Formation of the respiratory enzyme apparatus in plant cells. III. Antigenic spectra of mitochondria in the root tip of corn

By means of one- and two-dimensional (cross) immunoelectrophoresis, immunoelectro-diffusion and radial immunodiffusion, about 20 antigens were detected in mitochondria of meristem, zone of elongation and mature cells, among which some were identified by zymographic methods. The differentiation of root cells is not accompanied by qualitative changes in antigenic spectra of mitochondria and changes in the ratio of antigens (including glutamate and malate dehydrogenases) suggest that mature mitochondria develop from preexisting ones by gradual quantitative changes which are due to different rate of synthesis of constituent proteins.     

Different amounts of DNA in each mitochondrion in rice root

The DNA content of individual mitochondria in rice root cells was analyzed by fluorescence microscopy. Differences in DNA content were detected between individual mitochondria. Some mitochondria contained no detectable nucleoid (DNA-protein complexes). The percent of mitochondria with DAPI(4',6-Diamidino-2-phenylindole) -stained nucleoids varied over the length of the root (root base, 33%; middle portion of root, 41%; root tip, 91%). The mean amounts of DNA per mitochondrial nucleoid were equivalent to 46.4 kbp in the root base, 52.0 kbp in the middle portion of root and 124.2 kbp in the root tip. The amount of DNA in individual mitochondria and the ratio of mitochondria with visible nucleoids were higher in the root tip than in other parts of the root. The estimated amount of DNA in almost all of the observed mitochondria was smaller than the amount of DNA equivalent to the rice mitochondrial genome size (490 kbp), even in root tip.     

The development of structural changes in epidermal cells of Maize Roots During Water Stress

Ultrastructural changes following increasing periods of water stress induced by means of polyethylene glycol 4000 (from 5 min to 18 h) were investigated in young epidermal cells of the primary roots ofZea mays. The sensitivity of the individual cell components to water stress was considered according to the time sequence in which their alterations appeared. Endoplasmic reticulum (ER) and mitochondria proved to be the most sensitive cell components, their structure changing after 5 min of water stress. By 8 h of stress, the condensation of nuclear chromatin in some cells was apparent, preceding polyribosome degradation but not all the other changes of the stressed cells. Similar types of structural alterations appeared slightly earlier in the more differentiated epidermal cells. The rapid changes in the structure of ER and mitochondria coincided with the rapid decrease of water potential of the root tips and the immediate cessation of the root growth.     

Morphological differentiation of mitochondria in the early chick embryo: a stereological analysis

The morphological evolution of mitochondria in three cell types of chick embryo in neurulation was analyzed by stereological methods. Mitochondria, showing a random distribution, were characterized by moderate electron-dense matrices and normal cristae. The numerical density of mitochondria significantly increased in the neuroectoderm and epiblastic cells while their volume density remained unchanged. The mitochondria in mesoderm cells were ellipsoidal (axial ratio 2:1) at stages 5 and 8 although they underwent an elongation in neuroectoderm and epiblastic cells (axial ratio from 2:1 to 1.6:1). The individual size of "average mitochondria" in the mesoderm cells was smaller than in other cell types. The total V/S (volume/surface) ratio of mitochondria decreased during neurulation. These morphological changes have been discussed emphasizing the possible metabolical role of mitochondria during morphogenesis.     

Stability of membrane potential in heart mitochondria: single mitochondrion imaging

Mitochondrial membrane potential (delta psi(m)) plays an important role in cellular activity. Although delta psi(m) of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of delta psi(m). The current study is performed to investigate the factors that stabilize delta psi(m) in cells by observing delta psi(m) of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of delta psi(m) in cells.     

西洋参根的发育解剖学研究

西洋主根顶端的原分生组织由三群原始细胞组成。初生木质部为三原型。维管形成层产生的次生维管组织中薄壁细胞占主导地位;维管分子量少、聚集成群,分散在薄壁组织中。周皮加、周皮发生较迟,其木栓形成层由紧靠内皮层的皮层细胞产生。不同年龄西洋参主根随着龄龄的增加,周皮、次生真心皮部和木质部面积均呈增加趋势,但韧皮部与木质部面积比值自５：１下降至１：１。一年生根由中柱鞘产生初生分泌道,由维管形成层产生一圈次生分     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

Expression of the primary biliary cirrhosis antigens in yeast: Aspects of mitochondrial control

