Nodulation Genes Research Articles

Knockdown of GmD53a confers strigolactones mediated rhizobia interaction and promotes nodulation in soybean.

BackgroundStrigolactones (SLs) play a key role in modulating plant root growth, shoot branching, and plant-symbiont interaction. However, despite their significance, the components of SL biosynthesis and signaling in soybean and their role in soybean-rhizobia interaction is unknown.MethodsIn this study, we identified and functionally characterized the GmD53a from soybean. The GmD53a ORFs were amplified from root cDNA using primers for GmD53a RNA interference. To induce transgenic hairy roots of soybean, electric shock was used to transform pB7WG1WG2 vectors containing GmD53a knockdown and GUS into K599 strains of Agrobacterium rhizogenes. The hairy roots and nodules were collected and examined for root nodules ratio and RNA was extracted after 4 weeks of rhizobia inoculation.ResultsA tissue-specific expression assay showed that GmD53a was differentially expressed in plant parts, predominantly in the stem and nodule. Furthermore, its expression was significantly up-regulated during rhizobia infection and varied with nodule formation. The GmD53a-knockdown chimerical plants were produced to further check its role in soybean nodulation in comparison with control GUS. In knockdown lines, the GmD53a (suppressor of strigolactone MAX2) has a higher number of nodules compared to control lines. Furthermore, the expression levels of several nodulation genes essential for initiation and formation of nodules were altered in GmD53a-knockdown lines.ConclusionThe results revealed that SL biosynthesis and signaling are not conserved but also have close interaction between SL and legume rhizobia.

Genome wide analysis of DWARF27 genes in soybean and functional characterization of GmD27c reveals eminent role of strigolactones in rhizobia interaction and nodulation in Glycine max.

Strigolactones (SLs) are newly identified hormones and their biosynthesis is stimulated under phosphate deprivation and accomplished by the action of several enzymes, including the beta-carotene isomerase DWARF27 (D27). Expression of D27 is well renowned to respond to phosphate insufficiency. However, the identification and functional analysis of the carotenoid isomerase D27 genes are not elucidated in soybean. A total of six D27 genes were identified in the soybean genome and designated on the basis of chromosomal localization. According to the findings, these genes were irregularly distributed on chromosomes, and segmental repetition led to the expansion of the soybean GmD27 gene family. Based on a neighbor-joining phylogenetic tree, the predicted D27 proteins of soybean were divided into three clades. Based on RNA seq data analysis, GmD27 genes were differently expressed in various tissues but GmD27c was the highest. Therefore, GmD27c was chosen for the additional functional study due to its rather obvious transcription in nodulation and roots. RT-qPCR results showed that GmD27c was highly expressed in different nodule stages and in response to rhizobia infection. Functional characterization of GmD27c revealed that overexpression of GmD27c led to higher nodule number, while GmD27c knockdown caused fewer nodules compared to GUS control. Furthermore, GmD27c overexpressed and knockdown lines oppositely regulated the expression of numerous nodulation genes, which are vital for the development of nodules. This study not only discovered that SL biosynthesis and signaling pathway genes are conserved, but it also revealed that SL biosynthesis gene GmD27c and legume rhizobia have close interactions in controlling plant nodule number.

The nodD1 Gene of Sinorhizobium fredii HH103 Restores Nodulation Capacity on Bean in a Rhizobium tropici CIAT 899 nodD1/nodD2 Mutant, but the Secondary Symbiotic Regulators nolR, nodD2 or syrM Prevent HH103 to Nodulate with This Legume.

Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.

Spatiotemporal changes in gibberellin content are required for soybean nodulation.

