Immunocytochemical localization of glutathione precursors in plant cells

In plant cells, the antioxidant glutathione is synthesized out of its constituents cysteine, glutamate and glycine. As information about their subcellular and cellular distribution was not available in the past, the aim of the present study was to develop a technique that would allow visualization of their distribution in plant cells. With selective antibodies against cysteine, glutamate and glycine, it was possible, for the first time, to study the relative distribution of these components in meristematic root tip cells and mesophyll cells from older and younger leaves of Cucurbita pepo plants with the transmission electron microscope. These investigations revealed that high levels of cysteine accumulated in younger leaves. However, glutamate and glycine accumulated in the older ones, where cysteine levels were found to be significantly lower. On the subcellular level, glutamate was found in similar content in all investigated cellular compartments except for the cytosol, which showed up to 49% lower glutamate content (in younger leaves) than the other cell compartments. Levels of glycine were similar in all investigated cell compartments of one organ except vacuoles, which contained the lowest amounts of glycine. Cysteine was highest in plastids, the cytosol and in nuclei. When compared with younger leaves, glutamate and glycine were found to be accumulated in plastids (180 and 83%) and the cytosol (142 and 130%) of older leaves, whereas cysteine contents were found to be very low in the glutathione producing cell compartments of the leaves. These results indicate that low levels of cysteine might be a limitation for glutathione synthesis in older leaves. Cysteine and glycine were also detected in vacuoles where glutamate was absent. None of these components were detected in the apoplast. Therefore, these results indicate that glutathione degradation occurs within vacuoles or at the tonoplast, rather than at the plasmalemma or in the apoplast. Summing up, with the methods presented in this and a previous work, it is now possible to study the subcellular distribution of glutathione and its precursors in one plant sample simultaneously. The application of these methods on plant samples during abiotic and biotic stress situations should help us reveal possible limitations of glutathione synthesis during oxidative stress.