Transmembrane Electron Transport in Plasma Membrane Vesicles Loaded with an NADH-Generating System or Ascorbate

Abstract

Sugar beet (Beta vulgaris L.) leaf plasma membrane vesicles were loaded with an NADH-generating system (or with ascorbate) and were tested spectrophotometrically for their ability to reduce external, membrane-impermeable electron acceptors. Either alcohol dehydrogenase plus NAD* or 100 millimolar ascorbate was included in the homogenization medium, and right-side-out (apoplastic side-out) plasma membrane vesicles were subsequently prepared using two-phase partitioning. Addition of ethanol to plasma membrane vesicles loaded with the NADH-generating system led to a production of NADH inside the vesicles which could be recorded at 340 nanometers. This system was able to reduce 2,6-dichlorophenolindophenol-3'-sulfonate (DCIP-sulfonate), a strongly hydrophilic electron acceptor. The reduction of DCIP-sulfonate was stimulated severalfold by the K+ ionophore valinomycin, included to abolish membrane potential (outside negative) generated by electrogenic transmembrane electron flow. Fe3+-chelates, such as ferricyanide and ferric citrate, as well as cytochrome c, were not reduced by vesicles loaded with the NADH-generating system. In contrast, right-side-out plasma membrane vesicles loaded with ascorbate supported the reduction of both ferric citrate and DCIP-sulfonate, suggesting that ascorbate also may serve as electron donor for transplasma membrane electron transport. Differences in substrate specificity and inhibitor sensitivity indicate that the electrons from ascorbate and NADH were channelled to external acceptors via different electron transport chains. Transplasma membrane electron transport constituted only about 10% of total plasma membrane electron transport activity, but should still be sufficient to be of physiological significance in, e.g. reduction of Fea3 to Fe2+ for uptake.It has been suggested that the plant plasma membrane contains redox systems that transfer electrons from cytoplasmic donors to acceptors in the apoplast. One such transplasma membrane electron transport system is thought to be induced in roots of nongraminaceous plants during iron deficiency and to be involved in the reduction of Fe3" to Fe2" . 11 and 17). The nature of the electron donor(s) for the transplasma membrane electron transport system(s) is not known. Substantial evidence indicates, however, that NAD(P)H is such an electron donor (9, 11, 17).We (1) previously found that NADH-ferricyanide3 and NADH-Cyt c reductase activities were about 30% latent (i.e. could be measured only in the presence of a detergent, such as Triton X-100) with inside-out vesicles and about 80% latent with right-side-out vesicles. From these results, we concluded that both donor and acceptor sites for these activities were present on the cytoplasmic surface of the plasma membrane and that transplasma membrane electron transport from NADH to ferricyanide or Cyt c could at most constitute 30% of the total activities (1). Based on trypsin inhibition of NADH-Cyt c reductase activity (2, 21), H+-pumping capacity (21), and H+-ATPase latency using th...

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…RecAIR12 sensitivity to the redox potential, with two apparent midpoint redox potentials at +137 and +236 mV, is in line with that of partially purified PM cytochrome b from bean hypocotyls (+135 and +204 mV; Trost et al, 2000) and Arabidopsis leaves (+135 and +180 mV; Bérczi et al, 2003), and consistent with redox titrations of whole PM preparations (Asard et al, 1989;Askerlund and Larsson, 1991;Bérczi et al, 2003). This also indicates that AIR12 represents a major fraction of high potential and ascorbate-reducible cytochromes b of plant PM.…”

“…In whole cells or intact tissues, reduction of electron acceptors was found associated with apoplast acidification, membrane depolarization, or oxidation of internal electron donors, suggesting a transmembrane electron transfer chain fed by cytosolic reductants (Vuletić et al, 2005). PM vesicles loaded with physiological electron donors [ascorbate, NAD (P)H] were found to reduce impermeable electron acceptors (MDA, ferrichelates, ferricyanide) on the apoplastic side of the membrane (Askerlund and Larsson, 1991;Asard et al, 1992;Horemans et al, 1994;Menckhoff and Lü thje, 2004). In this way, PM redox systems may influence the redox state of the apoplast and provide a redox connection between apoplast and symplast.…”

“…In fact, in ascorbate-loaded PM vesicles, the totality of ascorbate-reducible cytochromes could be oxidized by an external electron acceptor such as ferricyanide or MDA (Asard et al, 1992;Horemans et al, 1994), and AIR12 certainly represents a major ascorbate-reducible cytochrome of plant PM. Trans-PM electron transport involving cytosolic NAD(P)H was also demonstrated, although it is not known whether a cytochrome was involved (Askerlund and Larsson, 1991;Menckhoff and Lü thje, 2004). In any case, because AIR12 is not a transmembrane protein, interactions with other redox partners would be needed to build up an electron route across the PM (Vuletić et al, 2005;Lü thje, 2008).…”

Transmembrane electron transport in ascorbate‐loaded plasma membrane vesicles from higher plants involves a b‐type cytochrome

“…The tissue homogenisation buffer was supplemented with 100 mM sodium ascorbate to include high concentrations of the reductant inside the plasma membrane vesicles [8,9]. The membrane-impermeable electron acceptors, ferricyanide and cytoehrome c,were rapidly reduced by the aseorbate-loaded membranes (Fig.…”

“…Recently a method became available for the prepara- tion of ascorbate-loaded plasma membrane vesicles, capable of electrogenic transmembrane electron transport [8,9]. These membrane fractions provide a tool to investigate the possible involvement of the high potential b-type cytochrome in trans-plasma membrane electron transport in plants.…”