Most plants live in environments where they are constantly exposed to one or combinations of various abiotic stressors, such as extreme temperatures, salinity, drought, and excessive light, which can severely limit plant growth and development. For many important crop plants, exposure to stress(es) ultimately results in a considerable reduction in potential yields (Atkinson and Urwin, 2012). The interaction between abiotic stressors and plants is complex, eliciting multiple morphological, physiological, biochemical and molecular changes that can ultimately result in varying degrees of stress adaptation, enabling some plants to grow and develop under environmentally induced stress. Because of the number of metabolic pathways involved, and the compexity of their regulation, it is often difficult for researchers to identify the major regulatory components involved in the abiotic stress responses of plants (Sharma et al., 2013). Plants subjected to stress often produce toxic aldehydes (Hoque et al., 2012a,b,c; Hoque M.A. et al., 2012; Mano, 2012), of which methylglyoxal (CH₃COCHO; MG) is the most ubiquitous. The reactive alpha-ketoaldehyde MG is cytotoxic to plant cells at high cellular concentrations, but it may act as an important signaling molecule at low concentrations (Yadav et al., 2005a,b; Singla-Pareek et al., 2006; Hossain et al., 2009; Kaur et al., 2015a,b). MG is produced in plant cells as a result of glycolysis, and its celluar concentrations are maintained at very low levels in the absence of any environmental stress (Kaur et al., 2015b). However, in response to abiotic stressors celluar concentrations of MG rapidly increase (Yadav et al., 2005a,b). Accumulation of MG can disrupt the normal functioning of cells, leading to alterations in metabolic behavior and, in some instances, the death of plants (Hossain et al., 2011). The glyoxalase pathway has evolved to enable plants, and other organisms, to withstand the detrimental effects of MG overproduction, by limiting the accumulation of MG in the cells under stress (Singla-Pareek et al., 2006, 2008; Alvarez Viveros et al., 2013). MG and the glyoxalases are now considered as potential markers for evaluating plant abiotic stress tolerance (Hossain et al., 2009; Kaur et al., 2014a,b,c; Nahar et al., 2015a). Although significant progress has been made in investigating MG metabolism and toxicity in plants, the role of MG as a signaling molecule in stress responses and the acquisition of stress tolerance in plants still remain unclear. In this review, we will summarize recent findings regarding MG metabolism and the glyoxalase system in plants under abiotic stress, evaluate the concept of MG signaling, and discuss the importance of MG metabolism in modulating plant abiotic stress responses and tolerance.

In plant cells, the cytosol, chloroplasts and mitochondria are all considered to be potential sites of MG production. However, the specific rate and sites of MG production vary depending upon the cell or tissue type, the plant organ (e.g. leaves or roots), and the physiological state of the whole plant (Kaur et al., 2015a,b). Spontaneous production of MG occurs as a consequence of glycolysis, in metabolically active plant cells, from the reaction of the triose sugar phosphates glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), both of which are photosynthetic intermediates (Yadav et al., 2005a; Takagi et al., 2014; Kaur et al., 2015a,b). This reaction is considered to be the principal route for MG formation under normal physiological conditions (Figure 1). Triose phosphates are unstable metabolites and show a high tendency to release an α-carbonyl proton, producing an enediolate phosphate intermediate that has a relatively low energy barrier for the elimination of phosphate groups (Richard, 1984). Thus, MG is formed by the deprotonation followed by the spontaneous β-elimination of the phosphate groups of triose phosphates (Richard, 1993). The enzymatic formation of MG occurs through the triose phosphate isomerase (TPI) that hydrolyzes G3P and DHAP, and removes phosphate to yield MG (Phillips and Thornalley, 1993). MG may also be formed by Amadori rearrangement during production of a Schiff base, which involves the reaction of the aldehyde groups of sugars with free amino acids or the amino acids of proteins (Vistoli et al., 2013). Other possible sources for MG formation include the auto-oxidation of surgars, as well as the metabolism of acetone and aminoacetone (Kalapos, 1999), although there is little evidence that these routes occur in plants.

