and current studies (Figs. 1, 3, 4) showed that Cd stress led to a decrease in seedling height, root length, FW, and DW of wheat seedlings in a dose dependent fashion, suggesting that Cd was toxic to the seedling growth. Interestingly, the negative effects of Cd were obviously ameliorated by MG was weakened by NAC, MG scavenger (Li et al., 2017b; Figs. 1, 3, 4). These results implied that MG could alleviate the toxicity of Cd in wheat seedlings, and the concerning possible mechanisms were dis- cussed as follows. In plants, GR and γ-ECS are the key enzymes in GSH biosynthetic pathway (May et al., 1998; Noctor et al., 1998; Jozefczak et al., 2012; Hasanuzzaman et al., 2017a). The former catalyzes the reduction of NADPH as co-factor. The latter catalyzes the de novo synthesis of GSH, that is, glutamine and cysteine are converted into a dipeptide, γ-glu- tamylcysteine (γ-Glu-Cys), which in turn synthesizes GSH, a tripeptide (γ-Glu-Cys-Gly), by glutathione synthetase (May et al., 1998; Noctor et al., 1998; Jozefczak et al., 2012; Hasanuzzaman et al., 2017a). During the process of plants respond and adapt to abiotic stress, in- cluding Cd stress, GR and γ-ECS are commonly stimulated, followed by inducing the accumulation of GSH (May et al., 1998; Noctor et al., 1998; Jozefczak et al., 2012; Hasanuzzaman et al., 2017a). In wheat seedlings, a low concentration of Cd stress increased GR activity, GSH content, and GSH/GSSG ratios; decreased the accumulation of MDA in wheat leaves; which in turn improved the resistance of wheat seedlings to a higher concentration of Cd (Lin et al., 2007). In addition, the results of Yannarelli et al. (2007) demonstrated that Cd stress up-regulated the activity of GR with distinctive isoforms, and this up-regulation was closely associated with post-translational modification, which became a primary defense mechanism against Cd-induced oxidative stress in wheat roots. In this study, the activities of GR and γ-ECS in both aboveground parts and roots of wheat seedlings were activated by Cd stress, which were further enhanced by administration of exogenous MG (Figs. 1 and 2), ultimately increased the content of GSH (Fig. 3B) and improved the growth of wheat seedlings under Cd stress (Figs. 1A, 3A). In contrast, the above positive effects induced by MG were wea- kened by MG scavenger (NAC), specific inhibitors of GR (DHBA and BCNU), and a specific inhibitor of GSH biosynthesis (BSO) and GSH scavenger (NEM) (Figs. 1–3). In addition, Cd-induced oxidative, as reflected by an increase in H2O2 and MDA, was ameliorated by MG (Fig. 6). This amelioration might be involved in the enhancement of GSH pool by MG (Figs. 1–3). In addition to the alleviation of oxidative stress, GSH also takes part in the detoxification of excessive MG. Due to dual role of MG, that is, cytotoxin at high dosage and signaling molecule at low concentration, its level in plant cells is closely controlled by GSH and glyoxalase system (Li, 2016; Hasanuzzaman et al., 2017a; Sankaranarayanan et al., 2017). Excessive MG can rapidly react with protein, lipid, DNA, RNA, and biomembrane, leading to MG stress, similar to oxidative stress in- duced by ROS. Therefore, MG homeostasis in plant cells is very im- portant. Generally, plants dependent on Gly I and Gly II to maintain MG balance or fluctuates at a certain range. Glyoxalase system, consisting of Gly I, Gly II, and GSH, minutely regulates plant growth, development, reproduction, and the formation of abiotic tolerance (Li, 2016; Hasanuzzaman et al., 2017a, 2017b). Gly I catalyzes the reaction of GSH with MG and forms S-D-lactoylglutahione (SLG), which is hydro- lyzed by Gly II and regenerates GSH (Li, 2016; Hasanuzzaman et al., 2017a, 2017b). In mung bean (Vigna radiata L.) seedlings, GSH or proline pretreatment could activate the activities of Gly I and Gly II, followed by weakening an increase in endogenous MG induced by salt stress (Nahar et al., 2015). Similarly, in wheat seedlings, treatment with nitric oxide (NO) improved the tolerance of seedlings to NaCl stress via activating Gly I, Gly II, and GSH pool (Hasanuzzaman et al., 2011). In the same way, in wheat seedlings, our previous study also indicated that MG treatment could mitigate a decrease in seed germination and subsequent seedling growth of wheat under NaCl stress by enhancing the glyoxalase and antioxidant systems (Li et al., 2017a). In the current study, Gly I and Gly II activities were increased in both acrial parts and roots of wheat seedlings under Cd stress, which were further enhanced by exogenous MG, thus alleviating the accumulation of endogenous MG and improving the growth of wheat seedlings (Figs. 4B, 5, 7). However, the enhanced Gly I activity by MG was eliminated by its inhibitors, IAS and SA, which was not obvious effect on Gly II (Figs. 4B, 5), indicating that the glyoxalase system plays a very important role in MG-alleviated Cd toxicity in wheat seedlings. 5.Conclusion In summary, Cd stress caused oxidative and MG stresses (an increase in H2O2, MDA, and endogenous MG), as well as the growth inhibition (a decrease in seedling height and root length) of wheat seedlings (Fig. 8). These injuries were ameliorated by exogenous MG via mobilizing GSH pool (stimulating GR and γ-ECS activities and accumulating GSH) and activating glyoxalase system (Gly I and Gly II) in wheat seedlings (Fig. 8). This study demonstrated that MG could alleviate the toxicity of Cd N-Ethylmaleimide by a coordinated induction of GSH pool and glyoxalase system in wheat seedlings.