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International audience; Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules.

Plants form beneficial symbioses with a wide variety of microorganisms. Among these, endophytes, arbuscular mycorrhizal fungi (AMF), and nitrogen-fixing rhizobia are some of the most studied and well understood symbiotic interactions. These symbiotic microorganisms promote plant nutrition and growth. In exchange, they receive the carbon and metabolites necessary for their development and multiplication. In addition to their role in plant growth and development, these microorganisms enhance host plant tolerance to a wide range of environmental stress. Multiple studies have shown that these microorganisms modulate the phytohormone metabolism in the host plant. Among the phytohormones involved in the plant defense response against biotic environment, salicylic acid (SA) plays an important role in activating plant defense. However, in addition to being a major actor in plant defense signaling against pathogens, SA has also been shown to be involved in plant–microbe symbiotic interactions. In this review, we summarize the impact of SA on the symbiotic interactions. In addition, we give an overview of the impact of the endophytes, AMF, and rhizobacteria on SA-mediated defense response against pathogens.

Reactive oxygen species such as hydrogen peroxide (H

Legumes can meet their nitrogen requirements through root nodule symbiosis, which could also trigger plant systemic resistance against pests. The pea aphid Acyrthosiphon pisum , a legume pest, can harbour different facultative symbionts (FS) influencing various traits of their hosts. It is therefore worth determining if and how the symbionts of the plant and the aphid modulate their interaction. We used different pea aphid lines without FS or with a single one ( Hamiltonella defensa , Regiella insecticola, Serratia symbiotica ) to infest Medicago truncatula plants inoculated with Sinorhizobium meliloti (symbiotic nitrogen fixation, SNF) or supplemented with nitrate (non-inoculated, NI). The growth of SNF and NI plants was reduced by aphid infestation, while aphid weight (but not survival) was lowered on SNF compared to NI plants. Aphids strongly affected the plant nitrogen fixation depending on their symbiotic status, suggesting indirect relationships between aphid- and plant-associated microbes. Finally, all aphid lines triggered expression of Pathogenesis-Related Protein 1 ( PR1 ) and Proteinase Inhibitor (PI) , respective markers for salicylic and jasmonic pathways, in SNF plants, compared to only PR1 in NI plants. We demonstrate that the plant symbiotic status influences plant–aphid interactions while that of the aphid can modulate the amplitude of the plant's defence response.

Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.

International audience

In the legume-rhizobia symbiosis, atmospheric dinitrogen (N2) fixation by rhizobia takes place in a specific root organ, called the nodule. During biological N2 fixation, the plant provides photosynthetic carbohydrates to bacteria and, in exchange, bacteria feed the plant with reduced nitrogen (N) under ammonia (NH3) form. Like most other organs, the root nodule has a limited lifespan, and eventually enters a senescence process characterized by a decline of N2 fixation and the coordinated death of both bacteria and plant cells. This senescence process characterized by a metabolic switch from a carbon sink to a nutrient source either results from nodule aging (developmental nodule senescence) or can be triggered prematurely by adverse environmental conditions. Accumulating evidence suggests that redox and hormone signaling contribute to the triggering of senescence. Indeed, nodule senescence is a developmentally programmed process in which antioxidants, reactive oxygen and N species (ROS/RNS), hormones and proteinases play key roles. This chapter reviews recent knowledge on nodule senescence and proposes models by which ROS, RNS, and likely hormones intervene in the orchestration of this process.

The interaction between legumes and bacteria of rhizobia type results in a beneficial symbiotic relationship characterized by the formation of new root organs, called nodules. Within these nodules the bacteria, released in plant cells, differentiate into bacteroids and fix atmospheric nitrogen through the nitrogenase activity. This mutualistic interaction has evolved sophisticated signaling networks to allow rhizobia entry, colonization, bacteroid differentiation and persistence in nodules. Nodule cysteine rich (NCR) peptides, reactive oxygen species (ROS), reactive nitrogen species (RNS), and toxin-antitoxin (TA) modules produced by the host plants or bacterial microsymbionts have a major role in the control of the symbiotic interaction. These molecules described as weapons in pathogenic interactions have evolved to participate to the intracellular bacteroid accommodation by escaping control of plant innate immunity and adapt the functioning of the nitrogen-fixation to environmental signalling cues.

Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.