Methylglyoxal detoxification involves the conversion of MG to less toxic molecules, thus limiting its detrimental effects. The major route for MG detoxification in plants is the glyoxalase pathway, whose prescence was demonstrated in plants over 20 years ago (Norton et al., 1990; Maiti et al., 1997). In plant cells, the glyoxalase pathway is present in the cytosol and organelles, with high levels of glyoxalase enzyme activity found in chloroplasts and mitochondria (Yadav et al., 2008; Rabbani and Thornalley, 2012). There are two main enzymes associated with the glyoxalase pathway; glyoxalase I (Gly I; lactoylglutathione lyase; EC 4.4.1.5) and glyoxalase II (Gly II; hydroxyacylglutathione hydrolase; EC 3.1.2.6). These enzymes function in tandem to transform MG, and other 2-oxoaldehydes, to 2-hydroxyacids with the release of glutathione (GSH) (Thornalley, 1990). The detoxification of MG involves two irreversible reactions catalyzed by glyoxalases. The first step involves the reaction of MG with GSH, resulting in the formation of hemithioacetal that is then converted to S-D-lactoylglutathione (SLG) in a reaction catalyzed by Gly I. In the second step, which is catalyzed by Gly II, GSH is regenerated and D-lactate is formed by the hydrolysis of SLG (Figure 1). D-lactate, which is also considered a toxic compound if overaccumulated, is converted into pyruvate by D-lactate dehydrogenase (Engqvist et al., 2009; Wienstroer et al., 2012). Pyruvate, the major catabolic product of MG, can enter the tricarboxylic acid (TCA) cycle via acetyl CoA (Figure 1). The availability of cellular GSH is an important factor for MG detoxification via the glyoxalase system as the lack of GSH restricts hemithioacetal formation, resulting in MG accumulation (Hossain et al., 2011). Recently, a novel glyoxalase enzyme, named glyoxalase III (Gly III), was detected in plants, providing a shorter route for MG detoxification (Ghosh et al., 2016). Gly III contains a DJ-1/PfpI domain, and the presence of this domain has been used to confirm the existence of Gly III-like proteins in various plant species. Conventional glyoxalases (Gly I and Gly II) detoxify MG by converting it to D-lactate, with the help of GSH, but Gly III is able to irreversibly convert MG to D-lactate in a single step, without the need for GSH (Figure 1).

In addition to the glyoxalase system, several other pathways contribute to the detoxification of MG in plants. Other enzymes, including NADPH-dependent reductases, such as the aldo-keto reductases and aldehyde/aldose reductases, involved in detoxifying reactive carbonyls (Yamauchi et al., 2010), can reduce MG to the corresponding alcohol (Simpson et al., 2009; Narawongsanont et al., 2012). Another pathway is the irreversible oxidation of reactive aldehydes, including MG, to their corresponding carboxylic acids, which is catalyzed by aldehyde dehydrogenases (Kirch et al., 2005). However, the glyoxalase system is the most efficient MG detoxification system in plants under normal physiological conditions (Ghosh et al., 2016), and this pathway is very important for plants under stress (Singla-Pareek et al., 2006; Alvarez Viveros et al., 2013).

Under normal metabolic conditions, plants usually maintain a lower level (30-75 μM) of MG (Yadav et al., 2005a; Hossain et al., 2009); however, an abrupt increase was observed in respone to abiotic stresses (Yadav et al., 2005a; Hossain et al., 2009; Mostofa et al., 2015a,b). Salt stress-induced inceases in MG levels were found in various plant species, including pumpkin (Cucurbita maxima L.) by 77%, tobacco (Nicotiana tabacum L., cv. BY-2) by 67% and potato (Solanum tuberosum L. cv. Taedong Valley) by 50%, compared with the respective controls (Hossain et al., 2009; Banu et al., 2010; Upadhyaya et al., 2011; Ghosh et al., 2014). Increased MG levels were also found in mung bean (Vigna radiata L.), Lepidium sativum and rice plants in response to drought (90–107%), and excessive Cd (60–260%) and Cu (106–156%) stresses, respectively, when compared with control counterparts (Nahar et al., 2015b; Mostofa et al., 2015b,c). These findings indicate that the increase in MG levels is a common response of plants to a variety of abiotic stressors, and that stress-induced MG could act as a generic signal molecule for plants under adverse environmental conditions.