The plant hormone gibberellin (GA) is required at different stages of legume nodule development, with its spatiotemporal distribution tightly regulated. Transcriptomic and bioinformatic analyses established that several key GA biosynthesis and catabolism enzyme encoding genes are critical to soybean (Glycine max) nodule formation. We examined the expression of several GA oxidase genes and used a Förster resonance energy transfer-based GA biosensor to determine the bioactive GA content of roots inoculated with DsRed-labelled Bradyrhizobium diazoefficiens. We manipulated the level of GA by genetically disrupting the expression of GA oxidase genes. Moreover, exogenous treatment of soybean roots with GA3 induced the expression of key nodulation genes and altered infection thread and nodule phenotypes. GmGA20ox1a, GmGA3ox1a, and GmGA2ox1a are upregulated in soybean roots inoculated with compatible B. diazoefficiens. GmGA20ox1a expression is predominately localized to the transient meristem of soybean nodules and coincides with the spatiotemporal distribution of bioactive GA occurring throughout nodule organogenesis. GmGA2ox1a exhibits a nodule vasculature-specific expression pattern, whereas GmGA3ox1a can be detected throughout the nodule and root. Disruptions in the level of GA resulted in aberrant rhizobia infection and reduced nodule numbers. Collectively, our results establish a central role for GAs in root hair infection by symbiotic rhizobia and in nodule organogenesis.

Phenolic Acids Induce Nod Factor Production in Lotus japonicus-Mesorhizobium Symbiosis.

In legume–rhizobia symbiosis, partner recognition and the initiation of symbiosis processes require the mutual exchange of chemical signals. Chemicals, generally (iso)flavonoids, in the root exudates of the host plant induce the expression of nod genes in rhizobia, and, thus, are called nod gene inducers. The expression of nod genes leads to the production of lipochitooligosaccharides (LCOs) called Nod factors. Natural nod gene inducer(s) in Lotus japonicus–Mesorhizobium symbiosis remain unknown. Therefore, we developed an LCO detection method based on ultra-high-performance liquid chromatography–tandem-quadrupole mass spectrometry (UPLC-TQMS) to identify these inducers and used it herein to screen 40 phenolic compounds and aldonic acids for their ability to induce LCOs in Mesorhizobium japonicum MAFF303099. We identified five phenolic acids with LCO-inducing activities, including p-coumaric, caffeic, and ferulic acids. The induced LCOs caused root hair deformation, and nodule numbers in L. japonicus inoculated with M. japonicum were increased by these phenolic acids. The three phenolic acids listed above induced the expression of the nodA, nodB, and ttsI genes in a strain harboring a multicopy plasmid encoding NodD1, but not that encoding NodD2. The presence of p-coumaric and ferulic acids in the root exudates of L. japonicus was confirmed by UPLC-TQMS, and the induction of ttsI::lacZ in the strain harboring the nodD1 plasmid was detected in the rhizosphere of L. japonicus. Based on these results, we propose that phenolic acids are a novel type of nod gene inducer in L. japonicus–Mesorhizobium symbiosis.

Modern concepts of the role of NOD gene polymorphism in the pathogenesis of tube infertility in patients with a past chlamydia infection

Modern concepts of the role of NOD gene polymorphism in the pathogenesis of tube infertility in patients with a past chlamydia infection

Symbiosis Contribution of Non-nodulating Bradyrhizobium cosmicum S23321 after Transferal of the Symbiotic Plasmid pDOA9.

The symbiotic properties of rhizobial bacteria are driven by the horizontal gene transfer of symbiotic genes, which are located in symbiosis islands or on plasmids. The symbiotic megaplasmid pDOA9 of Bradyrhizobium sp. DOA9, carrying the nod, nif, fix, and type three secretion system (T3SS) genes, has been conjugatively transferred to different Bradyrhizobium strains. In the present study, non-nodulating B. cosmicum S23321, which shows a close phylogenetic relationship with Bradyrhizobium sp. DOA9, but lacks symbiotic properties, was used to carry pDOA9 (annotated as chimeric S2:pDOA9). The results obtained showed that pDOA9 conferred symbiotic properties on S23321; however, nodulation phenotypes varied among the DOA9, chimeric ORS278:pDOA9, and S2:pDOA9 strains even though they all carried symbiotic pDOA9 plasmid. S23321 appeared to gain symbiotic nodulation from pDOA9 by processing nodulation genes and broadening the host range. The present results also showed the successful formation of active nodules in Arachis hypogaea (Dalbergoid) and Vigna radiata (Millitoid) by chimeric S2:pDOA9, while Crotalaria juncea (Genistoid) and Macroptilium atropurpureum (Millitoid) formed nodule-like structures. The formation of nodules and nodule-like structures occurred in a nod factor-dependent manner because the nod factor-lacking strain (S2:pDOA9ΩnodB) completely abolished nodulation in all legumes tested. Moreover, T3SS carried by S2:pDOA9 exerted negative effects on symbiosis with Crotalaria juncea, which was consistent with the results obtained on DOA9. T3SS exhibited symbiotic compatibility with V. radiata when nodulated by S23321. These outcomes implied that pDOA9 underwent changes during legume evolution that broadened host specificity and the compatibility of nodulation in a manner that was dependent on the chromosomal background of the recipient as well as legume host restrictions.

