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Electron transport across plasma membranes has been observed in vivo in several plant species and tissues after the application of ferricyanide (hexacyanoferrate III, HCF III). In the present work, a transmembrane electron flow was demonstrated in sealed and NAD(P)H-loaded right-side-out (apoplastic-side-out) plasma membrane vesicles isolated from maize (Zea mays L.) roots. HCF III was reduced at a rate of up to 126 nmol min(-1) mg(-1) protein by NADPH-loaded vesicles, while reduction rates with NADH-loaded vesicles were several-fold lower. Coincident with the reduction of HCF III, NAD(P)H oxidation was observed inside the vesicles. The dependence of reduction on K+ indicated an electrogenic transmembrane electron flow. Application of 100 microM calcium decreased HCF III reduction up to 66%, while pre-incubation with 200 microM warfarin or diphenylene iodonium inhibited transmembrane electron transport only weakly. Fe(3+)-EDTA was not reduced significantly by NADPH-loaded plasma membrane vesicles, whereas XTT was reduced at a rate of 765 pmol min(-1) mg(-1) protein. The results suggested a major function for NADPH in transmembrane electron flow and were discussed in conjunction with in vivo experiments.

Although several investigations have demonstrated a plasma membrane (PM)-bound peroxidase activity in plants, this study is the first, to our knowledge, to purify and characterize the enzymes responsible. Proteins were extracted from highly enriched and thoroughly washed PMs. Washing and solubilization procedures indicated that the enzymes were tightly bound to the membrane. At least two distinct peroxidase activities could be separated by cation exchange chromatography (pmPOX1 and pmPOX2). Prosthetic groups were identified in fractions with peroxidase activity by absorption spectra, and the corresponding protein bands were identified by heme staining. The activities of the peroxidase enzymes responded different to various substrates and effectors and had different thermal stabilities and pH and temperature optima. Because the enzymes were localized at the PM and were not effected by p-chloromercuribenzoate, they were probably class III peroxidases. Additional size exclusion chromatography of pmPOX1 revealed a single activity peak with a molecular mass of 70 kD for the native enzyme, whereas pmPOX2 had two activity peaks (155 and 40 kD). Further analysis of these fractions by a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with heme staining confirmed the estimated molecular masses of the size exclusion chromatography.

Proton-induced x-ray emission was used to investigate the penetration of compounds of the membrane-impermeant electron acceptors hexabromoiridate IV, hexachloroiridate IV, and hexacyanoferrate III into corn (Zea mays L.) roots. Maps of the heavy element distribution in cross-sections of fixed, epoxy-embedded roots showed for hexabromoiridate IV small amounts of Br in samples treated for 24 h with concentrations normally used in physiological experiments (0.02 mM). After treatment with high concentrations (0.8 mM) of these complexes, Fe and Ir as well as Br were found in root cross-sections. In samples taken at a distance of 5 mm behind the root tip, we found an even distribution of Fe, Ir, and Br over the whole cross-section. In samples taken 15 mm behind the root tip, about 99% of both Br and Ir was confined to the rhizodermal cell layer. The distribution did not change with the complex used. These data are consistent with the view that apoplastic diffusion of the electron acceptors was blocked by the hypodermal Casparian band.

The correlation of the effects of vitamin K3 and dicumarol (an anti-vitamin K in pharmaceutical applications) on the trans plasma membrane electrical potential difference of maize roots with the reduction of the artificial electron acceptors hexacyanoferrate (III) or hexabromoiridate (IV) and the concomitant enhancement of acidification of the incubation medium was investigated. Vitamin K3 depolarized the plasma membrane of Zea mays L. roots, while dicumarol had no significant effect on the membrane potential. Plants treated with vitamin K3 for 30 min followed by intense rinsing showed higher reduction of hexabromoiridate (IV) than hexacyanoferrate (III), as well as a stimulated acidification of the incubation medium. Depolarization of the plasma membrane by hexacyanoferrate (III) or hexabromoiridate (IV) decreased after an incubation with vitamin K3. Pretreatment with dicumarol caused an inhibition of hexacyanoferrate (III) reduction and medium acidification as well as depolarization by K3. The reduction of hexabromoiridate (IV) was not affected by dicumarol pretreatment. The proton secretion associated with the reduction was slightly lowered. According to our results, it seems possible that vitamin K3 acts as an electron acceptor for the plasmalemma electron transport system of maize roots whereas dicumarol appears to inhibit electron and proton transport.

The effects of vitamin K3 or dicumarol on plasma membrane bound hexacyanoferrate (III) and hexabromoiridate (IV) reductase activity and on the H + pumping rate were investigated. Incubation with vitamin K3 followed by intense rinsing stimulated the subsequent reduction of hexabromoiridate (IV) and hexacyanoferrate (III) as well as proton secretion induced by external electron-acceptors, while pretreatment with dicumarol inhibited proton secretion induced by redox activity and hexacyanoferrate (III) reduction rate, but not the effects of hexabromoiridate (IV). A 30 min incubation in 0-2 mM K3 or dicumarol, followed by rinsing, inhibited H+ secretion for about 2 d. Incubation for more than 12 h in OT mM dicumarol or 0-2 mM K3 caused lethal injury to the root cells.

Three artificial electron acceptors of different E'0 and charge, hexacyanoferrate (III) (K3Fe(CN)6), hexachloroiridate (IV) (K2IrCl6), and hexabromoiridate (IV) (K2IrBr6), were compared with respect to their rate of reduction by roots of Zea mays L., the concomitant proton secretion, and to the effect on plasmalemma depolarization. It has been shown that these plasma membrane impermeable electron acceptors were reduced by a plasmalemma reductase activity. At low concentrations proton secretion was slightly inhibited, at higher concentrations, however, the rate of proton secretion was stimulated. The root cell plasmalemma showed a transient depolarization after addition of all three electron acceptors. The depolarization was concentration-dependent for the iridate complexes but not for hexacyanoferrate (III). For both iridate complexes maximum depolarization was reached at 50 ??mol dm-3. A hypothetical model as an explanation of the redox dependent proton secretion will be given.