Fig. 4

Cytosolic Ca²⁺ levels and calcium signaling increase after Mn²⁺ addition. A The analysis of cytosolic Ca²⁺ levels via a Ca²⁺ fluorescent probe Fluo-3/AM. The T. reesei Rut-C30 and its derivative mutant strains were cultured in liquid minimal medium for 48–60 h with 0 or 10 mM final concentration of MnCl₂ (0 or 10 Mn, respectively). For detection, 50 μM Fluo-3/AM was used, and the intensity was monitored using Automatic Inverted Fluorescence Microscopy. Green fluorescence represents the free cytosolic Ca²⁺. DIC, differential interference contrast. B Comparative fluorescence ratio analysis of Mn²⁺ influence on cytosolic Ca²⁺ levels. The y-axis represents the Ca²⁺ fluorescence ratio measured by CLSM and the x-axis the different strains tested. The effects of Mn²⁺ on the expression levels of certain calcium signaling-related genes cam (C), cna1 (D) and crz1 (E) in T. reesei Rut-C30. 0 Mn, no MnCl₂ was added to the medium; 10 Mn, final concentration of 10 mM MnCl₂. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from untreated strains (*p < 0.05, **p < 0.01, Student’s t test). Different letters indicate significant differences between the columns (p < 0.05, according to Duncan’s multiple-range test)