Division of Physarum mitochondria during starvation

Microplasmodia of Physarum polycephalum used in this study form spherules after 18 h of starvation. Stereological morphometry revealed that between the 2nd and the 5th hour of starvation the number of mitochondria in 1 mm3 of cytoplasm rises from about 12 to 24 millions and the mean volume of mitochondria drops from circa 4.6 to 3.0 microns3. This denotes the synchronous division of mitochondria. The daughter mitochondria show an increase in density of the matrix and a decrease in condensation of the net of tubular cristae. The mitochondrial division, decrease in activity of the respiratory chain and maximum of its cyanide resistance occur at the same time.     

泥螺卵黄发生过程中线粒体的变化

利用透射电镜（TEM）技术研究了泥螺卵黄发生过程中线粒体的形态结构的变化特点,结果表明,从卵黄发生早期到晚期,卵母细胞内线粒体经历了从外部形态到内部结构的一系列变化。卵黄合成初期的卵母细胞内,线粒体多,结构典型,仅部分线粒体外膜破裂,嵴 和内膜逐渐消失,卵黄发生中期,线粒体基质空泡化,嵴和内膜消失,腔内充满颗粒状物质,最后演变成卵黄颗粒,随着卵母细胞的发育,卵黄颗粒的数量和直径逐渐增加,卵黄发生后期,卵质中胞器不发达,细胞质中充满卵黄颗粒,在卵黄颗粒之间仅有少量线粒体存在,提供细胞代谢所需的能量,此外,对线粒体在卵黄形成中的功能,去向及行为变化等 进行了讨论。     

Studies on ultrastructure and purification of isolated plant mitochondria

Sweetpotato mitochondria, that showed respiratory control, were studied with respect to ultrastructure. If fixed in media containing sucrose at 0.4 M, the cristae were dilated and the matrix was highly condensed. A more orthodox ultrastructural form was observed when the mitochondria were fixed in a medium containing sucrose at 0.25 M, i.e., the matrix was more expanded, the cristae were less dilated, and peripherally, the inner membrane element lay adjacent to the outer membrane element. These results are discussed in terms of a sucrose-accessible space (space between outer and inner membrane elements including intracristal space), and a space relatively inaccessible to sucrose (matrix). Ultrastructural shifts were not observed with change in metabolic steady state of the mitochondria. High resolution electron micrographs showed that the ultrastructure of sweetpotato mitochondria is very similar to that of animal mitochondria.

Purity and homogeneity of mitochondrial fractions were followed both by phase-contrast and electron microscopy. Preparations from sweetpotato, using older methods, were relatively homogeneous with respect to particle type and size, whereas avocado preparations contained a high proportion of chloroplasts and cellular debris. A method of purification involving sucrose-density-gradient centrifugation was developed. Purified mitochondria exhibited respiratory control and appeared similar to unpurified mitochondria under the electron microscope.

     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles.

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48-144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual "noncristate" mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of "noncristate" mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inhibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48–144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual “noncristate” mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of “noncristate” mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Growth and ultrastructural studies on the mitochondrial mutant of Coprinus lagopus

The cytoplasmic acu-10 mutant of Corprinus lagopus has a respiratory deficiency due to an altered cytochrome component and is slower growing than wild type. When growth of wild type and acu-10 monokaryons and dikaryons were compared on solid medium and in liquid culture the mutation was found to restrict growth of the dikaryon more severely than that of the monokaryon. Ultrastructural studies revealed that faster growth of the acu-10 monokaryon occurred at the expense of maintaining the cytoplasmic cell contents and with little increase in the numbers of mitochondria. Cells of the acu-10 dikaryon were comparatively unvacuolated and contained greatly increased numbers of mitochondria. Mitochondria in cells of the mutant had a typical orthodox conformation with clear matrix and well defined cristae. In contrast, mitochondria in wild type cells had a more compact and elongated shape with dense matrix and less obvious cristae. The observed difference in mitochondrial ultrastructure is interpreted as one of conformation rather than structure and is attributed to impaired ability of mutant mitochondria to carry out oxidative phosphorylation. In an old cell of the mutant the mitochondria showed signs of recovering the wild type conformation.     

