Localization of a protein, immunologically similar to a sucrose-binding protein from developing soybean cotyledons, on the plasma membrane of sieve-tube members of spinach leaves

Abstract

Immunocytochemical studies using antibodies raised against a 62-kDa membrane protein isolated from developing soybean (Glycine max (L.) Merr.) cotyledons were performed on leaf tissue of spinach (Spinacia oleracea L.). This 62-kDa protein was labeled by 6'-deoxy-6'-(4-azido-2-hydroxy)-benzamidosucrose (HABS), a photoaffinity sucrose analogue (K. G. Ripp et al., 1988, Plant Physiol.88, 1435-1445). Western-blot analysis of spinach plasma-membrane proteins indicated a cross-reactive polypeptide identical in molecular mass to that found in soybean. Indirect immunogold labeling of resin-embedded sections of fully expanded leaf tissue resulted in specific localization of colloidal gold on the sieve-tube plasma membrane. The label was uniform and, except for a few non-specific gold particles over the cell wall, all other cellular organelles and membrane systems were free of label. With the exception of occasional gold particles associated with the companion-cell plasma membrane, all other cell types of the leaf contained little or no label. Control sections treated with non-immune rabbit immunoglobulin-G were also essentially free of label. Immunogold labeling of young leaves, in which the phloem contained no mature sieve-tube members, were free of label for the 62-kDa protein. However, young leaf tissue in which mature or nearly mature sieve tubes could be identified, contained immunolabel associated with the sieve-tube plasma membranes. Similar results were obtained with mature leaf tissue of sugar beet (Beta vulgaris L.). The results of the immunocytochemical studies are consistent with the suggestion that the concentrating step in the phloem-loading process in this species may occur across the sieve-tube plasma membrane.