The mitochondria of 21 yeast strains were tested for the expression of primary biliary cirrhosis (PBC) specific antigens. The amounts of the antigens in the mitochondrial preparations varied with the strains. Genetic analysis of the strain differences in antigen expression indicated nuclear control which was complex. Those strains expressing the least amounts of antigens exhibited coagulating mitochondria in organellar preparations. Additional evidence relating expression of antigens to the physiological/structural state of mitochondria was that cells grown in the presence of the mitochondrial uncoupling agent, 2,4-dinitrophenol (DNP), failed to produce any antigens, and that glucose repression of mitochondria suppressed antigen expression. Blockage of mitochondrial protein synthesis either throughpetite mutation or by culture in the presence of erythromycin decreased the content of antigens in the mitochondria but did not competely block antigen production. The presence of the PBC antigen in the mitochondria of these cells with nonfunctional mitochondrial synthesizing machinery further indicates that these antigens are cytoplasmically synthesized. Analysis of the pre- and postmitochondrial fractions of all homogenates confirmed that the antigens are not only cytoplasmically synthesized but also have an extramitochondrial location in cells, probably in the plasma membrane.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells.

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2.In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".     

Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus. 2. Growth and proliferation of mitochondria in embryogenesis.

The dynamics of structural changes of the chondriome in the early development of the sea urchin Paracentrotus lividus was studied. Mature eggs and embryos at various stages of cleavage were used for quantitative and ultrastructural analysis based on computerized 3D reconstruction from serial ultrathin sections. The following structural transformations of the chondriome were shown to occur in the course of embryogenesis: (i) 15 min after fertilization, mitochondrial clusters disintegrate, and mitochondrial division is induced. At the stage of two blastomeres the population of mitochondria increases twofold; (ii) the mitochondria divide by means of the contraction of both outer and inner membranes. The forming furrow divides the "parental" mitochondrion into two equal "daughter" parts; (iii) at the four-cell stage the division ceases, and mitochondria start to grow, so that the mitochondrial length increases; (iv) cell differentiation further stimulates elongation of rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria changes; (v) in an unfertilised egg, the mitochondria are in a condensed form; after fertilisation all the mitochondria acquire a conventional form. Modern concepts of chondriome proliferation in eukaryotic cells are discussed.     

Influence of diet on the acyl composition of phospholipids in endothelial cells and mitochondria of rat brain

The effect of diet on phospholipid acyl groups of rat brain endothelial cells and mitochondria and of liver was determined. Rats were fed high-protein diets with a 41 linoleate/linolenate ratio but with 4.4%, 1.9%, or 0.8% of the caloric content provided by these essential fatty acids (cal % EFA) or were fed a fat-free diet. In capillary endothelial cells the greatest change occurred in the plasmalogen ethanolamine fraction, there being a significant reduction in then-3 series of acyl groups and increase in then-9 series as the cal % EFA was reduced. Other phospholipid fractions changed little. More pronounced changes occurred in brain mitochondria and liver phospholipids. The small changes in capillary endothelia with cal % EFA are in contrast to the great changes produced by a change in the linoleate/linolenate ratio. As the ratio is reduced, there is a progressive increase in then-3 series in all phospholipid fractions.     

Electron microscopic study of the mitochondria in the apical meristem of a wheet shoot in ontogeny]

The mitochondria of apical meristem cells in the wheat (Triticum aestivum L.) shoot were studied during ontogenesis using electron microscope and morphometrical methods. Changes in their structure were followed from the juvenile mitochondria of the seed embryonic ear cells. The parameters of the "average" mitochondrion, such as profile area, outer membrane length, were shown to differ relatively weakly during the periods with different meristem activity. Changes in the internal structure of the mitochondria having the developed system of crystae in the actively growing apices and those with weakly developed crystae in the resting seed or low active "waiting meristem" are much more pronounced. The relative volume of mitochondria, their number per unit of cytoplasm volume and total length of membranes suffer relatively insignificant changes during the vegetative phase and increase markedly during the prefloral phase when the apex is preparing itself for generative differentiation.     