Interactions between legumes and rhizobia lead to the establishment of a symbiotic relationship 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. Significant amounts of nitric oxide (NO) accumulate at different stages of nodule development, suggesting that NO performs specific signaling and/or metabolic functions during symbiosis. NO, which regulates nodule gene expression, accumulates to high levels in hypoxic nodules. NO accumulation is considered to assist energy metabolism within the hypoxic environment of the nodule via a phytoglobin–NO-mediated respiration process. NO is a potent inhibitor of the activity of nitrogenase and other plant and bacterial enzymes, acting as a developmental signal in the induction of nodule senescence. Hence, key questions concern the relative importance of the signaling and metabolic functions of NO versus its toxic action and how NO levels are regulated to be compatible with nitrogen fixation functions. This review analyses these paradoxical roles of NO at various stages of symbiosis, and highlights the role of plant phytoglobins and bacterial hemoproteins in the control of NO accumulation.

Under nitrogen-limiting conditions, legumes are able to form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce root nodules. These new root organs satisfy plant nitrogen needs by reducing atmospheric nitrogen to ammonium. However, the senescence of these organs disturbs the assimilation of nitrogen. In this study, we present different histological, biochemical, and genetic markers of the natural nodule senescence in Medicago truncatula over a 10-week period following bacterial inoculation. During aging the length and the weight of nodules increased, whereas the nitrogen-fixing capacity of the nodules decreased. The development of the nodule senescence zone correlated with a reduction in leghemoglobin levels without significant reduction in the total protein concentration of the nodule. In contrast, no difference in glutathione and homoglutathione concentration was detected at the onset of senescence at 6 and 8 weeks after bacterial infection. Furthermore, we observed a significant decrease in the relative transcription levels of Nodule Cysteine Rich 001, MtLb1, sucrose synthase 1, thioredoxin S1 and thioredoxin S2 genes, which are involved in nodule development and functioning, thus demonstrating that natural senescence impacts the transcription of genes involved in the expansion and the metabolism of the nitrogen-fixing zone. Finally, the induction of amine oxidase and cysteine protease CP6 transcription was unstable, suggesting that these two genes are related to senescence but are not robust gene markers of the natural senescence process. Considered together, our results define novel biochemical and genetic markers for natural nodule senescence and show that leghemoglobin gene transcription and protein concentration are robust markers that closely correlate with nitrogen fixation efficiency.

Leguminous plants can form a symbiotic relationship with Rhizobium bacteria, during which plants provide bacteria with carbohydrates and an environment appropriate to their metabolism, in return for fixed atmospheric nitrogen. The symbiotic interaction leads to the formation of a new organ, the root nodule, where a coordinated differentiation of plant cells and bacteria occurs. The establishment and functioning of nitrogen-fixing symbiosis involves a redox control important for both the plant-bacteria crosstalk and the regulation of nodule metabolism. In this review, we discuss the involvement of thioredoxin and glutaredoxin systems in the two symbiotic partners during symbiosis. The crucial role of glutathione in redox balance and S-metabolism is presented. We also highlight the specific role of some thioredoxin and glutaredoxin systems in bacterial differentiation. Transcriptomics data concerning genes encoding components and targets of thioredoxin and glutaredoxin systems in connection with the developmental step of the nodule are also considered in the model system Medicago truncatula⁻Sinorhizobium meliloti.

