Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria

Abstract

Exogenous NADPH oxidation by purified mitochondria from both potato tuber and Arum maculatum spadix was completely and irreversibly inhibited by sub-micromolar diphen~leneiodonium (DPI), while exogenous NADH oxidation was inhibited to only a small degree. Addition of DPI caused the collapse of the membrane potential generated by NADPH oxidation, while the potential generated by NADH was unaffected. We conclude that there are two distinct enzymes on the outer surface of the inner membrane of plant mitochondria, one specific for NADH, the other relatively specific for NADPH, with both enzymes linked to the electron transport chain.Key words: Diphenyleneiodonium; Electron transport chain; Mitochondria, plant; NAD(P)H dehydrogenase |. IntroductionUnlike mammalian mitochondria, the electron transport hain of plant mitochondria contains at least two NAD(P)H ,tehydrogenases in addition to Complex I [1]. One of these ~otenone-insensitive dehydrogenases is located on the outer , urface of the inner mitochondrial membrane, the other on the inner, matrix surface, and in the following we will refer to them ;s NDex and NDin, respectively.Due to the presence of NDex, plant mitochondria oxidize ~xogenous NADH and NADPH. The resulting electrons enter he electron transport chain at ubiquinone bypassing Complex i, the site of rotenone inhibition [1][2][3][4][5]. Compared to NADH ~xidation, NADPH oxidation has a lower pH optimum [6][7][8], s more sensitive to thiol reagents [7,9] and appears to require nore Ca 2÷ for activity [9][10][11][12]. NADH oxidation has been re~orted to be induced in red beetroots without a concomitant nduction of NADPH oxidation [13]. Likewise, mitochondria rom suspension-cultured cells of sugar beet oxidize NADH, ~ut exhibit very low rates of NADPH oxidation [14]. On the )asis of this circumstantial evidence it has been concluded that t is most likely that two separate enzymes exist, one for each ,:oenzyme [2,4,14]. However, no direct evidence has ever been r~resented to support this conclusion.Diphenyleneiodonium ( Materials and methodsMitochondria were prepared from potato (Solanum tuberosum L.) tubers according to [16]. Crude mitochondria were isolated from Arum maculatum spadices according to [17] and purified according to [18].Oxygen consumption was measured in a medium containing 0.3 M sucrose, 5 mM MOPS (pH 7.2), 5 mM KH:PO4, 2.5 mM MgCI2, 1 mM CaC12 and 0.4 ¢tM FCCP using an oxygen electrode (Rank Bros.). The membrane of the oxygen electrode was washed with 70% (v/v) ethanol between assays to avoid carry-over of DPI.Membrane potential measurements using 16 ]zM safranine as the probe were made according to [19]. The medium used was the same as that above except that FCCP was omitted, CaCI2 was 0.1 mM and 20 ,ug.mg 1 BSA was included.NAD(P)H oxidation with oxygen as the electron acceptor was assayed spectrophotometrically at 340 nm in the same medium used for the oxygen electrode measurements. Activities were calculated using an extinction coefficient for NAD(P)H of 6.2 mM -1 .cm -~, All ...

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“…To modify the levels of NAD(P)H and see how this affects both fluorescence and tube growth, we applied diphenyleneiodonium (DPI), which inhibits plasma membrane NAD(P)H oxidases and mitochondrial NAD(P)H dehydrogenases and other flavoproteins (Roberts et al, 1995;Baker et al, 1998;Møller, 2001), to germinated pollen tubes that were exhibiting growth oscillations (Fig. 5).…”

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