Fine Structural Alterations in Cell Particles During Chemical Carcinogenesis : II. Further Evidence for Their Involvement in the Mechanism of Carcinogenesis. The Swelling of Rat Liver Mitochondria During Feeding of Amino Azo Dyes

Swelling under carefully controlled conditions has been used to study alterations in the structure of rat liver mitochondria as a result of feeding azo dyes. The changes of the swelling properties of the mitochondria during feeding of the hepatocarcinogenic 3'-methyl-4-dimethylaminoazobenzene are essentially comparable to those observed previously with the microsomes, under the same dietary conditions. These alterations in mitochondrial swelling are not related to changes in the amount of these cell particulates per unit weight of tissue, during feeding of this azo dye. As with the microsomes, feeding of the isomeric but relatively noncarcinogenic 2-methyl-4-dimethylaminoazobenzene does not affect swelling. The structural differences between liver and hepatoma mitochondria show up not only in the rate and extent of swelling but also in the form of the curves of pH dependence. The influence of ketones and sulfhydryl compounds on the swelling of normal liver mitochondria were studied, with particular emphasis to the role of sulfhydryl groups in membrane permeability. The sudden steep rise in the tumor incidence in groups of rats fed 3'-methyl-4-dimethylaminoazobenzene for increasing intervals of time occurs at about 4 weeks. This time correlates with the point of the minimum swelling of microsomes and mitochondria isolated from the livers of rats fed this same dye. Thus, a correlation is established between the alterations of the swelling properties of these particulates and the carcinogenic process.     

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.     

A morphometric analysis of cellular differentiation in root caps ofCucurbita peop

In order to quantify the ultrastructural changes that occur during cellular differentiation in an “open” type of root cap, we have performed a morphometric analysis of the ultrastructures of calyptrogen, columella, and peripheral cells of the root cap ofCucurbita pepo. The relative volumes of nuclei, nucleoli, and mitochondria decrease as cells move (i.e., differentiate) through the root cap. Before cells are sloughed from the cap, the relative volume of the vacuole increases by 250%. The relative volumes of plastids and plastid starch increase as calyptrogen cells differentiate into columella cells, but decrease as columella cells differentiate into peripheral cells. Dictyosomal volumes increase only as columella cells differentiate into peripheral cells. These results indicate that the five cell types comprising the root cap ofC.pepo are each characterized by a unique structure, and that the ultrastructural changes associated with cellular differentiation in root caps are organelle specific. These results are discussed relative to the functions of the various cell types of the root cap.     

The fate of human sperm-derived mtDNA in somatic cells.

Inheritance of animal mtDNA is almost exclusively maternal, most likely because sperm-derived mitochondria are actively eliminated from the ovum, either at or soon after fertilization. How such elimination occurs is currently unknown. We asked whether similar behavior could be detected in somatic cells, by following the fate of mitochondria and mtDNAs after entry of human sperm into transformed cells containing mitochondria but lacking endogenous mtDNAs (rho0 cells). We found that a high proportion (10%-20%) of cells contained functioning sperm mitochondria soon after sperm entry. However, under selective conditions permitting only the survival of cells harboring functional mtDNAs, only approximately 1/10(5) cells containing sperm mitochondria survived and proliferated. These data imply that mitochondria in sperm can enter somatic cells relatively easily, but they also suggest that mechanisms exist to eliminate sperm-derived mtDNA from somatic cells, mechanisms perhaps similar to those presumed to operate in the fertilized oocyte.     

Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTracker™ Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD–redox system. Under the present experimental conditions these subsets show similar functional responses.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. Role of cardiolipin in fluidity of mitochondrial membranes

During temperature acclimation of Tetrahymena pyriformis, the changes in fluidity and composition of total lipids from three membrane fractions, mitochondria, pellicles and microsomes were studied by a spin-label technique using a stearate probe and thin-layer and gas-liquid chromatography. The increase of fluidity observed in microsomal and pellicular lipids following the temperature shift from 39 to 15 degrees C corresponds with the increase of the ratio of total unsaturated to saturated fatty acid content. However, despite the increase of this ratio, the fluidity of mitochondrial lipids was found to be constant up to 10 h after the temperature shift. The fluidity of total lipids of mitochondria isolated from Tetrahymena cells grown at 39 degrees C was not changed by removal of cardiolipin, whereas cardiolipin-depleted lipids of mitochondria from 15 degrees C-acclimated cells showed a decrease in fluidity. The re-addition of cardiolipin to the mitochondrial lipids depleted of cardiolipin restored the fluidity to the initial level, thereby confirming the rigidifying effect of cardiolipin in cold-acclimated cells. These results suggest that cardiolipin may be implicated in maintaining consistent fluidity of mitochondrial membranes against change in thermal environment.