Rhizobia cystathionine γ-lyase-derived H2S delays nodule senescence in soybean.

Hydrogen sulphide (H2S) is required for optimal establishment of soybean (Glycine max)-Sinorhizobium fredii symbiotic interaction, yet its role in regulating the nitrogen fixation-senescence transition remains poorly understood. A S. fredii cystathionine γ-lyase (CSE) mutant deficient in H2S synthesis showed early nodule senescence characterized by reduced nitrogenase activity, structural changes in nodule cells, and accelerated bacteroid death. In parallel, the CSE mutant facilitated the generation of reactive oxygen species (ROS) and elicited antioxidant responses. We observed that H2S-mediated persulfidation of cysteine C31/C80 in ascorbate peroxidase (APX) and C32 in APX2 modulated enzyme activity, thereby participating in hydrogen peroxide (H2O2) detoxification and delaying nodule senescence. Comparative transcriptomic analysis revealed a significant up-regulation of GmMYB128, an MYB transcription factor (TF), in the CSE mutant nodules. Functional analysis through overexpression and RNAi lines of GmMYB128 demonstrated its role as a positive regulator in nodule senescence. MYB128-OE inoculated with the CSE mutant strain exhibited a reduction in nitrogenase activity and a significant increase in DD15 expression, both of which were mitigated by NaHS addition. Changes at the protein level encompassed the activation of plant defenses alongside turnover in carbohydrates and amino acids. Our results suggest that H2S plays an important role in maintaining efficient symbiosis and preventing premature senescence of soybean nodules.

Dynamics of hemoglobins during nodule development, nitrate response, and dark stress in Lotus japonicus.

Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.

Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the in vivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.

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.

Persulfidation of plant and bacteroid proteins is involved in legume nodule development and senescence.

Legumes establish symbiosis with rhizobia forming nitrogen-fixing nodules. The central role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in nodule biology has been clearly established. Recently, hydrogen sulfide (H2S) and other reactive sulfur species (RSS) have emerged as novel signaling molecules in animals and plants. A major mechanism by which ROS, RNS, and RSS fulfil their signaling role is the post-translational modification of proteins. To identify possible functions of H2S in nodule development and senescence, we used the tag-switch method to quantify changes in the persulfidation profile of common bean (Phaseolus vulgaris) nodules at different developmental stages. Proteomic analyses indicate that persulfidation plays a regulatory role in plant and bacteroid metabolism and senescence. The effect of a H2S donor on nodule functioning and on several proteins involved in ROS and RNS homeostasis was also investigated. Our results using recombinant proteins and nodulated plants support a crosstalk among H2S, ROS and RNS, a protective function of persulfidation on redox-sensitive enzymes, and a beneficial effect of H2S on symbiotic nitrogen fixation. We conclude that the general decrease of persulfidation levels observed in plant proteins of aging nodules is one of the mechanisms that disrupt redox homeostasis leading to senescence.

Signaling in Legume-Rhizobia Symbiosis.

Legumes represent an important source of food protein for human nutrition and animal feed. Therefore, sustainable production of legume crops is an issue of global importance. It is well-known that legume-rhizobia symbiosis allows an increase in the productivity and resilience of legume crops. The efficiency of this mutualistic association strongly depends on precise regulation of the complex interactions between plant and rhizobia. Their molecular dialogue represents a complex multi-staged process, each step of which is critically important for the overall success of the symbiosis. In particular, understanding the details of the molecular mechanisms behind the nodule formation and functioning might give access to new legume cultivars with improved crop productivity. Therefore, here we provide a comprehensive literature overview on the dynamics of the signaling network underlying the development of the legume-rhizobia symbiosis. Thereby, we pay special attention to the new findings in the field, as well as the principal directions of the current and prospective research. For this, here we comprehensively address the principal signaling events involved in the nodule inception, development, functioning, and senescence.

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation.

Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA-sequencing (RNA-seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly-ß-hydroxybutyrate and disruption of mitochondria. RNA-seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb-deficient nodules had c. 30-50-fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post-translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.

Nitric oxide in plants: pro- or anti-senescence.

Senescence is a regulated process of tissue degeneration that can affect any plant organ and consists of the degradation and remobilization of molecules to other growing tissues. Senescent organs display changes at the microscopic level as well as modifications to internal cellular structure and differential gene expression. A large number of factors influencing senescence have been described including age, nutrient supply, and environmental interactions. Internal factors such as phytohormones also affect the timing of leaf senescence. A link between the senescence process and the production of nitric oxide (NO) in senescing tissues has been known for many years. Remarkably, this link can be either a positive or a negative correlation depending upon the organ. NO can be both a signaling or a toxic molecule and is known to have multiple roles in plants; this review considers the duality of NO roles in the senescence process of two different plant organs, namely the leaves and root nodules.

