JXB Advance Access originally published online on May 21, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1343-1349, June 1, 2004
© 2004 Oxford University Press
RESEARCH PAPER |
Transmembrane electron transport in sealed and NAD(P)H-loaded right-side-out plasma membrane vesicles isolated from maize (Zea mays L.) roots
Received 12 November 2003; Accepted 19 March 2004
1Universität Hamburg, Biozentrum Klein Flottbek und Botanischer Garten, Ohnhorststraße 18, D-22609 Hamburg, Germany
* To whom correspondence should be addressed. Fax: +49 40 42816 254. E-mail: s.luthje{at}botanik.uni-hamburg.de
Abbreviations: BPDS, bathophenanthroline disulphonate; Brij 58, polyoxyethylene 20 cetyl ether; DPI, diphenylene iodonium; FRE, ferric reductase; FRO, ferric reductase-oxidase; HCF III, ferricyanide; hexacyanoferrate III; ISO, inside-out; ROS, right-side-out; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide.
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 µM calcium decreased HCF III reduction up to 66%, while pre-incubation with 200 µM warfarin or diphenylene iodonium inhibited transmembrane electron transport only weakly. Fe3+-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.
Key words: Calcium, diphenylene iodonium, NAD(P)H-loaded vesicles, plasma membrane, transmembrane electron transport, Zea mays.
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