| 1. |
- Møller, Ian Max, et al.
(författare)
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Isolation of Highly Purified, Intact, and Functional Mitochondria from Potato Tubers Using a Two-in-One Percoll Density Gradient
- 2022
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Ingår i: Plant Mitochondria : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616529 - 9781071616536 ; 2363, s. 39-50
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Bokkapitel (refereegranskat)abstract
- The isolation of mitochondria from potato tubers (Solanum tuberosum L.) is described, but the methodology can easily be adapted to other storage tissues. After homogenization of the tissue, filtration and differential centrifugation, the key step is a Percoll density gradient centrifugation. The Percoll gradient contains two parts: a bottom part containing Percoll in 0.3 M sucrose, and a slightly less dense top part containing Percoll in 0.3 M mannitol. After centrifugation, a density gradient is formed that is almost linear in the central part, and this is where the band containing the purified intact mitochondria is formed. This method makes it possible to process large amounts of plant material (2–6 kg) and saves at least 1.5 h on the preparation time compared to methods where two consecutive purification methods are used. Nonetheless, it yields large amounts of mitochondria (50–125 mg protein) of very high purity, intactness and functionality.
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| 2. |
- Rasmusson, Allan G., et al.
(författare)
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Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin
- 2022
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Ingår i: Plant Mitochondria : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616529 - 9781071616536 ; 2363, s. 77-84
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Bokkapitel (refereegranskat)abstract
- We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.
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| 3. |
- Browse, John A., et al.
(författare)
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Respiration and Lipid Metabolism
- 2014. - 6th ed.
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Ingår i: Plant Physiology and Development. - 9781605352558 - 9781605354354 ; , s. 317-352
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Bokkapitel (refereegranskat)
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| 4. |
- Bykova, Natalia V., et al.
(författare)
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Two separate transhydrogenase activities are present in plant mitochondria
- 1999
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 265:1, s. 106-111
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Tidskriftsartikel (refereegranskat)abstract
- Inside-out submitochondrial particles from both potato tubers and pea leaves catalyze the transfer of hydride equivalents from NADPH to NAD+ as monitored with a substrate-regenerating system. The NAD+ analogue acetylpyridine adenine dinucleotide is also reduced by NADPH and incomplete inhibition by the complex I inhibitor diphenyleneiodonium (DPI) indicates that two enzymes are involved in this reaction. Gel-filtration chromatography of solubilized mitochondrial membrane complexes confirms that the DPI-sensitive TH activity is due to NADH-ubiquinone oxidoreductase (EC 1.6.5.3, complex I), whereas the DPI-insensitive activity is due to a separate enzyme eluting around 220 kDa. The DPI-insensitive TH activity is specific for the 4B proton on NADH, whereas there is no indication of a 4A-specific activity characteristic of a mammalian-type energy-linked TH. The DPI-insensitive TH may be similar to the soluble type of transhydrogenase found in, e.g., Pseudomonas. The presence of non-energy-linked TH activities directly coupling the matrix NAD(H) and NADP(H) pools will have important consequences for the regulation of NADP-linked processes in plant mitochondria.
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| 5. |
- Catania, Alessia, et al.
(författare)
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Arabidopsis thaliana alternative dehydrogenases : A potential therapy for mitochondrial complex i deficiency? Perspectives and pitfalls
- 2019
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Ingår i: Orphanet Journal of Rare Diseases. - : Springer Science and Business Media LLC. - 1750-1172. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Complex I (CI or NADH:ubiquinone oxidoreductase) deficiency is the most frequent cause of mitochondrial respiratory chain defect. Successful attempts to rescue CI function by introducing an exogenous NADH dehydrogenase, such as the NDI1 from Saccharomyces cerevisiae (ScNDI1), have been reported although with drawbacks related to competition with CI. In contrast to ScNDI1, which is permanently active in yeast naturally devoid of CI, plant alternative NADH dehydrogenases (NDH-2) support the oxidation of NADH only when the CI is metabolically inactive and conceivably when the concentration of matrix NADH exceeds a certain threshold. We therefore explored the feasibility of CI rescue by NDH-2 from Arabidopsis thaliana (At) in human CI defective fibroblasts. Results: We showed that, other than ScNDI1, two different NDH-2 (AtNDA2 and AtNDB4) targeted to the mitochondria were able to rescue CI deficiency and decrease oxidative stress as indicated by a normalization of SOD activity in human CI-defective fibroblasts. We further demonstrated that when expressed in human control fibroblasts, AtNDA2 shows an affinity for NADH oxidation similar to that of CI, thus competing with CI for the oxidation of NADH as opposed to our initial hypothesis. This competition reduced the amount of ATP produced per oxygen atom reduced to water by half in control cells. Conclusions: In conclusion, despite their promising potential to rescue CI defects, due to a possible competition with remaining CI activity, plant NDH-2 should be regarded with caution as potential therapeutic tools for human mitochondrial diseases.
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| 6. |
- Dotson, Bradley R., et al.