Drought-induced senescence of Medicago truncatula nodules involves serpin and ferritin to control proteolytic activity and iron levels.

Drought is a major constraint for legume growth and yield. Senescence of nitrogen-fixing nodules is one of the early drought responses and may cause nutrient stress in addition to water stress in legumes. For nodule senescence to function as part of a drought-survival strategy, we propose that the intrinsically destructive senescence process must be tightly regulated. Medicago truncatula protease inhibitor and iron scavenger-encoding genes, possibly involved in controlling nodule senescence, were identified. RNA interference (RNAi) lines were constructed in which expression of a serpin or ferritins was knocked down. Both wild-type and RNAi lines were subjected to drought stress and nodule activity and plant physiological responses were measured. Drought caused M. truncatula to initiate nodule senescence before plant growth was affected and before an increase in papain-like proteolytic activity and free iron levels was apparent. Knock-down expression of serpin6 and ferritins caused increased protease activity, free iron levels, early nodule senescence and reduced plant growth. The results suggest that M. truncatula nodule-expressed serpin6 and ferritins mediate ordered drought-induced senescence by regulating papain-like cysteine protease activity and free iron levels. This strategy may allow the drought-stressed plants to benefit maximally from residual nitrogen fixation and nutrient recovery resulting from break down of macromolecules.

Cell autonomous sanctions in legumes target ineffective rhizobia in nodules with mixed infections.

PREMISE OF THE STUDY: To maximize benefits from symbiosis, legumes must limit physiological inputs into ineffective rhizobia that nodulate hosts without fixing nitrogen. The capacity of legumes to decrease the relative fitness of ineffective rhizobia-known as sanctions-has been demonstrated in several legume species, but its mechanisms remain unclear. Sanctions are predicted to work at the whole-nodule level. However, whole-nodule sanctions would make the host vulnerable to mixed-nodule infections, which have been demonstrated in the laboratory and observed in natural settings. Here, we present and test a cell-autonomous model of legume sanctions that can resolve this dilemma. METHODS: We analyzed histological and ultrastructural evidence of sanctions in two legume species, Acmispon strigosus and Lotus japonicus. For the former, we inoculated seedlings with rhizobia that naturally vary in their abilities to fix nitrogen. In the latter, we inoculated seedlings with near-isogenic strains that differ only in the ability to fix nitrogen. KEY RESULTS: In both hosts, plants inoculated with ineffective rhizobia exhibited evidence for a cell autonomous and accelerated program of senescence within nodules. In plants that received mixed inoculations, only the plant cells harboring ineffective rhizobia exhibited features consistent with programmed cell death, including collapsed vacuoles, ruptured symbiosomes, and bacteroids that are released into the cytosol. These features were consistently linked with ultrastructural evidence of reduced survival of ineffective rhizobia in planta. CONCLUSIONS: Our data suggest an elegant cell autonomous mechanism by which legumes can detect and defend against ineffective rhizobia even when nodules harbor a mix of effective and ineffective rhizobial genotypes.

RbohB, a Phaseolus vulgaris NADPH oxidase gene, enhances symbiosome number, bacteroid size, and nitrogen fixation in nodules and impairs mycorrhizal colonization.

The reactive oxygen species (ROS) generated by respiratory burst oxidative homologs (Rbohs) are involved in numerous plant cell signaling processes, and have critical roles in the symbiosis between legumes and nitrogen-fixing bacteria. Previously, down-regulation of RbohB in Phaseolus vulgaris was shown to suppress ROS production and abolish Rhizobium infection thread (IT) progression, but also to enhance arbuscular mycorrhizal fungal (AMF) colonization. Thus, Rbohs function both as positive and negative regulators. Here, we assessed the effect of enhancing ROS concentrations, by overexpressing PvRbohB, on the P. vulgaris--rhizobia and P. vulgaris--AMF symbioses. We estimated superoxide concentrations in hairy roots overexpressing PvRbohB, determined the status of early and late events of both Rhizobium and AMF interactions in symbiont-inoculated roots, and analyzed the nodule ultrastructure of transgenic plants overexpressing PvRbohB. Overexpression of PvRbohB significantly enhanced ROS production, the formation of ITs, nodule biomass, and nitrogen-fixing activity, and increased the density of symbiosomes in nodules, and the density and size of bacteroides in symbiosomes. Furthermore, PvCAT, early nodulin, PvSS1, and PvGOGAT transcript abundances were elevated in these nodules. By contrast, mycorrhizal colonization was reduced in roots that overexpressed RbohB. Overexpression of PvRbohB augmented nodule efficiency by enhancing nitrogen fixation and delaying nodule senescence, but impaired AMF colonization.

VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle.

The soil bacterium Sinorhizobium meliloti can establish a nitrogen-fixing symbiosis with the model legume Medicago truncatula. The rhizobia induce the formation of a specialized root organ called nodule, where they differentiate into bacteroids and reduce atmospheric nitrogen into ammonia. Little is known on the mechanisms involved in nodule senescence onset and in bacteroid survival inside the infected plant cells. Although toxin-antitoxin (TA) systems have been shown to promote intracellular survival within host cells in human pathogenic bacteria, their role in symbiotic bacteria was rarely investigated. S. meliloti encodes several TA systems, mainly of the VapBC family. Here we present the functional characterization, through a multidisciplinary approach, of the VapBC10 TA system of S. meliloti. Following a mapping by overexpression of an RNase in Escherichia coli (MORE) RNA-seq analysis, we demonstrated that the VapC10 toxin is an RNase that cleaves the anticodon loop of two tRNASer. Thereafter, a bioinformatics approach was used to predict VapC10 targets in bacteroids. This analysis suggests that toxin activation triggers a specific proteome reprogramming that could limit nitrogen fixation capability and viability of bacteroids. Accordingly, a vapC10 mutant induces a delayed senescence in nodules, associated to an enhanced bacteroid survival. VapBC10 TA system could contribute to S. meliloti adaptation to symbiotic lifestyle, in response to plant nitrogen status.

Staying hungry: a roadmap to harnessing central regulators of symbiotic nitrogen fixation under fluctuating nitrogen availability.

Legumes have evolved specific inventions to enhance nitrogen (N) acquisition by establishing symbiotic interactions with N-fixing rhizobial bacteria. Because symbiotic N fixation is energetically costly, legumes have developed sophisticated mechanisms to ensure carbon-nitrogen balance, in a variable environment, both locally and at the whole plant level, by monitoring nodule number, nodule development, and nodular nitrogenase activity, as well as controlling nodule senescence. Studies of the autoregulation of nodulation and regulation of nodulation by nodule inception (NIN) and NIN-LIKE PROTEINs (NLPs) have provided great insights into the genetic mechanisms underlying the nitrate-induced regulation of root nodulation for adapting to N availability in the rhizosphere. However, many aspects of N-induced pleiotropic regulation remain to be fully explained, such as N-triggered senescence in mature nodules. Wang et al. determined that this process is governed by a transcriptional network regulated by NAC-type transcription factors. Characterization and dissection of these soybean nitrogen-associated NAPs (SNAPs) transcription factor-mastered networks have yielded a roadmap for exploring how legumes rewire nodule functions across a range of N levels, laying the foundation for enhancing the growth of N-deprived crops in agricultural settings.

Characteristics and Research Progress of Legume Nodule Senescence.

Delaying the nodule senescence of legume crops can prolong the time of nitrogen fixation and attenuate the lack of fertilizer in the later stage of legume crop cultivation, resulting in improved crop yield and reduced usage of nitrogen fertilizer. However, effective measures to delay the nodule senescence of legume crops in agriculture are relatively lacking. In the present review, we summarized the structural and physiological characteristics of nodule senescence, as well as the corresponding detection methods, providing technical support for the identification of nodule senescence phenotype. We then outlined the key genes currently known to be involved in the regulation of nodule senescence, offering the molecular genetic information for breeding varieties with delayed nodule senescence. In addition, we reviewed various abiotic factors affecting nodule senescence, providing a theoretical basis for the interaction between molecular genetics and abiotic factors in the regulation of nodule senescence. Finally, we briefly prospected research foci of nodule senescence in the future.

Gibberellins Inhibit Nodule Senescence and Stimulate Nodule Meristem Bifurcation in Pea (Pisum sativum L.).

The development of nitrogen-fixing nodules formed during Rhizobium-legume symbiosis is strongly controlled by phytohormones. In this study, we investigated the effect of gibberellins (GAs) on senescence of pea (Pisum sativum) symbiotic nodules. Pea wild-type line SGE, as well as corresponding mutant lines SGEFix--1 (sym40), SGEFix--2 (sym33), SGEFix--3 (sym26), and SGEFix--7 (sym27), blocked at different stages of nodule development, were used in the study. An increase in expression of the GA2ox1 gene, encoding an enzyme involved in GA deactivation (GA 2-oxidase), and a decrease in the transcript abundance of the GA20ox1 gene, encoding one of the enzymes involved in GA biosynthesis (GA 20-oxidase), were observed in analyzed genotypes during nodule aging. A reduction in the amount of bioactive GA3 was demonstrated by immunolocalization in the early senescent mutant and wild-type lines during aging of symbiotic nodules. Down-regulated expression of senescence-associated genes encoding cysteine proteases 1 and 15a, thiol protease, bZIP transcription factor, 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, and aldehyde oxidase was observed in the nodules of wild-type plants treated with exogenous GA3 relative to the untreated plants. GA3-treated plants also showed increases in nodule size and the nitrogen fixation zone, and decreases in the number of nodules and the senescence zone. Immunogold localization revealed higher levels of GA3 in the peribacteroid spaces in symbiosomes than in the matrix of infection threads. Furthermore, a decrease in GA3 label in mature and senescent symbiosomes in comparison with juvenile symbiosomes was observed. These results suggest a negative effect of GAs on the senescence of the pea symbiotic nodule and possible involvement of GAs in functioning of the mature nodule. Simultaneously, GA3 treatment led to nodule meristem bifurcation, indicating a possible role of GAs in nodule meristem functioning.

