The Regulation of Electron Partitioning between the Cytochrome and Alternative Pathways in Soybean Cotyledon and Root Mitochondria

Abstract

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on severa1 factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by a-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and lightgrown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which i s increased dramatically by either pyruvate or dithiothreitol. I n etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. I n addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration i s not present in the absence of inhibitors.The regulation of electron partitioning between the cyanide-sensitive Cyt pathway and the cyanide-resistant alternative pathway in plant mitochondria has long been of interest because of the nonphosphorylating nature of the latter pathway and its implications for ATP yield during

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

“…The inhibitor concentrations were obtained from titrations carried out in a liquid phase oxygen electrode and were similar to values used previously for these species (Lennon et al 1997;Gonzàlez-Meler, Matamala & Peñuelas 1998b). All stocks were freshly prepared before use.…”

“…Over the course of the experiment, each sample consumed at least 30% but no more than 50% of the initial oxygen. The r 2 values of all unconstrained linear regressions between -lnf and ln(R/R 0 ) (with at least five data points) were greater than the value of 0·995 considered minimally acceptable , 2000aLennon et al 1997;. During inhibitor treatments, either 1 mm KCN (in 1 mm TES, pH 8·0) or 6 mm…”

“…Mitochondrial mini-preparations were performed as described by Lennon et al (1997), and full mitochondrial isolations as described in Umbach & Siedow (1993). Protein concentrations were estimated by the method of Lowry et al (1951).…”

“…For alternative oxidase protein, samples were prepared with 100 mm DTT as a reductant in the sample buffer (Umbach & Siedow 1993;Umbach et al 1994). Proteins were transferred to nitrocellulose according to Towbin, Staehelin & Gordon (1979), and immunoblotting was performed as described by Lennon et al (1997) using the alternative oxidase monoclonal antibody (AOA) against the alternative oxidase protein from Sauromatum guttatum Schott (Elthon, Nickels & McIntosh 1989) at 1 : 1000 dilution (1 : 100 for G. sepium). For the ATP synthase immunoblots, polyclonal antibodies against the b-subunit of the mitochondrial ATP synthase (F 1 -ATP synthase) of Zea mays L. leaves were used at 1 : 1000 dilution.…”

“…The activity of the alternative pathway is thought to be dependent on: (i) the level of its substrate, reduced ubiquinone (Moore & Siedow 1991;Umbach, Wiskich & Siedow 1994); (ii) the level of alternative oxidase protein present (Gonzàlez-Meler et al 1998a; and (iii) the activation state of the alternative oxidase protein (Umbach & Siedow 1993, 1996 . Increases in protein level alone do not always result in increased alternative pathway activity (Lennon et al 1997;. This may be explained by the fact that alternative oxidase activity is subject to post-translational regulation involving the reduction state of a regulatory sulphhydryl-disulphide system (Umbach & Siedow 1993) and activation by a-keto acids, such as pyruvate (Millar et al 1993;Umbach et al 1994;Rhoads et al 1998).…”