The miR156b-GmSPL9d module modulates nodulation by targeting multiple core nodulation genes in soybean.

Summary Symbiotic nodulation is initiated in the roots of legumes in response to low nitrogen and rhizobial signal molecules and is dynamically regulated by a complex regulatory network that coordinates rhizobial infection and nodule organogenesis.It has been shown that the miR156‐SPL module mediates nodulation in legumes; however, conclusive evidence of how this module exerts its function during nodulation remains elusive.Here, we report that the miR156b‐GmSPL9d module regulates symbiotic nodulation by targeting multiple key regulatory genes in the nodulation signalling pathway of soybean. miR156 family members are differentially expressed during nodulation, and miR156b negatively regulates nodulation by mainly targeting soybean SQUAMOSA promoter‐binding protein‐like 9d (GmSPL9d), a positive regulator of soybean nodulation. GmSPL9d directly binds to the miR172c promoter and activates its expression, suggesting a conserved role of GmSPL9d. Furthermore, GmSPL9d was coexpressed with the soybean nodulation marker genes nodule inception a (GmNINa) and GmENOD40‐1 during nodule formation and development. Intriguingly, GmSPL9d can bind to the promoters of GmNINa and GmENOD40‐1 and regulate their expression.Our data demonstrate that the miR156b‐GmSPL9d module acts as an upstream master regulator of soybean nodulation, which coordinates multiple marker genes involved in soybean nodulation.

Overexpression of Terpenoid Biosynthesis Genes From Garden Sage (Salvia officinalis) Modulates Rhizobia Interaction and Nodulation in Soybean.

In legumes, many endogenous and environmental factors affect root nodule formation through several key genes, and the regulation details of the nodulation signaling pathway are yet to be fully understood. This study investigated the potential roles of terpenoids and terpene biosynthesis genes on root nodule formation in Glycine max. We characterized six terpenoid synthesis genes from Salvia officinalis by overexpressing SoTPS6, SoNEOD, SoLINS, SoSABS, SoGPS, and SoCINS in soybean hairy roots and evaluating root growth and nodulation, and the expression of strigolactone (SL) biosynthesis and early nodulation genes. Interestingly, overexpression of some of the terpenoid and terpene genes increased nodule numbers, nodule and root fresh weight, and root length, while others inhibited these phenotypes. These results suggest the potential effects of terpenoids and terpene synthesis genes on soybean root growth and nodulation. This study provides novel insights into epistatic interactions between terpenoids, root development, and nodulation in soybean root biology and open new avenues for soybean research.

Rhizobium sophorae is the dominant rhizobial symbiont of Vicia faba L. In North China

Faba bean (Vicia faba L.) is a major introduced grain-legume crop cultivated in China. In this study, rhizobia that nodulated faba bean grown in soils from three sites in North China (Hebei Province) were isolated and characterized. Firstly, isolates were categorized into genotypes by ribosomal IGS PCR-RFLP analysis, then representatives of the different IGS genotypes were further identified by phylogenetic analyses of 16S rRNA, housekeeping (atpD, recA) and nodulation (nodC) gene sequences. Rhizobial distribution based on the IGS genotype was related to the different soil physicochemical features by redundancy analysis. IGS typing and phylogenetic analyses of 16S rRNA and concatenated housekeeping gene sequences affiliated the 103 rhizobial strains isolated into four Rhizobium species/genospecies. A total of 69 strains of 3 IGS types were assigned to R. sophorae, 20 isolates of 5 IGS types to R. changzhiense and 9 isolates of 3 IGS types to R. indicum. The representative strain of the five remaining isolates (1 IGS type) was clearly separated from all Rhizobium type strains and was most closely related to defined genospecies according to the recently described R. leguminosarum species complex. Rhizobium sophorae strains (67% of total isolates) were common in all sites and shared an identical nodC sequence typical of faba bean symbionts belonging to symbiovar viciae. In this first study of rhizobia nodulating faba bean in Hebei Province, China, R. sophorae was found to be the dominant symbiont in contrast to other countries.

Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula.

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells.

Genomics and transcriptomics insights into luteolin effects on the beta-rhizobial strain Cupriavidus necator UYPR2.512.

Cupriavidus necator UYPR2.512 is a rhizobial strain that belongs to the Beta-subclass of proteobacteria, able to establish successful symbiosis with Mimosoid legumes. The initial steps of rhizobium-legumes symbioses involve the reciprocal recognition by chemical signals, being luteolin one of the molecules involved. However, there is a lack of information on the effect of luteolin in beta-rhizobia. In this work, we used long-read sequencing to complete the genome of UYPR2.512 providing evidence for the existence of four closed circular replicons. We used an RNA-Seq approach to analyse the response of UYPR2.512 to luteolin. One hundred and forty-five genes were differentially expressed, with similar numbers of downregulated and upregulated genes. Most repressed genes were mapped to the main chromosome, while the upregulated genes were overrepresented among pCne512e, containing the symbiotic genes. Induced genes included the nod operon and genes implicated in exopolysaccharides and flagellar biosynthesis. We identified many genes involved in iron, copper and other heavy metals metabolism. Among repressed genes, we identified genes involved in basal carbon and nitrogen metabolism. Our results suggest that in response to luteolin, C. necator strain UYPR2.512 reshapes its metabolism in order to be prepared for the forthcoming symbiotic interaction.

Regulation of Phosphorus Supply on Nodulation and Nitrogen Fixation in Soybean Plants with Dual-Root Systems

Phosphorus (P) is an important nutrient affecting nodulation and nitrogen fixation in soybeans. To further investigate the relationship of phosphorus with soybean nodulation and nitrogen fixation, the seedling grafting technique was applied in this study to prepare dual-root soybean systems for a sand culture experiment. From the unfolded cotyledon stage to the initial flowering stage, one side of each dual-root soybean system was irrigated with nutrient solution containing 1 mg/L, 31 mg/L, or 61 mg/L of phosphorus (phosphorus-application side), and the other side was irrigated with a phosphorus-free nutrient solution (phosphorus-free side), to study the effect of local phosphorus supply on nodulation and nitrogen fixation in soybean. The results are described as follows: (1) Increasing the phosphorus supply increased the nodules weight, nitrogenase activity, ureide content, number of bacteroids, number of infected cells, and relative expression levels of nodule nitrogen fixation key genes (GmEXPB2, GmSPX5, nifH, nifD, nifK, GmALN1, GmACP1, GmUR5, GmPUR5, and GmHIUH5) in root nodules on the phosphorus-application side. Although the phosphorus-application and phosphorus-free sides demonstrated similar changing trends, the phosphorus-induced increases were more prominent on the phosphorus-application side, which indicated that phosphorus supply systematically regulates nodulation and nitrogen fixation in soybean. (2) When the level of phosphorus supply was increased from 1 mg/L to 31 mg/L, the increase on the P– side root was significant, and nodule phosphorus content increased by 57.14–85.71% and 68.75–75.00%, respectively; ARA and SNA were 218.64–383.33% and 11.41–16.11%, respectively, while ureide content was 118.18–156.44%. When the level of phosphorus supply was increased from 31mg/L to 61mg/L, the increase in the regulation ability of root and nodule phosphorus content, ARA, SNA, and ureide content were low for roots, and the value for nodules was lower than when the phosphorus level increased from 1 mg/L to 31 mg/L. (3) A high-concentration phosphorus supply on one side of a dual-root soybean plant significantly increased the phosphorus content in the aboveground tissues, as well as the roots and nodules on both sides. In the roots on the phosphorus-free side, the nodules were prioritized for receiving the phosphorus transported from the aboveground tissues to maintain their phosphorus content and functionality.

