Protoplasma (1998) 203:186--193 PROTOPLASMA 9 Springer-Verlag 1998 Printed in Austria Orientation of NAHD-linked ferric chelate (turbo) reductase in plasma membranes from roots of Plantago lanceolata W. Schmidt* and M. Bartels Fachbereich Biologie, Carl yon Ossietzky Uuiversit~it Oldenburg, Oldenburg Received January 8, 1998 Accepted June 11, 1998 Summary. Plasma membrane vesicles isolated from roots of Planta- go lanceolata L. revealed approximately 70% right-side-out orienta- tion based on structure-linked latency with H+-ATPase as a marker. Incubation with 0.05% Brij 58 caused the formation of sealed inside- out vesicles, evidenced by assaying ATP-dependent proton pumping activity with the optical pH probe acridine orange. NADH-linked FeEDTA reductase activity was stimulated by including either Triton X-100 or Brij 58 in the assay medium. The activity of inverted (Brij- treated) vesicles was not further increased by the addition of Triton, suggesting that maximum activity was obtained in inside-out vesi- cles. Iron deficiency resulted in a ca. 2-fold increase in the specific activity of both ATPase and Fe(III) chelate reductase but did not cause significant alterations with respect to the effect of detergents. It is concluded that in vitro both donor and acceptor sites of NADH- FeEDTA reductase are located on the cytosolic face of the membrane and trans-oriented flow of electrons is not detectable in plasma mem- brane vesicles. Unlike Fe chelate reduction in vivo, the plasma mem- brane-bound reductase activity was insensitive towards application of the translation inhibitor cycloheximide prior to isolation of the membranes, implying the involvement of a regulatory enzyme in the electron transport in vivo. Keywords: Inside-out vesicles; Iron deficiency; Plantago lanceola- ta; Plasma membrane; Redox system. Abbreviations: BPDS bathophenanthroline disulfonate; BTP 1,3- bis[tris(hydroxymethyl)methylamino]-propane; PM plasma mem- brane. Introduction As in most eukaryotes, the iron uptake system of higher plants is composed of a plasma membrane- (PM)-bound Fe(III) reductase and an uptake system *Correspondence and reprints: Fachbereich Biologie, Carl yon Ossi- etzky Universitat Oldenburg, Postfach 2503, D-26111 Oldenburg, Federal Republic of Germany. for ferrous ions, both components being homeostati- cally regulated (Guerinot and Yi 1994, Klausner and Dancis 1994, Marschner and R6mheld 1994, Fox et al. 1996). While root-mediated ferric chelate reduc- tion is ubiquitous in higher plants, an increase in activity upon iron starvation has been demonstrated exclusively in dicotyledonous and nongraminaceous monocots (Strategy I plants), both at the level of PM vesicles and intact root tissue (Moog and Brtigge- mann 1994). In grasses (Poaceae), a high-capacity phytosiderophore uptake system is induced under conditions of low iron availability which is not cou- pled to extracellular reduction of ferric ions (Strategy II; Marschner and R6mheld 1994). The Fe-stress- inducible ferric chelate (turbo) redox system was sug- gested to be different from a constitutive oxido(stan- dard)reductase, not involved in physiologic iron uptake (Bienfait 1985). Although both (putative) redox systems are thought to mediate transmembrane electron flow in intact tissue, a vectorial orientation of the latter system has been questioned at the level of PM vesicles (Askerlund et al. 1988). The increase in specific activity of PM-bound enzymes caused by disrupting the permeability barri- er to hydrophilic substrates with detergents is usually interpreted in terms of unmasking latent active sites, and voted as evidence for transmembrane activity. However, a clear distinction between activity on the trans- or cis-surface of the membrane can only be made in a latency-free system, i.e., with vesicles of uniform orientation. Several techniques have been used to invert PM vesicles isolated by the aqueous