Glutathione is an essential metabolite for plant life best known for its role in the control of reactive oxygen species (ROS). Glutathione is also involved in the detoxification of methylglyoxal (MG) which, much like ROS, is produced at low levels by aerobic metabolism under normal conditions. While several physiological processes depend on ROS and MG, a variety of stresses can dramatically increase their concentration leading to potentially deleterious effects. In this review, we examine the structure and the stress regulation of the pathways involved in glutathione synthesis and degradation. We provide a synthesis of the current knowledge on the glutathione-dependent glyoxalase pathway responsible for MG detoxification. We present recent developments on the organization of the glyoxalase pathway in which alternative splicing generate a number of isoforms targeted to various subcellular compartments. Stress regulation of enzymes involved in MG detoxification occurs at multiple levels. A growing number of studies show that oxidative stress promotes the covalent modification of proteins by glutathione. This post-translational modification is called S-glutathionylation. It affects the function of several target proteins and is relevant to stress adaptation. We address this regulatory function in an analysis of the enzymes and pathways targeted by S-glutathionylation.

Nucleoside diphosphate kinase (NDPK) catalyzes the interconversion of nucleoside diphosphates and triphosphates using ATP as phosphate donor. This housekeeping enzyme is present in several subcellular compartments. The main isoform (NDPK1) is located in the cytosol and is highly expressed in meristems and provascular tissues. The manipulation of NDPK1 levels in transgenic potato roots demonstrates that this enzyme plays a key role in the transfer of energy between the cytosolic adenine and uridine nucleotide pools and in the distribution of carbon between starch and cellulose. Modulation of the expression of NDPK1 also alters the homeostasis of root respiration, glycolytic flux, reactive oxygen species production and growth. Herein, we propose a model summarizing the effects of the manipulation of NDPK1 levels on root metabolism. The model also accounts for G-quadruplex DNA binding, a moonlighting activity recently attributed to NDPK1, which possibly contributes to the metabolic phenotype of transgenic roots.

The glyoxalase pathway intermediate S-d-lactoylglutathione was recently implicated in protein post-translational modifications in animal systems. Here, we examined the spontaneous modification of the Arabidopsis thaliana cytosolic glyceraldehyde-3-phosphate dehydrogenase C1 (GAPC1) by this compound. Incubation of GAPC1 with S-d-lactoylglutathione resulted in the inhibition of enzyme activity. The inhibitory effect was concentration dependent and increased at alkaline pHs. Furthermore, the inhibition of GAPC1 by S-d-lactoylglutathione was favored by oxidative conditions and reversed by reduction with dithiothreitol. Analyses of the S-d-lactoylglutathione-treated protein by nanoLC-MS/MS revealed S-glutathionylation of its two Cys residues and N-lactoylation of six Lys residues. Protein structure predictions showed that the double S-glutathionylation is accommodated by the GAPC1 catalytic pocket, which likely explains enzyme inhibition. N-lactoylated sites overlap partially with previously reported N-acetylated sites at the surface of the GAPC1 tetramer. The efficiency of cytosolic glutaredoxin and thioredoxin isoforms was tested for reversing the S-d-lactoylglutathione-induced modification. In these assays, recovery of GAPC1 activity after inhibition by S-d-lactoylglutathione treatment was used as indicator of efficiency. The results show that both types of redoxins were able to reverse inhibition. We propose a model describing the mechanisms involved in the two types of post-translational modifications found on GAPC1 following exposure to S-d-lactoylglutathione. The possible involvement of these findings for the control over glycolytic metabolism is discussed.

In this study, the involvement of S,D-lactoylglutathione in the modification of the iso-form 1 of cytosolic glyceraldehyde 3P dehydrogenase (GAPC1) is examined. Incuba-tion of GAPC1 with S,D-lactoylglutathione resulted in inhibition of enzyme activity. The inhibitory effect was concentration-dependent and increased at alkaline pHs. Furthermore, inhibition of GAPC1 by S,D-lactoylglutathione was favored by oxidative conditions and reversed by reduction with dithiothreitol. Analyses of the S,D-lactoylglutathione-treated protein by nano LC-MS/MS revealed S-glutathionylation of its two Cys residues and N-lactoylation of six Lys residues. Pro-tein structure prediction showed that the double S-glutathionylation is accommodated by the GAPC1 catalytic pocket which likely explains enzyme inhibition. N-lactoylated sites overlap partially with previously reported N-acetylated sites at the surface of the GAPC1 tetramer. The efficiency of cytosolic glutaredoxin and thioredoxin isoforms was tested for reversing the S,D-lactoylglutathione-induced modification. In these as-says, recovery of GAPC1 activity after inhibition by S,D-lactoylglutathione treatment was used as indicator of efficiency. Results show that both types of redoxins were able to reverse inhibition. We propose a model describing the mechanisms involved in the two types of post-translational modification found on GAPC1 following exposure to S,D-lactoylglutathione. The possible involvement of these findings for the control over glycolytic metabolism is discussed.