Selection of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone 10 by a Cell Cloning Technique

Strains producing high levels of ubiquinone 10 were isolated from tobacco cell suspension cultures by a cell cloning technique, and the UQ content of these cell strains was analyzed by high performance liquid chromatography. Three strains with high UQ yields in suspension culture were selected and used for investigations on the necessary cultural conditions for UQ production. Sucrose concentration, 2,4-D concentration and culture temperature did not have marked effects on UQ formation. These results differed from those of the original cell lines. Furthermore, the addition of yeast extract to the medium promoted cell growth in these strains remarkably but did not enhance UQ production in the cell strains. According to these investigations, UQ production was increased to 15mg/liter and the UQ level to 1890 μg/g dry wt. of cells after 11 days of incubation.     

Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.     

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.     

High-voltage electron microscopy of human diploid fibroblasts during ageing in vitro. Morphometric analysis of mitochondria

Since recent studies have suggested a diminished mitochondrial functional capacity in late-passage ('old') compared to early-passage ('young') normal fibroblasts and fibroblasts from the Hutchinson-Gilford (progeria) syndrome of premature ageing, we analysed whole-cell preparations on the high voltage electron microscope to look for mitochondrial and related defects. All strains examined showed considerable heterogeneity in cell size and intracellular morphology. Mitochondria were readily seen in all cells, predominantly as long slender rods with frequent branching, but occasional circular and saccular forms were also evident. Various parameters of mitochondrial mass including mean number, weight, and total length of mitochondria per cell weight tended to increase in old and progeria cells, but only the former attained statistical significance due to the heterogeneity and consequent variance. A significant finding was the decreased width of mitochondria in old and progeria cells. Cystic blebs were evident in mitochondria of some cells with an apparent increase in old and progeria fibroblasts. These blebs appeared to be due to weakening of the inner membrane, allowing dilatation of the outer membrane which otherwise appeared intact. The number of osmiophilic inclusions per cell weight, particularly lipofuscin granules and autophagic vacuoles, was significantly increased in old and progeria cells. In conclusion, despite some morphological changes, mitochondria of old and progeria cells maintain a structurally and bioenergetically adequate mass compatible with continued cellular viability.     

The submitochondrial distribution of ubiquinone affects respiration in long-lived Mclk1+/? mice

Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1^+/− mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1^+/− mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ₁₀ was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3^+/− mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.     

Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.     

A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria.

A fusion protein was expressed in transgenic tobacco and yeast cells to examine the functional conservation of mechanisms for importing precursor proteins from the cytosol into mitochondria and chloroplasts. The test protein consisted of the mitochondrial leader peptide from the yeast precursor to cytochrome oxidase subunit Va (prC5) fused to the reporter protein chloramphenicol acetyltransferase. This protein, denoted prC5/CAT, was transported into the mitochondrial interior in yeast and tobacco cells. In both organisms, the mitochondrial form of prC5/CAT was smaller than the primary translation product, suggesting that proteolytic processing occurred during the transport process. prC5/CAT also was translocated into chloroplasts in vivo, accumulating to approximately the same levels as in plant mitochondria. However, accumulation of prC5/CAT in chloroplasts relative to mitochondria varied with the conditions under which plants were grown. The chloroplast form of prC5/CAT also appeared to have been proteolytically processed, yielding a mature protein of the same apparent size as that seen in mitochondria of either tobacco or yeast. Chloramphenicol acetyltransferase lacking a mitochondrial targeting peptide did not associate with either chloroplasts or mitochondria. The results demonstrated that in plant cells a single leader peptide can interact functionally with the protein translocation systems of both chloroplasts and mitochondria, and raised the possibility that certain native proteins might be shared between these two organelles.     

Engineering of ubiquinone biosynthesis using the yeast coq2 gene confers oxidative stress tolerance in transgenic tobacco

Ubiquinone (UQ), an electron carrier in the respiratory chain ranging from bacteria to humans, shows antioxidative activity in vitro, but its physiological role in vivo is not yet clarified in plants. UQ biosynthesis was modified by overexpressing the yeast gene coq2, which encodes p-hydroxybenzoate:polyprenyltransferase, to increase the accumulation of UQ-6 in yeast and UQ-10 in tobacco. The yeast and tobacco transgenic lines showed about a three- and six-fold increase in UQ, respectively. COQ2 polypeptide, the localization of which was forcibly altered to the endoplasmic reticulum, had the same or a greater effect as mitochondria-localized COQ2 on the increase in UQ in both the yeast and tobacco transformants, indicating that the UQ intermediate is transported from the endoplasmic reticulum to the mitochondria. Plants with a high UQ level are more resistant to oxidative stresses caused by methyl viologen or high salinity. This is attributable to the greater radical scavenging ability of the transgenic lines when compared with the wild type.     

