|Abstract:Glutathione (GSH)-deprived Dictyostelium discoideum accumulates methylglyoxal (MG) and reactive oxygen species (ROS) during vegetative growth. However, the reciprocal effects of the production and regulation of these metabolites on differentiation and cell motility are unclear. Based on the inhibitory effects of ?-glutamylcysteine synthetase (gcsA) disruption and GSH reductase (gsr) overexpression on aggregation and culmination, respectively, we overexpressed GSH-related genes encoding superoxide dismutase (Sod2), catalase (CatA), and Gcs, in D. discoideum. Wild-type KAx3 and gcsA-overexpressing (gcsA(OE)) slugs maintained GSH levels at levels of approximately 2.1-fold less than the reference GSH synthetase-overexpressing mutant; their GSH levels did not correlate with slug migration ability. Through prolonged KAx3 migration by treatment with MG and H2O2, we found that MG increased after the mound stage in this strain, with a 2.6-fold increase compared to early developmental stages; in contrast, ROS were maintained at high levels throughout development. While the migration-defective sod2- and catA-overexpressing mutant slugs (sod2(OE) and catA(OE)) decreased ROS levels by 50% and 53%, respectively, these slugs showed moderately decreased MG levels (36.2?5.8 and 40.7?1.6 nmol?g(-1) cells wet weight, P <0.05) compared to the parental strain (54.2?3.5 nmol?g(-1)). Importantly, defects in the migration of gcsA(OE) slugs decreased MG considerably (13.8?4.2 nmol?g(-1), P <0.01) along with a slight decrease in ROS. In contrast to the increase observed in migrating sod2(OE) and catA(OE) slugs by treatment with MG and H2O2, the migration of gcsA(OE) slugs appeared unaffected. This behavior was caused by MG-triggered Gsr and NADPH-linked aldolase reductase activity, suggesting that GSH biosynthesis in gcsA(OE) slugs is specifically used for MG-scavenging activity. This is the first report showing that MG upregulates slug migration via MG-scavenging-mediated differentiation.