Heterogeneity of plant mitochondrial responses underpinning respiratory acclimation to the cold in Arabidopsis thaliana leaves

In this study, we investigated whether changes in mitochondrial abundance, ultrastructure and activity are involved in the respiratory cold acclimation response in leaves of the cold-hardy plant Arabidopsis thaliana. Confocal microscopy [using plants with green fluorescence protein (GFP) targeted to the mitochondria] and transmission electron microscopy (TEM) were used to visualize changes in mitochondrial morphology, abundance and ultrastructure. Measurements of respiratory flux in isolated mitochondria and intact leaf tissue were also made. Warm-grown (WG, 25/ 20 degrees C day/night), 3-week cold-treated (CT) and cold-developed (CD) leaves were sampled. Although CT leaves exhibited some evidence of acclimation (as evidenced by higher rates of respiration at moderate measurement temperatures), it was only the CD leaves that were able to re-establish respiratory flux within the cold. Associated with the recovery of respiratory flux in the CD leaves were: (1) an increase in the total volume of mitochondria per unit volume of tissue in epidermal cells; (2) an increase in the ratio of cristae to matrix within mesophyll cell mitochondria; and (3) an increase in the capacity of the energy-producing cytochrome pathway in mitochondria isolated from whole leaf homogenates. Regardless of growth temperature, we found that contrasting cell types exhibited distinct differences in mitochondrial ultrastructure, morphology and abundance. Collectively, our data demonstrated the diversity and tissue-specific nature of mitochondrial responses that underpin respiratory acclimation to the cold, and revealed the heterogeneity of mitochondrial structure and abundance that exists within leaves.     

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO₂ by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the β-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall β-oxidation activity was assayed as the formation of [¹⁴C]acetyl-CoA from [¹⁴C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the β-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

During autophagy mitochondria elongate, are spared from degradation and sustain cell viability

A plethora of cellular processes, including apoptosis, depend on regulated changes in mitochondrial shape and ultrastructure. The role of mitochondria and of their morphology during autophagy, a bulk degradation and recycling process of eukaryotic cells' constituents, is not well understood. Here we show that mitochondrial morphology determines the cellular response to macroautophagy. When autophagy is triggered, mitochondria elongate in vitro and in vivo. During starvation, cellular cyclic AMP levels increase and protein kinase A (PKA) is activated. PKA in turn phosphorylates the pro-fission dynamin-related protein 1 (DRP1), which is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, possess more cristae, increased levels of dimerization and activity of ATP synthase, and maintain ATP production. Conversely, when elongation is genetically or pharmacologically blocked, mitochondria consume ATP, precipitating starvation-induced death. Thus, regulated changes in mitochondrial morphology determine the fate of the cell during autophagy.     

Sugar-Starvation-Induced Changes of Carbon Metabolism in Excised Maize Root Tips

Excised maize (Zea mays L.) root tips were used to study the early metabolic effects of glucose (Glc) starvation. Root tips were prelabeled with [1-13C]Glc so that carbohydrates and metabolic intermediates were close to steady-state labeling, but lipids and proteins were scarcely labeled. They were then incubated in a sugar-deprived medium for carbon starvation. Changes in the level of soluble sugars, the respiratory quotient, and the 13C enrichment of intermediates, as measured by 13C and 1H nuclear magnetic resonance, were studied to detect changes in carbon fluxes through glycolysis and the tricarboxylic acid cycle. Labeling of glutamate carbons revealed two major changes in carbon input into the tricarboxylic acid cycle: (a) the phosphoenolpyruvate carboxylase flux stopped early after the start of Glc starvation, and (b) the contribution of glycolysis as the source of acetyl-coenzyme A for respiration decreased progressively, indicating an increasing contribution of the catabolism of protein amino acids, fatty acids, or both. The enrichment of glutamate carbons gave no evidence for proteolysis in the early steps of starvation, indicating that the catabolism of proteins was delayed compared with that of fatty acids. Labeling of carbohydrates showed that sucrose turnover continues during sugar starvation, but gave no indication for any significant flux through gluconeogenesis.     

