Quantitative X-ray microanalysis of hydrogen peroxide within plant cells

Using quantitative X-ray microanalysis in combination with CeCl3-based cytochemical staining of hydrogen peroxide (H2O2) we have developed a new solution for quantification of H2O2 at the subcellular level. Quantitative X-ray microanalysis of plastic-embedded leaves of Populus euphratica Oliv. showed that the obtained cerium precipitates by CeCl3 staining were the mixture of cerium perhydroxides and cerium phosphate, in which the fractions of CePO4 were: (1) 52-74% in cell walls of fresh leaf segments, and (2) 34-70% in the cytoplasm in 10 mM H2O2-treated leaf segments that were previously freeze-dried. Taking the concentration of cerium phosphate as staining background, we reached the cellular concentration of cerium perhydroxides and the corresponding concentration of H2O2. Results showed that H2O2 was present in the cytoplasm of rehydrated leaf segments (29-58 mM), but in fresh leaves, H2O2 was observed in the walls of all measured cell types (17-74 mM).