Ajit Ghosh

Glyoxalases and stress tolerance in plants

The glyoxalase pathway is required for detoxification of cytotoxic metabolite MG (methylglyoxal) that would otherwise increase to lethal concentrations under adverse environmental conditions. Since its discovery 100 years ago, several roles have been assigned to glyoxalases, but, in plants, their involvement in stress response and tolerance is the most widely accepted role. The plant glyoxalases have emerged as multigene family and this expansion is considered to be important from the perspective of maintaining a robust defence machinery in these sessile species. Glyoxalases are known to be differentially regulated under stress conditions and their overexpression in plants confers tolerance to multiple abiotic stresses. In the present article, we review the importance of glyoxalases in plants, discussing possible roles with emphasis on involvement of the glyoxalase pathway in plant stress tolerance.

A unique Ni2+ -dependent and methylglyoxal-inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response.

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non-toxic metabolite D-lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn(2+) in the case of eukaryotes or Ni(2+) for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI-11.2) from Oryza sativa (rice) that requires Ni(2+) for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni(2+) coordination site despite containing two GLY I domains. The requirement of Ni(2+) as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI-11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI-11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na(+) /K(+) ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni(2+) - the heavy metal cofactor of OsGLYI-11.2, in relation to stress response and adaptation in plants.

A glutathione responsive rice glyoxalase II, OsGLYII-2, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti-oxidant pool.

Glyoxalase II (GLY II), the second enzyme of glyoxalase pathway that detoxifies cytotoxic metabolite methylglyoxal (MG), belongs to the superfamily of metallo-β-lactamases. Here, detailed analysis of one of the uncharacterized rice glyoxalase II family members, OsGLYII-2 was conducted in terms of its metal content, enzyme kinetics and stress tolerance potential. Functional complementation of yeast GLY II mutant (∆GLO2) and enzyme kinetics data suggested that OsGLYII-2 possesses characteristic GLY II activity using S-lactoylglutathione (SLG) as the substrate. Further, Inductively Coupled Plasma Atomic Emission spectroscopy and modelled structure revealed that OsGLYII-2 contains a binuclear Zn/Fe centre in its active site and chelation studies indicated that these are essential for its activity. Interestingly, reconstitution of chelated enzyme with Zn(2+), and/or Fe(2+) could not reactivate the enzyme, while addition of Co(2+) was able to do so. End product inhibition study provides insight into the kinetics of GLY II enzyme and assigns hitherto unknown function to reduced glutathione (GSH). Ectopic expression of OsGLYII-2 in Escherichia coli and tobacco provides improved tolerance against salinity and dicarbonyl stress indicating towards its role in abiotic stress tolerance. Maintained levels of MG and GSH as well as better photosynthesis rate and reduced oxidative damage in transgenic plants under stress conditions seems to be the possible mechanism facilitating enhanced stress tolerance.

Presence of unique glyoxalase III proteins in plants indicates the existence of shorter route for methylglyoxal detoxification

Glyoxalase pathway, comprising glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, is the major pathway for detoxification of methylglyoxal (MG) into D-lactate involving reduced glutathione (GSH). However, in bacteria, glyoxalase III (GLY III) with DJ-1/PfpI domain(s) can do the same conversion in a single step without GSH. Our investigations for the presence of DJ-1/PfpI domain containing proteins in plants have indicated the existence of GLY III-like proteins in monocots, dicots, lycopods, gymnosperm and bryophytes. A deeper in silico analysis of rice genome identified twelve DJ-1 proteins encoded by six genes. Detailed analysis has been carried out including their chromosomal distribution, genomic architecture and localization. Transcript profiling under multiple stress conditions indicated strong induction of OsDJ-1 in response to exogenous MG. A member of OsDJ-1 family, OsDJ-1C, showed high constitutive expression at all developmental stages and tissues of rice. MG depletion study complemented by simultaneous formation of D-lactate proved OsDJ-1C to be a GLY III enzyme that converts MG directly into D-lactate in a GSH-independent manner. Site directed mutagenesis of Cys-119 to Alanine significantly reduces its GLY III activity indicating towards the existence of functional GLY III enzyme in rice—a shorter route for MG detoxification.

Genome-wide analysis and expression profiling of glyoxalase gene families in soybean (Glycine max) indicate their development and abiotic stress specific response.