In plant cells, MG accumulation has been shown to correlate with increased levels of intracellular oxidative stress, due to the enhanced reactive oxygen species (ROS) production (Maeta et al., 2005; Kalapos, 2008). MG accumulation may indirectly result in increased ROS production by decreasing available GSH levels and by impairing the function of antioxidant enzymes in plants under oxidative stress. In addition, MG can function as a Hill oxidant and catalyze the photoreduction of O₂ to superoxide () in photosystem I (PSI) (Saito et al., 2011). The production of is deleterious as it can cause oxidative damage to cellular components.

Methylglyoxal is an α,β-dicarbonyl compound that can act both as a genotoxic and a glycation agent (Rabbani and Thornalley, 2014). MG has two functional groups; a ketone group and an aldehyde group, the latter being more reactive than the former (Leoncini, 1979). The dicarbonyl group within MG can readily react with the amine groups of proteins and nucleic acids, including DNA and RNA. The accumulation of MG is often called dicarbonyl stress, which has been implicated as a cause of tissue damage and aging (Rabbani and Thornalley, 2014). MG reacts with the amino acids lysine, cysteine and arginine producing glycated proteins, often referred to as advanced glycation end products (AGEs) (Ahmed and Thornalley, 2007), which can cause inactivation of proteins and oxidative damage to key cellular components (Thornalley, 2006). AGEs and dicarbonyl compounds, including MG, often accumulate in plant leaves upon exposure to high light or elevated CO₂ concentrations (Qiu et al., 2008; Bechtold et al., 2009). Thus, it appears that the increase in sugar accumulation and changes in the metabolic flux of sugars, which occur at high CO₂ concentrations, promote the production of MG and other reactive carbonyls, resulting in the accumulation of AGEs. In summary, excessive MG accumulation in plant cells under stress can inhibit cell proliferations, and cause the inactivation and/or degradation of proteins, inactivation of antioxidant defenses, leading to disruption of many cellular functions (Hoque et al., 2010; Hoque M.A. et al., 2012).

Indeed, MG showed toxicity to photosynthesis in the chloroplasts of spinach (Spinacia oleracea L.) (Mano et al., 2009), and the accumulation of MG in the pdtpi mutant, which lacks the plastid isoform of TPI, exhibited greatly reduced growth and increased chlorosis (Chen and Thelen, 2010). Yadav et al. (2005a) reported that accumulation of MG, as a result of salt stress, directly and adversely influenced plant developmental processes, such as seed germination and seedling growth, in tobacco plants. Similarly, Engqvist et al. (2009) found that Arabidopsis plants grown on MS medium supplemented with MG (0.1 or 1 mM MG) exhibited a significant reduction in shoot and root growth. Later, the same group reported a dose-dependent decrease in root and shoot growth of Arabidopsis, tomato and tobacco plants grown on MS medium containing 1 mM MG (Wienstroer et al., 2012). Furthermore, Hoque et al. (2012b) examined the inhibitory effects of MG on growth and development in Arabidopsis and suggested that 1 mM MG is toxic enough to significantly inhibit seed germination and root elongation in seedlings. However, concentrations lower than 0.1 mM MG had no influence on seed germination, but did reduced the rate of root elongation. In addition, concentrations of 1 mM MG or higher resulted in seedling chlorosis within 4 days of treatment. Recently, Kaur et al. (2015a) also reported that MG exposure caused a significant growth reduction in rice seedlings (Oryza sativa cv. IR4), with acute effects on root elongation in a concentration-dependent manner. The above findings highlight the growth inhibitory effects of MG on plants, and indicate that the MG levels causing toxic effects to plants vary depending on plant species, exposure time and perhaps age of plants.

The glyoxalase system is involved in various cellular functions, but the involvement of this system in plant stress responses and tolerance is regarded as its most significant role (Kaur et al., 2014a). The glyoxalase system regulates MG levels in plants under stress and regenerates GSH. GSH and a high GSH/GSSG ratio are required to help protect plants against oxidative stress (Yadav et al., 2005a,b; Noctor et al., 2012), and GSH is directly or indirectly required for the functioning of various antioxidant enzymes, including GST, glutathione peroxidase (GPX), and APX (Yadav et al., 2008). Several studies have shown close links between the antioxidant and glyoxalase systems in plants, suggesting a direct influence of the glyoxalase system on ROS detoxification (Yadav et al., 2005a; El-Shabrawi et al., 2010; Upadhyaya et al., 2011; Mostofa et al., 2014a, 2015a).

