Charanpreet Kaur

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.

Glyoxalase and Methylglyoxal as Biomarkers for Plant Stress Tolerance

Glyoxalases are known to play a very important role in abiotic stress tolerance. This two-step pathway detoxifies ubiquitously present cytotoxic metabolite methylglyoxal, which otherwise increases to lethal concentrations under various stress conditions. Methylglyoxal initiates stress-induced signaling cascade via reactive oxygen species, resulting in the modifications of proteins involved in various signal transduction pathways, that eventually culminates in cell death or growth arrest. The associated mechanism of tolerance conferred by over-expression of methylglyoxal-detoxifying glyoxalase pathway mainly involves lowering of methylglyoxal levels, thereby reducing subsequently induced cellular toxicity. Apart from abiotic stresses, expression of glyoxalases is affected by a wide variety of other stimuli such as biotic, chemical and hormonal treatments. Additionally, alterations in cellular milieu during plant growth and development also affect expression of glyoxalases. The multiple stress-inducible nature of these enzymes suggests a vital role for glyoxalases, associating them with plant defense mechanisms. In this context, we have summarized available transcriptome, proteome and genetic engineering- based reports in order to highlight the involvement of glyoxalases as important components of plant stress response. The role of methylglyoxal as signaling molecule is also discussed. Further, we examine the suitability of glyoxalases and methylglyoxal as potential markers for stress tolerance.

Episodes of horizontal gene-transfer and gene-fusion led to co-existence of different metal-ion specific glyoxalase I

Glyoxalase pathway plays an important role in stress adaptation and many clinical disorders. The first enzyme of this pathway, glyoxalase I (GlxI), uses methylglyoxal as a substrate and requires either Ni(II)/Co(II) or Zn(II) for activity. Here we have investigated the origin of different metal ion specificities of GlxI and subsequent pattern of inheritance during evolution. Our results suggest a primitive origin of single-domain Ni dependent GlxI [Ni-GlxI]. This subsequently evolved into Zn activated GlxI [Zn-GlxI] in deltaproteobacteria. However, origin of eukaryotic Zn-GlxI is different and can be traced to GlxI from Candidatus pelagibacter and Sphingomonas. In eukaryotes GlxI has evolved as two-domain protein but the corresponding Zn form is lost in plants/higher eukaryotes. In plants gene expansion has given rise to multiple two-domain Ni-GlxI which are differentially regulated under abiotic stress conditions. Our results suggest that different forms of GlxI have evolved to help plants adapt to stress.

Analysis of global gene expression profile of rice in response to methylglyoxal indicates its possible role as a stress signal molecule.

Methylglyoxal (MG) is a toxic metabolite produced primarily as a byproduct of glycolysis. Being a potent glycating agent, it can readily bind macromolecules like DNA, RNA, or proteins, modulating their expression and activity. In plants, despite the known inhibitory effects of MG on growth and development, still limited information is available about the molecular mechanisms and response pathways elicited upon elevation in MG levels. To gain insight into the molecular basis of MG response, we have investigated changes in global gene expression profiles in rice upon exposure to exogenous MG using GeneChip microarrays. Initially, growth of rice seedlings was monitored in response to increasing MG concentrations which could retard plant growth in a dose-dependent manner. Upon exposure to 10 mM concentration of MG, a total of 1685 probe sets were up- or down-regulated by more than 1.5-fold in shoot tissues within 16 h. These were classified into 10 functional categories. The genes involved in signal transduction such as, protein kinases and transcription factors, were significantly over-represented in the perturbed transcriptome, of which several are known to be involved in abiotic and biotic stress response indicating a cross-talk between MG-responsive and stress-responsive signal transduction pathways. Through in silico studies, we could predict 7–8 bp long conserved motif as a possible MG-responsive element (MGRE) in the 1 kb upstream region of genes that were more than 10-fold up- or down-regulated in the analysis. Since several perturbations were found in signaling cascades in response to MG, we hereby suggest that it plays an important role in signal transduction probably acting as a stress signal molecule.

OsSRO1a Interacts with RNA Binding Domain-Containing Protein (OsRBD1) and Functions in Abiotic Stress Tolerance in Yeast

SRO1 is an important regulator of stress and hormonal response in plants and functions by interacting with transcription factors and several other proteins involved in abiotic stress response. In the present study, we report OsRBD1, an RNA binding domain 1- containing protein as a novel interacting partner of OsSRO1a from rice. The interaction of OsSRO1a with OsRBD1 was shown in yeast as well as in planta. Domain–domain interaction study revealed that C-terminal RST domain of OsSRO1a interacts with the N-terminal RRM1 domain of OsRBD1 protein. Both the proteins were found to co-localize in nucleus. Transcript profiling under different stress conditions revealed co-regulation of OsSRO1a and OsRBD1 expression under some abiotic stress conditions. Further, co-transformation of both OsSRO1a and OsRBD1 in yeast conferred enhanced tolerance toward salinity, osmotic, and methylglyoxal treatments. Our study suggests that the interaction of OsSRO1a with OsRBD1 confers enhanced stress tolerance in yeast and may play an important role under abiotic stress responses in plants.

Methylglyoxal, Triose Phosphate Isomerase, and Glyoxalase Pathway: Implications in Abiotic Stress and Signaling in Plants

Methylglyoxal (MG) is a cytotoxic metabolite inevitably produced as a side product of primary metabolic pathways via both enzymatic and non-enzymatic reactions. In plants, spontaneous generation of MG through breakdown of triose sugars (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) is believed to be the major route for MG formation. MG is maintained at basal levels in plants under normal conditions that accumulate to higher concentrations under various stresses, probably as a general consequence of all abiotic stresses. The toxic effects of MG is due to its ability to induce oxidative stress in cells, either directly through increased generation of reactive oxygen species (ROS) or indirectly via advanced glycation end product (AGE) formation. Thus, elevated MG levels have implications in inhibition of growth and development in plants. To keep MG levels in check, the two-step glyoxalase pathway comprising glyoxalase I (GLYI) and glyoxalase II (GLYII) enzymes has evolved as the major MG-scavenging detoxification system that converts MG to d-lactate using glutathione as a cofactor in this process. Over-expression of glyoxalase pathway has been shown to confer tolerance to multiple stresses that works by controlling MG levels and maintaining glutathione homeostasis in plants. Moreover, increased activity of triose phosphate isomerase under different stresses that use up triose sugars via glycolysis further prevents MG levels from accumulating in the system along with increasing the energy status of plants. Considering the fact that MG levels are maintained at a threshold concentration in plants even under physiological conditions and also observed MG-dependent induction in expression of triose phosphate isomerase, a role for MG in signaling pathways is suggested. Here, we provide an insight to the role of MG and glyoxalases in plant stress response with special mention about the possible involvement of MG in signaling pathway.

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.

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.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni2+- and Zn2+-dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.