In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

An efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10⁸ cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.     

Respiratory activities in chloramphenicol-treated tobacco cells

Chloramphenicol (CAP) inhibited tobacco cell growth as shown by a reduction (34%) of cell mass 4 days after treatment. The rates of cell respiration were slightly higher than control under coupled conditions. However, CAP-treated cells showed a decreased maximal capacity of the cytochrome pathway (48%) and an increased maximal capacity of alternative path (56%) 4 days after treatment. In purified mitochondria, the rates of NADH or malate oxidation under state 4 conditions were not significantly changed by CAP treatment. However, the state 3 rates were 34–40% lower in CAP-treated than in control mitochondria. Succinate oxidation decreased by 31–46% under both state 4 and state 3 conditions after CAP treatment. The activities of complexes I, III, and IV, which contain mitochondrially encoded subunits, decreased by about 50% in CAP-treated mitochondria. There was also a decrease in the contents of mitochondrial cytochromes. Unexpectedly, the activities of complex II and the matrix-facing rotenone-insensitive NADH dehydrogenase, which are thought to be nuclear-encoded, also declined. The activities of external NADH dehydrogenase, NAD-linked malic enzyme, and fumarase remained unchanged after CAP treatment. There was a slight increase in the activity and protein level of alternative oxidase. An electrochemical gradient across the mitochondrial membranes was observed by Rhodamine 123 staining in CAP-treated cells. However, the morphology of most of the mitochondria changed from spherical to vermicular. A method for purifying a high yield of intact mitochondria from tobacco cell suspension cultures is described.     

Valine-Resistance, a Potential Marker in Plant Cell Genetics. II. Optimization of Uv Mutagenesis and Selection of Valine-Resistant Colonies Derived from Tobacco Mesophyll Protoplasts

The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.-Using cell densities of approximately 10( 4) cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Val(r)-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10(-6) per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10(-5) to 5 x 10(-4) resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F(1) double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were compared to those observed in wild-type cells. The results are considered in light of the amphidiploid structure of the tobacco genome.     

高温下线粒体小分子热激蛋白对柠檬酸合成酶、线粒体和花粉粒的保护作用

选用pGEX-6P-1表达载体,构建GST与线粒体小分子热激蛋白的融合蛋白表达载体,使用谷胱苷肽Sepharose-4B亲合柱,纯化大肠杆菌中表达的融合蛋白,利用ProScission蛋白酶,将线粒体小分子热激蛋白从融合蛋白中切出,获得纯化的线粒体小分子热激蛋白.分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性,并促进热变性的柠檬酸合成酶复性,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效.利用转基因方法,将线粒体小分子热激蛋白基因导入烟草,在CaMV35S启动子的驱动下,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照,说明线粒体小分子热激蛋白对线粒体具有保护作用.此外,转基因烟草花粉粒的高温萌发率也高于对照,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性,而且可以提高细胞的抗热能力.     

A high-yield preparative method for isolation of rat liver mitochondria

A differential centrifugation method for the preparation of rat liver mitochondria is described, which results in final mitochondrial yields of at least 35 to 40 mg of mitochondrial protein per gram wet weight of liver. These mitochondria are shown to be functionally and ultrastructurally intact. They exhibit acceptor control ratios of 6 to 8 and ADP/O ratios near 2.0 with succinate as a substrate. In addition, they appear homogeneous by electron microscopy criteria. This method can be used to prepare rat liver mitochondria in high yield on either a large- or a small-scale basis.     

