NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Glutathione-mediated thermomorphogenesis and heat stress responses in Arabidopsis thaliana.

In the context of climate change, the global rise of temperature and intense heat waves affect plant development and productivity. Among the molecular perturbations that high temperature induces in living cells is the accumulation of reactive oxygen species (ROS), which perturbs the cellular redox state. In plants, the dynamics of the cellular and subcellular redox state have been poorly investigated under high temperature. Glutathione plays a major role in maintaining the cellular redox state. We investigated its contribution in adaptation of Arabidopsis thaliana to contrasting high temperature regimes: high ambient temperature inducing thermomorphogenesis and heat stress affecting plant viability. Using the genetically encoded redox marker roGFP2, we show that high temperature regimes lead to cytoplasmic and nuclear oxidation and impact the glutathione pool. This pool is restored within a few hours, which probably contributes to plant adaptation to high temperatures. Moreover, low glutathione mutants fail to adapt to heat stress and to induce thermomorphogenesis, suggesting that glutathione is involved in both heat adaptation mechanisms. We also evaluate the transcriptomic signature in the two high temperature regimes and identified gene expression deviations in low glutathione mutants, which might contribute to their sensitivity to high temperature. Thus, we define glutathione as a major player in the adaptation of Arabidopsis to contrasting high temperature regimes.

Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress.

Heat stress induces misfolding and aggregation of proteins unless they are guarded by chaperone systems. Here, we examined the function of the glutaredoxin GRXS17, a member of thiol reductase families in the model plant Arabidopsis (Arabidopsis thaliana). GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin domain and three CGFS active-site motif-containing GRX domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione-dependent manner. As an Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, GRXS17 presented both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induced loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active-site cysteines in the corresponding thioredoxin domains. This oxidation led to a shift of GRXS17 to a high-molecular-weight complex and thus activated its holdase activity in vitro. Moreover, GRXS17 was specifically involved in plant tolerance to moderate high temperature and protected root meristematic cells from heat-induced cell death. Finally, GRXS17 interacted with a different set of proteins upon heat stress, possibly protecting them from heat injuries. Therefore, we propose that the Fe-S cluster enzyme GRXS17 is an essential guard that protects proteins against moderate heat stress, likely through a redox-dependent chaperone activity. We reveal the mechanism of an Fe-S cluster-dependent activity shift that converts the holoenzyme GRXS17 into a holdase, thereby preventing damage caused by heat stress.

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems.

A highly negative glutathione redox potential (EGSH ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing. Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.

Cytosolic Isocitrate Dehydrogenase from Arabidopsis thaliana Is Regulated by Glutathionylation.

NADP-dependent (Nicotinamide Adénine Dinucléotide Phosphate-dependent) isocitrate dehydrogenases (NADP-ICDH) are metabolic enzymes involved in 2-oxoglutarate biosynthesis, but they also supply cells with NADPH. Different NADP-ICDH genes are found in Arabidopsis among which a single gene encodes for a cytosolic ICDH (cICDH) isoform. Here, we show that cICDH is susceptible to oxidation and that several cysteine (Cys) residues are prone to S-nitrosylation upon nitrosoglutathione (GSNO) treatment. Moreover, we identified a single S-glutathionylated cysteine Cys363 by mass-spectrometry analyses. Modeling analyses suggest that Cys363 is not located in the close proximity of the cICDH active site. In addition, mutation of Cys363 consistently does not modify the activity of cICDH. However, it does affect the sensitivity of the enzyme to GSNO, indicating that S-glutathionylation of Cys363 is involved in the inhibition of cICDH activity upon GSNO treatments. We also show that glutaredoxin are able to rescue the GSNO-dependent inhibition of cICDH activity, suggesting that they act as a deglutathionylation system in vitro. The glutaredoxin system, conversely to the thioredoxin system, is able to remove S-nitrosothiol adducts from cICDH. Finally, NADP-ICDH activities were decreased both in a catalase2 mutant and in mutants affected in thiol reduction systems, suggesting a role of the thiol reduction systems to protect NADP-ICDH activities in planta. In line with our observations in Arabidopsis, we found that the human recombinant NADP-ICDH activity is also sensitive to oxidation in vitro, suggesting that this redox mechanism might be shared by other ICDH isoforms.

Comparison Between Fluorescent Probe and Ion-Selective Electrode Methods for Intracellular pH Determination in Leuconostoc mesenteroides.