Study of glucose starvation in excised maize root tips

Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4-dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. During starvation the sugar content sharply decreased for the first 20 hours and reached zero at 120 hours. Following root excision, proteins and lipids were continuously degraded and were virtually the only substrates for respiration and biosyntheses after 20 hours of starvation. Over the first 90 hours of starvation, enzymic activities related to sugar metabolic pathways and the Krebs cycle decreased to 20% or less of their initial activity. Starvation was reversible only for the first 80 to 90 hours. Between 80 and 100 hours, there was a sharp fall in intracellular osmolarity and a 25% loss in the dry weight. The irreversibility may be due, as in senescence, to a change in membrane selective permeability.     

Quantitative seasonal variation in mitochondrial ultrastructure of mesophyll cells ofDiapensia lapponica L. with reference to some effects of fixative osmolality

Summary Seasonal changes in the mitochondrial ultrastructure were examined in palisade parenchyma cells of a tuft-formingDiapensia lapponica L. collected at monthly intervals in Northern Finland. Quantitative analyses to measure volume and surface densities were conducted during different periods of growth (stages of growth, acclimation, winter period and deacclimation) in the annual cycle.The volume density was highest in the summer and lowest in the spring; the difference was significant with both fixatives used GA and GA/FA. The largest membrane area (the mitochondrial outer membrane and the cristal membranes together) was observed in the summer and autumn, and was significantly less in the winter and spring. This correlated with fewer mitochondria in the spring and a smaller number of cristae in the winter and spring. In the material fixed in GA/FA the distribution of length/width ratios of mitochondria was relatively uniform in all seasons. However, the mitochondrial ultrastructure had the most varied appearance during the winter. Hypertonie GA/FA solution did not cause significant differences either in the ultrastructure or the volume and surface densities of the mitochondria.Abbreviations GA glutaraldehyde - GA/FA glutaraldehyde/formaldehyde     

大鼠心肌线粒体缺血再灌损伤及SOD抗缺血再灌损伤作用的形态计量研究

研究了在体大鼠心肌缺血再灌模型心肌线粒体再灌损伤和SOD抗缺血再灌损伤的作用。实验分为:A组(缺血再灌组)B组(SOD组)和C组(假手术组)。电镜见A组线粒体高度肿胀、外膜缺损、嵴断裂溶解,出现无定形致密体、四膜嵴和杆状嵴等改变。B组线粒体轻度或中度肿胀、少数有轻度嵴溶解。个别线粒体内出现无定形致密体。三组线粒体超微结构立体计量数据比较,A组线粒体密度、线粒体体密度与肌原纤维体密度比率增高((P<0.01),线粒体比表面和比膜面降低(P<0.01)。自身动图像分析仪检测SDH反应灰度值A组呈强损伤反应,B组反应较轻,二者差别有显著性(P<0.05)。结果表明,心肌缺血再灌可致线粒体发生不可逆性损伤,SOD能减轻线粒体缺血再灌性损伤。     

Demonstration of plant adaptation syndrome in plants and possible molecular mechanisms of its realization under conditions of anaerobic stress

