The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

IMA peptides regulate root nodulation and nitrogen homeostasis by providing iron according to internal nitrogen status.

Legumes control root nodule symbiosis (RNS) in response to environmental nitrogen availability. Despite the recent understanding of the molecular basis of external nitrate-mediated control of RNS, it remains mostly elusive how plants regulate physiological processes depending on internal nitrogen status. In addition, iron (Fe) acts as an essential element that enables symbiotic nitrogen fixation; however, the mechanism of Fe accumulation in nodules is poorly understood. Here, we focus on the transcriptome in response to internal nitrogen status during RNS in Lotus japonicus and identify that IRON MAN (IMA) peptide genes are expressed during symbiotic nitrogen fixation. We show that LjIMA1 and LjIMA2 expressed in the shoot and root play systemic and local roles in concentrating internal Fe to the nodule. Furthermore, IMA peptides have conserved roles in regulating nitrogen homeostasis by adjusting nitrogen-Fe balance in L. japonicus and Arabidopsis thaliana. These findings indicate that IMA-mediated Fe provision plays an essential role in regulating nitrogen-related physiological processes.

Soluble humic acid suppresses plant immunity and ethylene to promote soybean nodulation.

Symbiotic nitrogen fixation (SNF) is a crucial process for nitrogen geochemical cycling and plant-microbe interactions. Water-soluble humic acid (WSHM), an active component of soil humus, has been shown to promote SNF in the legume-rhizobial symbiosis, but its molecular mechanism remains largely unknown. To reveal the SNF-promoting mechanism, we conducted transcriptomic analysis on soybean treated with WSHM. Our findings revealed that up- and downregulated differentially expressed genes (DEGs) were mainly involved in plant cell-wall/membrane formation and plant defence/immunity in the early stage, while the late stage was marked by the flavonoid synthesis and ethylene biosynthetic process. Further study on representative DEGs showed that WSHM could inhibit GmBAK1d-mediated immunity and BR signalling, thereby promoting rhizobial colonisation, infection, and nodulation, while not favoring pathogenic bacteria colonisation on the host plant. Additionally, we also found that the ethylene pathway is necessary for promoting the soybean nodulation by WSHM. This study not only provides a significant advance in our understanding of the molecular mechanism of WSHM in promoting SNF, but also provides evidence of the beneficial interactions among the biostimulator, host plant, and soil microbes, which have not been previously reported.

Rhizobium symbiotic efficiency meets CEP signaling peptides.

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the Medicago truncatula model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among CEP genes, MtCEP7 was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation.

Legume nodulation and nitrogen fixation require interaction of DnaJ-like protein and lipid transfer protein.

The lipid transport protein (LTP) product of the AsE246 gene of Chinese milk vetch (Astragalus sinicus) contributes to the transport of plant-synthesized lipids to the symbiosome membranes (SMs) that are required for nodule organogenesis in this legume. However, the mechanisms used by nodule-specific LTPs remain unknown. In this study, a functional protein in the DnaJ-like family, designated AsDJL1, was identified and shown to interact with AsE246. Immunofluorescence showed that AsDJL1 was expressed in infection threads (ITs) and in nodule cells and that it co-localized with rhizobium, and an immunoelectron microscopy assay localized the protein to SMs. Via co-transformation into Nicotiana benthamiana cells, AsDJL1 and AsE246 displayed subcellular co-localization in the cells of this heterologous host. Co-immunoprecipitation assays confirmed that AsDJL1 interacted with AsE246 in nodules. The essential interacting region of AsDJL1 was determined to be the zinc finger domain at its C-terminus. Chinese milk vetch plants transfected with AsDJL1-RNAi had significantly decreased numbers of ITs, nodule primordia and nodules as well as reduced (by 83%) nodule nitrogenase activity compared with the controls. By contrast, AsDJL1 overexpression led to increased nodule fresh weight and nitrogenase activity. RNAi-AsDJL1 also significantly affected the abundance of lipids, especially digalactosyldiacylglycerol, in early-infected roots and transgenic nodules. Taken together, the results of this study provide insights into the symbiotic functions of AsDJL1, which may participate in lipid transport to SMs and play an essential role in rhizobial infection and nodule organogenesis.

