Group VII ethylene response factors, MtERF74 and MtERF75, sustain nitrogen fixation in Medicago truncatula microoxic nodules.

Group VII ethylene response factors (ERF-VII) are plant-specific transcription factors (TFs) known for their role in the activation of hypoxia-responsive genes under low oxygen stress but also in plant endogenous hypoxic niches. However, their function in the microaerophilic nitrogen-fixing nodules of legumes has not yet been investigated. We investigated regulation and the function of the two Medicago truncatula ERF-VII TFs (MtERF74 and MtERF75) in roots and nodules, MtERF74 and MtERF75 in response to hypoxia stress and during the nodulation process using an RNA interference strategy and targeted proteolysis of MtERF75. Knockdown of MtERF74 and MtERF75 partially blocked the induction of hypoxia-responsive genes in roots exposed to hypoxia stress. In addition, a significant reduction in nodulation capacity and nitrogen fixation activity was observed in mature nodules of double knockdown transgenic roots. Overall, the results indicate that MtERF74 and MtERF75 are involved in the induction of MtNR1 and Pgb1.1 expression for efficient Phytogb-nitric oxide respiration in the nodule.

Role of Nitric Oxide of Bacterial Origin in the Medicago truncatula-Sinorhizobium meliloti Symbiosis.

Nitric oxide (NO) is a small ubiquitous gaseous molecule that has been found in many host-pathogen interactions. NO has been shown to be part of the defense arsenal of animal cells and more recently of plant cells. To fight this molecular weapon, pathogens have evolved responses consisting of adaptation to NO or degradation of this toxic molecule. More recently, it was shown that NO could also be produced by the pathogen and contributes likewise to the success of the host cell infection. NO is also present during symbiotic interactions. Despite growing knowledge about the role of NO during friendly interactions, data on the specificity of action of NO produced by each partner are scarce, partly due to the multiplicity of NO production systems. In the nitrogen-fixing symbiosis between the soil bacterium Sinorhizobium meliloti and the model legume Medicago truncatula, NO has been detected at all steps of the interaction, where it displays various roles. Both partners contribute to NO production inside the legume root nodules where nitrogen fixation occurs. The study focuses on the role of bacterial NO in this interaction. We used a genetic approach to identify bacterial NO sources in the symbiotic context and to test the phenotype in planta of bacterial mutants affected in NO production. Our results show that only denitrification is a source of bacterial NO in Medicago nodules, giving insight into the role of bacteria-derived NO at different steps of the symbiotic interaction. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Nitric Oxide Metabolic Pathway in Drought-Stressed Nodules of Faba Bean (Vicia faba L.).

Drought is an environmental stress that strongly impacts plants. It affects all stages of growth and induces profound disturbances that influence all cellular functions. Legumes can establish a symbiosis with Rhizobium-type bacteria, whose function is to fix atmospheric nitrogen in organs called nodules and to meet plant nitrogen needs. Symbiotic nitrogen fixation (SNF) is particularly sensitive to drought. We raised the hypothesis that, in drought-stressed nodules, SNF inhibition is partly correlated to hypoxia resulting from nodule structure compaction and an increased O2 diffusion barrier, and that the nodule energy regeneration involves phytoglobin-nitric oxide (Pgb-NO) respiration. To test this hypothesis, we subjected faba bean (Vicia faba L.) plants nodulated with a Rhizobium laguerreae strain to either drought or osmotic stress. We monitored the N2-fixation activity, the energy state (ATP/ADP ratio), the expression of hypoxia marker genes (alcohol dehydrogenase and alanine aminotransferase), and the functioning of the Pgb-NO respiration in the nodules. The collected data confirmed our hypothesis and showed that (1) drought-stressed nodules were subject to more intense hypoxia than control nodules and (2) NO production increased and contributed via Pgb-NO respiration to the maintenance of the energy state of drought-stressed nodules.

Nitrate reductases and hemoglobins control nitrogen-fixing symbiosis by regulating nitric oxide accumulation.

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.