Electron-microscopic examination of mitochondrial membrane ultrastructure in detached leaves of four-day-old wheat (Triticum aestivum L.) seedlings incubated under conditions of strict anoxia in the presence of exogenous glucose and cycloheximide or in the absence of these compounds revealed a paradoxical phenomenon: in the absence of exogenous glucose and cycloheximide, even a short-term (15–30 min) anaerobiosis resulted in a pathological destruction of mitochondria (swelling and the loss of cristae); however, a longer uninterrupted anaerobiosis (3–4 h) did not induce further mitochondria degradation but, in contrast, resulted in the recovery of their initial ultrastructure. Irreversible mitochondria degradation was observed only during subsequent still longer leaf anaerobic treatment (24–48 h). When, under conditions of strict anoxia, leaves were fed with glucose to stimulate glycolysis and ethanolic fermentation, we did not observe any signs of early destruction of mitochondrial ultrastructure and their swelling. Blockage of anaerobic protein synthesis with cycloheximide resulted in early destruction and subsequent irreversible degradation of mitochondria without any indications of their structural recovery. Based on the results of the experiments, we concluded that cell energy metabolism controlled byboth the presence of utilizable carbohydrates and also by the induction of anaerobic protein synthesis played a key role during early mitochondria destruction under extreme conditions of anaerobic stress, their subsequent recovery, and irreversible degradation during continuous long-term strict anoxia.     

A metabolic study of the regulation of proteolysis by sugars in maize root tips: effects of glycerol and dihydroxyacetone

Sugars, the main growth substrates of plants, act as physiological signals in the complex regulatory network of sugar metabolism. To investigate the function of different glycolytic steps in sugar sensing and signaling we compared the effects of carbon starvation with those of glucose, glycerol and dihydroxyacetone on carbon metabolism, proteolysis, and protease expression in excised maize (Zea mays L.) root tips. Respiration, soluble proteins, protein turnover and proteolytic activities were monitored as a function of time, along with in vitro and in vivo analysis of a variety of metabolites (sugars, amino and organic acids, phosphoesters, adenine nucleotides...) using ¹³C, ³¹P and ¹H NMR spectroscopy. Our results indicate that, in maize root tips, endopeptidase activities and protease expression are induced in response to a decrease in carbon supply to the upper part of the glycolytic pathway, i.e. at the hexokinase step. Proteolysis would be controlled downstream glycolysis, probably at the level of the respiratory substrate supply to mitochondria. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Ultrastructural zonal heterogeneity of hepatocytes and mitochondria within the hepatic acinus during liver regeneration after partial hepatectomy

BACKGROUND INFORMATION: Partial hepatectomy (70%) induces cell proliferation until the original mass of the liver is restored. In the first 24 h after partial hepatectomy, drastic changes in the metabolism of the remaining liver have been shown to occur. To evaluate changes in hepatocyte ultrastructure within the hepatic acinus during the liver regenerative process, we investigated, by light and electron microscopy observations on specimens taken 0 h, 24 h and 96 h after partial hepatectomy, the hepatocyte structure and ultrastructure in the periportal and pericentral area of the hepatic acinus, with a particular emphasis on mitochondria ultrastructure. Moreover, some biochemical events that could affect the mitochondria ultrastructure and function were investigated. RESULTS: We found that, 24 h after partial hepatectomy, mitochondria with altered ultrastructure were preferentially localized in the periportal area. Periportal hepatocytes showed also an increase in the number of peroxisomes, free ribosomes, lysosomes and autophagosomes. Altered mitochondria showed swelling, an ultrastructural index of increased membrane permeability, a reduction in the number of cristae, and a rarefied, often vacuoled, matrix. Consistently, an increase in the mitochondrial oxidized/reduced glutathione ratio was found as well as calcium release from mitochondria in a manner inhibited by cyclosporin A. Interestingly, light and electron microscopy analysis showed that the hepatocytes in the periportal area were the cells with the major structural attributes to proliferate. At 96 h after partial hepatectomy, the preferential zonation of altered mitochondria was lost and the normal mitochondrial membrane permeability properties were restored. CONCLUSIONS: We suggest that 24 h after partial hepatectomy, a preferential zonation of altered mitochondria in the periportal hepatocytes could be involved in the changes of metabolic and functional heterogeneity of the hepatocytes within the hepatic acinus during the regenerative process.     

