| 1. |
- Johansson, Fredrik I, et al.
(författare)
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Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria
- 2004
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Ingår i: Biochemical Journal. - 0264-6021. ; 380:1, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- The inner mitochondrial membrane is selectively permeable, which limits the transport of solutes and metabolites across the membrane. This constitutes a problem when intramitochondrial enzymes are studied. The channel-forming antibiotic AlaM (alamethicin) was used as a potentially less invasive method to permearbilize mitochondria and study the highly branched electron-transport chain in potato tuber (Solanum tuberosum) and pea leaf (Pisum sativum) mitochondria. We show that AlaM permeabilized the inner membrane of plant mitochondria to NAD(P)H, allowing the quantification of internal NAD(P)H dehydrogenarses as well as matrix enzymes in situ. AlaM was found to inhibit the electron-tran sport chain at the external Ca2+-dependent rotenone-insensitive NADH dehydrogenase and around complexes III and IV. Nevertheless, under optimal conditions, especially complex I-mediated NADH oxidation in AlaM-treated mitochondria was much higher than what has been previously measured by other techniques. Our results also show a difference in substrate specificities for complex I in mitochondria as compared with inside-out submitochondrial particles. AlaM facilitated the passage of cofactors to and from the mitochondrial matrix and allowed the determination of NAD(+) requirements of malate oxidation in situ. In summary, we conclude that AlaM provides the best method for quantifying NADH dehydrogenase activities and that AlaM will prove to be an important method to study enzymes under conditions that resemble their native environment not only in plant mitochondria but also in other membrane-enclosed compartments, such as intact cells, chloroplasts and peroxisomes.
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| 2. |
- Liu, Yunjun, et al.
(författare)
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A Redox-Mediated Modulation of Stem Bolting in Transgenic Nicotiana sylvestris Differentially Expressing the External Mitochondrial NADPH Dehydrogenase.
- 2009
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548. ; 150, s. 1248-1259
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Tidskriftsartikel (refereegranskat)abstract
- Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato NDB1 displayed early bolting whereas sense-suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance, but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+)-ratio was unaffected, the stem NADPH/NADP(+)-ratio was altered following the genetic modification and strongly correlated to the bolting phenotype. Metabolic profiling of the stem displayed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars and hexose phosphates. Consistent with the phenotype, the modified NDB1 level also affected expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+)-ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are present in green stems. The results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.
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| 3. |
- Michalecka, Agnieszka
(författare)
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Alternative NAD(P)H dehydrogenases in plant mitochondria - localisation, catalytic functions and physiological roles
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In addition to complex I, the plant mitochondrial electron transport chain possesses several alternative NAD(P)H dehydrogenases, not present in animals. These enzymes allow nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. The mitochondrial inner membrane was permeabilised with a channel-forming antibiotic, alamethicin, and the activity of internal NADH dehydrogenases was studied in the matrix. This technique revealed that changes occur in substrate specificity of complex I upon isolation of submitochondrial particles. Alamethicin permeabilisation was shown to be a reliable method for measurements of internal NADH dehydrogenases and soluble matrix proteins in their native environment of plant mitochondria. Three gene families encoding alternative NAD(P)H dehydrogenases were detected in Arabidopsis genome. There are two At-nda genes, four At-ndb genes and one At-ndc gene. The nda and ndb families are more closely related to fungal homologues, while the ndc family has its origin in cyanobacteria. Representative genes of all three families were all shown to encode proteins targeted to mitochondria. Expression of nda1 but not nda2 was shown to be light-dependent. Based on the sequence similarity between NDA proteins it is possible that they have the same submitochondrial localisation and enzymatic function. Most likely, NDA proteins are internal alternative NADH dehydrogenases. Expression of the St-ndb1 gene in transgenic Nicotiana sylvestris plants imposed specifically increased and decreased external NADPH oxidation in mitochondria isolated from different transgenic lines. A strict correlation to transcript and protein amounts allowed the assignment of St-NDB1 as an external NADPH dehydrogenase. As a consequence the St-ndb1 overexpressing transgenic plants had specifically increased protein levels for alternative oxidase and uncoupling protein, indicating crosstalk in regulation of the protein amount for the enzymes involved in non-energy-conserving pathways.
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| 4. |
- Michalecka, Agnieszka, et al.
(författare)
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Arabidopsis genes encoding mitochondrial type II NAD(P)H dehydrogenases have different evolutionary orgin and show distinct responses to light.
- 2003
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 133:2, s. 642-652
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Tidskriftsartikel (refereegranskat)abstract
- In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.
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| 5. |
- Michalecka, Agnieszka, et al.
(författare)
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Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic Nicotiana sylvestris
- 2004
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Ingår i: Plant Journal. - 1365-313X. ; 37:3, s. 415-425
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Tidskriftsartikel (refereegranskat)abstract
- The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD(P)H dehydrogenases, was introduced into Nicotiana sylvestris. Transgenic lines with high transcript and protein levels for St-NDB1 had up to threefold increased activity of external NADPH dehydrogenase in isolated mitochondria as compared to the wild type (WT). In two lines, the external NADPH dehydrogenase activity was instead 10-fold decreased, indicating that the corresponding N. sylvestris gene had been suppressed. Activities of external and internal rotenone-insensitive NADH dehydrogenases were unchanged in the transgenic lines. The results demonstrate that the St-ndb1 encodes an external dehydrogenase specific for NADPH and dependent on calcium for activity. Transgenic lines overexpressing St-ndb1 had specifically increased protein levels for alternative oxidase and uncoupling protein, as compared to the WT and one co-suppressing line. This indicates cross-talk in the expressional control, or metabolic conditions influencing it, for the different categories of energy-dissipating proteins that bypass oxidative phosphorylation. The potential effects of external NADPH oxidation on other cellular processes are discussed.
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