Pea Mitochondria Research Articles

Методические рекомендации в использовании антител против дегидринов в агробиотехнологиях

Detection of the expression levels of the dehydrin gene in plants is increasingly becoming a widespread technique for testing plant resistance to cold, drought, salinity or high heavy metal concentrations in soils. The simplest way to determine the content of dehydrins consists in using specific antibodies (antibody specificity is critical). The experience of dehydrin detection in various plants provided a basis for this work. In this study, we set out to compare the reactions of antibodies with reactive proteins derived from three most accessible sources, as well as to assess their specificity. The comparability of detected proteins in terms of their quantity and molecular weight was studied for several plants: 1) total and thermostable protein in scots pine needles; 2) mitochondrial protein in peas; 3) total and thermostable protein in winter wheat; 4) total and thermostable protein in arabidopsis. In all the cases, either slight or significant differences in the quantity and molecular weight of the detected protein groups were observed. These differences can be attributed to the three possible reasons: K-segment variability; different carrier proteins used by manufacturers; as well as the presence of shared reactive epitopes in some groups of proteins that are not dehydrins. The analysis of differences in the number of the groups detected using antibodies from different manufacturers reveals their amount to be minimal for cereal plants and becoming larger for dicotyledons and conifers. The largest number of the groups was detected in pea mitochondria. Some of the antibody-binding protein groups are definitely not dehydrins. The abovementioned differences are smaller for thermostable proteins. Thus, obtaining a thermostable protein fraction can be recommended as a simple procedure increasing the dehydrins detection level. The comparison of the dehydrin amounts should not be used as a measure of the relative plant resistance when comparing evolutionarily distant species, since the antibody reactivity to the dehydrins of distant species may differ significantly. At the same time, this method can be used in agrobiology for the molecular-level assessment of the initial or induced resistance of plant varieties (same or related species) to cold, drought, as well as other adverse conditions associated with water deficiency.

Ultrastructure and metabolic activity of pea mitochondria under clinorotation

Experimental data on the mitochondrial ultrastructure and tissue respiration in root apex as well as metabolic activity of the organelles isolated from pea seedling roots after 5-day of clinorotation are presented. It was shown that mitochondrial condensation in the distal elongation zone correlated with an increased rate of oxygen uptake on 7%. We also observed increase in rate of malate oxidation and respiratory control ratio increased simultaneously with a decreased in efficiency of oxidative phosphorylation. Such character of mitochondrial rearrangements in simulated microgravity is assumed to be a consequence of adaptation to these conditions.

Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria.

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.

Function and stress tolerance of seed mitochondria

Seeds of higher plant are desiccation tolerant, which suggests that their mitochondria exhibit particular properties. Insight into the function of seed mitochondria, especially in legume and model plants, has been fostered by the development of proteomics. Seed mitochondria are functional at the onset of imbibition, and their integrity and performance systematically improves during germination. This suggests that repair and biogenesis mechanisms exist, and this is supported by morphological and biochemical evidence. Seed mitochondria generate and operate in a hypoxic environment. They accumulate stress proteins, such as a small heat‐shock protein and a late embryogenesis abundant protein. The mitochondria of pea (Pisum sativum L.) seed also display a biased phospholipid composition likely to favour desiccation tolerance. These specific biochemical properties surely contribute to the remarkable tolerance of seed mitochondria to extreme temperatures. Recent progress towards the resolution of the seed mitochondrial proteome is discussed in light of the growing body of genomic data.

The pea mitochondrial atp6: RNA editing and similarity of presequences in the Vicieae tribe.

The atp6 gene has been identified as a single-copy sequence in the mitochondrial genome of the pea. An unexpected finding concerns the atp6 5' extension which is known to be poorly conserved at the sequence level, even between closely related plant species. We have shown that the presequences of ATP6 from the pea and other species belonging to the Vicieae tribe of Fabaceae (broad bean, hairy vetch) share a sequence similarity which extends to long 5' untranslated transcript termini. The reason for the observed conservation is not clear but may simply reflect the close phylogenetic relationship of species from the Vicieae tribe. The result of editing analysis indicates the occurrence of fully and partially edited transcripts of atp6 in the pea mitochondria. The majority of the editing sites are targeted to the last transmembrane domain of the pea ATP6, important in proton translocation and interactions with other subunits of ATP synthase.

