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The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship between plant and bacteria. This is characterized by the formation of a new organ, the nodule, which facilitates the fixation of atmospheric nitrogen (N2) by nitrogenase through the creation of a hypoxic environment. Nitric oxide (NO) accumulates at each stage of the symbiotic process. NO is involved in defense responses, nodule organogenesis and development, nitrogen fixation metabolism, and senescence induction. During symbiosis, either successively or simultaneously, NO regulates gene expression, modulates enzyme activities, and acts as a metabolic intermediate in energy regeneration processes via phytoglobin-NO respiration and the bacterial denitrification pathway. Due to the transition from normoxia to hypoxia during nodule formation, and the progressive presence of the bacterial partner in the growing nodules, NO production and degradation pathways change during the symbiotic process. This review analyzes the different source and degradation pathways of NO, and highlights the role of nitrate reductases and hemoproteins of both the plant and bacterial partners in the control of NO accumulation.

Summary In legumes, phytoglobins (Phytogbs) are known to regulate nitric oxide (NO) during early phase of the nitrogen‐fixing symbiosis and to buffer oxygen in functioning nodules. However, their expression profile and respective role in NO control at each stage of the symbiosis remain little‐known.We first surveyed the Phytogb genes occurring in Medicago truncatula genome. We analyzed their expression pattern and NO production from inoculation with Sinorhizobium meliloti up to 8 wk post‐inoculation. Finally, using overexpression and silencing strategy, we addressed the role of the Phytogb1.1‐NO couple in the symbiosis.Three peaks of Phytogb expression and NO production were detected during the symbiotic process. NO upregulates Phytogbs1 expression and downregulates Lbs and Phytogbs3 ones. Phytogb1.1 silencing and overexpression experiments reveal that Phytogb1.1‐NO couple controls the progression of the symbiosis: high NO concentration promotes defense responses and nodular organogenesis, whereas low NO promotes the infection process and nodular development. Both NO excess and deficiency provoke a 30% inhibition of nodule establishment. In mature nodules, Phytogb1.1 regulates NO to limit its toxic effects while allowing the functioning of Phytogb‐NO respiration to maintain the energetic state.This work highlights the regulatory role played by Phytogb1.1–NO couple in the successive stages of symbiosis., See also the Commentary on this article by Singh et al., 227: 5–7.

Plant glutathione peroxidases (Gpx) catalyse the reduction of various peroxides, such as hydrogen peroxide (H2O2), phospholipid hydroperoxides and peroxynitrite, but at the expense of thioredoxins rather than glutathione. A main function of plant Gpxs is the protection of biological membranes by scavenging phospholipid hydroperoxides, but some Gpxs have also been associated with H2O2 sensing and redox signal transduction. Nitric oxide (NO) is not only known to induce the expression of Gpx family members, but also to inhibit Gpx activity, presumably through the S-nitrosylation of conserved cysteine residues. In the present study, the effects of NO-donors on both the activity and S-nitrosylation state of purified Medicago truncatula Gpxl1 were analyzed using biochemical assay measurements and a biotin-switch/mass spectrometry approach. MtGpx1 activity was only moderately inhibited by the NO donors diethylamine-NONOate and S-nitrosoglutathione, and the inhibition may be reversed by DTT. The three conserved Cys of MtGpx1 were found to be modified through S-nitrosylation and S-glutathionylation, although to different extents, by diethylamine-NONOate and S-nitrosoglutathione, or by a combination of diethylamine-NONOate and reduced glutathione. The regulation of MtGpx1 and its possible involvement in the signaling process is discussed in the light of these results.

International audience; Leguminous biological nitrogen fixation (BNF) is very sensitive to environmental fluctuations. It is still contentious how BNF is regulated under stress conditions. The local or systemic control of BNF and the role played by reactive oxygen species (ROS) in such regulation have still not been elucidated completely. Cadmium, which belongs to the so-called heavy metals, is one of the most toxic substances released into the environment. The mechanisms involved in Cd toxicity are still not completely understood but the overproduction of ROS is one of its characteristic symptoms. In this work, we used a split-root system approach to study nodule BNF and the antioxidant machinerys response to the application of a mild Cd treatment on one side of a nodulated Medicago truncatula root system. Cd induced the majority of nodule antioxidants without generating any oxidative damage. Cd treatment also provoked BNF inhibition exclusively in nodules directly exposed to Cd, without provoking any effect on plant shoot biomass or chlorophyll content. The overall data suggest that the decline in BNF was not due to a generalized breakdown of the plant but to control exerted through leghaemoglobin/oxygen availability, affecting nitrogenase function.