Iron-induced nitric oxide leads to an increase in the expression of ferritin during the senescence of Lotus japonicus nodules.

Iron is an essential nutrient for legume-rhizobium symbiosis and accumulates abundantly in the nodules. However, the concentration of free iron in the cells is strictly controlled to avoid toxicity. It is known that ferritin accumulates in the cells as an iron storage protein. During nodule senescence, the expression of the ferritin gene, Ljfer1, was induced in Lotus japonicus. We investigated a signal transduction pathway leading to the increase of Ljfer1 in the nodule. The Ljfer1 promoter of L. japonicus contains a conserved Iron-Dependent Regulatory Sequence (IDRS). The expression of Ljfer1 was induced by the application of iron or sodium nitroprusside, which is a nitric oxide (NO) donor. The application of iron to the nodule increased the level of NO. These data strongly suggest that iron-induced NO leads to increased expression of Ljfer1 during the senescence of L. japonicus nodules.

Ethylene, a Hormone at the Center-Stage of Nodulation.

Nodulation is the result of a beneficial interaction between legumes and rhizobia. It is a sophisticated process leading to nutrient exchange between the two types of symbionts. In this association, within a nodule, the rhizobia, using energy provided as photosynthates, fix atmospheric nitrogen and convert it to ammonium which is available to the plant. Nodulation is recognized as an essential process in nitrogen cycling and legume crops are known to enrich agricultural soils in nitrogenous compounds. Furthermore, as they are rich in nitrogen, legumes are considered important as staple foods for humans and fodder for animals. To tightly control this association and keep it mutualistic, the plant uses several means, including hormones. The hormone ethylene has been known as a negative regulator of nodulation for almost four decades. Since then, much progress has been made in the understanding of both the ethylene signaling pathway and the nodulation process. Here I have taken a large view, using recently obtained knowledge, to describe in some detail the major stages of the process. I have not only reviewed the steps most commonly covered (the common signaling transduction pathway, and the epidermal and cortical programs), but I have also looked into steps less understood (the pre-infection step with the plant defense response, the bacterial release and the formation of the symbiosome, and nodule functioning and senescence). After a succinct review of the ethylene signaling pathway, I have used the knowledge obtained from nodulation- and ethylene-related mutants to paint a more complete picture of the role played by the hormone in nodule organogenesis, functioning, and senescence. It transpires that ethylene is at the center of this effective symbiosis. It has not only been involved in most of the steps leading to a mature nodule, but it has also been implicated in host immunity and nodule senescence. It is likely responsible for the activation of other hormonal signaling pathways. I have completed the review by citing three studies which makes one wonder whether knowledge gained on nodulation in the last decades is ready to be transferred to agricultural fields.

Antioxidant activities in mycorrhizal soybean plants under drought stress and their possible relationship to the process of nodule senescence.

• The mechanisms by which the mycorrhizal symbiosis protects soybean ( Glycine max ) plants against premature nodule senescence induced by drought stress is investigated here by evaluating the activity of a set of antioxidant enzymes in relation to nodule senescence. • Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activity was determined in well watered or drought-stressed soybean plants inoculated with Bradyrrhizobium japonicum alone or in combination with Glomus mosseae . • In roots, only GR activity was higher in mycorrhizal than in non-mycorrhizal plants. The other antioxidant activities were similar, or lower (APX), in droughted, mycorrhizal plants than in the corresponding nonmycorrhizal ones. Similarly, in nodules, SOD, CAT and APX activities were lower in droughted, mycorrhizal plants than in nonmycorrhizal plants whereas, again, GR activity was higher in nodules from mycorrhizal plants. • We propose that the consistently higher GR activity in roots and nodules of mycorrhizal plants might have contributed to decreased oxidative damage to biomolecules, which are involved in premature nodule senescence. Additional drought-avoidance mechanisms induced by the AM symbiosis might also contribute to the lower oxidative stress in mycorrhizal plants.