Effects of glucose starvation on mitochondrial subpopulations in the meristematic and submeristematic regions of maize root

Mitochondria isolated from 3-mm long maize (Zea mays L. var Dea) root tips were found to be heterogeneous on Percoll density gradients. The ultrastructure of these isolated mitochondria correlated well with that of mitochondria observed in situ and was consistent with the existence of mitochondria at different stages of maturation during cell development. The mitochondria of higher density presented an ultrastructure with many cristae and a dense matrix. These mitochondria showed classic respiratory properties, although with low ADP/O ratios. In contrast, the mitochondria of lower density showed few cristae and a clear matrix and did not seem to be fully functional because their rate of respiration was low and showed weak respiratory control. Lower- and higher- density mitochondria were shown to be differentially affected during the first stages of glucose starvation. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria, whereas lower- and intermediate-density mitochondria were absent in the mitochondrial preparations from glucose-starved maize root tips and were not observed in situ. Quantitatively, there was a decrease of the total mitochondrial pool when expressed as the amount of mitochondrial protein per root tip. However, this decrease affected low- and intermediate-density mitochondria, but not higher-density mitochondria. Thus, it was shown that a significant pool of functional mitochondria is maintained in maize root tips during the first stages of glucose starvation. The reasons for these apparently selective effects of glucose starvation on mitochondria are discussed in relation to effects on mitotic and differentiation processes.     

The temperature dependence of State IV respiration, the calcium uptake system, and the activity of the calcium ionophore A23187 in mitochondria from endo- and ectothermic animals.

1. Arrhenius plots of State IV respiratory activity of liver mitochondria from both rainbow trout and rat were linear over the temperature range 5-35 degrees C. 2. Calcium uptake was monitored by stimulation of oxygen consumption and by calcium electrode recording, with quite comparable results. Rainbow trout gave the usual linear Arrhenius plot but this plot for rat mitochondria exhibited two well-defined inflections or discontinuities. 3. The temperature dependence of the activity of the ionophore A23187 was investigated by measuring the increase in oxygen uptake following a sub-maximal dose of this drug. Again a linear relation was found for rainbow trout, but in this case the rat curves showed only a single inflection point. 4. These results are discussed in relation to other work on the effects of lipid phase transitions on mitochondrial membrane-associated systems.     

Environmentally induced changes in mitochondria and endoplasmic reticulum of Saccharomyces carlsbergensis yeast

The effects of culture environment on the volume density and surface density of mitochondria and endoplasmic reticulum in a facultative yeast were studied. When compared with cells grown aerobically on a nonrepressive substrate, cells grown in the absence of oxygen showed a sharp reduction in both volume density of mitochondria and surface density of the inner mitochondrial membrane (imm) in the remaining mitochondrial profiles. Use of fermentable (repressive) substrates under aerobic conditions restricted the volume density of mitochondria to a much greater extent than the surface density of imm. The range of mitochondrial volume densities in these experiments was 4-11%. Surface density of endoplasmic reticulum (ER) was sensitive to growth rate and in particular to changes in oxygen tension, showing large fluctuations during both anaerobic and aerobic adaptation. These fluctuations in ER are discussed in relation to the known role of this organelle in lipid metabolism.     

BIOCHEMICAL EFFECTS OF VICTORIN ON OAT TISSUES AND MITOCHONDRIA

Victorin, the pathotoxin produced by the plant pathogenic fungus Helminthosporium victoriae, causes changes in respiration and permeability which are typical of diseased plant tissues. To provide information on the site and mode of action of this toxin, the effects of victorin on mitochondria were studied and the nature and quantities of materials released from victorin-treated tissues were determined. Victorin added to isolated mitochondria had no effect on release of electrolytes or on oxidative-phosphorylative capacity. Hence the high respiratory rate found in victorin-treated tissues does not appear to be the result of a direct effect of the toxin on the respiratory centers. With mitochondria extracted from tissue pretreated with victorin, electrolyte release was unaffected but oxygen uptake was slightly higher and phosphate esterification considerably lower than controls. Thus the toxin produces indirect effects on mitochondrial activity, but the relation of these effects to tissue respiration remains undetermined. Victorin-treated tissue lost much larger quantities of certain organic and inorganic materials than control tissue with the most striking difference in the amount of potassium released.     

Ionophoretic properties and mitochondrial effects of cereulide: the emetic toxin of B. cereus.

The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black-lipid membranes in KCl solutions in an ionophore-like manner. It formed adducts with K+, Na+, and NH4+ but the conductance was highly selective for K+ in relation to Na+ and H+ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K+. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K+, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO3- but not in NaNO3-containing media, less in acetate. In NaNO3 media addition of the cereulide caused a transient diffusion potential which was reduced by adding K+. It is concluded that the toxic effects of cereulide are due to it being a K+ ionophore.     

Glucagon treatment of rats inhibits the accumulation of lysophospholipids by liver mitochondria during preparation and subsequent incubation

1) Rat liver mitochondria isolated from rats exposed to³²P_i for 24 h and treated with glucagon for 15 min before sacrifice contained less lysophosphatidylcholine and lysophosphatidylethanolamine than those from control animals. 2) Incubation of the mitochondria at 37°C for 15 min increased the lysophosphatidylcholine concentration of control mitochondria but not of those from glucagon-treated animals. 3) Lysophosphatidylethanolamine showed little change during in vitro incubation of mitochondria and this was not affected by glucagon treatment. 4) These results are discussed in relation to the effects of glucagon treatment on mitochondrial function in situ and in vitro.     

Inhibition of energy metabolism by benzoxazolin-2-one

The effects of the title compound (BOA) on energy-linked reactions in mitochondria were studied. BOA inhibited electron transfer between the flavin and ubiquinone in Complex I, and ATP synthesis at the F1 moiety of the ATPase complex. These results are discussed in relation to the toxicity of BOA towards a wide range of aerobic organisms.     

Cation permeability induced by spermine and polybrene in rat liver mitochondria

The effect of the polybasic substances Polybrene and spermine on the passive and active transport of monovalent cations in mitochondria was studied. These agents were found to stimulate the low amplitude swelling of mitochondria. Volume oscillations were induced by addition of substrate in the presence of spermine. In conditions where weak oscillations were obtained without these substances, oscillations were stimulated and their frequencies increased in the presence of Polybrene and spermine. Their effects were maximal with 100–300 moles spermine per litre and 3–5 mg Polybrene per litre. These results are discussed in relation to an interaction of the agents studied with membrane negative charges which may be important regulators of ion transport.     

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: a 31P-NMR and NAD(P)H fluorescence study

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO2), and extra-mitochondrial phosphates. Alterations in NADH and QO2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO2; (2) when QO2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO2. To investigate this further, effects of changing substrates at constant QO2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.     

Ethanol feeding to rats reversibly decreases hepatic carnitine palmitoyltransferase activity and increases enzyme sensitivity to malonyl-CoA

The effects of prolonged ethanol feeding on both carnitine palmitoyltransferase I activity and enzyme sensitivity to inhibition by malonyl-CoA were studied in rat liver, heart, skeletal muscle and kidney cortex mitochondria. Heart and skeletal muscle enzymes showed the highest specific activity and sensitivity to malonyl-CoA. Carnitine palmitoyltransferase I in extrahepatic tissues showed no changes on ethanol feeding. Only the liver enzyme activity was altered after long term ethanol administration, by suffering a progressive decrease in activity and a parallel increase in sensitivity to malonyl-CoA. These alterations reversed after 10 days of ethanol withdrawal. These results are discussed in relation to the control of carnitine palmitoyltransferase I and the effects of ethanol on fatty acid metabolism.     

The inhibition by fluorocitrate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1. The effects of synthetic fluorocitrate were studied on: (a) the oxidation of citrate and cis-aconitate by rat liver mitochondria; (b) the activity of the aconitate hydratase found in the liver cell sap; (c) the activity of the aconitate hydratase solubilized from liver mitochondria. 2. Fluorocitrate was found to be a potent inhibitor of oxidation of citrate but only a weak inhibitor of oxidation of cis-aconitate: 6.7mum-fluorocitrate (containing 4% of the inhibitory isomer) caused 94% inhibition of the oxidation of citrate (2mm) whereas 1.0mm-fluorocitrate was necessary to provoke the same inhibition when cis-aconitate (2mm) was the substrate. The degree of inhibition varied in relation to the respiratory state of mitochondria when fluorocitrate was added. The inhibition could be partially reversed by cis-aconitate. 3. The aconitate hydratase extracted from the mitochondria was much less inhibited by fluorocitrate than was the mitochondria-bound enzyme, and the aconitate hydratase found in the cell sap was even less sensitive. 0.3mm-Fluorocitrate was required to cause 50% inhibition of the reaction citrate-->cis-aconitate, catalysed by the aconitate hydratase extracted from the mitochondria, and 1.2m-fluorocitrate for the extramitochondrial enzyme. For both enzymes the reaction citrate-->cis-aconitate was 2-3 times more sensitive to fluorocitrate than was the reaction isocitrate-->cis-aconitate. The inhibition was of the competitive type for both reactions.     

Relationship between the conformation of isolated rat liver mitochondria and their susceptibility to rabbit reticulocyte lipoxygenase]

Lipoxygenase from rabbit reticulocytes cause disruption of mitochondrial membranes and peroxidation of their lipids as judged by electronmicroscopy, release of matrix enzymes and formation of malonyldialdehyde. Without substrate mitochondria become orthodox and strong lysis by lipoxygenase appears. The lysis is prevented by ATP or ADP plus succinate; in this case mitochondria remain condensed or partly condensed. The protection by substrate was even observed in the presence of 2,4-DNP, although the mitochondria were transformed to the condensed state. Lysis was more pronounced in hypotonic than in hypertonic sucrose, condensed mitochondria are also attacked. No relation seems to exist between lipoxygenase attack and the conformational state of mitochondria. Lysis of mitochondria is dependent on the susceptibility of the fatty acid moiety of phospholipids, which may be influenced by both metabolic and structural events via alteration of protein-lipid interactions.     

The effects of zinc and lanthanum on calcium uptake by mitochondria and fragmented sarcoplasmic reticulum of frog skeletal muscle

Calcium uptake by mitochondria and fragmented sarcoplasmic reticulum (FSR) isolated from frog skeletal muscle was studied. These fractions were characterized by electron microscopy, succinic dehydrogenase assay and by using mitochondrial inhibitors. With high (100 μM) Ca in the medium, the Ca accumulating capacity of the two fractions was similar. Zinc in concentrations of 5–10 μM in the medium had no effect on Ca uptake by either fraction whereas higher concentration of Zn (25 μM) reduced Ca uptake in both fractions. Five micromolar lanthanum lowered Ca uptake by 70% in mitochondria but had no effect on Ca uptake by FSR. With 10 and 25 μM La, Ca uptake by FSR decreased by 12 and 20% respectively. Addition of La (5 μM) to Ca-loaded mitochondria had no effect indicating that La could only interfere with the Ca binding step and was unable to release Ca that was already stored. In the medium that originally contained low (10 μM) Ca FSR was able to reduce the Ca concentration below 0.1 μM. In contrast mitochondria, although possessing an equal capacity for Ca uptake were unable to accumulate Ca from the medium when Ca was lowered to approximately 4 μM. Presence of 5–10 μM La in the low Ca medium had no effect on the total amount of Ca taken up by FSR in two minutes but reduced the rate of Ca uptake significantly. The relation of the effects of Zn and La on the isolated fractions to their reported effects on the contractile response of skeletal muscle is discussed.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

Activity of mitochondria isolated and purified from dwarf and tall,light-grown pea seedlings

Mitochondria from dwarf and tall light-grown peas have been separated from contaminating chloroplasts and other organelles on discontinuous sucrose gradients. A comparison between activities of the mitochondria from the two cultivars of light-grown pea seedlings was made. It was observed that both respiratory control (R.C.) indices and phosphate to oxygen (P : O) ratios were superior in mitochondria from the tall cultivar than the dwarf cultivar. The results are discussed in relation to energy status and possible gibberellic acid content in the two cultivars.     

Salt tolerance in Suaeda maritima (L.) Dum: A COMPARISON OF MITOCHONDRIA ISOLATED FROM GREEN TISSUES OF SUAEDA AND PISUM

Mitochondria were isolated from green leaves and stems of theglycophyte Pisum sativum and the halophyte Suaeda maritima.The preparations oxidized malate, succinate, and 2-oxoglutarateas well as externally added NADH. Acceptor control ratios wereabout 2.8 for mitochondria from Pisum and 1.8 for mitochondriafrom Suaeda oxidizing malate+pyruvate in 125 mM sodium chloride.The mitochondrial fraction was contaminated with chloroplastfragments which resulted in relatively low rates of oxygen uptakewhen these were expressed on a protein basis. The addition of sodium chloride at concentrations greater than200 mM considerably reduced the rates of oxygen uptake by bothspecies in the presence and absence of phosphate acceptor (ADP).Acceptor control ratios were reduced and there was a markeddecline in the ADP/O ratio. Sucrose at equivalent molar concentrationshad a much less drastic effect on the mitochondria. There was no significant difference in the effects of thesetwo solutes on mitochondria from the two species and the similarityof response is discussed in relation to the cytoplasmic ioncontent of the halophyte.     

EPR studies of higher plant mitochondria. I Ubisemiquinone and its relation to alternative respiratory oxidations.

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.     

Morphologic, biochemical and functional properties of the mitochondria of the rabbit gastric mucosa]

Purified fractions of the rabbit gastric mucosa and liver mitochondria were isolated. Electrone microscopy and functional studies of mitochondria showed the most intact mitochondria of gastric mucosa to be localized in gradient fractions of p=1.16-1.18, and those of the liver in fractions with p=1.19-1.20. The gastric mitochondria differed from the liver mitochondria by specific localization in oxyntic cells, abundance of crists, small matrix volume, presence of high respiratory control at lesser values of pH, high respiratory activity, great amount of cytochromes of electron transport chain in inner mitochondria membrane, two-fold excess of cytochroma a in relation to b and c, absence of b5 and by presence of great amount of phosphathydilaethanolamine and unsaturated fatty acids.     

Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells

Summary. The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.Correspondence and reprints: Arbeitsgruppe Pflanzenphysiologie, Fachbereich Zellbiologie, Universität Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.     

The effect of pressure on fetal and neonatal liver mitochondrial membranes

The effects of pressure on late fetal and neonatal rat liver mitochondria have been investigated. High hydrostatic pressure, as produced by isopycnic centrifugation in sucrose and glycogen gradients, altered the mitochondrial membranes of 1- and 7-day-old rats. Most of the mitochondrial enzymes, chosen for their known submitochondrial location, had a trimodal distribution in the sucrose gradients. In the glycogen gradients, a shift of the mitochondria to a lower density was noticed. Fetal liver mitochondria were resistant to the hydrostatic pressure exerted during isopycnic centrifugation experiments under different conditions such as sucrose and glycogen density gradients. The submitochondrial compartment tracer enzymes exhibited an unimodal distribution. Experimental temperatures set at 15 degrees C had a protective effect from hydrostatic pressure alterations in the neonatal liver mitochondria, whereas no effects were noticeable in the fetal mitochondria. Experiments in a hydraulic compression chamber showed that outer membranes of fetal mitochondria were more fragile and the inner membranes more resistant to compression than in the early stages after birth.