The Phaseolus vulgaris Receptor-Like Kinase PvFER1 and the Small Peptides PvRALF1 and PvRALF6 Regulate Nodule Number as a Function of Nitrate Availability.

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

Local and systemic targets of the MtCLE35-SUNN pathway in the roots of Medicago truncatula.

CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION-related) peptides are systemic regulators of legume-rhizobium symbiosis that negatively control the number of nitrogen-fixing nodules. CLE peptides are produced in the root in response to rhizobia inoculation and/or nitrate treatment and are transported to the shoot where they are recognized by the CLV1-like (CLAVATA1-like) receptor kinase. As a result, a shoot-derived signaling pathway is activated that inhibits subsequent nodule development in the root. In Medicago truncatula, MtCLE35 is activated in response to rhizobia and nitrate treatment and the overexpression of this gene systemically inhibits nodulation. The inhibitory effect of MtCLE35 overexpression is dependent on the CLV1-like receptor kinase MtSUNN (SUPER NUMERIC NODULES), suggesting that MtSUNN could be involved in the reception of the MtCLE35 peptide. Yet little is known about the downstream genes regulated by a MtCLE35-activated response in the root. In order to identify genes whose expression levels could be regulated by the MtCLE35-MtSUNN pathway, we performed a MACE-Seq (Massive Analysis of cDNA Ends) transcriptomic analysis of MtCLE35-overexpressing roots. Among upregulated genes, the gene MtSUNN that encodes a putative receptor of MtCLE35 was detected. Moreover, we found that MtSUNN, as well as several other differentially expressed genes, were upregulated locally in MtCLE35-overexpressing roots whereas the MtTML1 and MtTML2 genes were upregulated systemically. Our data suggest that MtCLE35 has both local and systemic effects on target genes in the root.

The Medicago SymCEP7 hormone increases nodule number via shoots without compromising lateral root number.

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood. In Medicago (Medicago truncatula), a subset of mature C-TERMINALLY ENCODED PEPTIDE (CEP) hormones can systemically promote nodule number, but all CEP hormones tested to date negatively regulate lateral root number. Here we showed that Medicago CEP7 produces a mature peptide, SymCEP7, that promotes nodulation from the shoot without compromising lateral root number. Rhizobial inoculation induced CEP7 in the susceptible root nodulation zone in a Nod factor-dependent manner, and, in contrast to other CEP genes, its transcription level was elevated in the ethylene signaling mutant sickle. Using mass spectrometry, fluorescence microscopy and expression analysis, we demonstrated that SymCEP7 activity requires the COMPACT ROOT ARCHITECTURE 2 receptor and activates the shoot-to-root systemic effector, miR2111. Shoot-applied SymCEP7 rapidly promoted nodule number in the pM to nM range at concentrations up to five orders of magnitude lower than effects mediated by root-applied SymCEP7. Shoot-applied SymCEP7 also promoted nodule number in White Clover (Trifolium repens) and Lotus (Lotus japonicus), which suggests that this biological function may be evolutionarily conserved. We propose that SymCEP7 acts in the Medicago shoot to counter balance the autoregulation pathways induced rapidly by rhizobia to enable nodulation without compromising lateral root growth, thus promoting the acquisition of nutrients other than nitrogen to support their growth.

The small peptide CEP1 and the NIN-like protein NLP1 regulate NRT2.1 to mediate root nodule formation across nitrate concentrations.

Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N2-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.

A mutant-based analysis of the establishment of Nod-independent symbiosis in the legume Aeschynomene evenia.

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca2+/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.

Progress in the Self-Regulation System in Legume Nodule Development-AON (Autoregulation of Nodulation).

