Immunogold Localization of Chloroplast Protein in Spinach Leaf Mesophyll, Epidermis, and Guard Cells

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Abstract
Immunogold labelling on ultrathin sections of Lowicryl K4M embedded lower epidermis of spinach (Spinacia oleracea L.) with adhering mesophyll cells has been applied to localize five proteins, namely ribulose‐1,5‐bisphosphate carbocylase/oxygenase (EC 4.1.1.39), chloroplast coupling factor (EC 3.6.1.34), NADP‐malate dehydrogenase (EC 1.1.1.82), chloroplast fructose‐1,6‐bisphosphatase (EC 3.1.3.11) and thioredoxin m by electron microscopy in stomata. Comparative data of labelling densities of the localized proteins in chloroplasts of spongy parenchyma, epidermis and guard cells are presented. All five proteins could be detected in guard cell chloroplasts at levels little different from those found in chloroplasts of spongy parenchyma and of epidermis cells. The two light‐activated enzymes NADP‐malate dehydrogenase and fructose‐1,6‐bisphosphatase are reduced in stomata to about one third of the mesophyll concentration. Aldehyde‐sensitivity of the inspected enzymes and suitability of cryosections for immunolocalization studies on plant tissues, in comparison with resin‐embedded specimens, was investigated. Fixation with 2% glutaraldehyde resulted in no detectable decrease of antigenicity. Cryosections were found to be less suitable for immunogold labelling, since labelling densities were lower than on resin. Only labelling of the thylakoid‐bound coupling factor was comparable using both techniques.