BackgroundGlyoxalase pathway consists of two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII) which detoxifies a highly cytotoxic metabolite methylglyoxal (MG) to its non-toxic form. MG may form advanced glycation end products with various cellular macro-molecules such as proteins, DNA and RNA; that ultimately lead to their inactivation. Role of glyoxalase enzymes has been extensively investigated in various plant species which showed their crucial role in salinity, drought and heavy metal stress tolerance. Previously genome-wide analysis of glyoxalase genes has been conducted in model plants Arabidopsis and rice, but no such study was performed in any legume species.ResultsIn the present study, a comprehensive genome database analysis of soybean was performed and identified a total of putative 41 GLYI and 23 GLYII proteins encoded by 24 and 12 genes, respectively. Detailed analysis of these identified members was conducted including their nomenclature and classification, chromosomal distribution and duplication, exon-intron organization, and protein domain(s) and motifs identification. Expression profiling of these genes has been performed in different tissues and developmental stages as well as under salinity and drought stresses using publicly available RNAseq and microarray data. The study revealed that GmGLYI-7 and GmGLYII-8 have been expressed intensively in all the developmental stages and tissues; while GmGLYI-6, GmGLYI-9, GmGLYI-20, GmGLYII-5 and GmGLYII-10 were highly abiotic stress responsive members.ConclusionsThe present study identifies the largest family of glyoxalase proteins to date with 41 GmGLYI and 23 GmGLYII members in soybean. Detailed analysis of GmGLYI and GmGLYII genes strongly indicates the genome-wide segmental and tandem duplication of the glyoxalase members. Moreover, this study provides a strong basis about the biological role and function of GmGLYI and GmGLYII members in soybean growth, development and stress physiology.

A nuclear‐localized rice glyoxalase I enzyme, OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

&NA; The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni2+ or Zn2+ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast‐localized GLYI enzyme, OsGLYI‐8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI‐8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI‐8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady‐state kinetics with a low‐affinity and a high‐affinity substrate‐binding component. Loss of AtGLYI‐2, the closest Arabidopsis ortholog of OsGLYI‐8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI‐2 or OsGLYI‐8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus. Significance Statement Methylglyoxal, a toxic byproduct of glycolysis, increases under abiotic stress and is detoxified primarily by glyoxalases. Previously studied glyoxalase I (GLYI) enzymes are cytoplasmic metalloproteins. Here, we demonstrate a nucleus‐localized rice glyoxalase I, OsGLYI‐8, that detoxifies methylglyoxal in a metal‐independent but Zn2+/Mn2+‐stimulated manner. As Arabidopsis mutant of its homolog exhibits severe growth retardation in the presence of methylglyoxal or salinity stress, we suggest that nuclear detoxification of methylglyoxal might protect DNA from damage, especially under stress conditions.

Genome-wide identification and expression analysis of glutathione S-transferase gene family in tomato: Gaining an insight to their physiological and stress-specific roles.

Glutathione S-transferase (GST) refers to one of the major detoxifying enzymes that plays an important role in different abiotic and biotic stress modulation pathways of plant. The present study aimed to a comprehensive genome-wide functional characterization of GST genes and proteins in tomato (Solanum lycopersicum L.). The whole genome sequence analysis revealed the presence of 90 GST genes in tomato, the largest GST gene family reported till date. Eight segmental duplicated gene pairs might contribute significantly to the expansion of SlGST gene family. Based on phylogenetic analysis of tomato, rice, and Arabidopsis GST proteins, GST family members could be further divided into ten classes. Members of each orthologous class showed high conservancy among themselves. Tau and lambda are the major classes of tomato; while tau and phi are the major classes for rice and Arabidopsis. Chromosomal localization revealed highly uneven distribution of SlGST genes in 13 different chromosomes, where chromosome 9 possessed the highest number of genes. Based on publicly available microarray data, expression analysis of 30 available SlGST genes exhibited a differential pattern in all the analyzed tissues and developmental stages. Moreover, most of the members showed highly induced expression in response to multiple biotic and abiotic stress inducers that could be harmonized with the increase in total GST enzyme activity under several stress conditions. Activity of tomato GST could be enhanced further by using some positive modulators (safeners) that have been predicted through molecular docking of SlGSTU5 and ligands. Moreover, tomato GST proteins are predicted to interact with a lot of other glutathione synthesizing and utilizing enzymes such as glutathione peroxidase, glutathione reductase, glutathione synthetase and γ-glutamyltransferase. This comprehensive genome-wide analysis and expression profiling would provide a rational platform and possibility to explore the versatile role of GST genes in crop engineering.