Cell cycle-regulated, microtubule-independent organelle division in Cyanidioschyzon merolae

Mitochondrial and chloroplast division controls the number and morphology of organelles, but how cells regulate organelle division remains to be clarified. Here, we show that each step of mitochondrial and chloroplast division is closely associated with the cell cycle in Cyanidioschyzon merolae. Electron microscopy revealed direct associations between the spindle pole bodies and mitochondria, suggesting that mitochondrial distribution is physically coupled with mitosis. Interconnected organelles were fractionated under microtubule-stabilizing condition. Immunoblotting analysis revealed that the protein levels required for organelle division increased before microtubule changes upon cell division, indicating that regulation of protein expression for organelle division is distinct from that of cytokinesis. At the mitochondrial division site, dynamin stuck to one of the divided mitochondria and was spatially associated with the tip of a microtubule stretching from the other one. Inhibition of microtubule organization, proteasome activity or DNA synthesis, respectively, induced arrested cells with divided but shrunk mitochondria, with divided and segregated mitochondria, or with incomplete mitochondrial division restrained at the final severance, and repetitive chloroplast division. The results indicated that mitochondrial morphology and segregation but not division depend on microtubules and implied that the division processes of the two organelles are regulated at distinct checkpoints.     

CORTISONE-INDUCED ALTERATIONS IN MITOCHONDRIAL FUNCTION AND STRUCTURE

The effects of cortisone treatment on oxygen consumption, oxidative phosphorylation, and fine structure of rat liver mitochondria have been studied. Male rats weighing 125 g were treated for 6 days with 5 mg of cortisone acetate or isotonic saline. On the 7th day, sections of liver were excised and processed for light and electron microscopy. Mitochondrial respiration and oxidative phosphorylation were studied with mitochondria isolated from these livers. Cortisone treatment is responsible for a 14–40% decrease in the amount of oxygen consumed per mg of mitochondrial protein when succinate, α-ketoglutarate, or β-hydroxybutyrate are used as substrates, or with ascorbate and N,N,N¹,N¹-tetramethyl p-phenylenediamine as electron donors. In addition, oxidative phosphorylation is uncoupled with a lowering of the P:O ratios. Randomly selected liver cells have been analyzed by quantitative morphometric techniques. The average mitochondrial volume is increased fourfold in the peripheral and midzonal regions with a commensurate decrease in the number of mitochondria per cell. These alterations are present throughout the hepatic lobule, but are most marked in midzonal cells. The total mitochondrial volume per cell and the per cent of the total cytoplasmic volume occupied by mitochondria remains relatively unaltered, as does the total amount of cristae surface per cell. While the mitochondria are enlarged, they are not "swollen." The relationships between the steroid hormone treatment and the alterations in mitochondrial function and structure are discussed.     

液体发酵法筛选樟芝优良诱变新菌株的研究

对3个樟芝野生菌株和5个物理诱变菌株进行液体发酵,通过测定发酵液黏度变化情况,观测培养性状、菌球及粗提物产量和多糖、蛋白质及三萜含量,从中筛选出适宜液体发酵的多糖、三萜及蛋白质高产优良菌株。试验结果显示：供试的8个樟芝菌株生长曲线基本一致,其中,菌株327和Gg生长速度较快,培养的第9–10天开始出现菌丝自溶现象;经过筛选,327菌株为多糖和蛋白质高产菌株,多糖和蛋白质的产量分别比出发菌株提高238.20%和10.33%;Gg为三萜高产菌株,三萜产量比出发菌株提高57.86%。野生樟芝菌株经物理诱变效果明显。     

Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur.

Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5' regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial beta-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of beta-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cell cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0-24 h) cultivation periods compared with cells grown in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.     

Measurements of in Vivo Ubiquinone Reduction Levels in Plant Cells

A method is described for the determination of in vivo ubiquinone (UQ) reduction levels in nongreen tissues by extraction and subsequent detection of ubiquinone-10 and ubiquinol-10 with high-performance liquid chromatography. In Petunia hybrida cell suspensions UQ reduction remained at a stable level of about 60%, despite the changing conditions during the batch culture (from excess sugar to starvation) and the concomitant variations in respiration. Also, in the presence of uncoupler, which causes a large increase in respiration via both the cytochrome pathway and the alternative pathway, UQ reduction levels stayed at 60%. In mitochondria isolated from these cells, activity of the alternative pathway was only observed at UQ reduction levels higher than 80%. It is proposed that in vivo the relationship between UQ reduction and the activity of the alternative oxidase is modulated by mechanisms such as thiol modifications and accumulation of organic acids. Accordingly, pyruvate concentration in P. hybrida cells increased in the presence of uncoupler.     

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.