International audience; Background: Broad bean fits sustainable agriculture model due to symbiosis with Rhizobium, the seeds being a good source of energy, proteins, polyphenols, and fiber. The large amount of broad bean biomass residues can be employed for biofuel production, thus valorizing the overall production process. This research was aimed to investigate on the effects of farming management, such as greenhouse cultivation and appropriate planting time on the qualities of broad bean seeds and residual biomass for conversion into biofuel. The related balances of energy gain associated to both ethanol yield and nitrogen fertilizer saving due to Rhizobium nitrogen fixation were assessed. Methods: Research was carried out on broad bean in Portici, province of Naples, southern Italy, based on the factorial combination of two farming systems (open field, greenhouse) and five planting times: 27 September and 11 October, to obtain early production; 25 October, which fell in the usual period for broad bean planting in the province area; and 8 November and 22 November, for late production. For each of these cultivation conditions, the quality of seeds, in terms of protein, fiber and antioxidant concentrations, and of crop residual biomass were determined. In addition, the energy yield as ethanol production from residual biomass and nitrogen fertilizer saving due to Rhizobium atmospheric fixation were assessed. Results and discussion: The highest plant nitrogen uptake was recorded under the fourth planting time in open field and the third in greenhouse, the average accumulation attaining 87% in residual biomass, 7.4% in pods, and 5.6% in seeds. Seed protein content was 12.6% higher in greenhouse than in open field and 16.2% higher under the latest planting time compared to the earliest one. Seed polyphenol concentration was higher in open field than in greenhouse and with the two earliest planting times. Greenhouse grown biomass showed higher values of lignin, hemicellulose and pectin, compared to open field, whereas the opposite trend was for cellulose. Lignin showed a decrease from the first to the last crop cycle, opposite to cellulose, and glucose was the most represented monosaccharide. Both the highest theoretical ethanol and overall energy production were highest with the fourth planting time. Conclusions: Greenhouse management enabled broad bean plants to accumulate higher proteins in seeds, but open field conditions resulted in better residual biomass quality for ethanol and Rhizobium-depending energy production.

International audience; Background: Nitrogen-fixing symbiosis between Rhizobium bacteria and legumes leads to the formation of a new organ, the root nodule. The development of the nodule requires the differentiation of plant root cells to welcome the endosymbiotic bacterial partner. This development includes the formation of an efficient vascular tissue which allows metabolic exchanges between the root and the nodule, the formation of a barrier to oxygen diffusion necessary for the bacterial nitrogenase activity and the enlargement of cells in the infection zone to support the large bacterial population. Inside the plant cell, the bacteria differentiate into bacteroids which are able to reduce atmospheric nitrogen to ammonia needed for plant growth in exchange for carbon sources. Nodule functioning requires a tight regulation of the development of plant cells and bacteria. Scope of the review: Nodule functioning requires a tight regulation of the development of plant cells and bacteria. The importance of redox control in nodule development and N-fixation is discussed in this review. The involvement of reactive oxygen and nitrogen species and the importance of the antioxidant defense are analyzed. Major conclusions: Plant differentiation and bacterial differentiation are controlled by reactive oxygen and nitrogen species, enzymes involved in the antioxidant defense and antioxidant compounds. General significance: The establishment and functioning of nitrogen-fixing symbiosis involve a redox control important for both the plant-bacteria crosstalk and the consideration of environmental parameters. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

Glutathione (GSH) is a major antioxidant molecule in plants. It is involved in regulating plant development and responses to abiotic and biotic environment changes. In leguminous plants, a GSH homolog, homoglutathione is also found. Most legumes can develop a symbiotic interaction with soil bacteria of the rhizobium family under nitrogen deficiency. This symbiosis allows the reduction of atmospheric nitrogen by the bacteria in plant organs called root nodules. In this chapter, we summarize studies that describe the synthesis and the roles of GSH and hGSH in the nitrogen-fixing symbiosis.

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen-fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down-regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down-regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.

The symbiotic interaction between legumes and bacteria of Rhizobium type leads to the formation of new organs, called nodules, which provides a niche for bacterial nitrogen (N2) fixation. In the nodules, bacteria differentiate into bacteroids able to fix atmospheric N2 through nitrogenase activity. As nitrogenase is strongly inhibited by oxygen, N2-fixation is made possible thanks to microaerophilic conditions prevailing in the nodules, characterized by a balance between low oxygen contents and adjusted nitric oxide (NO) contents. NO was shown to be produced by both the plant and bacterial partners during symbiosis, from early interaction steps between the plant and the bacteria to N2-fixing and senescence steps in mature nodules. NO is required for an optimal establishment of the symbiotic interaction. Transcriptomic analysis at early stage of the symbiosis showed that NO regulates about 2030 genes including genes involved in the induction of cell dedifferentiation and organogenesis, and in the repression of plant defense reactions, favouring the establishment of the plant–microbe interaction. In mature nodules, NO was found to act as both an inhibitor of N2-fixation, a regulator of nitrogen metabolism, a beneficial metabolic intermediate for the maintenance of the energy metabolism under hypoxic conditions and a signal triggering nodule senescence. The present review provides an overview of NO sources and degradation pathways and of its multifaceted functions throughout the whole symbiotic process. It additionally analyses NO crosstalks with reactive oxygen species and hormones and presents particularly promising issues to decipher the roles of NO in N2-fixing symbioses.

