Juan J. Lázaro

Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

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.

Thermodynamics of the dimer-decamer transition of reduced human and plant 2-cys peroxiredoxin.

Isothermal titration calorimetry (ITC) is a powerful technique for investigating self-association processes of protein complexes and was expected to reveal quantitative data on peroxiredoxin oligomerization by directly measuring the thermodynamic parameters of dimer-dimer interaction. Recombinant classical 2-cysteine peroxoredoxins from Homo sapiens, Arabidopsis thaliana, and Pisum sativum as well as a carboxy-terminally truncated variant were subjected to ITC analysis by stepwise injection into the reaction vessel under various redox conditions. The direct measurement of the decamer-dimer equilibrium of reduced peroxiredoxin revealed a critical concentration in the very low micromolar range. The data suggest a cooperative assembly above this critical transition concentration where a nucleus facilitates assembly. The rather abrupt transition indicates that assembly processes do not occur below the critical transition concentration while oligomerization is efficiently triggered above it. The magnitude of the measured enthalpy confirmed the endothermic nature of the peroxiredoxin oligomerization. Heterocomplexes between peroxiredoxin polypeptides from different species were not formed. We conclude that a functional constraint conserved the dimer-decamer transition with highly similar critical transition concentrations despite emerging sequence variation during evolution.

High-Yield Expression of Pea Thioredoxin m and Assessment of Its Efficiency in Chloroplast Fructose-1,6-Bisphosphatase Activation

A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schurmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37–45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.

Dissecting the integrative antioxidant and redox systems in plant mitochondria. Effect of stress and S-nitrosylation

Mitochondrial respiration provides the energy needed to drive metabolic and transport processes in cells. Mitochondria are a significant site of reactive oxygen species (ROS) production in plant cells, and redox-system components obey fine regulation mechanisms that are essential in protecting the mitochondrial integrity. In addition to ROS, there are compelling indications that nitric oxide can be generated in this organelle by both reductive and oxidative pathways. ROS and reactive nitrogen species play a key role in signaling but they can also be deleterious via oxidation of macromolecules. The high production of ROS obligates mitochondria to be provided with a set of ROS scavenging mechanisms. The first line of mitochondrial antioxidants is composed of superoxide dismutase and the enzymes of the ascorbate-glutathione cycle, which are not only able to scavenge ROS but also to repair cell damage and possibly serve as redox sensors. The dithiol-disulfide exchanges form independent signaling nodes and act as antioxidant defense mechanisms as well as sensor proteins modulating redox signaling during development and stress adaptation. The presence of thioredoxin (Trx), peroxiredoxin (Prx) and sulfiredoxin (Srx) in the mitochondria has been recently reported. Cumulative results obtained from studies in salt stress models have demonstrated that these redox proteins play a significant role in the establishment of salt tolerance. The Trx/Prx/Srx system may be subjected to a fine regulated mechanism involving post-translational modifications, among which S-glutathionylation and S-nitrosylation seem to exhibit a critical role that is just beginning to be understood. This review summarizes our current knowledge in antioxidative systems in plant mitochondria, their interrelationships, mechanisms of compensation and some unresolved questions, with special focus on their response to abiotic stress.

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Purification and heterogeneity

Abstract The enzyme fructose- 1,6-diphosphatase (FDPase), involved in the reductive cycle of the pentose phosphate pathway, has been purified from spinach leaves by heating (30 min at 60°), “salting out” with ammonium sulphate (between 30–70% of saturation), filtration through Sephadex G-100 and G-200, fractionation on DEAE-52 cellulose and preparative electrophoresis on polyacrylamide gel. Filtration through DEAE-cellulose led to the isolation of two active fractions (fractions I and II) with very close MWs and isoelectric points. By electrophoresis on acrylamide gel, both fractions gave two active fractions (fractions I a -I b and II a -II b ). The fractions with low electrophoretic migration rate—I b and II b —are stable in acid and neutral pH, have a MW between 90 000 and 110 000 and constitute the native form of the photosynthetic enzyme. The fractions of faster migration rate—I a and II a -originate from the corresponding fractions I b and II b under alkaline conditions, show half the MW of the respective fractions, and behave as subunits of the original dimer form. Measured by electrofocusing, the four active fractions have isoclectric points in the range 4·10–4.30.

The donor side of Photosystem II as the copper-inhibitory binding site. Fluorescence and polarografic studies*

We have measured, under Cu (II) toxicity conditions, the oxygen-evolving capacity of spinach PS II particles in the Hill reactions H2O→SiMo (in the presence and absence of DCMU) and H2O→PPBQ, as well as the fluorescence induction curve of Tris-washed spinach PS II particles. Cu (II) inhibits both Hill reactions and, in the first case, the DCMU-insensitive H2O → SiMo activity. In addition, the variable fluorescence is lowered by Cu (II). We have interpreted our results in terms of a donor side inhibition close to the reaction center. The same polarographic and fluorescence measurements carried out at different pHs indicate that Cu (II) could bind to amino acid residues that can be protonated and deprotonated. In order to reverse the Cu (II) inhibition by a posterior EDTA treatment, in experiments of preincubation of PS II particles with Cu (II) in light we have demonstrated that light is essential for the damage due to Cu (II) and that this furthermore is irreversible.

Binding site on pea chloroplast fructose-1,6-bisphosphatase involved in the interaction with thioredoxin

When we compare the primary structures of the six chloroplast fructose-1,6-bisphosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceding the redox regulatory cysteines cluster can be observed. This region is a good candidate for binding of FBPase to its physiological modulator thioredoxin (Td), as this association shows clear differences between species. Using a cDNA clone for pea chloroplast FBPase as template, we have amplified by PCR a DNA insert coding for a 19 amino acid fragment (149Pro-167Gly), which was expressed in pGEMEX-1 as a fusion protein. This protein strongly interacts with pea Td m, as shown by ELISA and Superose 12 gel filtration, depending on pH of the medium. Preliminary assays have shown inhibition of FBPase activity in the presence of specific IgG against the 19 amino acid insert. Surprisingly the fusion protein enhances the FBPase activation in competitive inhibition experiments carried out with FBPase and Td. These results show the fundamental role played by this domain in FBPase-Td binding, not only as docking point for Td, but also by inducing some structural modification in the Td molecule. Taking as model the structural data recently published for spinach photosynthetic FBPase [29], this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.

Cloning, structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme.

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3′-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the native chloroplast enzyme.

An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in leaf crude extracts

An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in crude extracts of leaves is proposed. It is based on the ELISA technique, and offers two modifications. A non-competitive technique has a higher sensitivity and is the right option for samples of low fructose-1,6-bisphosphatase content. However, this method is not sufficiently specific when the total protein is higher than 5 μg/cm3; so, despite its lower sensitivity, in these circumstances a competitive technique is more suitable. Thus photosynthetic fructose-1,6-bisphosphatase can be measured without interferences from the gluconeogenic cytosolic enzyme of the photosynthetic cell or from a non-specific phosphatase present in the chloroplast.