International audience; Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

International audience; Our study demonstrated that the NAPDH oxidase gene MtRbohE is expressed in arbusculated cells and plays a role in arbuscule development. Plant NADPH oxidases, known as respiratory burst oxidase homologs (RBOH), belong to a multigenic family that plays an important role in the regulation of plant development and responses to biotic and abiotic stresses. In this study, we monitored the expression profiles of five Rboh genes (MtRbohA, MtRbohB, MtRbohE, MtRbohG, MtRbohF) in the roots of the model species Medicago truncatula upon colonization by arbuscular mycorrhizal fungi. A complementary cellular and molecular approach was used to monitor changes in mRNA abundance and localize transcripts in different cell types from mycorrhizal roots. Rboh transcript levels did not drastically change in total RNA extractions from whole mycorrhizal and non-mycorrhizal roots. Nevertheless, the analysis of laser microdissected cells and Agrobacterium rhizogenes-transformed roots expressing a GUS transcriptional fusion construct highlighted the MtRbohE expression in arbuscule-containing cells. Furthermore, the down regulation of MtRbohE by an RNAi approach generated an altered colonization pattern in the root cortex, when compared to control roots, with fewer arbuscules and multiple penetration attempts. Altogether our data indicate a transient up-regulation of MtRbohE expression in cortical cells colonized by arbuscules and suggest a role for MtRbohE in arbuscule accommodation within cortical cells.

International audience; Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules.

International audience; The symbiotic interaction between legumes and nitrogen fixing rhizobium bacteria leads to the formation of a new organ, the nodule. Early steps of the interaction are characterized by the production of bacterial Nod factors, the reorientation of root-hair tip growth, the formation of an infection thread (IT) in the root hair, and the induction of cell division in inner cortical cells of the root, leading to a nodule primordium formation. Reactive oxygen species (ROS) and nitric oxide (NO) have been detected in early steps of the interaction. ROS/NO are determinant signals to arbitrate the specificity of this mutualistic association and modifications in their content impair the development of the symbiotic association. The decrease of ROS level prevents root hair curling and ITs formation, and that of NO conducts to delayed nodule formation. In root hairs, NADPH oxidases were shown to produce ROS which could be involved in the hair tip growth process. The use of enzyme inhibitors suggests that nitrate reductase and NO synthase-like enzymes are the main route for NO production during the early steps of the interaction. Transcriptomic analyses point to the involvement of ROS and NO in the success of the infection process, the induction of early nodulin gene expression, and the repression of plant defense, thereby favoring the establishment of the symbiosis. The occurrence of an interplay between ROS and NO was further supported by the finding of both S-sulfenylated and S-nitrosylated proteins during early symbiotic interaction, linking ROS/NO production to a redox-based regulation of the symbiotic process.

Legumes interact symbiotically with bacteria of the Rhizobiaceae to form nitrogen-fixing root nodules. We investigated the contribution of the three glutaredoxin (Grx)-encoding genes present in the Sinorhizobium meliloti genome to this symbiosis. SmGRX1 (CGYC active site) and SmGRX3 (CPYG) recombinant proteins displayed deglutathionylation activity in the 2-hydroethyldisulfide assay, whereas SmGRX2 (CGFS) did not. Mutation of SmGRX3 did not affect S. meliloti growth or symbiotic capacities. In contrast, SmGRX1 and SmGRX2 mutations decreased the growth of free-living bacteria and the nitrogen fixation capacity of bacteroids. Mutation of SmGRX1 led to nodule abortion and an absence of bacteroid differentiation, whereas SmGRX2 mutation decreased nodule development without modifying bacteroid development. The higher sensitivity of the Smgrx1 mutant strain as compared with wild-type strain to oxidative stress was associated with larger amounts of glutathionylated proteins. The Smgrx2 mutant strain displayed significantly lower levels of activity than the wild type for two iron-sulfur-containing enzymes, aconitase and succinate dehydrogenase. This lower level of activity could be associated with deregulation of the transcriptional activity of the RirA iron regulator and higher intracellular iron content. Thus, two S. meliloti Grx proteins are essential for symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and the regulation of iron metabolism.

• The plant plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), appear to play crucial roles in plant growth and development. They are involved in important processes, such as root hair growth, plant defence reactions and abscisic acid signalling. • Using sequence similarity searches, we identified seven putative RBOH-encoding genes in the Medicago truncatula genome. A phylogenetic reconstruction showed that Rboh gene duplications occurred in legume species. We analysed the expression of these MtRboh genes in different M. truncatula tissues: one of them, MtRbohA, was significantly up-regulated in Sinorhizobium meliloti-induced symbiotic nodules. • MtRbohA expression appeared to be restricted to the nitrogen-fixing zone of the functional nodule. Moreover, using S. meliloti bacA and nifH mutants unable to form efficient nodules, a strong link between nodule nitrogen fixation and MtRbohA up-regulation was shown. MtRbohA expression was largely enhanced under hypoxic conditions. Specific RNA interference for MtRbohA provoked a decrease in the nodule nitrogen fixation activity and the modulation of genes encoding the microsymbiont nitrogenase. • These results suggest that hypoxia, prevailing in the nodule-fixing zone, may drive the stimulation of MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning

International audience; AtNoa1/Rif1 (formerly referred to as AtNos1) has been shown to modulate nitric oxide (NO) content in Arabidopsis. As NO generation in the legume-rhizobium symbiosis has been shown, the involvement of an AtNoa1/Rif1 orthologue from Medicago truncatula (MtNoa1/Rif1) during its symbiotic interaction with Sinorhizobium meliloti has been studied. The expression of MtNoa1/Rif1 appeared to occur mainly in nodule vascular bundles and the meristematic zone. Using an RNA interference strategy, transgenic roots exhibiting a significantly decreased level of MtNoa1/Rif1 expression were analysed. NO production was assessed using a fluorescent probe, and the symbiotic capacities of the composite plants upon infection with Sinorhizobium meliloti were determined. The decrease in MtNoa1/Rif1 expression level resulted in a decrease in NO production in roots, but not in symbiotic nodules, indicating a different regulation of NO synthesis in these organs. However, it significantly lowered the nodule number and the nitrogen fixation capacity of the functional nodules. Although having no influence on NO production in nodules, MtNOA1/RIF1 significantly affected the establishment and the functioning of the symbiotic interaction. The impairment of plastid functioning may explain this phenotype.

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodule's enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially draughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal. © 2007 American Society of Plant Biologists.

International audience; The specific interaction between legumes and Rhizobium-type bacteria leads to the establishment of a symbiotic relationship characterized by the formation of new differentiated organs named nodules, which provide a niche for bacterial nitrogen (N2) fixation. In the nodules, bacteria differentiate into bacteroids with the ability to fix atmospheric N2 via nitrogenase activity. As nitrogenase is strongly inhibited by oxygen, N2 fixation is made possible by the microaerophilic conditions prevailing in the nodules. Increasing evidence has shown the presence of NO during symbiosis, from early interaction steps between the plant and the bacterial partners to N2-fixing and senescence steps in mature nodules. Both the plant and the bacterial partners participate in NO synthesis. NO was found to be required for the optimal establishment of the symbiotic interaction. Transcriptomic analysis at an early stage of the symbiosis showed that NO is potentially involved in the repression of plant defence reactions, favouring the establishment of the plant-microbe interaction. In mature nodules, NO was shown to inhibit N2 fixation, but it was also demonstrated to have a regulatory role in nitrogen metabolism, to play a beneficial metabolic function for the maintenance of the energy status under hypoxic conditions, and to trigger nodule senescence. The present review provides an overview of NO sources and multifaceted effects from the early steps of the interaction to the senescence of the nodule, and presents several approaches which appear to be particularly promising in deciphering the roles of NO in N2-fixing symbioses.

JBIC Journal of Biological Inorganic Chemistry, 10 (8), ISSN:0949-8257, ISSN:1432-1327

KatB is the only catalase-peroxidase identified so far in Sinorhizobium meliloti. It plays a housekeeping role, as it is expressed throughout all the growth phases of the free-living bacterium and also during symbiosis. This paper describes the functional and structural characterization of the KatB mutants Gly303Ser, Trp95Ala, Trp95Phe, Tyr217Leu, Tyr217Phe and Met243Val carried out by optical and electron spin resonance spectroscopy. The aim of this work was to investigate the involvement of these residues in the catalatic and/or peroxidatic reaction and falls in the frame of the open dispute around the factors that influence the balance between catalatic and peroxidatic activity in heme enzymes. The Gly303 residue is not conserved in any other protein of this family, whereas the Trp95, Tyr217 and Met243 residues are thought to form an intrinsic cofactor that is likely to play a role in intramolecular electron transfer. Spectroscopic investigations show that the Gly303Ser mutant is almost similar to the wild-type KatB and should not be involved in substrate binding. Mutations on Trp95, Tyr217 and Met243 clear out the catalatic activity completely, whereas the peroxidatic activity is maintained or even increased with respect to that of the wild-type enzyme. The k (cat) values obtained for these mutants suggest that Trp95 and Tyr217 form a huge delocalized system that provides a pathway for electron transfer to the heme. Conversely, Met243 is likely to be placed close to the binding site of the organic molecules and plays a crucial role in substrate docking.

A kinetic study of the reaction of two soybean leghemoglobins (components a and c) with hydrogen peroxide to form the oxidized compound (leghemoglobin IV) has been carried out over the pH range 2.5--10. Three different ionization processes of leghemoglobins with pKa values of 3,4.7 +/- 0.2 and 8.2 +/- 0.1 are required to explain the rate/pH profiles. Protonation of the former group and ionization of the latter cause a decrease in the rate of reaction of the hemoproteins with H2O2. The results are compared to those obtained for the reactions of plant peroxidases and myoglobin with H2O2. The results obtained from the kinetic study of cyanide binding to soybean leghemoglobins indicate that CN- is the reactive species. Two ionization processes of leghemoglobins with pKa values of 4.7 +/- 0.2 and 8.2 +/- 0.1 affect the reaction rates. The association and dissociation rate constants corresponding to nicotinic acid binding to leghemoglobins a and c have been measured over the pH range 2.5--7. The dissociation rate constant is affected by ionization of a group with pKa less than 2.5 for both leghemoglobin-nicotinate complexes. In this pH range the association rate constant is only affected by ionization of a group with pKa value of 4.7 +/- 0.2. The analysis of these results shows that both ionization processes corresponding to ring nitrogen atom of the ligand (pKa approximately equal to 4.9) and to a heme-linked group (pKa approximately equal to 4.7 +/- 0.2) influence the association rate constant. Furthermore, it appears that in the binding site of leghemoglobins the pKa value corresponding to ionization of the ring nitrogen atom of nicotinic acid is shifted from the normal value of 4.9 to a value of less than 2.5. This pecularity might explain the exceptional reactivity of leghemoglobins for nicotinic acid, over a large pH range. For both cyanide and nicotinic acid binding reactions, the ionizable group of leghemoglobins with pKa value of 4.7 +/- 0.2 seems to act as an electrostatic gate. When the group is deprotonated, it restricts the access of anion ligands to the heme pocket. For all the three reactions studied, leghemoglobin a reacts about twice as fast as leghemoglobin c.

Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti. The recombinant proteins were overexpressed in Escherichia coli and purified in order toachieve a spectral and kinetic characterization. The three proteins contain heme b with high-spin Fe(III). KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k c a t = 2400 s - 1 ) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K M = 1.6 mM). KatA and KatC are acidic monofunctional homotetrameric catalases. Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H 2 O 2 (apparent K M values of 160 and 150 mM). However, the turnover rate of KatA (k c a t = 279000 s - 1 ) exceeds that of KatC (k c a t = 3100 s - 1 ) significantly. The kinetic parameters are in good agreement with the physiological role of these heme proteins. KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H 2 O 2 affinity but the highest k c a t /K M value (1.75 x 10 6 M - 1 s - 1 ), in agreement with the hydrogen peroxide inducibility of the encoding gene. Moreover, the lower catalytic efficiency of KatC (2.1 × 10 4 M - 1 s - 1 ) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).

Haem oxygenases (HO) are ubiquitous enzymes catalysing the oxidative degradation of haem into biliverdin, iron and carbon monoxide. Whereas animal HOs participate in multiple cellular functions including haemoglobin catabolism, antioxidant defence and iron homeostasis, to date, plant HOs have so far only been involved in phytochrome metabolism. The expression of the HO1 gene was studied in Medicago sativa, especially during the interaction with its symbiotic partner, Sinorhizobium meliloti. Transcript accumulation was higher in mature root nodules than in roots and leaves and was correlated to HO1 protein immunodetection. The analysis of HO1 expression following alfalfa root inoculation with S. meliloti indicates that transcripts do not accumulate during the early steps of symbiosis, but rather in the mature nodules. These results correlate with the expression of the leghaemoglobin gene, which encodes the major haem-containing protein present in the nodule. Contrary to its animal counterpart, alfalfa HO1 was not induced by pro- oxidant compounds including H(2)O(2), paraquat and sodium nitroprusside, suggesting that it is not involved in the antioxidant defence. The results suggest that HO1 could play a role in the alfalfa mature nodule and its involvement in leghaemoglobin metabolism is hypothesized.

Summary • The expression of a cysteine protease (CP) gene during soybean (Glycine max) root nodule development has been studied. In 5-wk-old nodules, when N2 fixation began to decline, the CP gene appeared to be expressed in the periphery of the central infected tissue which, in determinate nodules, is considered as a developmental zone. • Concomitantly, events related to programmed cell death (PCD) and accumulation of significant amounts of H2O2 in the apoplasm were observed in this zone. In 7.5-wk-old nodules, the zone that exhibited CP expression, PCD events and H2O2 accumulation enlarged towards the centre of the organ. • In 10-wk-old nodules, which are largely senescent, a widespread expression of the CP gene was observed in all of the central infected tissue; this was associated with the presence of large amounts of hydrogen peroxide (H2O2) in the cytoplasmic and apoplastic compartments, and the existence of a necrotic zone. • It is proposed that the developmental zone of the nodule is undergoing a PCD process, involving both a CP and H2O2. This can be viewed as the onset of the nodule senescence process and could be under the control of a plant signal.

Reactive oxygen species are generated in the first steps of the Rhizobium–legume symbiosis. Superoxide radicals and hydrogen peroxide have been detected in infection threads and there is also evidence of the presence of nitric oxide in young alfalfa nodules. Moreover, rhizobial mutants, with a reduced antioxidant defense, exhibit an impaired capacity to nodulate. The oxidative burst generated in response to symbiotic infection can be consistent with rhizobia being initially perceived as invaders by the plant; in this framework, it may be correlated with the existence of abortive infections. However, the burst appears to be also involved in the expression of early nodulins associated with successful infections. Thus, in parallel to its involvement in defense processes, a positive role for the oxidative burst (including nitric oxide) in the establishment of the symbiotic interaction can also be proposed. The burst could trigger the expression of plant and/or bacterial genes which are essential for the nodulation process. In this framework, glutathione and homoglutathione could be key intermediates for gene expression, via the modification of the redox balance. Thus, the oxidative burst may have a dual role in the establishment of the symbiosis. © 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.

Several markers of oxidative stress were measured in 2- to 10-week-old soybean (Glycine max [L.] Merr.) nodules. There were increases in peroxides, protein carbonyls and modified DNA base concentrations with nodule age. The catalytic iron content also increased significantly during nodule ageing. Iron contained in the peribacteroid space was effective in promoting lipid peroxidation and this might contribute to the degradation of the peribacteroid membrane in senescing nodules. The concentration of the oxidized forms of glutathione and homoglutathione increased significantly during nodule development and the concentration of reduced glutathione and homoglutathione decreased during senescence. Taken together, these results are consistent with the development of oxidative stress in senescing nodules. Significant DNA and protein damage also occurred in the first days of nodule development, suggesting that an earlier period of oxidative stress might occur in the period over which the symbiosis becomes established.

A gamma-ECS cDNA from Medicago truncatula was isolated using an Arabidopsis thaliana cDNA as probe. The analysis of the amino acid sequence deduced from this cDNA revealed 80% identity with the gamma-ECS from A. thaliana and Brassica juncea and suggested a plastidial localisation for the enzyme. Gamma-ECS activity and high level of GSH were detected in the gamma-ECS-deficient E. coli strain expressing a fusion protein containing the M. truncatula gamma-ECS protein. Southern blot analysis suggests that gamma-ECS is encoded by a small multigenic family in M. truncatula and shows that homologous genes are present in two other leguminous plants, Medicago sativa and Pisum sativum. Gamma-ECS gene expression was analysed by Northern blot in seedlings, plantlets and mature plants.

Summary Glutathione (GSH) and homoglutathione (hGSH) were quantified inMedicago truncatuladuring plant development. hGSH was detectable only 48 h after seed germination whereas GSH was present in the dry seeds, indicating that only GSH is used for sulphur storage in seeds. The hGSH was detectable only in the underground part of mature plants whereas GSH was present in all the organs. γ-EC synthetase (γ-ECS) and GSH synthetase (GSHS) activities were found in roots and leaves whereas hGSH synthetase (hGSHS) was found only in roots. Full-length cDNA encoding γ-ECS and two partial cDNAs (gshs1andgshs2) showing high identity with GSHS were isolated inM. truncatula. High γ-ECS activity was detected in protein extracts of a γ-ECS-deficientE. colistrain expressing theM. truncatulaγ-ECS. Northern blot analysis showed that the γ-ECS gene was similarly expressed in all the mature plant organs tested, whereasgshs1had a higher expression in leaves and flowers andgshs2was preferentially expressed in roots and nodules. We hypothesise thatgshs1andgshs2encode a GSHS and an hGSHS, respectively.

Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules of the soybean plants, in a manner analogous to that recently described for Lupinus albus. This Lb-NO complex is present at lower concentrations in older nodules, and is almost completely absent from senescent nodules. Exposure of young and mature nodules to oxidant stress, in the form of hydrogen peroxide, results in changes in the EPR spectra, with the loss of the signals from the Lb-NO complex and appearance of absorptions similar to those from untreated senescent nodules. These results suggest that there are characteristic changes in both the metal ion complexes and radicals present in intact root nodules of different ages, and support the theory that nitric oxide and other radicals play a significant role in determining the nitrogen fixing activity of root nodules; the modulatory activity of NO. may involve regulation of gene activity.

International audience; Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2)O(2)), play an important role in signalling in various cellular processes. The involvement of H(2)O(2) in the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction raises questions about its effect on gene expression. A transcriptome analysis was performed on inoculated roots of M. truncatula in which ROS production was inhibited with diphenylene iodonium (DPI). In total, 301 genes potentially regulated by ROS content were identified 2 d after inoculation. These genes included MtSpk1, which encodes a putative protein kinase and is induced by exogenous H(2)O(2) treatment. MtSpk1 gene expression was also induced by nodulation factor treatment. MtSpk1 transcription was observed in infected root hair cells, nodule primordia and the infection zone of mature nodules. Analysis with a fluorescent protein probe specific for H(2)O(2) showed that MtSpk1 expression and H(2)O(2) were similarly distributed in the nodule infection zone. Finally, the establishment of symbiosis was impaired by MtSpk1 downregulation with an artificial micro-RNA. Several genes regulated by H(2)O(2) during the establishment of rhizobial symbiosis were identified. The involvement of MtSpk1 in the establishment of the symbiosis is proposed.

[Abstract] Medicago truncatula serves as a model plant for legume genetics and genomics. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We generated approximately 135.5 million high-quality 36-bp reads, which were then aligned with the M. truncatula genome sequence (Mt3.0 version) and with sequences from a custom splice-junction database, for the detection of transcribed regions and splicing sites. Mapping and analysis of the reads conducted to the detection of 37,333 expressed transcription units (TUs), 1,670 had never been described before and were functionally annotated. We identified 7,595 new transcribed regions, mostly corresponding to 5’ and 3’ UTR extensions and new exons associated with 5,264 previously annotated genes. We also assessed the complexity in the nodulation transcriptome by performing a Cufflinks analysis to determine the frequency of the various alternatively spliced forms. Thus, we identified 23,164 different transcripts derived from 6,587 genes. Finally, we carried out a differential expression analysis, which provided a comprehensive view of transcriptional reprogramming during nodulation. All Illumina sequence data have been deposited in the NCBI short-read archive, and Sangersequenced PCR products have been deposited in GenBank (SRA048731). Assembled contigs longer than 200 bp have been deposited at TSA (JR366937-JR375780). Coverage data are available at http://ddlab.sci.univr.it/cgi-bin/gbrowse/medicago/.

International audience; The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible with nitrogenase activity. The present review analyses the different roles of NO in functioning nodules, and discusses the role of plant and bacterial (flavo)hemoglobins in the control of NO level in nodules.

Reaction of H2O2 with ferric leghemoglobin (metLb, the monomeric, oxygen-carrying, heme protein from root nodules of nitrogen-fixing plants) has been previously shown to generate an iron(IV)-oxo (ferryl) species and at least one protein radical. The latter has been suggested to be a tyrosine-derived phenoxyl radical present at Tyr-133 in the soybean protein and Tyr-138 in the lupin protein. To obtain further information on these protein radicals and their potential interaction with the physiologically important peribacteroid membrane (which surrounds the microsymbiont in vivo), EPR spin trapping studies have been carried out with soybean metLb. Evidence has been obtained for at least two additional protein-derived radicals in addition to the phenoxyl radical; these radicals are transient and reactive in nature. These species are carbon-centered, and at least one is a tertiary species (.CR1R2R3); these radicals may be side chain- or alpha-carbon-derived, their exact sites have not been determined. Some of these radicals are on the protein surface and may be key intermediates in the formation of protein dimers. These radicals have been shown to be capable of reacting with peribacteroid membrane fractions, with the consequent generation of lipid-derived radicals. The formation of such radicals may result in the depletion of membrane antioxidants and the initiation of lipid peroxidation. This transfer of damage from the heme center via the protein surface to neighboring membranes may be of considerable biological significance; the destruction of this membrane is one of the earliest observable events in root nodule senescence and is associated with the loss of nitrogen-fixing activity.

During the Legume-Rhizobium symbiosis, hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) appear to play an important signaling role in the establishment and the functioning of this interaction. Modifications of the levels of these reactive species in both partners impair either the development of the nodules (new root organs formed on the interaction) or their N(2)-fixing activity.NADPH oxidases (Noxs) have been recently described as major sources of H(2)O(2) production, via superoxide anion dismutation, during symbiosis. Nitrate reductases (NR) and electron transfer chains from both partners were found to significantly contribute to NO production in N(2)-fixing nodules. Both S-sulfenylated and S-nitrosylated proteins have been detected during early interaction and in functioning nodules, linking reactive oxygen species (ROS)/NO production to redox-based protein regulation. NO was also found to play a metabolic role in nodule energy metabolism.H(2)O(2) may control the infection process and the subsequent bacterial differentiation into the symbiotic form. NO is required for an optimal establishment of symbiosis and appears to be a key player in nodule senescence.A challenging question is to define more precisely when and where reactive species are generated and to develop adapted tools to detect their production in vivo. To investigate the role of Noxs and NRs in the production of H(2)O(2) and NO, respectively, the use of mutants under the control of organ-specific promoters will be of crucial interest. The balance between ROS and NO production appears to be a key point to understand the redox regulation of symbiosis.

Medicago truncatula is one of the most studied model plants. Nevertheless, the genome of this legume remains incompletely determined. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We detected 37,333 expressed transcription units; to our knowledge, 1,670 had never been described before and were functionally annotated. We identified 7,595 new transcribed regions, mostly corresponding to 5′ and 3′ untranslated region extensions and new exons associated with 5,264 previously annotated genes. We also inferred 23,165 putative transcript isoforms from 6,587 genes and measured the abundance of transcripts for each isoform, which suggests an important role for alternative splicing in the generation of proteome diversity in M. truncatula. Finally, we carried out a differential expression analysis, which provided a comprehensive view of transcriptional reprogramming during nodulation. In particular, depletion of nitric oxide in roots inoculated with Sinorhizobium meliloti greatly increased our understanding of the role of this reactive species in the optimal establishment of the symbiotic interaction, revealing differential patterns of expression for 2,030 genes and pointing to the inhibition of the expression of defense genes.

Reaction of the ferric form of leghaemoglobin with hydrogen peroxide has been previously shown to give rise to an iron(IV)-oxo (ferryl) species, and a protein radical. Inclusion of a variety of thiol compounds in this system is shown to lead to rapid loss of the iron(IV)-oxo species and the regeneration of the ferric form and/or the formation of novel sulf species formed by nucleophilic attack of the thiol group on the tetrapyrrole ring. The reduction process also results in the generation of thiyl radicals which have been detected by EPR spin trapping. The relative yields of the products produced by these two competing pathways is shown to be highly dependent on the steric and electronic characteristics of the thiol compound. Evidence has also been obtained, in the absence of hydrogen peroxide, for both the reduction of the ferric form of the protein to the oxy-ferrous form, via a process believed to involve the deoxy-ferrous species, and the formation of sulf-leghaemoglobin species. Both of these pathways are again highly dependent on the structure of the thiol, and the former also results in the generation of thiyl radicals. Inclusion of the sulfide anion in place of the organic thiols results in somewhat different behaviour, in that this species appears to both reduce the iron centre and form a complex with the iron atom. This ligation process is reversible, and the sulfide complex is shown to react readily with both strong oxidizing and reducing agents. The behaviour of this protein, which is structurally related to myoglobin, is dramatically different to that demonstrated by myoglobin; this is rationalized in terms of the much more open heme site of leghaemoglobins, and the presence of an electronic gate which hinders access by negatively charged molecules. The contribution of these processes to the maintenance of the leghaemoglobin proteins in the oxy-ferrous form in vivo and the binding of oxygen is discussed.

International audience; Reactive oxygen species such as hydrogen peroxide (H2O2), play a crucial role as signaling molecules in the establishment and functioning of the nitrogen-fixing legume-Rhizobium symbiosis. The regulation of protein function through oxidative modification has emerged as an important molecular mechanism modulating various biological processes. Protein cysteine residues are known to be sensitive targets of H2O2, in a posttranslational modification called sulfenylation. We trapped and identified sulfenylated proteins in the Medicago truncatula-Sinorhizobium meliloti symbiosis, by combining the use of chemical and genetic probes with mass spectrometry analysis. We identified 44 M. truncatula proteins sulfenylated in inoculated roots (two days post infection, 2 dpi) and 65 such proteins in the functioning symbiotic organ, the nodule (four weeks post infection, 4 wpi); 18 proteins were identified at both time points. However, the largest functional groups at 2 dpi and 4 wpi were different: redox state-linked proteins early in the interaction and proteins involved in amino-acid and carbohydrate metabolism in the nodule. Twenty proteins from S. meliloti, including some directly involved in nitrogen fixation, were also identified as sulfenylated. These results suggest that sulfenylation may regulate the activity of proteins playing major roles in the development and functioning of the symbiotic interaction. (c) 2012 Elsevier B.V. All rights reserved.

International audience; Reactive oxygen species (ROS) are highly reactive molecules able to damage cellular components but they also act as cell signalling elements. ROS are produced by many different enzymatic systems. Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), are the most thoroughly studied enzymatic ROS-generating systems and our understanding of their involvement in various plant processes has increased considerably in recent years. In this review we discuss their roles as ROS producers during cell growth, plant development and plant response to abiotic environmental constraints and biotic interactions, both pathogenic and symbiotic. This broad range of functions suggests that RBOHs may serve as important molecular 'hubs' during ROS-mediated signalling in plants.

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes., Author Summary Parasitic nematodes are microscopic worms that cause major diseases of plants, animals and humans. During compatible interactions, root-knot nematodes (RKN) induce the formation of galls in which redifferentiation of root cells into multinucleate and hypertrophied giant cells is essential for nematode growth and reproduction. The importance of glutathione (GSH), a major antioxidant molecule involved in plant development, in plant microbe interaction and in abiotic stress response, was analyzed during the plant-RKN interaction. Our analyses demonstrated that the gall development and functioning are characterized by an adapted GSH metabolism and that depletion of GSH content impairs nematode reproduction and modified sex ratio. This phenotype is linked to specific modifications of carbon metabolism which do not occur in uninfected roots indicating a peculiar metabolism of this neoformed organ. This first metabolomic analysis during the plant-RKN interaction highlights the regulatory role played by GSH in this pathogenic interaction and completes our vision of the role of GSH during plant-pathogen interactions. RKN sex ratio modification has previously been observed under unfavorable nematode feeding conditions suggesting that the GSH-redox system could be a general sensor of gall fitness in natural conditions.

International audience; # The establishment and development of plant–microorganism interactions involve impressive transcriptomic reprogramming of target plant genes. The symbiont (Sinorhizobium meliloti) and the root knot-nematode pathogen (Meloidogyne incognita) induce the formation of new root organs, the nodule and the gall, respectively. # • Using laser-assisted microdissection, we specifically monitored, at the cell level, Medicago gene expression in nodule zone II cells, which are preparing to receive rhizobia, and in gall giant and surrounding cells, which play an essential role in nematode feeding and constitute the typical root swollen structure, respectively. # • We revealed an important reprogramming of hormone pathways and C1 metabolism in both interactions, which may play key roles in nodule and gall neoformation, rhizobia endocytosis and nematode feeding. Common functions targeted by rhizobia and nematodes were mainly down-regulated, whereas the specificity of the interaction appeared to involve up-regulated genes. # • Our transcriptomic results provide powerful datasets to unravel the mechanisms involved in the accommodation of rhizobia and root-knot nematodes. Moreover, they raise the question of host specificity and the evolution of plant infection mechanisms by a symbiont and a pathogen

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction

GSH is able to reduce soybean (Glycine max) ferryl-leghaemoglobin [Lb(IV)] formed by the reaction of ferric or ferrous Lb with H2O2; in both cases, ferric Lb is obtained and GSH is incapable of reducing ferric Lb to ferrous Lb. Furthermore, the addition of GSH before H2O2 to ferric Lb prevents side reactions which lead to a species whose spectrum differs markedly from that of Lb(IV). These reactions are likely to occur in vivo, as high GSH concentrations have been detected in soybean nodules. The GSH-dependent reduction of Lb(IV) is associated with the oxidation of GSH to GSSG. E.s.r. experiments show that the glutathione thiyl radical (GS.) is formed during this reaction. In the case of ferric Lb, both ferryl Lb and a globin-derived radical previously described appear to be involved in the formation of GS. Both of these processes may be protective and can help account for the exclusive presence of ferrous (oxygenated or not) Lb in functioning nodules.

The root nodules of leguminous plants contain an oxygen-carrying protein which is somewhat similar to myoglobin. Reaction of the Fe3+ form of this protein (metleghaemoglobin; MetLb) with H2O2 is known to generate a ferryl [iron(IV)-oxo] species. This intermediate, which is analogous to Compound II of peroxidases and ferryl myoglobin, is one oxidizing equivalent above the initial level. In the present study it is shown that the second oxidizing equivalent from the peroxide is rapidly transferred into the surrounding protein, generating a protein radical which has been detected by e.p.r. spectroscopy; this reaction is analogous to that observed with metmyoglobin. An identical protein-derived species is observed with all three forms of MetLb tested (a, c1, c3) and with a number of other hydroperoxides and two-electron oxidants. This latter result, the observation that the concentration of this species is not affected by certain hydroxyl-radical scavengers, and the loss of the radical when the oxy or deoxy forms are used, demonstrate that this species is formed by electron transfer within the protein rather than by the generation and subsequent reaction of hydroxyl radicals (and related species from the other hydroperoxides). The e.p.r. signal of this species, which decays rapidly with a half-life of approx. 40 s, is consistent with the formation of a sterically constrained tyrosine-derived phenoxyl radical; protein-iodination experiments lend support to this assignment. Reaction between the radical and a number of other compounds has been observed, demonstrating that it is at least partially exposed on the surface of the protein. Analysis of the protein structure suggest that the radical may be centred on a tyrosine residue present at position 132 in the protein; this residue is close to the haem prosthetic group, which would facilitate rapid electron transfer.

The formation of hydroxyl radicals in Fenton-type reactions involving ferric-EDTA chelate was enhanced by flavonoids. The efficiency was in the order: myricetin > quercetin > catechin > morin > kaempferol; flavone had no effect. Spectral modifications indicated that flavonoids were oxidized during these reactions. When ATP and citrate were used as iron chelators, the level of hydroxyl radical formation was not influenced by the addition of flavonoids. In the presence of ascorbate, flavonoids had little or no effect in assays containing EDTA chelate. In contrast, they were inhibitors of hydroxyl radical formation with ATP and citrate chelates. Based on these results, the role of the iron chelator on the influence of flavonoids on hydroxyl radical formation in Fenton-type reactions is discussed.

Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M r thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-β-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.