Complex cis-elements determine an RNA editing site in pea mitochondria.

The cis-requirements for the first editing site in the atp9 mRNA from pea mitochondria were investigated in an in vitro RNA editing system. Template RNAs deleted 5' of -20 are edited correctly, but with decreased efficiency. Deletions between -20 and the edited nucleotide abolish editing activity. Substitution of the sequences 3' of the editing site has little effect, which suggests that the major determinants reside upstream. Stepwise mutated RNA sequences were used as templates or competitors that divide the cis-elements into several distinct regions. In the template RNAs, mutation of the sequence between -40 and -35 reduces the editing activity, while the region from -15 to -5 is essential for the editing reaction. In competition experiments the upstream region can be titrated, while the essential sequence near the editing site is largely resistant to excess competitor. This observation suggests that either one trans-factor attaches to these separate cis-regions with different affinities or two distinct trans-factors bind to these sequences, and one of which is present in limited amounts, whereas the other one is more abundant in the lysate.

Biochemical and immunochemical evidences for the presence of lipoxygenase in plant mitochondria.

In this paper, both biochemical and immunochemical evidence for the presence of lipoxygenase (LOX) in plant mitochondria is presented. Highly purified pea (Pisum sativum L., cv. Alaska) mitochondria show LOX activity, evaluated as conjugated diene formation, oxygen consumption, and hydroperoxide formation. Both 9- and 13-hydroperoxy-octadecadienoic acids are produced by the oxidation of linoleic acid. LOX activity is particularly evident in swollen mitochondria; it is inhibited by nordihydroguaiaretic acid, a pea anti-LOX B antibody, and has two pH optima (6.0 and 7.5). A mitochondrial protein of approximately 97 kDa cross-reacts with a pea seed anti-LOX B antibody. This reaction is detectable in both soluble (matrix fraction) and membrane-bound (submitochondrial particles) proteins. Considering that pea mitochondria were extracted from actively growing stems that were differentiating tube elements, it is suggested that the presence of LOX in these organelles may be related to their degradation linked to xylem differentiation.

A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor

We have identified a novel protein on the outer membrane of Arabidopsis thaliana mitochondria. This protein displays 67% sequence identity with the 64 kDa translocase of the outer envelope membrane of chloroplasts (Toc). A mitochondrial localisation for this protein was determined by (i) its presence in the proteome of highly purified Arabidopsis mitochondria, (ii) Western blot analysis with antibodies to Toc64 from pea that indicate its presence in Arabidopsis and pea mitochondria, (iii) green fluorescent protein fusion proteins that indicate an exclusive mitochondrial localisation for this protein, and (iv) expression profiles in various tissue types and during development that are more similar to translocase of the outer mitochondrial membrane components than to chloroplastic Toc components. Thus Arabidopsis mitochondria contain a protein with high sequence identity to a plastid protein import receptor.

Subcellular localization of two types of ferrochelatase in cucumber.

It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.

Effect of electron-transport inhibitors on the generation of reactive oxygen species by pea mitochondria during succinate oxidation.

The effect of inhibitors of the cytochrome pathway and alternative oxidase on the rate of respiration and generation of reactive oxygen species by pea mitochondria was studied. Respiration of mitochondria from pea cotyledons was inhibited by 70-80% by salicylhydroxamate (SHAM). The rate of hydrogen peroxide production by pea cotyledon mitochondria during succinate oxidation was 0.15 nmol/min per mg protein. SHAM considerably accelerated the hydrogen peroxide production. The SHAM-dependent H2O2 production was stimulated by 2 micro M antimycin A and inhibited by 5 mM KCN and 1 micro M myxothiazol. The study of the rate of O2*- generation by pea mitochondria using EPR spin traps and epinephrine oxidation showed that H2O2 accumulation can be accounted for by a significant increase in the rate of O2*- production.

