C cortex, PL photobiont layer, Pho photobiont, M medulla, Hy fungal hyphae ROS generation, chlorophyll autofluorescence and lipid peroxidation selleck during lichen rehydration Although several works

have described an extracellular oxidative burst during rehydration in some lichen species, virtually nothing is known about intracellular ROS production and its relationship to abiotic stress. In order to determine whether intracellular ROS release follows the rehydration of R. farinacea thalli, 10 μM of the fluorescent probe DFCH2-DA was added to the deionized water Copanlisib supplier used for rehydration. The samples were observed by fluorescence and confocal microscopy 3-4 h after rehydration. The presence of 2′,7′- dichlorofluorescin (DCF), the fluorescent oxidation product of DCFH2, indicated the intracellular production of free radicals during lichen rehydration

(Figure 2B-D). DCF was especially concentrated in the lichen cortex. No significant green autofluorescence was detected in the absence of the probe (Figure 2A). Confocal microscopy showed discrete points of green fluorescence around several large photobionts (Figure 2E), probably due to mycobiont hyphae tips. Figure 2 ROS click here in rehydrated R. farinacea thalli. Thalli of R. farinacea rehydrated with deionized water and 10 μM DCFH2-DA and observed 3-4 h post-rehydration. A, B, C, D ROS content, as revealed by the green fluorescence emission of DCF under a fluorescence microscope (magnification: 400× for A, B and 1000× for C, D); E overlay of confocal microscopy images reveals ROS distribution around some of the photobionts (green fluorescence); F overlay of confocal microscopy images of ROS content of R. farinacea thalli that had

been rehydrated with c-PTIO 200 μM, arrows point to photobionts photobleached by the confocal laser during the observation (oxPho). Red fluorescence is due to the photobiont’s chlorophyll in all cases. Each micrograph is representative of several images corresponding to independent Niclosamide samples. C cortex, M medulla, PL photobiont layer, Pho photobiont, oxPho photobleached photobiont, Hy fungal hyphae A fluorometric kinetics of intracellular free radical production in Ramalina farinacea thalli was performed in order to confirm microscopical data. Figure 3A demonstrates that the rate of intracellular free radical production in recently rehydrated thalli was much higher than the rate of intracellular free radical production in thalli kept in the hydrated state during the previous 24 h. Furthermore, intracellular release of free radicals during rehydration under physiological conditions was biphasic with an initial exponential phase of 20 min followed by a linear phase (Figure 3B). Chlorophyll autofluorescence was simultaneously recorded since this parameter is a surrogate of the levels and integrity of this molecule and therefore of the photosynthetic status of the cell.