Nitric oxide production is involved in maintaining energy state in Alfalfa (Medicago sativa L.) nodulated roots under both salinity and flooding.

MAIN CONCLUSION: In Medicago sativa nodulated roots, NR-dependent NO production is involved in maintaining energy state, presumably through phytoglobin NO respiration, under both salinity and hypoxia stress. The response to low and average salinity stress and to a 5 day-long flooding period was analyzed in M. sativa nodulated roots. The two treatments result in a decrease in the biological nitrogen fixation capacity and the energy state (evaluated by the ATP/ADP ratio), and conversely in an increase nitric oxide (NO) production. Under salinity and hypoxia treatments, the use of either sodium tungstate, an inhibitor of nitrate reductase (NR), or carboxy-PTIO, a NO scavenger, results in a decrease in NO production and ATP/ADP ratio, meaning that NR-dependent NO production participates to the maintenance of the nodulated roots energy state.

Medicago truncatula Phytoglobin 1.1 controls symbiotic nodulation and nitrogen fixation via the regulation of nitric oxide concentration.

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.

Recommendations on terminology and experimental best practice associated with plant nitric oxide research.

Nitric oxide (NO) emerged as a key signal molecule in plants. During the last two decades impressive progress has been made in plant NO research. This small, redox-active molecule is now known to play an important role in plant immunity, stress responses, environmental interactions, plant growth and development. To more accurately and robustly establish the full spectrum of NO bioactivity in plants, it will be essential to apply methodological best practice. In addition, there are some instances of conflicting nomenclature within the field, which would benefit from standardization. In this context, we attempt to provide some helpful guidance for best practice associated with NO research and also suggestions for the cognate terminology.

Regulating the regulator: nitric oxide control of post-translational modifications.

Nitric oxide (NO) is perfectly suited for the role of a redox signalling molecule. A key route for NO bioactivity occurs via protein S-nitrosation, and involves the addition of a NO moiety to a protein cysteine (Cys) thiol (-SH) to form an S-nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants that are linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post-translational modifications including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues.

Nitric oxide signaling, metabolism and toxicity in nitrogen-fixing symbiosis.

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.

Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions.

The interaction between legumes and rhizobia leads to the establishment of a beneficial symbiotic relationship. Recent advances in legume - rhizobium symbiosis revealed that various reactive oxygen and nitrogen species including nitric oxide (NO) play important roles during this process. Nodule development occurs with a transition from a normoxic environment during the establishment of symbiosis to a microoxic environment in functional nodules. Such oxygen dynamics are required for activation and repression of various NO production and scavenging pathways. Both the plant and bacterial partners participate in the synthesis and degradation of NO. However, the pathways of NO production and degradation as well as their cross-talk and involvement in the metabolism are still a matter of debate. The plant-originated reductive pathways are known to contribute to the NO production in nodules under hypoxic conditions. Non-symbiotic hemoglobin (phytoglobin) (Pgb) possesses high NO oxygenation capacity, buffers and scavenges NO. Its operation, through a respiratory cycle called Pgb-NO cycle, leads to the maintenance of redox and energy balance in nodules. The role of Pgb/NO cycle under fluctuating NO production from soil needs further investigation for complete understanding of NO regulatory mechanism governing nodule development to attain optimal food security under changing environment.

Post-translational modifications of Medicago truncatula glutathione peroxidase 1 induced by nitric oxide.

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 Gpx1 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.

Nitric oxide: a multifaceted regulator of the nitrogen-fixing symbiosis.

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.

(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula.

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.

Involvement of papain and legumain proteinase in the senescence process of Medicago truncatula nodules.

