Real-time monitoring of subcellular H(2)O(2) distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H(2)O(2)) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H(2)O(2) sensor that allows H(2)O(2) monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H(2)O(2) sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔC(R) to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H(2)O(2) produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H(2)O(2) levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H(2)O(2) levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H(2)O(2) gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H(2)O(2) biology in plant cells.