The intracellular pH (pHin) of Leuconostoc mesenteroides subsp. mesenteroides 19D was evaluated by two different methods, fluorescent probe and ion-selective electrode. Two fluorescent probes 5 (and-6)-carboxyfluorescein diacetate succinimidyl ester (cFDASE) and 5 (and-6)-carboxy-2',7'-dichlorofluorescein diacetate succinimidyl ester (cDCFDASE) were tested to evaluate the intracellular pH (pHin) of living cells at a medium pH (pHex) ranged from 5.0 to 6.5. Salicylic acid was used as a probe for the ion-selective electrode method. Cells kept 60-80% of cFDASE probe at all pHex values against 5-10% of cDCFDASE probe at pHex ≤ 6.0. The pHin values measured by the ion-selective electrode were higher by 0.1-0.6 pH units at pHex ranged from 5.0 to 6.5 than those determinated by fluorescent probe method. The possibility to study the intracellular pH at a wide external pH range using a single probe, and the simplicity of the material and experimental protocol may make the ion-selective electrode method most useful and easy to measure the intracellular pH of lactic acid bacteria compared with the other techniques like fluorescent probes.

Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria.

Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: "What regulates flux through this pathway in vivo?" Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis: the NADP-TRX reductase a and b double mutant (ntra ntrb) and the mitochondrially located thioredoxin o1 (trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when (13)C-glucose, (13)C-malate, or (13)C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.

Arabidopsis glutaredoxin S17 and its partner, the nuclear factor Y subunit C11/negative cofactor 2α, contribute to maintenance of the shoot apical meristem under long-day photoperiod.

Glutaredoxins (GRXs) catalyze the reduction of protein disulfide bonds using glutathione as a reductant. Certain GRXs are able to transfer iron-sulfur clusters to other proteins. To investigate the function of Arabidopsis (Arabidopsis thaliana) GRXS17, we applied a strategy combining biochemical, genetic, and physiological approaches. GRXS17 was localized in the nucleus and cytosol, and its expression was elevated in the shoot meristems and reproductive tissues. Recombinant GRXS17 bound Fe2S2 clusters, a property likely contributing to its ability to complement the defects of a Baker's yeast (Saccharomyces cerevisiae) strain lacking the mitochondrial GRX5. However, a grxs17 knockout Arabidopsis mutant exhibited only a minor decrease in the activities of iron-sulfur enzymes, suggesting that its primary function is as a disulfide oxidoreductase. The grxS17 plants were sensitive to high temperatures and long-day photoperiods, resulting in elongated leaves, compromised shoot apical meristem, and delayed bolting. Both environmental conditions applied simultaneously led to a growth arrest. Using affinity chromatography and split-Yellow Fluorescent Protein methods, a nuclear transcriptional regulator, the Nuclear Factor Y Subunit C11/Negative Cofactor 2α (NF-YC11/NC2α), was identified as a GRXS17 interacting partner. A mutant deficient in NF-YC11/NC2α exhibited similar phenotypes to grxs17 in response to photoperiod. Therefore, we propose that GRXS17 interacts with NF-YC11/NC2α to relay a redox signal generated by the photoperiod to maintain meristem function.

NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells.

Thioredoxins (TRX) are key components of cellular redox balance, regulating many target proteins through thiol/disulfide exchange reactions. In higher plants, TRX constitute a complex multigenic family whose members have been found in almost all cellular compartments. Although chloroplastic and cytosolic TRX systems have been largely studied, the presence of a nuclear TRX system has been elusive for a long time. Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms. In contrast to mammals, which harbor a unique NRX, angiosperms generally possess multiple NRX organized in three subfamilies. Here, we show that Arabidopsis thaliana has two NRX genes (AtNRX1 and AtNRX2), respectively, belonging to subgroups I and III. While NRX1 harbors typical TRX active sites (WCG/PPC), NRX2 has atypical active sites (WCRPC and WCPPF). Nevertheless, both NRX1 and NRX2 have disulfide reduction capacities, although NRX1 alone can be reduced by the thioredoxin reductase NTRA. We also show that both NRX1 and NRX2 have a dual nuclear/cytosolic localization. Interestingly, we found that NTRA, previously identified as a cytosolic protein, is also partially localized in the nucleus, suggesting that a complete TRX system is functional in the nucleus. We show that NRX1 is mainly found as a dimer in vivo. nrx1 and nrx2 knockout mutant plants exhibit no phenotypic perturbations under standard growth conditions. However, the nrx1 mutant shows a reduced pollen fertility phenotype, suggesting a specific role of NRX1 at the haploid phase.

