Regulation of alternative pathway activity in plant mitochondria: nonlinear relationship between electron flux and the redox poise of the quinone pool

The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not apparent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respiratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regulation of respiratory flux via the alternative pathway.     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.     

Measurement of the redox state of the ubiquinone pool in Rhodobacter capsulatus membrane fragments

The dependence of the respiratory rate on the redox poise of the quinone pool was investigated in wild type and mutant membranes of Rhodobacter capsulatus. A linear relationship has been found between these two parameters only when succinate was oxidized by the bc1 complex. Conversely, a marked nonlinear relationship was observed between the Q-pool reduction level and the respiratory rate when O2 uptake occurred via the alternative oxidase. In addition, it was found that this latter pathway was not engaged until Q-pool reduction level reached approximately 25%. These results are discussed within the framework of a homogeneous pool regulating both photosynthetic and respiratory fluxes.     

Possible existence of an intermediate pool of ubiquinone in rat heart mitochondria

1. The existence of an intermediate pool of ubiquinone in intact mitochondria of rat heart was investigated. 2. The incorporation of [3H-methyl]S-adenosylmethionine into ubiquinone-9 was not influenced by the co-synthesis of the intermediate, 3-nonaprenyl-4-hydroxybenzoate. 3. In the intermediate-depleted mitochondria, the synthetic rate of the intermediate, 3-nonaprenyl-4-hydroxybenzoate was similar to that of ubiquinone. 4. The possible existence of 3-nonaprenyl-4-hydroxybenzoate as a metabolic pool under physiological condition is discussed.     

Light-enhanced dark respiration in mesophyll protoplasts from leaves of pea

The respiratory oxygen uptake by mesophyll protoplasts of pea (Pisum sativum cv Arkel) was stimulated up to threefold after 15 minutes of illumination at an intensity of 1250 microeinsteins per square meter per second in the presence of 5 millimolar bicarbonate at 30°C. The extent of light-enhanced dark respiration (LEDR) increased progressively with duration of preillumination. The LEDR exhibited two phases. The initial high rate of respiration decreased in about 10 minutes to a lower steady value similar to that before illumination. The promotion of LEDR by the presence of bicarbonate and inhibition by glyceraldehyde or 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that LEDR was dependent on products of photosynthetic carbon assimilation/electron transport. Thus, the photosynthetic products exert a markedly quick influence on dark respiration in mesophyll protoplasts.     

The mechanism of reduction of the ubiquinone pool in photosynthetic bacteria at different redox potentials.

(1) A flash number dependency of flash-induced absorbance changes was observed with whole cells of Rhodospirillum rubrum and chromatophores of R. rubrum and Rhodopseudomonas sphaeroides wild type and the G1C mutant. The oscillatory behavior was dependent on the redox potential; it was observed under oxidizing conditions only. Absorbance difference spectra measured after each flash in the 275--500 nm wavelength region showed that a molecule of ubiquinone, R, is reduced to the semiquinone (R-) after odd-numbered flashes and reoxidized after even-numbered flashes. The amount of R reduced was approximately one molecule per reaction center. (2) The flash number dependency of the electrochromic shift of the carotenoid spectrum was studied with chromatophores of Rps. sphaeroides wild type and the G1C mutant. At higher values of the ambient redox potential a relatively slow phase with a rise time of 30 ms was observed after even-numbered flashes, in addition to the fast phase (completed within 0.2 ms) occurring after each flash. Evidence was obtained that the slow phase represents the formation of an additional membrane potential during a dark reaction that occurs after flashes with an even number. This reaction is inhibited by antimycin A, whereas the oscillations of the R/R- absorbance changes remain unaffected. At low potentials (E = 100 mV) no oscillations of the carotenoid shift were observed: a fast phase was followed by a slow phase (antimycin-sensitive) with a half-time of 3 ms after each flash. (3) The results are discussed in terms of a model for the cyclic electron flow as described by Prince and Dutton (Prince, R.C. and Dutton, P.L. (1976) Bacterial Photosynthesis Conference, Brussels, Belgium, September 6--9, Abstr. TB4) employing the so-called Q-cycle.     

Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

An analysis of the control of phosphorylation-coupled respiration in isolated plant mitochondria

The control of phosphorylation-coupled respiration in isolated turnip (Brassica rapa) mitochondria was investigated according to the principles of metabolic control analysis as developed by H. Kacser and J. A. Burns ([1973] Symp Soc Exp Biol 32: 65-104) and R. Heinrich and T. A. Rapoport ([1974] Eur J Biochem 42: 97-105). Inhibitor titration studies were used to determine quantitatively the amount of control exerted by four individual processes—cytochrome bc₁, cytochrome oxidase, H⁺-ATPase, and the adenine nucleotide carrier—on respiratory flux under ADP-excess (state 3) and ADP-limited (state 4) conditions with a range of respiratory substrates. Under state 3 conditions control strength was found to be distributed between cytochrome oxidase, cytochrome bc₁, and H⁺-ATPase in decreasing order of importance. The adenine nucleotide carrier exerted no control on respiratory flux under these conditions. Control strength at each step was found to vary with different substrates and with the respiratory flux as altered by ADP supply, i.e. virtually zero control strength at cytochrome oxidase and cytochrome bc₁ under state 4 conditions.     