Effect of actinorhizal root exudates on the proteomes of Frankia soli NRRL B-16219, a strain colonizing the root tissues of its actinorhizal host via intercellular pathway

Frankia and actinorhizal plants exchange signals in the rhizosphere leading to specific mutual recognition of partners and nitrogen-fixing nodule organogenesis. Frankia soli strain NRRL B-16219, from the Elaeagnus specificity group, colonizes the root tissues of its actinorhizal host through direct intercellular penetration of root epidermis cells and cortex. Here, we studied the early proteogenomic response of strain NRRL B-16219 to treatment with root exudates from compatible Elaeagnus angustifolia, and incompatible Ceanothus thyrsiflorus and Coriaria myrtifolia, host plants grown in nitrogen depleted hydroponic medium. Next-generation proteomics was used to identify the main Frankia proteins differentially expressed in response to the root exudates. No products of the nod genes present in B-16219 were detected. Proteins specifically upregulated in presence of E. angustifolia root exudates include those connected to nitrogen fixation and assimilation (glutamate synthetase, hydrogenase and squalene synthesis), respiration (oxidative phosphorylation and citric acid cycle pathways), oxidative stress (catalase, superoxide dismutase, and peroxidase), proteolysis (proteasome, protease, and peptidase) and plant cell wall degrading proteins involved in the depolymerization of celluloses (endoglucanase, glycosyltransferase, beta-mannanases, glycoside hydrolase and glycosyl hydrolase). Proteomic data obtained in this study will help link signaling molecules/factors to their biosynthetic pathways once those factors have been fully characterized.

Bradyrhizobium altum sp. nov., Bradyrhizobium oropedii sp. nov. and Bradyrhizobium acaciae sp. nov. from South Africa show locally restricted and pantropical nodA phylogeographic patterns

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

Transcriptomic Responses of Rhizobium phaseoli to Root Exudates Reflect Its Capacity to Colonize Maize and Common Bean in an Intercropping System.

Corn and common bean have been cultivated together in Mesoamerica for thousands of years in an intercropping system called “milpa,” where the roots are intermingled, favoring the exchange of their microbiota, including symbionts such as rhizobia. In this work, we studied the genomic expression of Rhizobium phaseoli Ch24-10 (by RNA-seq) after a 2-h treatment in the presence of root exudates of maize and bean grown in monoculture and milpa system under hydroponic conditions. In bean exudates, rhizobial genes for nodulation and degradation of aromatic compounds were induced; while in maize, a response of genes for degradation of mucilage and ferulic acid was observed, as well as those for the transport of sugars, dicarboxylic acids and iron. Ch24-10 transcriptomes in milpa resembled those of beans because they both showed high expression of nodulation genes; some genes that were expressed in corn exudates were also induced by the intercropping system, especially those for the degradation of ferulic acid and pectin. Beans grown in milpa system formed nitrogen-fixing nodules similar to monocultured beans; therefore, the presence of maize did not interfere with Rhizobium–bean symbiosis. Genes for the metabolism of sugars and amino acids, flavonoid and phytoalexin tolerance, and a T3SS were expressed in both monocultures and milpa system, which reveals the adaptive capacity of rhizobia to colonize both legumes and cereals. Transcriptional fusions of the putA gene, which participates in proline metabolism, and of a gene encoding a polygalacturonase were used to validate their participation in plant–microbe interactions. We determined the enzymatic activity of carbonic anhydrase whose gene was also overexpressed in response to root exudates.

Enhancing Symbiotic Nitrogen Fixation and Soybean Growth through Co-Inoculation with Bradyrhizobium and Pseudomonas Isolates

The present study was undertaken to explore acidotolerant rhizobial and non-rhizobial bacteria associated with root nodules of soybean (Glycine max L.). Genotypic and phenotypic characterization regarding nitrogen fixation, nodulation and other potentially plant growth-promotion traits were performed in several isolates. Influences of bacterial inoculation on symbiotic nitrogen fixation and soybean growth were evaluated through flask and pot experiments in a greenhouse. The highest nitrogen-fixing activity was recorded in isolate Bra6, which was closely related to Bradyrhizobium diazoefficiens based on 16S rDNA, nifH, and nodD gene sequences. All the non-rhizobial Pseudomonas isolates possessed multiple plant growth-promoting traits, with various hydrolytic patterns toward plant constituents. In sterile water agar-containing flasks, Bra6 + Pse2 treatment significantly (p < 0.05) increased the number of nodules, fresh weight, and dry weight of both root and shoot. This also led to the increment of most of the nutrients in the soybean plant compared with the uninoculated control or sole inoculation of Bra6. In non-sterile strongly acidic soil-containing pots, co-inoculation with Bra6 and various Pseudomonas isolates showed distinctively positive effects on symbiotic nitrogen fixation and soybean growth. The highest symbiotic nitrogen-fixing activity; root and shoot biomass; as well as N, P, K, Ca, Mg, S, Mn, Cu, and Zn contents of soybean plant were observed in Bra6 + Pse2 treatment. Synergistic symbiosis occurred through co-inoculation with Bradyrhizobium and Pseudomonas isolates, which further enhanced nutrients’ acquisition and growth of soybean in the strongly acidic soils.

