Simultaneous isolation of intact mitochondria and chloroplasts from a single pulping of plant tissue

Rödiger, Anja; Baudisch, Bianca; Klösgen, Ralf Bernd

doi:10.1016/j.jplph.2009.11.013

Cited by 48 publications

(43 citation statements)

References 15 publications

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“…The two enzyme assays confirmed that the purified mitochondria are not contaminated by the cytosolic fraction nor from integral, undisrupted plastids, since in that case the G6PDH enzymatic activity should have been detected also in the mitochondrial fraction. Furthermore, the plastid marker Toc33 protein (Rödiger et al, 2010) accumulated only in the P fraction (Figure 2C), whereas porin (Balk and Leaver, 2001) accumulated, as expected, in the M fraction and, to lesser extent, in the P fraction (Figure 2C). …”

Section: Resultssupporting

confidence: 63%

“…SDS-PAGE was performed according to Vigani et al (2009) with the following antibodies: spinach anti-Toc33 (Rödiger et al, 2010) at 1:1000 dilution, maize anti-porin (Balk and Leaver, 2001) at 1:2000 dilution, Arabidopsis anti-ATFER1 (Murgia et al, 2007) at 1:2000 and an anti-rabbit conjugated with alkaline phosphatase as secondary antibody. Protein quantification was determined according to Vigani et al (2009).…”

Section: Methodsmentioning

confidence: 99%

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Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots

Vigani¹,

Tarantino²,

Murgia³

2013

Front. Plant Sci.

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In plants, intracellular Fe trafficking must satisfy chloroplasts' and mitochondrial demands for Fe without allowing its accumulation in the organelles in dangerous redox-active forms. Protein ferritin is involved in such homeostatic control, however its functional role in mitochondria, differently from its role in chloroplasts, is still matter of debate. To test ferritin functionality as a 24-mer Fe-storage complex in mitochondria, cucumber seedlings were grown under different conditions of Fe supply (excess, control, deficiency) and mitochondria were purified from the roots. A ferritin monomer of around 25 KDa was detected by SDS-PAGE in Fe-excess root mitochondria, corresponding to the annotated Csa5M215130/XP_004163524 protein: such a monomer is barely detectable in the control mitochondria and not at all in the Fe-deficient ones. Correspondingly, the ferritin 24-mer complex is abundant in root mitochondria from Fe-excess plants and it stores Fe as Fe(III): such a complex is also detectable, though to a much smaller extent, in control mitochondria, but not in Fe-deficient ones. Cucumber ferritin Csa5M215130/XP_004163524 is therefore a functional Fe(III)-store in root mitochondria and its abundance is dependent on the Fe nutritional status of the plant.

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Section: Resultssupporting

confidence: 63%

Section: Methodsmentioning

confidence: 99%

Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots

Vigani¹,

Tarantino²,

Murgia³

2013

Front. Plant Sci.

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“…Leaf water status was determined by measuring the RWC of leaves, as previously described (Wang and Vanlerberghe, 2013). Separate chloroplast and mitochondrial fractions for immunoblotting were isolated simultaneously from 40 g fresh weight of leaf tissue using the protocol developed by Rödiger et al (2010). Chl a and b were determined according to Arnon (1949) using leaf samples that had been snap-frozen in liquid N 2 .…”

Section: Other Methodsmentioning

confidence: 99%

Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum

et al. 2014

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The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum 'Petit Havana SR1' wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate droughtstressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO 2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.

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“…radiolabelled precursor proteins of (A) GrpE, (B) EF-tu, both from Arabidopsis thaliana, (C) mtrieske from potato, and (D) Fnr from spinach, were obtained by coupled in vitro transcription/ translation of the corresponding cDna clones and incubated for 20 min at 25 °C with freshly isolated intact mitochondria (lanes M) or chloroplasts (lanes C) from pea in the presence or absence of competitor protein dissolved in urea. 8 the concentration of competitor protein present in each assay (given in µm) is indicated above the lanes. Standard import reactions (lanes control) were performed in the absence of both competitor and urea.…”

mentioning

confidence: 99%

Organelle import of proteins with dual targeting properties into mitochondria and chloroplasts takes place by the general import pathways

Langner¹,

Baudisch

Klösgen³

2014

Plant Signaling & Behavior

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Simultaneous isolation of intact mitochondria and chloroplasts from a single pulping of plant tissue

Cited by 48 publications

References 15 publications

Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots

Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots

Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum

Organelle import of proteins with dual targeting properties into mitochondria and chloroplasts takes place by the general import pathways

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