Temperature-dependent changes in Photosystem II heterogeneity of attached leaves under high light

Attached leaves of pumpkin ( Cucurbita pepo L. cv. Jattiläismeloni) were exposed to high light intensity at room temperature (ca 23°C) and at 1°C. Fluorescence parameters and electron transport activities measured from isolated thylakoids indicated faster photoinhibition of PSII at low temperature. Separation of the α and β components of the complementary area above the fluorescence induction curve of dichlorophenyl-dimethylurea-poisoned thylakoids revealed that at low temperature only the α-centers declined during exposure to high light intensity while the content of functional β-centers remained constant. Freeze-fracture electron microscopy showed no decrease in the density of particles on the appressed exoplasmic fracture face, indicating that the photoinhibited α-centers remained in the appressed membranes at 1°C. Because of the function of the repair and protective mechanisms of PSII, strong light induced less photoinhibition at room temperature, but more complicated changes occurred in the α/β-heterogeneity of PSII. During the first 30 min at high light intensity the decrease in α-centers was almost as large as at 1°C, but in contrast to the situation at low temperature the decrease in α-centers was compensated for by a significant increase in PSIIβ-centers. Changes in the density and size of freeze-fracture particles suggest that this increase in β-centers was due to migration of phosphorylated light-harvesting complex from appressed to non-appressed thylakoid membranes while the PSII core remained in the appressed membranes. This situation, however, was only transient and was followed by a rapid decrease in the functionalβ-centers.     

The effect of nucleotides and inhibitors on respiration in isolated wheat mitochondria

The effect of mono-, di-, and trinucleoside phosphates and respiratory inhibitors on respiration in winter wheat (Triticum aestivum L. cv. Rideau) mitochondria has been examined. When added during state 4 respiration, subsequent to addition of ADP, all of the dinucleotides stimulated oxidation and induced respiratory control with all substrates examined. Similar results were obtained with AMP, but other mononucleotides and all trinucleotides did not affect the rate of oxidation. Nucleoside diphosphates did not stimulate respiration when added prior to the addition of ADP, but subsequent addition of AMP, ADP, or ATP re-established coupled respiration in the presence of the dinucleotides.     

Cytochrome b558 monitors the steady state redox state of the ubiquinone pool in the aerobic respiratory chain of Escherichia coli

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome o complex and the cytochrome d complex. These both function as ubiquinol-8 oxidases and reduce molecular oxygen to water. Electron flux is funneled from a variety of dehydrogenases, such as succinate dehydrogenase, through ubiquinone-8, to either of the terminal oxidases. A strain was examined which lacks the intact cytochrome d complex, but which overproduces one of the two subunits of this complex, cytochrome b558. This cytochrome, in the absence of the other subunit of the oxidase complex, does not possess catalytic activity. It is shown that the extent of reduction of cytochrome b558 in the E. coli membrane monitors the extent of reduction of the quinone pool in the membrane. The activity of each purified oxidase was examined in phospholipid vesicles as a function of the amount of ubiquinone-8 incorporated in the bilayer. A ratio of ubiquinol-8:phospholipid as low as 1:200 is sufficient to saturate each oxidase. The maximal turnover of the oxidases in the reconstituted system is considerably faster than observed in E. coli membranes, demonstrating that the rate-limiting step in the E. coli respiratory chain is at the dehydrogenases which feed electrons into the system.     

Wy-14,643 and fenofibrate inhibit mitochondrial respiration in isolated rat cardiac mitochondria

We investigated the direct effects of two selective PPARalpha ligands, fenofibrate and Wy-14,643, on mitochondrial respiratory function using isolated rat cardiac mitochondria. Isolated left ventricular mitochondria were incubated with increasing concentrations of fenofibrate or Wy-14,643 (10, 100, and 500 microM) and mitochondrial respiration determined using: malate/glutamate (complex I), succinate (complex II) and palmitoyl-l-carnitine as oxidative substrates. Our data show that acute exposure to Wy-14,643 and fenofibrate differentially perturb cardiac mitochondrial respiration i.e., fenofibrate more potently inhibited mitochondrial respiration and bioenergetic capacity compared to Wy-14,643. Moreover, we found that both agents increased uncoupling of mitochondrial oxidative phosphorylation.     

Greening-related lipid changes in leaves,protoplasts and a plasmamembrane-enriched fraction of pea

Light-induced changes in the membrane lipid compositions were studied in pea leaves and in protoplasts and a plasmamembrane-enriched fraction (PMEF)* of pea leaves. PC, PE, PI, PG, PA, MGDG, DGDG and SL were identified as the glycerolipids. The relative levels of various membrane lipids changed due to light-induced greening. There was an increase in the galactolipids of leaves and leaf protoplasts. The galactolipid constituent of the PMEF was very low and showed no change. Among the plasmamembrane phospholipids, PI increased with a concomitant decrease in PC.