(författare)
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The antibiotic peptaibol alamethicin from Trichoderma permeabilises Arabidopsis root apical meristem and epidermis but is antagonised by cellulase-induced resistance to alamethicin
- 2018
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Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Trichoderma fungi live in the soil rhizosphere and are beneficial for plant growth and pathogen resistance. Several species and strains are currently used worldwide in co-cultivation with crops as a biocontrol alternative to chemical pesticides even though little is known about the exact mechanisms of the beneficial interaction. We earlier found alamethicin, a peptide antibiotic secreted by Trichoderma, to efficiently permeabilise cultured tobacco cells. However, pre-treatment with Trichoderma cellulase made the cells resistant to subsequent alamethicin, suggesting a potential mechanism for plant tolerance to Trichoderma, needed for mutualistic symbiosis. Results: We here investigated intact sterile-grown Arabidopsis thaliana seedlings germinated in water or growth medium. These could be permeabilised by alamethicin but not if pretreated with cellulase. By following the fluorescence from the membrane-impermeable DNA-binding probe propidium iodide, we found alamethicin to mainly permeabilise root tips, especially the apical meristem and epidermis cells, but not the root cap and basal meristem cells nor cortex cells. Alamethicin permeabilisation and cellulase-induced resistance were confirmed by developing a quantitative in situ assay based on NADP-isocitrate dehydrogenase accessibility. The combined assays also showed that hyperosmotic treatment after the cellulase pretreatment abolished the induced cellulase resistance. Conclusion: We here conclude the presence of cell-specific alamethicin permeabilisation, and cellulase-induced resistance to it, in root tip apical meristem and epidermis of the model organism A. thaliana. We suggest that contact between the plasma membrane and the cell wall is needed for the resistance to remain. Our results indicate a potential mode for the plant to avoid negative effects of alamethicin on plant growth and localises the point of potential damage and response. The results also open up for identification of plant genetic components essential for beneficial effects from Trichoderma on plants.
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| 7. |
- Fan, Kaijian, et al.
(författare)
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The pentatricopeptide repeat protein EMP603 is required for the splicing of mitochondrial Nad1 intron 2 and seed development in maize
- 2021
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 72:20, s. 6933-6948
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Tidskriftsartikel (refereegranskat)abstract
- Intron splicing is an essential event in post-transcriptional RNA processing in plant mitochondria, which requires the participation of diverse nuclear-encoded splicing factors. However, it is presently unclear how these proteins cooperatively take part in the splicing of specific introns. In this study, we characterized a nuclear-encoded mitochondrial P-type pentatricopeptide repeat (PPR) protein named EMP603. This protein is essential for splicing of intron 2 in the Nad1 gene and interacts with the mitochondria-localized DEAD-box RNA helicase PMH2-5140, the RAD52-like proteins ODB1-0814 and ODB1-5061, and the CRM domain-containing protein Zm-mCSF1. Further study revealed that the N-terminal region of EMP603 interacts with the DEAD-box of PMH2-5140, the CRM domain of Zm-mCSF1, and OBD1-5061, but not with OBD1-0814, whereas the PPR domain of EMP603 can interact with ODB1-0814, ODB1-5061, and PMH2-5140, but not with Zm-mCSF1. Defects in EMP603 severely disrupt the assembly and activity of mitochondrial complex I, leading to impaired mitochondrial function, and delayed seed development. The interactions revealed between EMP603 and PMH2-5140, ODB1-0814, ODB1-5061, and Zm-mCSF1 indicate a possible involvement of a dynamic 'spliceosome-like' complex in intron splicing, and may accelerate the elucidation of the intron splicing mechanism in plant mitochondria.
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| 8. |
- Fredlund, Kenneth M., et al.
(författare)
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Oxidation of external NAD(P)H by purified mitochondria from fresh and aged red beetroots (Beta vulgaris L.)
- 1991
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 97:1, s. 99-103
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria were isolated from fresh beetroots (Beta vulgaris L. cvs Rubria and Nina) by differential centrifugation followed by Percoll gradient centrifugation. These purified mitochondria oxidized external NADH, although relatively slowly (20-40 versus 100-120 nanomoles oxygen per minute times milligram protein for NADH and succinate oxidation, respectively), with respiratory control ratios of two to three and ADP/O ratios of 1.2 to 1.6. NADPH was also oxidized, but even more slowly and with little or no coupling. The optimum for both NADH and NADPH oxidation by fresh beetroot mitochondria was pH 6. The rate of external NADH oxidation by isolated mitochondria was enhanced threefold during storage of the intact tubers at 10°C for 12 weeks. The optimum of the induced NADH oxidation was approximately pH 6.8. Succinate and malate oxidation only increased by 30% during the same period and NADPH oxidation was constant. This is strong evidence that NADH and NADPH oxidation are catalyzed by different enzymes at least in beetroots. Activity staining of nondenaturing polyacrylamide gels with NADH and Nitro Blue Tetrazolium did not show differences in banding pattern between mitochondria isolated from fresh and stored beetroots. The induction is discussed in relation to physiological aging processes.
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| 9. |
- Glab, Bartosz, et al.
(författare)
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Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS
- 2016
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 291:48, s. 25066-25076
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Tidskriftsartikel (refereegranskat)abstract
- Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).
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| 10. |
- Hao, Meng-Shu, et al.
(författare)
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The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:9
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Tidskriftsartikel (refereegranskat)abstract
- NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K-0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O-2 and decylubiquinone as electron acceptors. The K-0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K-0.5(Ca2+) for NADPH oxidation was high (approximate to 100 mu M), yet 20-fold lower K-0.5(Ca2+) values were determined at pH 6.8. Lower K-0.5(Ca2+) values were observed with decylubiquinone than with O-2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.
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