66 research outputs found
A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly
An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe(2+) alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.This work was supported in part by the Biotechnology and Biological Sciences Research Council Grant BB/H00288X/1
A Mitochondrial calcium dynamics - checks and balances of energy physiology
Mitochondria conserve the energy released from metabolic redox reactions and supply the cell with ATP. When the rate of respiratory metabolism does not match ATP demand active regulation of mitochondrial function is essential. For plants particularly sophisticated regulation strategies can be expected, to ensure maintenance of homeostasis in the presence of frequent environmental changes. Yet, the mechanisms by which such control is achieved in vivo are poorly understood.
Calcium acts as a key regulator of mitochondrial energy metabolism in mammals by modulating the activity TCA cycle dehydrogenases. Calcium flux into the matrix is controlled by the recently identified mitochondrial uniporter complex. Plants contain homologues of components of the uniporter, but their function has been unclear. To understand how mitochondrial calcium dynamics are regulated and what their impact is on energy metabolism, we have combined reverse genetics with in vivo sensing of calcium. Fluorescent protein sensors and quantitative confocal imaging allow monitoring of mitochondrial energy physiology in living Arabidopsis tissues. We have found that several homologues of components of the mitochondrial calcium uniporter complex localize to mitochondria in Arabidopsis. Mutant lines have shown severely altered mitochondrial calcium levels and abnormal organellar calcium transients, providing a novel genetic handle on the dissection of the role of calcium regulation in plant mitochondria. We will discuss the specific impact of de-regulated mitochondrial calcium on the physiological network and the function of plant mitochondria
Photosynthesis-dependent H₂O₂ transfer from chloroplasts to nuclei provides a high-light signalling mechanism
Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression
Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors
Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically-encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, named MetSOx and MetROx for their ability to detect S and R-forms of MetO, respectively, were utilized for targeted analysis of protein oxidation, regulation and repair, as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO, and examining responses to physiological stimuli
Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge.
In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A+SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H(2)O(2) treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge
Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge.
In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A+SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H(2)O(2) treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge
Thiol switches in mitochondria: Operation and physiological relevance.
Mitochondria are a major source of reactive oxygen species (ROS) in the cell, particularly of superoxide and hydrogen peroxide. A number of dedicated enzymes regulate the conversion and consumption of superoxide and hydrogen peroxide in the intermembrane space and the matrix of mitochondria. Nevertheless, hydrogen peroxide can also interact with many other mitochondrial enzymes, particularly those with reactive cysteine residues, modulating their reactivity in accordance with changes in redox conditions. In this review we will describe the general redox systems in mitochondria of animals, fungi and plants and discuss potential target proteins that were proposed to contain regulatory thiol switches
Shining a light on NAD- and NADP-based metabolism in plants
The pyridine nucleotides nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] simultaneously act as energy transducers, signalling molecules, and redox couples. Recent research into photosynthetic optimisation, photorespiration, immunity, hypoxia/oxygen signalling, development, and post-harvest metabolism have all identified pyridine nucleotides as key metabolites. Further understanding will require accurate description of NAD(P)(H) metabolism, and genetically encoded fluorescent biosensors have recently become available for this purpose. Although these biosensors have begun to provide novel biological insights, their limitations must be considered and the information they provide appropriately interpreted. We provide a framework for understanding NAD(P)(H) metabolism and explore what fluorescent biosensors can, and cannot, tell us about plant biology, looking ahead to the pressing questions that could be answered with further development of these tools
The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide 'flashes'.
The properties of a cpYFP [circularly permuted YFP (yellow fluorescent protein)] reported to act as a superoxide sensor have been re-examined in Arabidopsis mitochondria. We have found that the probe has high pH sensitivity and that dynamics in the cpYFP signal disappeared when the matrix pH was clamped by nigericin. In contrast, genetic and pharmacological manipulation of matrix superoxide had no detectable effect on the cpYFP signal. These findings question the existence of superoxide flashes in mitochondria
Biological control of weeds: an analysis of introductions, rates of establishment and estimates of success, worldwide
The foremost document that comprehensively reports on biological control introductions against weeds—‘Biological control of weeds: a world catalogue of agents and their target weeds’—has been updated and now includes all deliberate releases made through 2012. It includes data on 1555 intentional releases of 468 biological control agent species used against 175 species of target weeds in 48 plant families, in 90 countries. For 55 (31.4%) of the target weed species, only one biocontrol agent was introduced. The largest number of agent species (44) was introduced for the biological control of Lantana camara (Verbenaceae). Three insect orders (Coleoptera, Lepidoptera and Diptera) comprised about 80% of all biocontrol agent species released and releases made. Of the 468 biocontrol agent species introduced, 332 (70.9%) established in at least one instance. Of the 313 species, for which impact could be categorized, 172 (55.0%) caused medium, variable or heavy levels of damage (impacts). Of all releases made through 2012, 982 (63.2%) led to establishment. Forty-two releases were judged too early post-release to categorize impact, leaving 940 releases for which impact analyses were conducted. Similar to agent species, approximately half of the established releases (503 or 53.5%) caused medium, variable or heavy levels of damage on the target weeds, and almost a quarter of releases (225 or 23.9%) caused heavy impact. Across all countries and regions, 65.7% of the weeds targeted for biological control experienced some level of control. These data indicate the value of this practice, on its own, or as a supplement to other methods, in the management of invasive plants
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