Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance to deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigate functions of the dugB and dugC genes in the model fungus Aspergillus nidulans. These genes are orthologues of dug2-3 involved in cytosolic GSH degradation in the yeast. The deletion (Δ) of dugB, dugC, or both resulted in a moderate increase in the GSH content in the mycelia grown on glucose, reduced conidia production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding MAP kinase pathway elements (e.g. steC, sskB, hogA, mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g. flbA,C,E, nosA, rosA, nsdC,D) were down-regulated in the ΔdugBΔdugC mutant. Deletion of dugB and/or dugC slowed down the depletion of GSH pools under carbon starvation. It also reduced accumulation of reactive oxygen species, decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions - in contrast to mycotoxin production - were controlled at post-transcriptional level. We suggest that GSH connects starvation and redox regulation to each other: Cells utilize GSH as stored carbon source during starvation. The reduction of GSH content alters the redox state activating regulatory pathways responsible for carbon starvation stress responses. Importance Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, secondary metabolite production (including mycotoxin formation) as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity or oxidative stress tolerance of a fungal strain is manipulated.

Relative transcriptions of Aspergillus nidulans dug1-3 (orthologes of Saccharomyces cerevisiae DUG -deficient in utilization of glutathione -pathway genes) and ggtA encoding g-glutamyl transpeptidase were studied under conditions inducing glutathione degradation. GgtA was induced in all cases when glutathione levels decreased, but addition of yeast extract, which moderated glutathione degradation, enhanced its induction. Although dug2 showed constitutive transcription, dug1 and dug3 were induced by carbon and nitrogen starvation and yeast extract did not caused significant changes in their relative transcription. The in silico reconstructed DUG pathway of A. nidulans is a promising candidate for cytosolic GSH degradation induced by carbon/nitrogen stress.