RT-PCR analysis of 5' to 3'-end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria.

Gene expression in plant mitochondria is still inadequately analyzed. To learn more about transcription and RNA processing in plant mitochondria, the 5'- and 3'-RNA extremities and the promoters of the cytochrome oxidase gene (cox2) were analyzed in pea. Both 5' and 3' ends of cox2 transcripts were examined by RT-PCR across the ligation site of circularized mitochondrial RNA as template. This approach identified 5' ends a few nucleotides shorter than three major 5' ends mapped by primer extension analysis. Presumably, only monophosphate 5' ends derived from processing can be ligated. In vitro transcription assays using a homologous mitochondrial protein extract from pea strongly suggest the major 5' ends to derive from transcription initiation. The cDNA analysis of the head-to-tail ligated cox2 mRNA identified 3' ends within a thymidine stretch approximately 300 nt downstream of the reading frame in a sequence segment that was not present in the previous investigation of this gene. Nuclease S1 protection experiments confirmed this newly identified 3' terminus and corroborated the validity of this technique in mRNA end analysis. The general use of the circularized RNA (CR)-RT-PCR approach for the simultaneous analysis of the 5' and 3' extremities of mRNA molecules is discussed.

Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria

Using in vitro import assays into purified mitochondria and chloroplasts we found that Arabidopsis ferrochelatase-I and ferrochelatase-II were not imported into mitochondria purified from Arabidopsis (or several other plants) but were imported into pea leaf chloroplasts. Other dual targeted proteins could be imported into purified mitochondria from Arabidopsis. As only two ferrochelatase genes are present in the completed Arabidopsis genome, the presence of ferrochelatase activity in plant mitochondria needs to be re-evaluated. Previous reports of Arabidopsis ferrochelatase-I import into pea mitochondria are due to the fact that pea leaf (and root) mitochondria appear to import a variety, but not all chloroplast proteins. Thus pea mitochondria are not a suitable system to either study dual targeting, or to distinguish between isozymes present in mitochondria and chloroplasts.

AN INFLUENCE OF ANTISERUM AGAINST WINTER WHEAT STRESS UNCOUPLING PROTEIN, CSP 310, ON ENERGETIC ACTIVITY OF SOME PLANT SPECIES MITOCHONDRIA

It is determined that addition of an anti-CSP 310 antiserum to isolated mitochondria of cereals (winter rye, winter wheat, and maize) caused an increasing of mitochondrial respiratory control. In a similar manner, addition of this antiserum to isolated pea mitochondria did not cause this effect. It is shown that coupling effect of antiserum is not dependent upon the presence of bovine serum albumin in mitochondria incubation media. Therefore, these results show that the mechanism of oxidation and phosphorylation uncoupling, and participation of immunochemically related CSP 310 proteins that can be precipitated by anti-CSP 310 antiserum is specific for cereals.

Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth

Acyl-CoA dehydrogenase activity has been measured in homogenates of post-imbibition to 14-day-old hydroponically grown pea seeds at daily intervals, using C 4 , C 12 and C 16 acyl-CoA substrates. The activity peaks of the different chain-length acyl-CoA dehydrogenases did not transpose at all points and the ratios of the chain-length activities were not constant. It therefore has to be concluded that more than one dehydrogenase is present in pea mitochondria. There was a post-imbibition initial surge of activity with short- and mid-chain-length substrates. The C 16- handling enzyme first peaked at 3–4 days, which coincided with the onset of plumule unfurling and greening. Further peaks were observed with all three substrates, coinciding with secondary root formation and leaf enlargement and later with cotyledon degeneration. Overall activity showed that the long-chain acyl-CoA dehydrogenase was much more active than the short-chain acyl-CoA dehydrogenase.

Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth

Acyl-CoA dehydrogenase activity has been measured in homogenates of post-imbibition to 14-day-old hydroponically grown pea seeds at daily intervals, using C(4), C(12) and C(16) acyl-CoA substrates. The activity peaks of the different chain-length acyl-CoA dehydrogenases did not transpose at all points and the ratios of the chain-length activities were not constant. It therefore has to be concluded that more than one dehydrogenase is present in pea mitochondria. There was a post-imbibition initial surge of activity with short- and mid-chain-length substrates. The C(16)-handling enzyme first peaked at 3-4 days, which coincided with the onset of plumule unfurling and greening. Further peaks were observed with all three substrates, coinciding with secondary root formation and leaf enlargement and later with cotyledon degeneration. Overall activity showed that the long-chain acyl-CoA dehydrogenase was much more active than the short-chain acyl-CoA dehydrogenase.

Hydrogen peroxide generation by higher plant mitochondria oxidizing complex I or complex II substrates

The generation of H 2O 2 by isolated pea stem mitochondria, oxidizing either malate plus glutamate or succinate, was examined. The level of H 2O 2 was almost one order of magnitude higher when mitochondria were energized by succinate. The succinate-dependent H 2O 2 formation was abolished by malonate, but unaffected by rotenone. The lack of effect of the latter suggests that pea mitochondria were working with a proton motive force below the threshold value required for reverse electron transfer. The activation by pyruvate of the alternative oxidase was reflected in an inhibition of H 2O 2 formation. This effect was stronger when pea mitochondria oxidized malate plus glutamate. Succinate-dependent H 2O 2 formation was ca. four times lower in Arum sp. mitochondria (known to have a high alternative oxidase) than in pea mitochondria. An uncoupler (FCCP) completely prevented succinate-dependent H 2O 2 generation, while it only partially (40–50%) inhibited that linked to malate plus glutamate. ADP plus inorganic phosphate (transition from state 4 to state 3) also inhibited the succinate-dependent H 2O 2 formation. Conversely, that dependent on malate plus glutamate oxidation was unaffected by low and stimulated by high concentrations of ADP. These results show that the main bulk of H 2O 2 is formed during substrate oxidation at the level of complex II and that this generation may be prevented by either dissipation of the electrochemical proton gradient (uncoupling and transition state 4-state 3), or preventing its formation (alternative oxidase). Conversely, H 2O 2 production, dependent on oxidation of complex I substrate, is mainly lowered by the activation of the alternative oxidase.

Protein synthesis by isolated pea mitochondria is dependent on the activity of respiratory complex II.

In isolated pea (Pisum sativum L.) mitochondria incorporation of 35S-methionine into newly synthesised proteins was influenced by the presence of site-specific inhibitors of the respiratory electron-transport chain. These effects were not produced by changes in the rate of respiratory electron transport itself nor by changes in ATP concentration. Protein synthesis was inhibited by inhibitors of ubiquinone reduction but not by inhibitors of ubiquinol oxidation. By the use of additional inhibitors at specific sites of the respiratory chain, different oxidation-reduction states were obtained for the different complexes in the electron-transport chain. It was found that electron transport through succinate:ubiquinone oxidoreductase (respiratory complex II) was specifically required for protein synthesis, even when all the other conditions for protein synthesis were satisfied. We suggest that a subunit of complex II, or a component closely associated with complex II, is involved in a regulatory system that couples electron transport to protein synthesis.

Characterization of DNA-Binding Proteins from Pea Mitochondria

We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed.

3'-Inverted repeats in plant mitochondrial mRNAs are processing signals rather than transcription terminators.

A number of mRNAs in plant mitochondria contain inverted repeats at their 3'-termini. These have been discussed as potential transcription terminators or, alternatively, as post-transcriptional processing and stability signals of longer precursor RNAs. In vitro transcription in a pea mitochondrial lysate now shows that transcription proceeds almost unimpeded through these inverted repeat structures. To investigate their potential function in mRNA processing, we developed an in vitro processing system from pea mitochondria. This in vitro system correctly processes synthetic precursor mRNAs containing the pea atp9 double stem-loop structure, yielding the same 3'-termini observed in vivo. Analysis of the in vitro-generated products and of the processivity of the reaction suggests exonucleolytic degradation up to the stem-loop. The inverted repeat structures found at the 3'-termini of mRNAs in plant mitochondria are thus recognized as processing and most likely also stabilizing signals in transcript maturation, but do not terminate transcription.