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.

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.

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.

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.

International audience; Climate change is increasingly impacting the water deficit over the world. Because of drought and the high pressure of the rising human population, water is becoming a scarce and expensive commodity, especially in developing countries. The identification of crops presenting a higher acclimation to drought stress is thus an important objective in agriculture. The present investigation aimed to assess the adaptation of three Vicia faba genotypes, Aguadulce (AD), Luz d'Otonio (LO) and Reina Mora (RM) to water deficit. Multiple physiological and biochemical parameters were used to analyse the response of the three genotypes to two soil water contents (80% and 40% of field capacity). A significant lower decrease in shoot, root and nodule dry weight was observed for AD compared to LO and RM. The better growth performance of AD was correlated to higher carbon and nitrogen content than in LO and RM under water deficit. Leaf parameters such as relative water content, mass area, efficiency of photosystem II and chlorophyll and carotenoid content were significantly less affected in AD than in LO and RM. Significantly higher accumulation of proline was correlated to the higher performance of AD compared to LO and RM. Additionally, the better growth of AD genotype was related to an important mobilisation of antioxidant enzyme activities such as ascorbate peroxidase and catalase. Taken together, these results allow us to suggest that AD is a water deficit tolerant genotype compared to LO and RM. Our multiple physiological and biochemical analyses show that nitrogen content, leaf proline accumulation, reduced leaf hydrogen peroxide accumulation and leaf antioxidant enzymatic activities (ascorbate peroxidase, guaiacol peroxidase, catalase and polyphenol oxidase) are potential biological markers useful to screen for water deficit resistant Vicia faba genotypes.

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.

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.

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.

10 Págs., 2 Figs. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CCBY). The .pdf document is protected by copyright and was first published by Frontiers., In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS) so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed us to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, that their spatio-temporally distribution is correlated with the corresponding (homo)glutathione synthetase activities, and that they are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume-rhizobial symbiosis., Research from our laboratories has been funded by grants from MAE and CNRS (Envimedprogram) to Pierre Frendo and the Spanish Ministry of Economy and Competitivity (AGL2011-24524, cofunded by FEDER) to Manuel Becana.

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen-fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down-regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down-regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.

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.

International audience; Clubroot disease affects all Brassicaceae spp. and is caused by the obligate biotroph pathogen Plasmodiophora brassicae. The development of galls on the root system is associated with the establishment of a new carbon metabolic sink. Here, we aimed to deepen our knowledge of the involvement of primary metabolism in the Brassica napus response to clubroot infection. We studied the dynamics and the diversity of the metabolic responses to the infection. Root system metabotyping was carried out for 18 rapeseed genotypes displaying different degrees of symptom severity, under inoculated and noninoculated conditions at 42 days postinoculation (dpi). Clubroot susceptibility was positively correlated with clubroot-induced accumulation of several amino acids. Although glucose and fructose accumulated in some genotypes with minor symptoms, their levels were negatively correlated to the disease index across the whole set of genotypes. The dynamics of the metabolic response were studied for the susceptible genotype 'Yudal,' which allowed an "early" metabolic response (established from 14 to 28 dpi) to be differentiated from a "late" response (from 35 dpi). We discuss the early accumulation of amino acids in the context of the establishment of a nitrogen metabolic sink and the hypothetical biological role of the accumulation of glutathione and S-methylcysteine.

