Immunocytolocalization of Plasma Membrane

The localization of plasma membrane H+-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific antiATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a 'two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H+ currents, which leave nongrowing parts and enter growing tips. The major ATPase of plant plasma membranes is a H+ pump, which seems to play a central role in plant physiology. The proton gradient generated by the enzyme is the driving force for active nutrient transport, and the pH changes resulting from proton pumping may be involved in growth control (21). At the level of whole plants, the loading of root xylem with inorganic nutrients and the loading of leaf phloem with organic nutrients seem to depend on active transport processes driven by the H+-ATPase (3, 12, 16). Knowledge about the distribution of plasma membrane H+-ATPase in plant tissues is essential for understanding the pathway and mechanism of nutrient transport (3, 12, 16). Previous approaches to this problem consisted of the cytochemical staining for ATP hydrolysis by lead-induced precipitation of the released Pi (7, 25-27). However, it has recently been demonstrated that the plasma membrane H+-ATPase is inactivated by both the lead and the fixatives used in the cytochemical procedure and that the measured ATP hydrolysis is catalyzed by a molybdate-sensitive phosphatase (13). Clearly, a more specific approach to ATPase localization was needed. We have expressed in Escherichia coli the carboxyl-terminal domain of a cloned ATPase gene (18) and generated specific polyclonal antibody against the enzyme. By utilizing this antibody and either frozen or paraffin sections ofplant tissues, we found that the ATPase is highly enriched in vascular tissues ' Present address: Instituto de Recursos Naturales y Agrobiologia, Avda. Reina Mercedes s/n, 41080 Sevilla, Spain. and root epidermis and endodermis. The implications of this distribution for current models of nutrient transport in plants