Genome-Wide Identification of Glyoxalase Genes in Medicago truncatula and Their Expression Profiling in Response to Various Developmental and Environmental Stimuli

Glyoxalase is an evolutionary highly conserved pathway present in all organisms. Conventional glyoxalase pathway has two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII) that act sequentially to detoxify a highly cytotoxic compound methylglyoxal (MG) to D-lactate with the help of reduced glutathione. Recently, proteins with DJ-1/PfpI domain have been reported to perform the same conversion in a single step without the help of any cofactor and thus termed as “unique glyoxalase III” enzyme. Genome-wide analysis of glyoxalase genes have been previously conducted in Arabidopsis, rice and Soybean plants, but no such study was performed for one of the agricultural important model legume species, Medicago truncatula. A comprehensive genome-wide analysis of Medicago identified a total of putative 29 GLYI, 14 GLYII genes, and 5 glyoxalase III (DJ-1) genes. All these identified genes and their corresponding proteins were analyzed in detail including their chromosomal distribution, gene duplication, phylogenetic relationship, and the presence of conserved domain(s). Expression of all these genes was analyzed in different tissues as well as under two devastating abiotic stresses- salinity and drought using publicly available transcript data. This study revealed that MtGLYI-4, MtGLYII-6, and MtDJ-1A are the constitutive members with a high level of expression at all 17 analyzed tissues; while MtGLYI-1, MtGLYI-11, MtGLYI-5, MtGLYI-7, and MtGLYII-13 showed tissue-specific expression. Moreover, most of the genes displayed similar pattern of expression in response to both salinity and drought stress, irrespective of stress duration and tissue type. MtGLYI-8, MtGLYI-11, MtGLYI-6, MtGLYI-16, MtGLYI-21, and MtGLYII-9 showed up-regulation, while MtGLYI-17 and MtGLYI-7/9 showed down-regulation in response to both stresses. Interestingly, MtGLYI-14/15 showed completely opposite pattern of expression in these two stresses. This study provides an initial basis about the physiological significance of glyoxalase genes in plant development and stress response of Medicago that could be explored further.

Comprehensive genome-wide analysis of Glutathione S-transferase gene family in potato (Solanum tuberosum L.) and their expression profiling in various anatomical tissues and perturbation conditions

Glutathione S-transferases (GSTs) are ubiquitous enzymes which play versatile functions including cellular detoxification and stress tolerance. In this study, a comprehensive genome-wide identification of GST gene family was carried out in potato (Solanum tuberosum L.). The result demonstrated the presence of at least 90 GST genes in potato which is greater than any other reported species. According to the phylogenetic analyses of Arabidopsis, rice and potato GST members, GSTs could be subdivided into ten different classes and each class is found to be highly conserved. The largest class of potato GST family is tau with 66 members, followed by phi and lambda. The chromosomal localization analysis revealed the highly uneven distribution of StGST genes across the potato genome. Transcript profiling of 55 StGST genes showed the tissue-specific expression for most of the members. Moreover, expression of StGST genes were mainly repressed in response to abiotic stresses, while largely induced in response to biotic and hormonal elicitations. Further analysis of StGST genes promoter identified the presence of various stress responsive cis-regulatory elements. Moreover, one of the highly stress responsive StGST members, StGSTU46, showed strong affinity towards flurazole with lowest binding energy of -7.6kcal/mol that could be used as antidote to protect crop against herbicides. These findings will facilitate the further functional and evolutionary characterization of GST genes in potato.

Glyoxalase Pathway and Drought Stress Tolerance in Plants

The ubiquitously present glyoxalase pathway consists of two enzymes, Glyoxalase I and Glyoxalase II, which act in a stepwise manner and catalyze the detoxification of a highly cytotoxic metabolite methylglyoxal to d-lactate with the help of glutathione. Methylglyoxal (MG) is generated endogenously through different enzymatic and nonenzymatic reactions and is a potent glycating agent. It inhibits cell division and forms various degrees of irreversible adducts with cellular macromolecules such as nucleic acids, lipids, and proteins. MG along with reactive oxygen species (ROS) has been shown to accumulate in plant cells in response to various abiotic stresses including drought and their accumulation results in an imbalance in different cellular metabolic processes. Plants being sessile organisms have evolved various mechanisms that permit them to cope with and withstand various degrees of stress. The glyoxalase pathway is one such mechanism which acts to control excessive accumulation of MG and ROS in the system, either directly or in cooperation with other pathways involved in stress response. In response to drought, transcript and protein levels of glyoxalases are altered which is suggestive of their involvement in stress response. MG has also been shown to induce stress-responsive signaling cascades related to drought and even regulates stomatal movements. Here, we discuss the role of the plant glyoxalase pathway with respect to drought stress adaptation.