Highly purified mitochondria from potato (S&men tuberosum L. cv. Bintje) tubers were subfractionated into a matrix fraction, an inner membrane fraction and an outer membrane fraction with minimal cross-contamination. When the matrix and inner membrane fractions were incubated with [$'P]ATP only one and three prominent phosphoproteins were detected after SDS-PAGE and autoradiography, respectively. In contrast, more than 20 phosphoproteins could be labelled in the outer membrane fraction, the main ones at 12, l&26,43, 58, 60, 65, 74 and 110 kDa. Only one band, at 18 kDa, was detectable when the labelling was done in the presence of EGTA. We conclude that the outer membrane of plant mitochondria contains at least one Ca*'-dependent protein kinase and more than 20 endogenous substrates.

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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Inside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with [Q Q-32 P]ATP. Proteins were separated by SDS^PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of [Q Q-32 P]ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins. ß

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP + ratio was unaffected, the stem NADPH/NADP + ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP + ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.