Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

The plasma-membrane H(+)-ATPase (EC 3.6.1.35) of maize (Zea mays L.) coleoptiles and enclosed leaves has been localized at the optical-microscope level utilizing paraffin sections and a specific monoclonal antibody. Both in coleoptiles and in leaves the stomatal guard cells and the phloem of vascular bundles are the tissues most enriched in ATPase. The enrichment in guard cells is relevant to active ion transport during stomata opening. Considering the postulated activation of coleoptile ATPase by auxin, it is remarkable that the ATPase is not enriched in the coleoptile epidermis, where most of the auxin receptor is located.

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The proton pumping ATPase in the plasma membrane of Elodea canadensis is believed to playa major role in inorganic carbon acquisition. To investigate potentially different carbon uptake strategies within the same plant. plasma membrane H+-ATPase distribution and polar current patterns were investigated in Elodea leaves and stems. Specific activity of plasma membrane H+-ATPase in leaf microsomal fractions was tenfold higher than in stem derived microsomes. Probing western blots with a monoclonal antibody specific for plasma membrane W -ATPase, yielded strongly visible double bands at 100 kDa in leaf microsome preparations. whereas little antigen was detected in analogous stem microsome preparations.Several accumulated arguments reveal the PM-H+-ATPase as the ATP-dependent H+-extruding system in Elodea densa leaves (Marre et al., 1988(Marre et al., , 1989 Trockner and Marre, 1988). This enzyme would thus be responsible for acidification at the

When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.