The formation and development of legumes nodules requires a lot of energy. Legumes must strictly control the number and activity of nodules to ensure efficient energy distribution. The AON system can limit the number of rhizobia infections and nodule numbers through the systemic signal pathway network that the aboveground and belowground parts participate in together. It can also promote the formation of nodules when plants are deficient in nitrogen. The currently known AON pathway includes four parts: soil NO3- signal and Rhizobium signal recognition and transmission, CLE-SUNN is the negative regulation pathway, CEP-CRA2 is the positive regulation pathway and the miR2111/TML module regulates nodule formation and development. In order to ensure the biological function of this important approach, plants use a variety of plant hormones, polypeptides, receptor kinases, transcription factors and miRNAs for signal transmission and transcriptional regulation. This review summarizes and discusses the research progress of the AON pathway in Legume nodule development.

A legume-specific novel type of phytosulfokine, PSK-δ, promotes nodulation by enhancing nodule organogenesis.

Phytosulfokine-α (PSK-α), a tyrosine-sulfated pentapeptide with the sequence YSO3IYSO3TQ, is widely distributed across the plant kingdom and plays multiple roles in plant growth, development, and immune response. Here, we report a novel type of phytosulfokine, PSK-δ, and its precursor proteins (MtPSKδ, LjPSKδ, and GmPSKδ1), specifically from legume species. The sequence YSO3IYSO3TN of sulfated PSK-δ peptide is different from PSK-α at the last amino acid. Expression pattern analysis revealed PSK-δ-encoding precursor genes to be expressed primarily in legume root nodules. Specifically, in Medicago truncatula, MtPSKδ expression was detected in root cortical cells undergoing nodule organogenesis, in nodule primordia and young nodules, and in the apical region of mature nodules. Accumulation of sulfated PSK-δ peptide in M. truncatula nodules was detected by LC/MS. Application of synthetic PSK-δ peptide significantly increased nodule number in legumes. Similarly, overexpression of MtPSKδ in transgenic M. truncatula markedly promoted symbiotic nodulation. This increase in nodule number was attributed to enhanced nodule organogenesis induced by PSK-δ. Additional genetic evidence from the MtPSKδ mutant and RNA interference assays suggested that the PSK-δ and PSK-α peptides function redundantly in regulating nodule organogenesis. These results suggest that PSK-δ, a legume-specific novel type of phytosulfokine, promotes symbiotic nodulation by enhancing nodule organogenesis.

Plant circadian clock control of Medicago truncatula nodulation via regulation of nodule cysteine-rich peptides.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules, we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcription factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of nodule-specific cysteine-rich peptides (NCRs) that have the LHY-bound conserved evening element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.

Role of ethylene in effective establishment of the peanut-bradyrhizobia symbiotic interaction.

Ethylene has been implicated in nitrogen fixing symbioses in legumes, where rhizobial invasion occurs via infection threads (IT). In the symbiosis between peanut (Arachis hypogaea L.) and bradyrhizobia, the bacteria penetrate the root cortex intercellularly and IT are not formed. Little attention has been paid to the function of ethylene in the establishment of this symbiosis. The aim of this article is to evaluate whether ethylene plays a role in the development of this symbiotic interaction and the participation of Nod Factors (NF) in the regulation of ethylene signalling. Manipulation of ethylene in peanut was accomplished by application of 1-aminocyclopropane-1-carboxylic acid (ACC), which mimics applied ethylene, or AgNO3, which blocks ethylene responses. To elucidate the participation of NF in the regulation of ethylene signalling, we inoculated plants with a mutant isogenic rhizobial strain unable to produce NF and evaluated the effect of AgNO3 on gene expression of NF and ethylene responsive signalling pathways. Data revealed that ethylene perception is required for the formation of nitrogen-fixing nodules, while addition of ACC does not affect peanut symbiotic performance. This phenotypic evidence is in agreement with transcriptomic data from genes involved in symbiotic and ethylene signalling pathways. NF seem to modulate the expression of ethylene signalling genes. Unlike legumes infected through IT formation, ACC addition to peanut does not adversely affect nodulation, but ethylene perception is required for establishment of this symbiosis. Evidence for the contribution of NF to the modulation of ethylene-inducible defence gene expression is provided.

