Despite its ubiquitous presence, the role of glyoxalase I has not been well investigated in plants. In order to find out its physiological functions, we have cloned and characterized a cDNA from Brassica juncea encoding glyoxalase I (Gly I) and made transgenic tobacco plants harbouring Gly I in both sense and antisense orientation. The transgenic nature of the plants was confirmed by Southern blotting, and the estimated number of genes inserted ranged from one to six. The transcript and protein levels of glyoxalase I were also monitored in transgenic plants. The expression of glyoxalase I in B. juncea was upregulated in response to salt, water and heavy metal stresses. In response to a high concentration of salt, the transcript level averaged threefold higher in 72 h, and an increase in the protein was also seen by immunoblotting. The transgenic plants over-expressing glyoxalase I showed significant tolerance to methylglyoxal and high salt, as tested in detached leaf disc senescence assay. A comparison of plants expressing high and low levels of glyoxalase I showed that the tolerance to different salt concentrations was correlated with the degree of glyoxalase I expression. Our results suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions.

Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress.

Abiotic stresses cause extensive losses to agricultural production worldwide. In this study, the effects of various abiotic stresses on the upregulation of methylglyoxal levels and glyoxalase I activities in pumpkin seedlings (Cucurbita maxima Duch.) were investigated. Most of the stresses caused significant increases in methylglyoxal level and glyoxalase I activity, white light causing the highest induction followed by salinity, chemical, drought, and heavy metal stresses. We showed that accumulation of methylglyoxal in plants under various stressful conditions is a common phenomenon, and methylglyoxal could therefore act as a signal for plants to respond to stress. The stress-induced increases in methylglyoxal level, glyoxalase I activity and Gly I transcript found in the present study suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions and showed that the glyoxalase pathway is the main detoxification pathway of methylglyoxal in plants. The multistress response of glyoxalase I gene indicates its future utility in developing tolerance to various stresses in crop plants. A cDNA encoding glyoxalase I has been isolated, subcloned and nucleotide sequence was determined. The pumpkin glyoxalase I cDNA consists of 975-bp nucleotides encoding a polypeptide of 185 amino acids having a predicted molecular weight of 20,772.14 Da. Based on the number of amino acids, it is categorized as short-type glyoxalase I and the nucleotide sequence of pumpkin glyoxalase I showed significant homology with other known glyoxalase I sequences of plants.

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Stress-induced changes of methylglyoxal level and glyoxalase I activity in pumpkin seedlings and cDNA cloning of glyoxalase I gene.

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.

Genome-wide analysis of rice and Arabidopsis identifies two glyoxalase genes that are highly expressed in abiotic stresses

The present work aimed to study the activities of glyoxalase system enzymes, glyoxalase I (G I) and glyoxalase II (G II), as well as the expression of their genes in human breast carcinoma. Samples of tumoral tissue and normal counterparts were drawn from several patients during surgery. They served either for preparing extracts to be used in enzyme activity evaluations or for RNA extraction and subsequent northern blot analysis. A far higher activity level of G I and G II occurs in the tumor compared with pair-matched normal tissue, as shown by both spectrophotometrical assay and electrophoretic pattern. Such increased activities of G I and G II likely result from an enhanced enzyme synthesis as a consequence of increased expression of the respective genes in the tumoral tissue, as evidenced by northern blot. The present findings confirm a key-role of glyoxalase system to detoxify cytotoxic methylglyoxal and modulate S-D-lactoylglutathione levels in tumor cells. Moreover, they suggest a possible employment of GI inhibitors as anti-cancer drugs.

Expression of glyoxalase I and II in normal and breast cancer tissues.

Methylglyoxal (MG) is a highly reactive cytotoxic alpha-oxoaldehyde compound and is formed endogenously via different enzymatic and non-enzymatic reactions. In plants MG is detoxified mainly via the glyoxalase system that is comprised of two enzymes, glyoxalase I and glyoxalase II. Glyoxalase I converts MG to S-D-lactoylglutathione by utilizing glutathione, while glyoxalase II converts S-D-lactoylglutathione to D-lactic acid, and during this reaction glutathione is regenerated. The presence and characterization of both glyoxalase I and II has been reported in many plants and the genes encoding these have been cloned and found to be regulated under various environmental conditions. In plants, MG has been found to be present during normal growth conditions and it accumulates to higher levels under various environmental stresses. Abiotic and heavy metal stresses induce reactive oxygen species (ROS) and MG. Overexpression of the glyoxalase pathway in transgenic tobacco and rice plants has been found to check an increase of ROS and MG under stress conditions by maintaining glutathione homeostasis and antioxidant enzyme levels. There is also evidence that in addition to glyoxalase, other pathways, such as the aldose reductase pathway, may also be involved in MG detoxification in plants. To unravel the role of MG and the glyoxalase pathway in signal transduction during environmental stress conditions in plants is a topic of future research interest. In this paper we review work on plant glyoxalases especially with respect to their role under abiotic stresses.

An overview on the role of methylglyoxal and glyoxalases in plants.

Glyoxalase-I activity in growingDatura callus showed 184% increase with the age of the culture. Spermidine increased the enzyme activity together with DNA and protein synthesis. With the addition of mitotic inhibitors, vinblastine and methylglyoxal in the growth medium, the enzyme activity was inhibited by 92 percent and 50 percent respectively, at the most effective concentration and the callus growth was also reduced. Similar results were obtained with specific glyoxalase I inhibitors, iso-ascorbate and squaric acid.

Correlation of glyoxalase I activity with cell proliferation inDatura callus culture

Activation of glyoxalase I during the cell division cycle and its homology with auxin regulated genes

Red light-mediated changes in inositol phospholipids and phosphatidylcholine in Brassica hypocotyls

The presence of the enzyme glyoxalase was reported by Neuberg (1) and Dakin and Dudley in 1913 (2). Since then, it has been isolated from many animal systems and from yeast. However, except for one casual report, its existence has not been reported in higher plants.

Sumita Pal

Papers

Correlation of glyoxalase I activity with cell proliferation inDatura callus culture

Activation of glyoxalase I during the cell division cycle and its homology with auxin regulated genes

Red light-mediated changes in inositol phospholipids and phosphatidylcholine in Brassica hypocotyls

Effect of Squaric Acid and Isoascorbate on Glyoxalase-I Cell Division and DNA Synthesis in Datura Callus