GNRH induces activation of Leydig-like cells in Pleurodeles waltlii. A morphometric study

The ultrastructure of the interstitial cells of the glandular tissue of Pleurodeles waltlii was studied in testis of animals obtained in early breeding season (January) under gonadotropic releasing hormone (GNRH) treatments and controls. These cells (parenchymal or Leydig-like cells) displayed the structural characteristics of steroid-producing cells. GNRH administration for 24 hours induced a significant decrease of both medial volume and volume density of lipid droplets. On the other hand, cell volume, nucleus, mitochondria, mitochondrial cristae and tubules of smooth endoplasmic reticulum were increased. The surface density of mitochondrial cristae was also increased.     

Mitochondrial biogenesis during germination in maize embryos

Mitochondrial biogenesis and metabolism were investigated during maize (Zea mays) seed germination. Mitochondria from dry and imbibed seed exhibited NADH-dependent O(2) uptake that was completely inhibited by KCN and antimycin A. Mitochondria in the dry seed had a lower rate of succinate-dependent O(2) uptake relative to that measured in imbibed and germinated seed. The activities of the tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, NAD-malic enzyme, and citrate synthase, are similarly low in mitochondria from dry seed and this correlates with a lower relative abundance of the mitochondrial matrix-located citrate synthase and pyruvate dehydrogenase complex E1alpha-subunit polypeptides. Electron microscopy revealed that mitochondria in the dry seed have a poorly developed internal membrane structure with few cristae; following 24 h of germination the mitochondria developed a more normal structure with more developed cristae. The mitochondria from maize embryos could be fractionated into two subpopulations by Suc density gradient centrifugation: one subpopulation of buoyant density equivalent to 22% to 28% (w/w) Suc; the other equivalent to 37% to 42% (w/w) Suc. These two subpopulations had different activities of specific mitochondrial enzymes and contained different amounts of specific mitochondrial proteins as revealed by western-blot analysis. Both subpopulations from the dry embryo were comprised of poorly developed mitochondria. However, during imbibition mitochondria in the heavy fraction (37%-42% [w/w] Suc) progressively acquired characteristics of fully functional mitochondria found in the germinated seedling in terms of structure, enzymic activity, and protein complement. In contrast, mitochondria in the light fraction (22% to 28% [w/w] Suc) show no significant structural change during imbibition and the amounts of specific mitochondrial proteins decreased significantly during germination.     

The effect of age on mitochondrial ultrastructure.

The ultrastructure of perfused livers and of mitochondrial fractions from 6-month-old and 30-month-old C57/BL mice were studied. In old mice the liver cell mitochondria were enlarged and rounded with a light 'foamy', vacuolated matrix, short cristae and a loss of dense granules. Quantitative studies showed a 60% increase in the mean size and an increased proportion of larger mitochondria in intact 30-month-old perfused livers. Endothelial and Kupffer cell mitochondria were smaller than those of the parenchymal cells. Mitochondria in pellets prepared from 6- and 30-month-old livers were rounded and condensed, although there were a few larger and 'foamy' mitochondria in the preparations from old mice. Up to 47% of large mitochondria in the old livers were lost during cell fractionation.     

The dynamics of structural modifications of mitochondria at the early stages of sea urchin embryonic development.

The organization of the chondriome and the ultrastructure of mitochondria have been studied in eggs and embryos of the sea urchin Paracentrotus lividus. The egg chondriome is characterized by an arrangement in well-delimited clusters. Analysis of mitochondrial clusters on electron micrographs of ultrathin serial sections shows two kinds of mitochondria of different shapes, the rod-shaped and the spherical. The egg mitochondria have a dense matrix and a well-ordered arrangement of cristae which, in rod-shaped variety, are perpendicular to the major axis. Cell division is accompanied by significant changes in intracellular distribution of mitochondria and in their structure. At the stage of 2-4 blastomeres, the clusters break up and numerous mitochondrial rods show signs of fragmentation; most of the observable mitochondria are of spherical shape. At the same time, the matrix becomes less dense, and the orderly arrangement of the cristae disappears. From the blastula to the gastrula stage, the observed modifications are reversed: the number of spherical-shaped mitochondria decreases, while that of the rod-shaped increases; the diameter of the latter is almost equal to the initial diameter of the spherical forms, the matrix becomes dense again and the cristae resume their orderly arrangement.