Mesorhizobium ciceri as biological tool for improving physiological, biochemical and antioxidant state of Cicer aritienum (L.) under fungicide stress.

Fungicides among agrochemicals are consistently used in high throughput agricultural practices to protect plants from damaging impact of phytopathogens and hence to optimize crop production. However, the negative impact of fungicides on composition and functions of soil microbiota, plants and via food chain, on human health is a matter of grave concern. Considering such agrochemical threats, the present study was undertaken to know that how fungicide-tolerant symbiotic bacterium, Mesorhizobium ciceri affects the Cicer arietinum crop while growing in kitazin (KITZ) stressed soils under greenhouse conditions. Both in vitro and soil systems, KITZ imparted deleterious impacts on C. arietinum as a function of dose. The three-time more of normal rate of KITZ dose detrimentally but maximally reduced the germination efficiency, vigor index, dry matter production, symbiotic features, leaf pigments and seed attributes of C. arietinum. KITZ-induced morphological alterations in root tips, oxidative damage and cell death in root cells of C. arietinum were visible under scanning electron microscope (SEM). M. ciceri tolerated up to 2400 µg mL-1 of KITZ, synthesized considerable amounts of bioactive molecules including indole-3-acetic-acid (IAA), 1-aminocyclopropane 1-carboxylate (ACC) deaminase, siderophores, exopolysaccharides (EPS), hydrogen cyanide, ammonia, and solubilised inorganic phosphate even in fungicide-stressed media. Following application to soil, M. ciceri improved performance of C. arietinum and enhanced dry biomass production, yield, symbiosis and leaf pigments even in a fungicide-polluted environment. At 96 µg KITZ kg-1 soil, M. ciceri maximally and significantly (p ≤ 0.05) augmented the length of plants by 41%, total dry matter by 18%, carotenoid content by 9%, LHb content by 21%, root N by 9%, shoot P by 11% and pod yield by 15% over control plants. Additionally, the nodule bacterium M. ciceri efficiently colonized the plant rhizosphere/rhizoplane and considerably decreased the levels of stressor molecules (proline and malondialdehyde) and antioxidant defence enzymes viz. ascorbate peroxidise (APX), guaiacol peroxidise (GPX), catalase (CAT) and peroxidises (POD) of C. arietinum plants when inoculated in soil. The symbiotic strain effectively colonized the plant rhizosphere/rhizoplane. Conclusively, the ability to endure higher fungicide concentrations, capacity to secrete plant growth modulators even under fungicide pressure, and inherent features to lower the level of proline and plant defence enzymes makes this M. ciceri as a superb choice for augmenting the safe production of C. arietinum even under fungicide-contaminated soils.

Bradyrhizobium diazoefficiens Requires Chemical Chaperones To Cope with Osmotic Stress during Soybean Infection.

