Effects of rotenoids on isolated plant mitochondria

The effects of several rotenoids have been studied on potato (Solanum tuberosum L.) tuber and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls mitochondria. The selective inhibition of mitochondrial complex I is characterized by several tests: (a) no effect can be observed on exogenous NADH or succinate oxidation; (b) malate oxidation is inhibited at pH 7.5; (c) one-third decrease of ADP/O ratio appears during malate oxidation at pH 6.5 or during α-ketoglutarate, citrate, or pyruvate oxidation at a pH about 7; (d) during malate oxidation at pH 6.5, a transient inhibition appears which can be maintained by addition of exogenous oxaloacetate; (e) in potato mitochondria, the inhibition of malate oxidation disappears at pH 6.5 when NAD⁺ is added. Then, a one-third decrease of the ADP/O ratio can be measured.

Such a selective inhibition of complex I is obtained with deguelin, tephrosin, elliptone, OH-12 rotenone, and almost all the rotenoids extracted from Derris roots. The presence of the rings A, B, C, D, E seems to be necessary for the selective inhibition. Opening of the E ring and hydroxylation of the 9 position (rot-2′-enoic acid) give a rotenoid derivative with multisite inhibitory activities on flavoproteins, which are quite comparable to those of common flavonoids such as kaempferol (Ravanel et al. 1982 Plant Physiol 69: 375-378).

     

Effects of freezing on isolated plant mitochondria

Mitochondria isolated from spinach leaves (Spinacia oleracea L.) and potato tubers (Solanum tuberosum L.) were partly injured when subjected to freezing for 2 to 4 h at-25°C in salt solutions in the absence of cryoprotectants. Damage was manifested by the inactivation of respiratory properties and increase in the permeability of the mitochondrial membranes. Decrease in respiratory control indicated that the control mechanism of the electron transport chain was influenced by freezing. Oxidative phosphorylation was only slightly more affected than electron transport. The inactivation of the membrane systems was caused by an increase in the concentration of membrane-toxic solutes. This was confirmed by treatment of the organelles at 0°C in solutions of high salt concentrations. When sugar was present in the course of freezing, mitochondria were partly or completely protected. On a molar basis, sucrose was more effective in membrane protection than glucose. Under certain conditions amino acids, e.g., proline and hydroxyproline, also stabilized isolated mitochondria during freezing.Abbreviations BSA bovine albumin - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - PVP polyvinyl pyrrolidone - RC respiratory control - Tris tris (hydroxymethyl) aminomethane     

Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue

Apoptosis in myocardial tissue slices was induced by extended incubation under anoxic conditions. Mitochondria were isolated from the studied tissue. A new method of isolation of mitochondria in special conditions by differential centrifugation at 1700, 10,000, and 17,000g resulted in three fractions of mitochondria. According to the data of electron microscopy the heavy mitochondrial fraction (1700g) consisted of mitochondrial clusters only, the middle mitochondrial fraction (10,000g) consisted of mitochondria with typical for isolated mitochondria ultrastructure, and the light fraction consisted of small mitochondria (2 or 3 cristae) of various preservation. The heavy fraction contained unusual structural elements that we detected earlier in apoptotic myocardial tissue—small electron-dense mitochondria incorporated in bigger mitochondria. The structure of small mitochondria from the light fraction corresponded to that of the small mitochondria from these unusual elements—mitochondrion in mitochondrion. The most important functions of isolated mitochondria are strongly inhibited when apoptosis is induced in our model. The detailed study of the activities of the two fractions of the apoptotic mitochondria showed that the system of malate oxidation is completely altered, the activity of cytochrome c as electron carrier is partly inhibited, while succinate oxidase activity is completely preserved (complexes II, III, and IV of the respiration chain). Succinate oxidase activity was accompanied by high permeability of the internal membrane for protons: the addition of uncoupler did not stimulate respiration. ATP synthesis in mitochondria was inhibited. We demonstrated that in our model of apoptosis cytochrome c remains in the intermembrane space, and, consequently, is not involved in the cascade of activation of effector caspases. The possible mechanisms of induction of apoptosis during anoxia are discussed.     