An increase in glyoxalase enzyme activities occurs in plants in response to many different stressors, including osmotic stress, extremes of temperature, heavy metals and exposure to stress-related hormones, including MeJA, ABA and salicylic acid (SA) (Hossain and Fujita, 2009; Hossain et al., 2009). Transcriptomic and proteomic analyses of various plant species have improved our knowledge and understanding of the roles of glyoxalases in plant stress responses and tolerance (Singla-Pareek et al., 2003, 2006; Hossain et al., 2009; Lin et al., 2010; Mustafiz et al., 2011). Plant glyoxalase genes (Gly I and Gly II) have been cloned from various plant species and characterized in detail. The expression of Gly I and Gly II genes has been shown to be up-regulated in many plant species by a diverse range of environmental cues, and plants overexpressing either Gly I or Gly II showed enhanced plant abiotic stress tolerance (Singla-Pareek et al., 2003, 2006, 2008; Lin et al., 2010; Alvarez Viveros et al., 2013; Wu et al., 2013; Kaur et al., 2014a,c). The genetic manipulation of the glyoxalase system in plants has successfully contributed to improved tolerance to multiple abiotic stresses, such as salinity, heavy metals and MG treatments (Table 1). Transgenic plants overexpressing glyoxalase pathway genes have lower MG and ROS levels when exposed to stress, because they have better GSH homeostasis and retain better antioxidant enzyme functionality. Thus, glyoxalase enzyme levels can be used as phenomic biomarkers to indicate degrees of stress tolerance, and plants with high glyoxalase enzyme levels are potentially tolerant to a wide range of abiotic stresses (Kaur et al., 2014c). In Table 1, we summarized most of the successful transgenic studies that showed that transgenic plants, including important crop plants, overexpressing individually or together Gly I and Gly II have increased stress tolerance.

In addition to the transgenic approach, alternative methods, such as treatments of seeds prior to sowing and/or plants with exogenous chemicals, e.g., plant growth regulators, osmoprotectants, signaling molecules etc., can also alter the glyoxalase system in plants, thereby improving stress tolerance (Table 2). For instance, treatment of rice seedlings with Ca has been shown to increase the activities of Gly I and Gly II, contributing to the reduction in As- and Cd- induced growth inhibition (Rahman et al., 2015a,b). Mostofa and Fujita (2013) reported that a SA pre-treatment of rice seedlings under Cu stress alleviated Cu-toxicity by increasing the capacity of both antioxidant and glyoxalase systems. Table 2 lists most of the important studies in which chemical treatments were used to influence the glyoxalase system, leading to enhanced stress tolerance.

Recent studies of MG metabolism have revealed many important functions of MG related to stress responses and tolerance in plants. The excessive accumulation of MG in plants is an inevitable stress, but MG can stimulate the components of different stress-protection pathways (Figure 2; Engqvist et al., 2009; Hoque et al., 2012a,b,c; Wienstroer et al., 2012; Kaur et al., 2015a), which could be considered as an acclimation/adaptation process. The glyoxalase pathway scavenges MG and confers tolerance to multiple stresses; and thus, MG levels and glyoxalase pathway are closely associated with abiotic stress tolerance in plants. The signaling roles of MG in up-regulating stress-responsive pathways and its potential to active multiple pathways have made MG a suitable marker for abiotic stress tolerance in plants. Recent progress made by genome-wide and in silico analyses has revealed intricate regulatory networks associated with MG signaling, which control gene expression, protein modification and the metabolite composition of plants. Further omic studies investigating the roles of MG would be worthwhile to improve our understanding of multiple abiotic stress tolerance. In-depth understanding of the interactions of MG with Ca²⁺, ROS, NO, H₂S, plant hormones, TFs, and the glyoxalase system, as well as with other MG detoxification systems in different subcellular compartments will reveal more regulatory roles for MG in plant abiotic stress responses and tolerance.

TH and MH conceived the idea. TH, MH, MM, DB, MF, and L-ST wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.