High resolution imaging of subcellular glutathione concentrations by quantitative immunoelectron microscopy in different leaf areas of Arabidopsis

Glutathione is an important antioxidant and redox buffer in plants. It fulfills many important roles during plant development, defense and is essential for plant metabolism. Even though the compartment specific roles of glutathione during abiotic and biotic stress situations have been studied in detail there is still great lack of knowledge about subcellular glutathione concentrations within the different leaf areas at different stages of development. In this study a method is described that allows the calculation of compartment specific glutathione concentrations in all cell compartments simultaneously in one experiment by using quantitative immunogold electron microscopy combined with biochemical methods in different leaf areas of Arabidopsis thaliana Col-0 (center of the leaf, leaf apex, leaf base and leaf edge). The volume of subcellular compartments in the mesophyll of Arabidopsis was found to be similar to other plants. Vacuoles covered the largest volume within a mesophyll cell and increased with leaf age (up to 80% in the leaf apex of older leaves). Behind vacuoles, chloroplasts covered the second largest volume (up to 20% in the leaf edge of the younger leaves) followed by nuclei (up to 2.3% in the leaf edge of the younger leaves), mitochondria (up to 1.6% in the leaf apex of the younger leaves), and peroxisomes (up to 0.3% in the leaf apex of the younger leaves). These values together with volumes of the mesophyll determined by stereological methods from light and electron micrographs and global glutathione contents measured with biochemical methods enabled the determination of subcellular glutathione contents in mM. Even though biochemical investigations did not reveal differences in global glutathione contents, compartment specific differences could be observed in some cell compartments within the different leaf areas. Highest concentrations of glutathione were always found in mitochondria, where values in a range between 8.7 mM (in the apex of younger leaves) and 15.1 mM (in the apex of older leaves) were found. The second highest amount of glutathione was found in nuclei (between 5.5 mM and 9.7 mM in the base and the center of younger leaves, respectively) followed by peroxisomes (between 2.6 mM in the edge of younger leaves and 4.8 mM in the base of older leaves, respectively) and the cytosol (2.8 mM in the edge of younger and 4.5 mM in the center of older leaves, respectively). Chloroplasts contained rather low amounts of glutathione (between 1 mM and 1.4 mM). Vacuoles had the lowest concentrations of glutathione (0.01 mM and 0.14 mM) but showed large differences between the different leaf areas. Clear differences in glutathione contents between the different leaf areas could only be found in vacuoles and mitochondria revealing that glutathione in the later cell organelle accumulated with leaf age to concentrations of up to 15 mM and that concentrations of glutathione in vacuoles are quite low in comparison to the other cell compartments.