The symbiotic interaction between legumes and Rhizobiaceae leads to the formation of new root organs called nodules. Within the nodule, Rhizobiaceae differentiate into nitrogen-fixing bacteroids. However, this symbiotic interaction is time-limited as a result of the initiation of a senescence process, leading to a complete degradation of bacteroids and host plant cells. The increase in proteolytic activity is one of the key features of this process. In this study, we analysed the involvement of two different classes of cysteine proteinases, MtCP6 and MtVPE, in the senescence process of Medicago truncatula nodules. Spatiotemporal expression of MtCP6 and MtVPE was investigated using promoter- ß-glucuronidase fusions. Corresponding gene inductions were observed during both developmental and stress-induced nodule senescence. Both MtCP6 and MtVPE proteolytic activities were increased during stress-induced senescence. Down-regulation of both proteinases mediated by RNAi in the senescence zone delayed nodule senescence and increased nitrogen fixation, while their early expression promoted nodule senescence. Using green fluorescent protein fusions, in vivo confocal imaging showed that both proteinases accumulated in the vacuole of uninfected cells or the symbiosomes of infected cells. These data enlighten the crucial role of MtCP6 and MtVPE in the onset of nodule senescence.

Control of the rhizobium-legume symbiosis by the plant nitrogen demand is tightly integrated at the whole plant level and requires inter-organ systemic signaling.

Symbiotic nodules formed on legume roots with rhizobia fix atmospheric N2. Bacteria reduce N2 to NH4 + that is assimilated into amino acids by the plant. In return, the plant provides photosynthates to fuel the symbiotic nitrogen fixation. Symbiosis is tightly adjusted to the whole plant nutritional demand and to the plant photosynthetic capacities, but regulatory circuits behind this control remain poorly understood. The use of split-root systems combined with biochemical, physiological, metabolomic, transcriptomic, and genetic approaches revealed that multiple pathways are acting in parallel. Systemic signaling mechanisms of the plant N demand are required for the control of nodule organogenesis, mature nodule functioning, and nodule senescence. N-satiety/N-deficit systemic signaling correlates with rapid variations of the nodules' sugar levels, tuning symbiosis by C resources allocation. These mechanisms are responsible for the adjustment of plant symbiotic capacities to the mineral N resources. On the one hand, if mineral N can satisfy the plant N demand, nodule formation is inhibited, and nodule senescence is activated. On the other hand, local conditions (abiotic stresses) may impair symbiotic activity resulting in plant N limitation. In these conditions, systemic signaling may compensate the N deficit by stimulating symbiotic root N foraging. In the past decade, several molecular components of the systemic signaling pathways controlling nodule formation have been identified, but a major challenge remains, that is, to understand their specificity as compared to the mechanisms of non-symbiotic plants that control root development and how they contribute to the whole plant phenotypes. Less is known about the control of mature nodule development and functioning by N and C nutritional status of the plant, but a hypothetical model involving the sucrose allocation to the nodule as a systemic signaling process, the oxidative pentose phosphate pathway, and the redox status as potential effectors of this signaling is emerging. This work highlights the importance of organism integration in plant biology.

Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules.

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Up-regulation of leucine aminopeptidase-A in cadmium-treated tomato roots.

The effects of cadmium (Cd) on aminopeptidase (AP) activities and Leucine-AP (LAP) expression were investigated in the roots of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 10 days in the presence of 0.3-300 µM Cd and compared to control plants grown in the absence of Cd. AP activities were measured using six different p-nitroanilide (p-NA) substrates. Leu, Met, Arg, Pro and Lys hydrolyzing activities increased in roots of Cd-treated plants, while Phe-pNA cleavage was not enhanced after Cd treatments. The use of peptidase inhibitors showed that most of the Leu-pNA hydrolyzing activity was related to one or several metallo-APs. Changes in Lap transcripts, protein and activities were measured in the roots of 0 and 30-µM Cd-treated plants. LapA transcript levels increased in Cd-treated roots, whereas LapN RNAs levels were not modified. To assess amount of Leu-pNA hydrolyzing activity associated with the hexameric LAPs, LAP activity was measured following immunoprecipitation with a LAP polyclonal antiserum. LAP activity increased in Cd-treated roots. There was a corresponding increase in LAP-A protein levels detected in 2D-immunoblots. The role of LAP-A in the proteolytic response to Cd stress is discussed.

Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis.

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.

MtNOA1/RIF1 modulates Medicago truncatula-Sinorhizobium meliloti nodule development without affecting its nitric oxide content.

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.