Phosphate starvation responsive GmSPX5 mediates nodule growth through interaction with GmNF-YC4 in soybean (Glycine max).

Phosphorus (P) deficiency adversely affects nodule development as reflected by reduced nodule fresh weight in legume plants. Though mechanisms underlying nodule adaptation to P deficiency have been studied extensively, it remains largely unknown which regulator mediates nodule adaptation to P deficiency. In this study, GUS staining and quantitative reverse transcription-PCR analysis reveal that the SPX member GmSPX5 is preferentially expressed in soybean (Glycine max) nodules. Overexpression of GmSPX5 enhanced soybean nodule development particularly under phosphate (Pi) sufficient conditions. However, the Pi concentration was not affected in soybean tissues (i.e., leaves, roots, and nodules) of GmSPX5 overexpression or suppression lines, which distinguished it from other well-known SPX members functioning in control of Pi homeostasis in plants. Furthermore, GmSPX5 was observed to interact with the transcription factor GmNF-YC4 in vivo and in vitro. Overexpression of either GmSPX5 or GmNF-YC4 significantly upregulated the expression levels of five asparagine synthetase-related genes (i.e., GmASL2-6) in soybean nodules. Meanwhile, yeast one-hybrid and luciferase activity assays strongly suggested that interactions of GmSPX5 and GmNF-YC4 activate GmASL6 expression through enhancing GmNF-YC4 binding of the GmASL6 promoter. These results not only demonstrate the GmSPX5-GmNF-YC4-GmASL6 regulatory pathway mediating soybean nodule development, but also considerably improve our understanding of SPX functions in legume crops.

Analysis of the expression of polyamine biosynthesis genes in nodules of the garden pea (Pisum sativum L.) and the effect of exogenous treatment with polyamines on their development

BACKGROUND: Polyamines are acting as signaling molecules during adaptation to stressful environment and as regulators of plant development. In plants, polyamines are represented mainly by putrescine, spermidine and spermine. The concentration of polyamines in symbiotic nodules of some legumes is 510 times higher than in the other organs, which indicates their important role in the formation and functioning of symbiotic nodules.
 MATERIALS AND METHODS: We analyzed the expression of genes encoding polyamine biosynthesis enzymes in symbiotic nodules, as well as the effect of exogenous polyamines on the nodule number and the average nodule weight in wild-type SGE plants and symbiotic pea mutants SGEFix-1 (sym40-1) and SGEFix-2 (sym33-3).
 RESULTS: The comparable expression level of arginine decarboxylase gene (PsADC) was observed in all analyzed nodules, whereas the expression level of ornithine decarboxylase gene (PsODC), was highly increased in nodules of SGEFix-2 (sym33-3) mutant. Treatment of the root system with a 0.1 mM solution of polyamines mixture led to an increase in the average weight of the nodule in wild-type plants and in the SGEFix-2 (sym33-3) mutant plants.
 CONCLUSIONS: It was shown that the main pathway of putrescine synthesis in wild-type pea symbiotic nodules is the arginine pathway, while the ornithine pathway is probably associated with activation of plant defense reactions. Polyamines acting, apparently, through ethylene, affect the functioning of the nodule meristem.

Evolutionary and functional analysis of two-component system in chickpea reveals CaRR13, a TypeB RR, as positive regulator of symbiosis.

SummaryThe critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo‐nodules even in the absence of rhizobia. Hence, a genome‐wide analysis of the cytokinin signalling, two‐component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule‐deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay‐based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea.