A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence.

Legumes establish symbioses with rhizobia by forming nitrogen-fixing nodules. Nitrate is a major environmental factor that affects symbiotic functioning. However, the molecular mechanism of nitrate-induced nodule senescence is poorly understood. Comparative transcriptomic analysis reveals an NAC-type transcription factor in Lotus japonicus, LjNAC094, that acts as a positive regulator in nitrate-induced nodule senescence. Stable overexpression and mutant lines of NAC094 were constructed and used for phenotypic characterization. DNA-affinity purification sequencing was performed to identify NAC094 targeting genes and results were confirmed by electrophoretic mobility shift and transactivation assays. Overexpression of NAC094 induces premature nodule senescence. Knocking out NAC094 partially relieves nitrate-induced degradation of leghemoglobins and abolishes nodule expression of senescence-associated genes (SAGs) that contain a conserved binding motif for NAC094. Nitrate-triggered metabolic changes in wild-type nodules are largely affected in nac094 mutant nodules. Induction of NAC094 and its targeting SAGs was almost blocked in the nitrate-insensitive nlp1, nlp4, and nlp1 nlp4 mutants. We conclude that NAC094 functions downstream of NLP1 and NLP4 by regulating nitrate-induced expression of SAGs. Our study fills in a key gap between nitrate and the execution of nodule senescence, and provides a potential strategy to improve nitrogen fixation and stress tolerance of legumes.

A collection of novel Lotus japonicus LORE1 mutants perturbed in the nodulation program induced by the Agrobacterium pusense strain IRBG74.

The Lotus japonicus population carrying new Lotus retrotransposon 1 (LORE1) insertions represents a valuable biological resource for genetic research. New insertions were generated by activation of the endogenous retroelement LORE1a in the germline of the G329-3 plant line and arranged in a 2-D system for reverse genetics. LORE1 mutants identified in this collection contributes substantially to characterize candidate genes involved in symbiotic association of L. japonicus with its cognate symbiont, the nitrogen-fixing bacteria Mesorhizobium loti that infects root nodules intracellularly. In this study we aimed to identify novel players in the poorly explored intercellular infection induced by Agrobacterium pusense IRBG74 sp. For this purpose, a forward screen of > 200,000 LORE1 seedlings, obtained from bulk propagation of G329-3 plants, inoculated with IRBG74 was performed. Plants with perturbed nodulation were scored and the offspring were further tested on plates to confirm the symbiotic phenotype. A total of 110 Lotus mutants with impaired nodulation after inoculation with IRBG74 were obtained. A comparative analysis of nodulation kinetics in a subset of 20 mutants showed that most of the lines were predominantly affected in nodulation by IRBG74. Interestingly, additional defects in the main root growth were observed in some mutant lines. Sequencing of LORE1 flanking regions in 47 mutants revealed that 92 Lotus genes were disrupted by novel LORE1 insertions in these lines. In the IM-S34 mutant, one of the insertions was located in the 5´UTR of the LotjaGi5g1v0179800 gene, which encodes the AUTOPHAGY9 protein. Additional mutant alleles, named atg9-2 and atg9-3, were obtained in the reverse genetic collection. Nodule formation was significantly reduced in these mutant alleles after M. loti and IRBG74 inoculation, confirming the effectiveness of the mutant screening. This study describes an effective forward genetic approach to obtain novel mutants in Lotus with a phenotype of interest and to identify the causative gene(s).

Proteome and Physiological Characterization of Halotolerant Nodule Endophytes: The Case of Rahnella aquatilis and Serratia plymuthica.

Bacterial endophytes were isolated from nodules of pea and fava bean. The strains were identified and characterized for plant beneficial activities (phosphate solubilization, synthesis of indole acetic acid and siderophores) and salt tolerance. Based on these data, four strains of Rahnella aquatilis and three strains of Serratia plymuthica were selected. To shed light on the mechanisms underlying salt tolerance, the proteome of the two most performant strains (Ra4 and Sp2) grown in the presence or not of salt was characterized. The number of proteins expressed by the endophytes was higher in the presence of salt. The modulated proteome consisted of 302 (100 up-regulated, 202 down-regulated) and 323 (206 up-regulated, 117 down-regulated) proteins in Ra4 and Sp2, respectively. Overall, proteins involved in abiotic stress responses were up-regulated, while those involved in metabolism and flagellum structure were down-regulated. The main up-regulated proteins in Sp2 were thiol: disulfide interchange protein DsbA, required for the sulfur binding formation in periplasmic proteins, while in Ra4 corresponded to the soluble fraction of ABC transporters, having a role in compatible solute uptake. Our results demonstrated a conserved response to salt stress in the two taxonomically related species.

Nodules of wild legumes as unique natural hotspots of antibiotic resistance genes.

Root nodules (RN) of legumes have distinct microenvironment from their symbiotic roots and surrounding soils. The rhizobia can withstand the host-produced phytoalexins and antimicrobial compounds. We thus hypothesize that the wild legume RN may develop unique natural resistome and be antibiotic resistance gene (ARG) hotspots. In this study, in comparison with rhizosphere soil (RS) and bulk soil (BS), we characterized the feature of antibiotic resistance in the RN of two wild legumes, Medicago polymorpha and Astragalus sinicus, by metagenomics. It was shown that the total relative abundance of ARGs followed the order of RN > RS > BS for both legumes. ARGs encoding antibiotic efflux pump predominated in all samples with increased proportion from BS to RN samples for both legumes. Totally 275 ARG subtypes were detected, and diversity of ARGs in RN was significantly lower than in BS samples for both legumes. 32 and 25 unique ARGs subtypes were detected in RN of both legumes. Bacterial community played a key role in shaping nodule-associated resistome because both ARG profiles and bacterial community differed greatly among BS, RS and RN. Rhizobia potentially hosted 10 and 15 ARGs subtypes for both legumes. The number and proportion of plasmid- and ARG-carrying contigs (ACCs) were higher in RN than in BS. Host tracking analysis of plasmid-ACCs suggests that proportion of rhizobial bacteria identified as their hosts decreased from BS to RN samples. No plasmid-ACCs with multiple ARGs were observed in BS samples, whereas they were detected in RN samples of both legumes. Our study showed that even wild legume nodules are unique natural ARG hotspots and enough attention should be paid to the dissemination risk of ARGs posed by globally produced legume crops.

Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases.

Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.

Oxygen regulatory mechanisms of nitrogen fixation in rhizobia.

Rhizobia are α- and ß-proteobacteria that form a symbiotic partnership with legumes, fixing atmospheric dinitrogen to ammonia and providing it to the plant. Oxygen regulation is key in this symbiosis. Fixation is performed by an oxygen-intolerant nitrogenase enzyme but requires respiration to meet its high energy demands. To satisfy these opposing constraints the symbiotic partners cooperate intimately, employing a variety of mechanisms to regulate and respond to oxygen concentration. During symbiosis rhizobia undergo significant changes in gene expression to differentiate into nitrogen-fixing bacteroids. Legumes host these bacteroids in specialized root organs called nodules. These generate a near-anoxic environment using an oxygen diffusion barrier, oxygen-binding leghemoglobin and control of mitochondria localization. Rhizobia sense oxygen using multiple interconnected systems which enable a finely-tuned response to the wide range of oxygen concentrations they experience when transitioning from soil to nodules. The oxygen-sensing FixL-FixJ and hybrid FixL-FxkR two-component systems activate at relatively high oxygen concentration and regulate fixK transcription. FixK activates the fixNOQP and fixGHIS operons producing a high-affinity terminal oxidase required for bacterial respiration in the microaerobic nodule. Additionally or alternatively, some rhizobia regulate expression of these operons by FnrN, an FNR-like oxygen-sensing protein. The final stage of symbiotic establishment is activated by the NifA protein, regulated by oxygen at both the transcriptional and protein level. A cross-species comparison of these systems highlights differences in their roles and interconnections but reveals common regulatory patterns and themes. Future work is needed to establish the complete regulon of these systems and identify other regulatory signals.

Nodule bacteria from the cultured legume Phaseolus dumosus (belonging to the Phaseolus vulgaris cross-inoculation group) with common tropici phenotypic characteristics and symbiovar but distinctive phylogenomic position and chromid.

Phaseolus dumosus is an endemic species from mountain tops in Mexico that was found in traditional agriculture areas in Veracruz, Mexico. P. dumosus plants were identified by ITS sequences and their nodules were collected from agricultural fields or from trap plant experiments in the laboratory. Bacteria from P. dumosus nodules were identified as belonging to the phaseoli-etli-leguminosarum (PEL) or to the tropici group by 16S rRNA gene sequences. We obtained complete closed genomes from two P. dumosus isolates CCGE531 and CCGE532 that were phylogenetically placed within the tropici group but with a distinctive phylogenomic position and low average nucleotide identity (ANI). CCGE531 and CCGE532 had common phenotypic characteristics with tropici type B rhizobial symbionts. Genome synteny analysis and ANI showed that P. dumosus isolates had different chromids and our analysis suggests that chromids have independently evolved in different lineages of the Rhizobium genus. Finally, we considered that P. dumosus and Phaseolus vulgaris plants belong to the same cross-inoculation group since they have conserved symbiotic affinites for rhizobia.

Sulfate is transported at significant rates through the symbiosome membrane and is crucial for nitrogenase biosynthesis.

Legume-rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced ("fixed") to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20-fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant-microbe interaction that is most relevant for agriculture and soil fertility.

A Third Class: Functional Gibberellin Biosynthetic Operon in Beta-Proteobacteria.

The ability of plant-associated microbes to produce gibberellin A (GA) phytohormones was first described for the fungal rice pathogen Gibberella fujikuroi in the 1930s. Recently the capacity to produce GAs was shown for several bacteria, including symbiotic alpha-proteobacteria (α-rhizobia) and gamma-proteobacteria phytopathogens. All necessary enzymes for GA production are encoded by a conserved operon, which appears to have undergone horizontal transfer between and within these two phylogenetic classes of bacteria. Here the operon was shown to be present and functional in a third class, the beta-proteobacteria, where it is found in several symbionts (ß-rhizobia). Conservation of function was examined by biochemical characterization of the enzymes encoded by the operon from Paraburkholderia mimosarum LMG 23256T. Despite the in-frame gene fusion between the short-chain alcohol dehydrogenase/reductase and ferredoxin, the encoded enzymes exhibited the expected activity. Intriguingly, together these can only produce GA9, the immediate precursor to the bioactive GA4, as the cytochrome P450 (CYP115) that catalyzes the final hydroxylation reaction is missing, similar to most α-rhizobia. However, phylogenetic analysis indicates that the operon from ß-rhizobia is more closely related to examples from gamma-proteobacteria, which almost invariably have CYP115 and, hence, can produce bioactive GA4. This indicates not only that ß-rhizobia acquired the operon by horizontal gene transfer from gamma-proteobacteria, rather than α-rhizobia, but also that they independently lost CYP115 in parallel to the α-rhizobia, further hinting at the possibility of detrimental effects for the production of bioactive GA4 by these symbionts.

Sulfur Transport and Metabolism in Legume Root Nodules.

Sulfur is an essential nutrient in plants as a constituent element of some amino acids, metal cofactors, coenzymes, and secondary metabolites. Not surprisingly, sulfur deficiency decreases plant growth, photosynthesis, and seed yield in both legumes and non-legumes. In nodulated legumes, sulfur supply is positively linked to symbiotic nitrogen fixation (SNF) and sulfur starvation causes three additional major effects: decrease of nodulation, inhibition of SNF, and slowing down of nodule metabolism. These effects are due, at least in part, to the impairment of nitrogenase biosynthesis and activity, the accumulation of nitrogen-rich amino acids, and the decline in leghemoglobin, ferredoxin, ATP, and glucose in nodules. During the last decade, some major advances have been made about the uptake and metabolism of sulfur in nodules. These include the identification of the sulfate transporter SST1 in the symbiosomal membrane, the finding that glutathione produced in the bacteroids and host cells is essential for nodule activity, and the demonstration that sulfur assimilation in the whole plant is reprogrammed during symbiosis. However, many crucial questions still remain and some examples follow. In the first place, it is of paramount importance to elucidate the mechanism by which sulfur deficiency limits SNF. It is unknown why homoglutahione replaces glutathione as a major water-soluble antioxidant, redox buffer, and sulfur reservoir, among other relevant functions, only in certain legumes and also in different tissues of the same legume species. Much more work is required to identify oxidative post-translational modifications entailing cysteine and methionine residues and to determine how these modifications affect protein function and metabolism in nodules. Likewise, most interactions of antioxidant metabolites and enzymes bearing redox-active sulfur with transcription factors need to be defined. Solving these questions will pave the way to decipher sulfur-dependent mechanisms that regulate SNF, thereby gaining a deep insight into how nodulated legumes adapt to the fluctuating availability of nutrients in the soil.

Function of glutathione peroxidases in legume root nodules.

Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function.

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.

Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and NO2- by alternative means to nitrate reductase, probably via a ˙NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.

Thiol-based redox signaling in the nitrogen-fixing symbiosis.

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 N 2 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.

Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress.

The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O(2) and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and 'truncated' (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O(2) affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O(2) scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.

An alternative nitrogenase is not expressed in molybdenum-deficient legume root nodules.

Three legume root nodule bacteria systems (Medicago polymorpha L. -Rhizobium meliloti, Ornithopus sativus Brot. -Bradyrhizobium lupini and Trifolium subterraneum L.- Rhizobium leguminosarum by. trifolii) were grown in solution culture under conditions likely to lead to the production of alternative nitrogenases (molybdenum-deficient, or molybdenum-deficient but supplemented with vanadium). Addition of 1 µM molybdenum produced significant responses in both nodule and top weights while 2 µM vanadium did not. Ethane, which is produced as well as ethylene when acetylene is reduced by vanadium nitrogenase or nitrogenase-3 from Azotobacter, was not found in significant amounts during assays of acetylene reduction in either molybdenum-deficient or molybdenum-deficient, vanadium-supplemented treatments, suggesting that no non-molybdenum nitrogenase was produced by these root nodule bacteria.