Biological nitrogen fixation (BNF) is the biological process by which the atmospheric nitrogen (N2) is converted to ammonia by an enzyme called nitrogenase. It is the major source of the biosphere nitrogen and as such has an important ecological and agronomical role, accounting for 65 % of the nitrogen used in agriculture worldwide. The most important source of fixed nitrogen is the symbiotic association between rhizobia and legumes. The nitrogen fixation is achieved by bacteria inside the cells of de novo formed organs, the nodules, which usually develop on roots, and more occasionally on stems. This mutualistic relationship is beneficial for both partners, the plant supplying dicarboxylic acids as a carbon source to bacteria and receiving, in return, ammonium. Legume symbioses have an important role in environment-friendly agriculture. They allow plants to grow on nitrogen poor soils and reduce the need for nitrogen inputs for leguminous crops, and thus soil pollution. Nitrogen-fixing legumes also contribute to nitrogen enrichment of the soil and have been used from Antiquity as crop-rotation species to improve soil fertility. They produce high protein-containing leaves and seeds, and legumes such as soybeans, groundnuts, peas, beans, lentils, alfalfa and clover are a major source of protein for human and animal consumption. Most research concentrates on the two legume-rhizobium model systems Lotus-Mesorhizobium loti and Medicago-Sinorhizobium meliloti, with another focus on the economically-important Glycine max (soybean) -Bradyrhizobium japonicum association. The legume genetic models Medicago truncatula and Lotus japonicus have a small genome size of ca. 450 Mbp while Glycine max has a genome size of 1,115 Mbp, and all are currently targets of large-scale genome sequencing projects (He et al., 2009; Sato et al., 2008; Schmutz et al., 2010). The complete genome sequence of their bacterial partners has been established (Galibert et al., 2001; Kaneko et al., 2000; Kaneko et al., 2002; Schneiker-Bekel et al., 2011).

Cyclophilins (Cyp) are ubiquitous proteins with peptidyl-prolyl cis-trans isomerase activity that catalyses rotation of X-Pro peptide bonds and facilitates the folding of proteins; these enzymes are believed to play a role in in vivo protein folding. During development of normal bean plants, Cyp transcripts are first detected three days after beginning of germination and are present in all plant tissues examined. In a general way, higher amounts of Cyp mRNAs are found in developing tissues. Cyp mRNA accumulates in alfalfa mosaic virus-infected bean leaves and after ethephon and salicylic acid treatments. In response to a localized chemical treatment Cyp mRNA accumulation is observed in the untreated parts of the plants; however these changes in mRNA levels are restricted to the aerial part of the plant. A comparative study of Cyp mRNA accumulation in bean and maize in response to various external stimuli shows striking differences in profiles between the two plants. For instance, in response to heat shock, maize Cyp mRNA significantly accumulates, whereas no remaining mRNA is observed a few hours after the beginning of the heat stress in bean. Differences in mRNA accumulation profiles are also observed upon salt stress which induces the response earlier in maize than in bean, whereas the opposite situation is observed when plants are cold-stressed. All these findings further suggest that cyclophilin might be a stress-related protein.

The cDNA clone (CHEM4) coding for a stress-induced protein has been isolated from a mercuric chloride-treated maize (Zea mays L.) cDNA library by differential screening. Nucleotide sequence analysis of the 694-bp-cDNA insert predicts an open reading frame of 522 nucleotides encoding a mature protein of 149 amino acids and a signal peptide of 25 amino acid residues. The sequence of CHEM4-encoded protein shows 50 to 60% sequence identity with thaumatin, bifunctional α-amylase/trypsin inhibitor from maize and thaumatin-like (TL) proteins from dicotyledonous plants. Higher sequence identities are found to wheat PWIR2 and barley Hv-1b TL-proteins. CHEM4-mRNA is not only induced by mercuric chloride treatment but also by other stresses such as high salt concentration, high temperatures, UV light, wounding and polluted rainwater. CHEM4 genes appear to be highly inducible by UV light and mercuric chloride, moderately by wounding, salt stress, polluted rainwater and heat shock and very weakly by cooling.

In a search for stress-related genes, a differential screening of a mercuric chloride-treated maize cDNA library has been performed. Among the positive clones identified, one, designated as CHEM 7, has been found to be expressed at a significantly higher level in stressed plants than in non-stressed plants. The sequence of CHEM 7 showed complete identity to maize (Zea mays L.) cyclophilin (CyP) described earlier. CHEM 7 was used as a probe to screen a chemically-stressed bean cDNA library. DNA sequencing of the longest positive clone revealed a high homology with maize CyP cDNA. Time course studies of CyP mRNA accumulation upon chemical stress in maize and bean (Phaseolus vulgaris L.) showed that in maize maximum amounts of mRNA are found 6–7 h after treatment, whereas in bean there is a rather long delay before mRNA synthesis is stimulated and the maximum accumulation is only reached 48 h after the onset of stress. Southern blot analysis suggests that maize CyP is encoded by a small family of genes (6–7) but there is only a single CyP gene in bean. CyP mRNA synthesis is not only stimulated by a mercuric chloride treatment but also by other abiotic stresses such as heat-shock, wounding, salt stress and low temperature.