Thioredoxin and glutaredoxin systems in plants: molecular mechanisms, crosstalks, and functional significance.

Thioredoxins (Trx) and glutaredoxins (Grx) constitute families of thiol oxidoreductases. Our knowledge of Trx and Grx in plants has dramatically increased during the last decade. The release of the Arabidopsis genome sequence revealed an unexpectedly high number of Trx and Grx genes. The availability of several genomes of vascular and nonvascular plants allowed the establishment of a clear classification of the genes and the chronology of their appearance during plant evolution. Proteomic approaches have been developed that identified the putative Trx and Grx target proteins which are implicated in all aspects of plant growth, including basal metabolism, iron/sulfur cluster formation, development, adaptation to the environment, and stress responses. Analyses of the biochemical characteristics of specific Trx and Grx point to a strong specificity toward some target enzymes, particularly within plastidial Trx and Grx. In apparent contradiction with this specificity, genetic approaches show an absence of phenotype for most available Trx and Grx mutants, suggesting that redundancies also exist between Trx and Grx members. Despite this, the isolation of mutants inactivated in multiple genes and several genetic screens allowed the demonstration of the involvement of Trx and Grx in pathogen response, phytohormone pathways, and at several control points of plant development. Cytosolic Trxs are reduced by NADPH-thioredoxin reductase (NTR), while the reduction of Grx depends on reduced glutathione (GSH). Interestingly, recent development integrating biochemical analysis, proteomic data, and genetics have revealed an extensive crosstalk between the cytosolic NTR/Trx and GSH/Grx systems. This crosstalk, which occurs at multiple levels, reveals the high plasticity of the redox systems in plants.

A dicotyledon-specific glutaredoxin GRXC1 family with dimer-dependent redox regulation is functionally redundant with GRXC2.

The major known function of glutaredoxins (Grxs) is to reduce disulphide bridges. Recently, some have also been shown to interact with iron-sulphur clusters. These can be classified in two subgroups: class II Grx are found in all living organisms and are implicated in assembly of iron-sulphur clusters, while class I Grx are represented by only two members known to form a holodimer structure containing a cluster in vitro, but with an unknown function different from class II. Here, we report that in eukaryotic plants, GRXC1 (class I) orthologs are exclusively present in dicotyledonous plants, suggesting a specific function. Indeed, in Arabidopsis thaliana, reducing activity of recombinant GRXC1 is regulated by redox-dependent stability of the cluster. In planta, GRXC1 has been found predominantly in a holodimeric form, indicating the presence of the cluster in vivo. This suggests that GRXC1 acts as a redox sensor, reducing downstream pathways under oxidative conditions. GRXC2, the closest homolog of GRXC1, is unable to form a cluster in vitro. Knock-out mutants in grxc1 or grxc2 are aphenotypic, but the double mutant produces a lethal phenotype at an early stage after pollinization, suggesting that GRXC1 and GRXC2 share redundant and vital functions.

Glutaredoxins and thioredoxins in plants.

During the 70s and 80s two plant thioredoxin systems were identified. The chloroplastic system is composed of a ferredoxin-dependent thioredoxin, with two thioredoxin types (m and f) regulating the activity of enzymes implicated in photosynthetic carbon assimilation. In the cytosol of heterotrophic tissues, an NADP dependent thioredoxin reductase and a thioredoxin (h) were identified. The first plant glutaredoxin was only identified later, in 1994. Our view of plant thioredoxins and glutaredoxins was profoundly modified by the sequencing programs which revealed an unexpected number of genes encoding not only the previously identified disulfide reductases, but also numerous new types. At the same time it became clear that plant genomes encode chloroplastic, cytosolic and mitochondrial peroxiredoxins, suggesting a major role for redoxins in anti-oxidant defense. Efficient proteomics approaches were developed allowing the characterization of numerous thioredoxin target proteins. They are implicated in different aspects of plant life including development and adaptation to environmental changes and stresses. The most important challenge for the next years will probably be to identify in planta which redoxin reduces which target, a question which remains unsolved due to the low specificities of redoxins in vitro and the numerous redundancies which in most cases mask the phenotype of redoxin mutants.

Inactivation of thioredoxin reductases reveals a complex interplay between thioredoxin and glutathione pathways in Arabidopsis development.

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

Regulation of reactive oxygen species production by a 14-3-3 protein in elicited tobacco cells.

The regulation of the system responsible for the production of reactive oxygen species (ROS) during plant-micro-organism interaction is still largely unknown. The protein NtrbohD has been recently demonstrated as the plasma membrane oxidase responsible for ROS production in elicited tobacco cells. Here, its C-terminus part was used as a bait in a two-hybrid screen in order to identify putative regulators of this system. This led to the isolation of a cDNA coding for a member of the 14-3-3 protein family. The corresponding transcript was induced after infiltration of tobacco leaves with the fungal elicitor cryptogein. Tobacco cells transformed with an antisense construct of this 14-3-3 no longer accumulated ROS, which constitutes a functional validation of the two-hybrid screen. This work provides new insights to the understanding of the regulation of ROS production in a signalling context and gives a new light to the possible role of 14-3-3 proteins in plant-micro-organisms interactions.

Evolution of redoxin genes in the green lineage.

The availability of the Arabidopsis genome revealed the complexity of the gene families implicated in dithiol disulfide exchanges. Most non-green organisms present less dithiol oxidoreductase genes. The availability of the almost complete genome sequence of rice now allows a systematic search for thioredoxins, glutaredoxins and their reducers. This shows that all redoxin families previously defined for Arabidopsis have members in the rice genome and that all the deduced rice redoxins fall within these families. This establishes that the redoxin classification applies both to dicots and monocots. Nevertheless, within each redoxin type the number of members is not the same in these two higher plants and it is not always possible to define orthologues between rice and Arabidopsis. The sequencing of two unicellular algae (Chlamydomonas and Ostreococcus) genomes are almost finished. This allowed us to follow the origin of the different gene families in the green lineage. It appears that most thioredoxin and glutaredoxin types, their chloroplastic, mitochondrial and cytosolic reducers are always present in these unicellular organisms. Nevertheless, striking differences appear in comparison to higher plant redoxins. Some thioredoxin types are not present in these algal genomes including thioredoxins o, clot and glutaredoxins CCxC. Numerous redoxins, including the cytosolic thioredoxins, do not fit with the corresponding higher plant classification. In addition both algae present a NADPH-dependent thioredoxin reductase with a selenocysteine which is highly similar to the animal thioredoxin reductases, a type of thioredoxin reductase not present in higher plants.

Determination of total ribonucleotide pool in plant materials by high-pH anion-exchange high-performance liquid chromatography following extraction with potassium hydroxide.

A new, improved method that only requires a potassium hydroxide extraction procedure is presented for the analysis of a full nucleotide pool in plant materials. Quantification was performed by high-pH anion-exchange chromatography (HPAEC) with UV detection after a potassium hydroxide extraction, and allowed the quantification of 13 linear ribonucleotides in a single run. The method has been validated by comparison of six extraction methods and also by measurement of the intracellular nucleotide levels of three plant species (cell cultures and leaves). The evolution of the nucleotide pool of Nicotiana tabacum cell culture during growth has also been measured, and showed an increase in the pool until the fifth day, where the growth rate reaches a maximum, after which a decrease was observed.

Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis.

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.

Effect of reducing agents on the acidification capacity and the proton motive force of Lactococcus lactis ssp. cremoris resting cells.

Reducing agents are potential inhibitors of the microbial growth. We have shown recently that dithiothreitol (DTT), NaBH(4) and H(2) can modify the proton motive force of resting cells of Escherichia coli by increasing the membrane protons permeability [Eur. J. Biochem. 262 (1999) 595]. In the present work, the effect of reducing agents on the resting cells of Lactococcus lactis ssp. cremoris, a species widely employed in dairy processes was investigated. DTT did not affect the acidification nor the DeltapH, in contrast to the effect previously reported on E. coli. The DeltaPsi was slightly increased (30 mV) at low pH (pH 4) in the presence of 31 mM DTT or 2.6 mM NaBH(4). In the case of Na(2)S(2)O(4), small amounts (0.9 mM) drastically decreased the acidification range and this product was shown to abolish the DeltapH. These results are discussed in terms of the diversity of action of the chemical reagents and strain sensitivity.