Ananda Mustafiz

Genome-wide analysis of rice and Arabidopsis 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.

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.

Metabolic Engineering of Glyoxalase Pathway for Enhancing Stress Tolerance in Plants

Glyoxalase system consists of two enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I detoxifies methylglyoxal (MG), a cytotoxic byproduct of glycolysis, to S-lactoylglutathione (SLG) where it uses one molecule of reduced glutathione. Subsequently, SLG is converted to lactate by Gly II and one molecule of reduced glutathione is recycled back into the system. The level of MG, which is produced ubiquitously in all living organisms, is enhanced upon exposure to different abiotic stresses in plants. Overexpression of glyoxalase pathway genes in transgenic plants has been found to keep a check on the MG level under stress conditions, regulate glutathione homeostasis, and the transgenic plants are able to survive and grow under various abiotic stresses.

Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice

As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min−1mg−1 which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG.

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.

Arabidopsis thaliana Contains Both Ni2+ and Zn2+ Dependent Glyoxalase I Enzymes and Ectopic Expression of the Latter Contributes More towards Abiotic Stress Tolerance in E. coli

The glyoxalase pathway is ubiquitously found in all the organisms ranging from prokaryotes to eukaryotes. It acts as a major pathway for detoxification of methylglyoxal (MG), which deleteriously affects the biological system in stress conditions. The first important enzyme of this system is Glyoxalase I (GLYI). It is a metalloenzyme which requires divalent metal ions for its activity. This divalent metal ion can be either Zn2+ as found in most of eukaryotes or Ni2+ as seen in prokaryotes. In the present study, we have found three active GLYI enzymes (AtGLYI2, AtGLYI3 and AtGLYI6) belonging to different metal activation classes coexisting in Arabidopsis thaliana. These enzymes have been found to efficiently complement the GLYI yeast mutants. These three enzymes have been characterized in terms of their activity, metal dependency, kinetic parameters and their role in conferring tolerance to multiple abiotic stresses in E. coli and yeast. AtGLYI2 was found to be Zn2+ dependent whereas AtGLYI3 and AtGLYI6 were Ni2+ dependent. Enzyme activity of Zn2+ dependent enzyme, AtGLYI2, was observed to be exceptionally high (~250–670 fold) as compared to Ni2+ dependent enzymes, AtGLYI3 and AtGLYI6. The activity of these GLYI enzymes correlated well to their role in stress tolerance. Heterologous expression of these enzymes in E. coli led to better tolerance against various stress conditions. This is the first report of a higher eukaryotic species having multiple active GLYI enzymes belonging to different metal activation classes.

Transcript Profiling Reveals the Presence of Abiotic Stress and Developmental Stage Specific Ascorbate Oxidase Genes in Plants

Abiotic stress and climate change is the major concern for plant growth and crop yield. Abiotic stresses lead to enhanced accumulation of reactive oxygen species (ROS) consequently resulting in cellular damage and major losses in crop yield. One of the major scavengers of ROS is ascorbate (AA) which acts as first line of defense against external oxidants. An enzyme named ascorbate oxidase (AAO) is known to oxidize AA and deleteriously affect the plant system in response to stress. Genome-wide analysis of AAO gene family has led to the identification of five, three, seven, four, and six AAO genes in Oryza sativa, Arabidopsis, Glycine max, Zea mays, and Sorghum bicolor genomes, respectively. Expression profiling of these genes was carried out in response to various abiotic stresses and during various stages of vegetative and reproductive development using publicly available microarray database. Expression analysis in Oryza sativa revealed tissue specific expression of AAO genes wherein few members were exclusively expressed in either root or shoot. These genes were found to be regulated by both developmental cues as well as diverse stress conditions. The qRT-PCR analysis in response to salinity and drought stress in rice shoots revealed OsAAO2 to be the most stress responsive gene. On the other hand, OsAAO3 and OsAAO4 genes showed enhanced expression in roots under salinity/drought stresses. This study provides lead about important stress responsive AAO genes in various crop plants, which could be used to engineer climate resilient crop plants.

Glyoxalase System: A Glutathione-Dependent Pathway for Abiotic Stress Tolerance in Plants

Due to their sessile nature, plants have to go through various adverse environmental conditions. Abiotic stresses, such as salinity, drought, flooding, cold, heat, etc. have been the major environmental factors contributing to the decreased yield of important crop plants. Abiotic stress leads to an abrupt increase in the contents of methylglyoxal (MG) in plants. MG is a potent cytotoxin, and readily reacts with major macromolecules of the cell to form advanced glycation end-products (AGEs ). MG detoxification is principally carried out by the glyoxalase (GLY) system, which consists of two enzymes, GLYI and GLYII. GLYI acts upon the non-enzymatically formed complex of MG and a molecule of reduced glutathione (GSH), leading to the production of S-D-lactoylglutathione (SLG). GLYII, then, catalyzes the conversion of SLG to D-lactate giving GSH back to the system, thereby maintaining GSH homeostasis. The glyoxalase pathway keeps a check on the elevation of the MG level and helps in maintaining a higher “reduced to oxidized” GSH ratio. The glyoxalase pathway has been directly correlated to abiotic stress tolerance. Overexpression of GLY enzymes confers improved abiotic stress tolerance in plants. This chapter provides insights into the importance of the glyoxalase pathway in stress response and sheds light on the dependence of the glyoxalase pathway on GSH as the key player in regulating the pathway.

GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance

Methylglyoxal(MG) is a potent cytotoxin that is produced as a byproduct of various metabolic reactions in the cell. The major enzymes for MG detoxification are Glyoxalase I(GLYI), Glyoxalase II(GLYII) and D-lactate dehydrogenase(D-LDH). These three enzymes work together and convert MG into D-pyruvate, which directly goes to TCA cycle. Here, a comparative study of the ability of MG detoxification of these three enzymes has been done in both E. coli and yeast. Ectopic expression of these three genes from Arabidopsis in E. coli in presence of different abiotic stress revealed the contribution of each of these genes in detoxifying MG. Yeast mutants of MG detoxification enzymes were also grown in different stress conditions to record the effect of each gene. These mutants were also used for complementation assays using the respective MG detoxifying genes from Arabidopsis in presence of various stress conditions. The MG content and the corresponding growth of cells was measured in all the bacterial as well as yeast strains. This study reveals differential contribution of MG detoxification enzymes in mitigating MG levels and alleviating stress in both prokaryotes as well as eukaryotes. GLYI and D-LDH were found to be key enzymes in MG detoxification under various abiotic stresses.

Second Messengers: Central Regulators in Plant Abiotic Stress Response

Plants differ from animals by lacking the ability to escape from their environmental conditions. Plants adapt to the seasonal as well as nonseasonal perturbations by means of stress-responsive genes. Manipulation of such genes has been shown to provide abiotic stress tolerance in plants. Since abiotic stress is a polygenic trait, overexpression of single stress-responsive gene would not serve the purpose of getting stress-tolerant plants. So, the focus needs to be shifted towards the “master regulators” which are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses by modulating gene expression machinery. In plants, there are various second messengers including calcium, ROS, phosphoinositides, cyclic nucleotides, etc., which are known to initiate the downstream signaling cascade leading to response against different, multiple, and simultaneous ambient cues. A better understanding of these elements will allow us to engineer a particular stress-responsive pathway, to achieve better stress-tolerant plants.