Interspecific transfer of isolated plant mitochondria by microinjection

Summary Data on isolation, purification and transfer of mitochondria from a cytoplasmic male sterile line of the Ogura type of Raphanus sativus to a male fertile line of Brassica napus are reported. Microinjection has been used for the transfer of the donor mitochondria to the recipient protoplasts. The injected protoplasts were identified and followed individually throughout their development using a computerized microscope stage which greatly enhanced the number of injections (five-fold). The transferred donor mitochondria were stably maintained during several successive cell divisions, revealing that they were viable and functional. Several calluses were obtained from injected protoplasts without using any selection pressure. Restriction fragment length analysis of seventeen calluses, using mitochondrial DNA probes, indicated that three contained the donor Ogura type mitochondria. No recombinant types of mitochondria have been observed. Flow cytometric and karyotype analyses of the calluses revealed the presence of similar amount of DNA and chromosome number as those of the recipient plants of B.napus. The application of microinjection for the manipulation of cytoplasmic composition is discussed.Abbreviations ATPase adenosine triphosphate phosphohydrolase - BSA bovine serum albumin - CMS cytoplasmic male sterility - DIOC6(3) 3,3-dihexyloxacarbocyanine-iodide - mt mitochondria(I) - PEG polyethylene glycol - RFLP restriction fragment length polymorphism - UV ultraviolet     

Free-flow electrophoresis for purification of plant mitochondria by surface charge

Sample purity is the key for a successful in-depth analysis of any given subcellular proteome. The suitability of free-flow electrophoresis to assist conventional, centrifugation-based techniques in the preparation of plant mitochondria from green and non-green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.     

Studies of transcription in isolated wheat mitochondria and organelle extracts

RNA synthesis has been studied in purified wheat embryo mitochondria and in a mitochondrial lysate depleted of endogeneous DNA. Both systems work optimally at pH 8.5 and at 25°C. Magnesium is a better cofactor than manganese; moreover, in purified mitochondria the latter cation seems to abolish the stimulatory effect of magnesium. The RNA polymerase activity present in the mitochondrial lysate is inhibited strongly by heparin and actinomycin D while alpha-amanitin and rifampicin do not affect the enzyme activity. A low molecular weight form (50 kDa) as well as a larger form (150 kDa) of the wheat mitochondrial RNA polymerase have been obtained by gel filtration. Several DNA templates can be used by the mitochondrial lysate; the best template seems to be poly d(AT) but the mitochondrial DNAs from yeast and wheat, as well as single stranded M13 DNA, can be used efficiently as templates. Multiple RNA species, between 1 and 6 kb in size, were synthesized ‘in organello’.     

Effect of brief vascular perfusion on the ultrastructure and activity of mitochondria isolated from the rat heart

Summary Mitochondria isolated from heart tissue after a 1-min perfusion with Hanks medium were found to have significantly lower rates of State-3 respiration and respiratory control ratios compared to mitochondria isolated from non-perfused hearts. Examination of the mitochondrial preparations by electron microscopy revealed that a large proportion of the mitochondria isolated from perfused heart tissue were swollen and broken compared to mitochondria from non-perfused hearts.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

Cristae formation-linking ultrastructure and function of mitochondria

Mitochondria are double-membrane enclosed eukaryotic organelles with a central role in numerous cellular functions. The ultrastructure of mitochondria varies considerably between tissues, organisms, and the physiological state of cells. Alterations and remodeling of inner membrane structures are evident in numerous human disorders and during apoptosis. The inner membrane is composed of two subcompartments, the cristae membrane and the inner boundary membrane. Recent advances in electron tomography led to the current view that these membrane domains are connected by rather small tubular structures, termed crista junctions. They have been proposed to regulate the dynamic distribution of proteins and lipids as well as of soluble metabolites between individual mitochondrial subcompartments. One example is the release of cytochrome c upon induction of apoptosis. However, only little is known on the molecular mechanisms mediating the formation and maintenance of cristae and crista junctions. Here we review the current knowledge of the factors that determine cristae morphology and how the latter is linked to mitochondrial function. Further, we formulate several theoretical models which could account for the de novo formation of cristae as well as their propagation from existing cristae.