FIGURE 1: Increased hydrogen peroxide generation and release by P. anserina cultures during aging and PQ-stress.

(A) After 2 days of ascospore germination, fungal mycelia were either directly challenged with 20 µM PQ (juv) or grown for 11 days on M2 medium to obtain a senescent stage. The cultures were subsequently incubated for 4 days on M2 with 20 µM PQ. As a control, both age stages (juv: 6 days; sen: 17 days) were cultivated on PQ-free M2 medium. After this time, DAB-staining-solution was poured on the cultures. The reaction with secreted H₂O₂ leads to the formation of a dark brown pigment.

(B) Quantitative measurement of H₂O₂ release. P. anserina cultures were essentially grown as described in (A). Instead of on M2, cultures were grown on PASM medium until reaching a final age of 6 days (juv), 11 days (ma) and 15 days (sen), respectively. Concentrations of 0 µM, 10 µM and 20 µM PQ were administered. Bars indicate mean ± standard error of H₂O₂ release relative to juvenile untreated cultures. n = 16 – 41. Asterisks indicate pV < 0.01.

(C and D) Relative changes in H₂O₂ concentration in the cytoplasm. P. anserina strains producing the redox sensitive HyPer protein, localized to the cytoplasm, were germinated for 2 days on germination medium and grown for 4 days on M2 agar plates. The mycelium was punched out of the agar, transferred to a 96 well plate and soaked in 60 µl water. Subsequently, H₂O₂ (C) or PQ (D) was added at the indicated times (arrows) to a final concentration of 0.5 mM, 5 mM and 50 mM or 1.3 µM, 2.5 µM and 25 µM, respectively. The fluorescence of the mycelium after exitation at 488 ± 5 nm (oxidized HyPer) and 420 ± 5 nm (reduced HyPer) was measured at 530 ± 20 nm. The graphs show the ratio of oxidized to reduced HyPer normalized to a untreated HyPer strain.