When engaging in symbiosis with legume hosts, rhizobia are confronted with environmental changes, including nutrient availability and stress exposure. Genetic circuits allow responding to these environmental stimuli to optimize physiological adaptations during the switch from the free-living to the symbiotic life style. A pivotal regulatory system of the nitrogen-fixing soybean endosymbiont Bradyrhizobium diazoefficiens for efficient symbiosis is the general stress response (GSR), which relies on the alternative sigma factor σEcfG However, the GSR-controlled process required for symbiosis has not been identified. Here, we demonstrate that biosynthesis of trehalose is under GSR control, and mutants lacking the respective biosynthetic genes otsA and/or otsB phenocopy GSR-deficient mutants under symbiotic and selected free-living stress conditions. The role of trehalose as a cytoplasmic chemical chaperone and stress protectant can be functionally replaced in an otsA or otsB mutant by introducing heterologous genetic pathways for biosynthesis of the chemically unrelated compatible solutes glycine betaine and (hydroxy)ectoine. Alternatively, uptake of exogenously provided trehalose also restores efficient symbiosis and tolerance to hyperosmotic and hyperionic stress of otsA mutants. Hence, elevated cytoplasmic trehalose levels resulting from GSR-controlled biosynthesis are crucial for B. diazoefficiens cells to overcome adverse conditions during early stages of host infection and ensure synchronization with root nodule development.IMPORTANCE The Bradyrhizobium-soybean symbiosis is of great agricultural significance and serves as a model system for fundamental research in bacterium-plant interactions. While detailed molecular insight is available about mutual recognition and early nodule organogenesis, our understanding of the host-imposed conditions and the physiology of infecting rhizobia during the transition from a free-living state in the rhizosphere to endosymbiotic bacteroids is currently limited. In this study, we show that the requirement of the rhizobial general stress response (GSR) during host infection is attributable to GSR-controlled biosynthesis of trehalose. Specifically, trehalose is crucial for an efficient symbiosis by acting as a chemical chaperone to protect rhizobia from osmostress during host infection.

The influence of ethylene, gibberellins and brassinosteroids on energy and nitrogen-fixation metabolites in nodule tissue.

Legume nodules are a unique plant organ that contain nitrogen-fixing rhizobial bacteria. For this interaction to be mutually beneficial, plant and bacterial metabolism must be precisely co-ordinated. Plant hormones are known to play essential roles during the establishment of legume-rhizobial symbioses but their role in subsequent nodule metabolism has not been explored in any depth. The plant hormones brassinosteroids, ethylene and gibberellins influence legume infection, nodule number and in some cases nodule function. In this paper, the influence of these hormones on nodule metabolism was examined in a series of well characterised pea mutants with altered hormone biosynthesis or response. A targeted set of metabolites involved in nutrient exchange and nitrogen fixation was examined in nodule tissue of mutant and wild type plants. Gibberellin-deficiency had a major negative impact on the level of several major dicarboxylates supplied to rhizobia by the plant and also led to a significant deficit in the amino acids involved in glutamine-aspartate transamination, consistent with the limited bacteroid development and low fixation rate of gibberellin-deficient na mutant nodules. In contrast, no major effects of brassinosteroid-deficiency or ethylene-insensitivity on the key metabolites in these pathways were found. Therefore, although all three hormones influence infection and nodule number, only gibberellin is important for the establishment of a functional nodule metabolome.

Sustainable effect of a symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti on nodulation and photosynthetic traits of four leguminous plants under low moisture stress environment.

Sustainable effect of a nitrogen-fixing bacterium Sinorhizobium meliloti on nodulation and photosynthetic traits (phenomenological fluxes) in four leguminous plants species under low moisture stress (20-25% soil moisture content) environment was studied. Sinorhizobium meliloti was isolated from fenugreek (Trigonella foenum-graecum) root nodules, and later, it was cultured and purified. Nodulation and photosynthetic ability in the presence of S. meliloti were tested in four leguminous plant species, that is, kidney bean (cv. lobia-2000), black bean (cv. NM-97), mung bean (cv. NM-2006) and chickpea (cv. Pb-2008). Plants of each species were grown in sterilized soil that was previously treated with 25 ml suspension containing S. meliloti at 41 × 106  CFU ml-1  kg-1 pot. One-month-old plants were subjected to low soil moisture stress conditions for 15 days, and soil moisture contents were maintained to 20-25% throughout the experimental period. The ability to fix nitrogen, nodule formation, and their subsequent effect on phenomenological fluxes in low moisture treated legumes were studied.

Synthetase of the methyl donor S-adenosylmethionine from nitrogen-fixing α-rhizobia can bind functionally diverse RNA species.

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti. The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans-sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme.