Role In Nodulation Research Articles

Nodule-specific PLAT domain proteins are expanded in the Medicago lineage and required for nodulation.

Symbiotic nitrogen fixation in legumes is mediated by an interplay of signaling processes between plant hosts and rhizobial symbionts. In legumes, several secreted protein families have undergone expansions and play key roles in nodulation. Thus, identifying lineage-specific expansions (LSEs) of nodulation-associated genes can be a strategy to discover candidate gene families. Using bioinformatic tools, we identified 13 LSEs of nodulation-related secreted protein families, each unique to either Glycine, Arachis or Medicago lineages. In the Medicago lineage, nodule-specific Polycystin-1, Lipoxygenase, Alpha Toxin (PLAT) domain proteins (NPDs) expanded to five members. We examined NPD function using CRISPR/Cas9 multiplex genome editing to create Medicago truncatula NPD knockout lines, targeting one to five NPD genes. Mutant lines with differing combinations of NPD gene inactivations had progressively smaller nodules, earlier onset of nodule senescence, or ineffective nodules compared to the wild-type control. Double- and triple-knockout lines showed dissimilar nodulation phenotypes but coincided in upregulation of a DHHC-type zinc finger and an aspartyl protease gene, possible candidates for the observed disturbance of proper nodule function. By postulating that gene family expansions can be used to detect candidate genes, we identified a family of nodule-specific PLAT domain proteins and confirmed that they play a role in successful nodule formation.

Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen

Coumestrol (CMS), a coumestan isoflavone, plays key roles in nodulation through communication with rhizobia, and has been used as phytoestrogens for hormone replacement therapy in humans. Because CMS content is controlled by multiple genetic factors, the genetic basis of CMS biosynthesis has remained unclear. We identified soybean genotypes with consistently high (Daewonkong) or low (SS0903-2B-21-1-2) CMS content over 2 years. We performed RNA sequencing of leaf samples from both genotypes at developmental stage R7, when CMS levels are highest. Within the phenylpropanoid biosynthetic pathway, 41 genes were tightly connected in a functional co-expression gene network; seven of these genes were differentially expressed between two genotypes. We identified 14 candidate genes involved in CMS biosynthesis. Among them, seven were annotated as encoding oxidoreductases that may catalyze the transfer of electrons from daidzein, a precursor of CMS. Two of the other genes, annotated as encoding a MYB domain protein and a MLP–like protein, may increase CMS accumulation in response to stress conditions. Our results will help to complete our understanding of the CMS biosynthetic pathway, and should facilitate development of soybean cultivars with high CMS content that could be used to promote the fitness of plants and human beings.

An MAP kinase interacts with LHK1 and regulates nodule organogenesis in Lotus japonicus.

Symbiosis receptor-like kinase (SymRK) is a key protein mediating the legume-Rhizobium symbiosis. Our previous work has identified an MAP kinase kinase, SIP2, as a SymRK-interacting protein to positively regulate nodule organogenesis in Lotus japonicus, suggesting that an MAPK cascade might be involved in Rhizobium-legume symbiosis. In this study, LjMPK6 was identified as a phosphorylation target of SIP2. Stable transgenic L. japonicus with RNAi silencing of LjMPK6 decreased the numbers of nodule primordia (NP) and nodule, while plants overexpressing LjMPK6 increased the numbers of nodule, infection threads (ITs), and NP, indicating that LjMPK6 plays a positive role in nodulation. LjMPK6 could interact with a cytokinin receptor, LHK1 both in vivo and in vitro. LjMPK6 was shown to compete with LHP1 to bind to the receiver domain (RD) of LHK1and to downregulate the expression of two LjACS (1-aminocyclopropane-1-carboxylic acid synthase) genes and ethylene levels during nodulation. This study demonstrated an important role of LjMPK6 in regulation of nodule organogenesis and ethylene production in L. japonicus.

Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus.

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical β-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root’s stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

Co-existence of Rhizobia and Diverse Non-rhizobial Bacteria in the Rhizosphere and Nodules of Dalbergia odorifera Seedlings Inoculated with Bradyrhizobium elkanii, Rhizobium multihospitium–Like and Burkholderia pyrrocinia–Like Strains

Rhizobia induce root nodules and fix atmospheric N2 for most legume species in exchange for carbon. However, the diverse endophytic non-rhizobial bacteria in legume nodules that co-exist with rhizobia are often ignored because they are difficult to cultivate using routine cultivation approaches. To enhance our understanding of the incidence and diversity of legume–bacteria associations, a high-throughput sequencing analysis of bacterial 16S rRNA genes was used to examine the bacterial community in the rhizospheres and root nodules of Dalbergia odorifera seedlings that were uninoculated or inoculated with Bradyrhizobium elkanii H255, Rhizobium multihospitium–like HT221, or Burkholderia pyrrocinia–like H022238, in two growth media (nitrogen [N]-supplied soil or N-omitted potting mix). Seedlings inoculated with Bradyrhizobium had significantly more nodules than seedlings in the other inoculation conditions, regardless of growth media. Using the 15N natural abundance method, it was shown that the inoculated plants had significantly higher N2 fixation efficiency (48–57%) and specific nodule activity [269–313 μg N mg−1 of dry weight (dwt) nodule] compared to the uninoculated plants (203 μg N mg−1 dwt nodule). The 16S rRNA gene analysis showed that there was generally a higher bacterial diversity in the rhizosphere than in the nodules in the corresponding condition. Both rhizobial inoculation and media status significantly altered the bacterial communities in the rhizospheres and nodules (P < 0.05), with the exception of the inoculated soil rhizospheres. Regarding non-rhizobial bacteria, three genera, i.e., Lactococcus, Bacillus, and Pseudomonas, were consistently enriched in the rhizosphere and Bradyrhizobium, Chloroplast norank (which belongs to Cyanobacteria), and Lactococcus were commonly found in the nodules. In contrast, common rhizobial genera (including Rhizobium, Mesorhizobium, and Burkholderia) were only present in the nodules at low relative abundances (0.01–3.41%). Regarding non-rhizobial bacteria, 32 genera were found in the nodules, with non-rhizobial bacteria being predominant in the N omitted potting mix (with a relative abundance of 56–87%). This study suggests that legume nodules are inhabited by a high diversity of non-rhizobial bacteria, which may play a vital role in nodulation and N2 fixation in the host plants.

Heterogeneity in the expression and subcellular localization of POLYOL/MONOSACCHARIDE TRANSPORTER genes in Lotus japonicus.

Polyols can serve as a means for the translocation of carbon skeletons and energy between source and sink organs as well as being osmoprotective solutes and antioxidants which may be involved in the resistance of some plants to biotic and abiotic stresses. Polyol/Monosaccharide transporter (PLT) proteins previously identified in plants are involved in the loading of polyols into the phloem and are reported to be located in the plasma membrane. The functions of PLT proteins in leguminous plants are not yet clear. In this study, a total of 14 putative PLT genes (LjPLT1-14) were identified in the genome of Lotus japonicus and divided into 4 clades based on phylogenetic analysis. Different patterns of expression of LjPLT genes in various tissues were validated by qRT-PCR analysis. Four genes (LjPLT3, 4, 11, and 14) from clade II were expressed at much higher levels in nodule than in other tissues. Moreover, three of these genes (LjPLT3, 4, and 14) showed significantly increased expression in roots after inoculation with Mesorhizobium loti. Three genes (LjPLT1, 3, and 9) responded when salinity and/or osmotic stresses were applied to L. japonicus. Transient expression of GFP-LjPLT fusion constructs in Arabidopsis and Nicotiana benthamiana protoplasts indicated that the LjPLT1, LjPLT6 and LjPLT7 proteins are localized to the plasma membrane, but LjPLT2 (clade IV), LjPLT3, 4, 5 (clade II) and LjPLT8 (clade III) proteins possibly reside in the Golgi apparatus. The results suggest that members of the LjPLT gene family may be involved in different biological processes, several of which may potentially play roles in nodulation in this nitrogen-fixing legume.

Foliar Application of Sodium Molybdate Enhanced Nitrogen Uptake and Translocation in Soybean Plants by Improving Nodulation Process Under Salt Stress

Abstract Soil salinity with different harmful effects on plant growth and productivity is one of the main reasons in diminishing biological nitrogen fixation and nitrogen assimilation in legume plants. Molybdate has a key role on nitrogen metabolism of plants and can be has a beneficial effect on it. Thus, this experiment was conducted to evaluate the effects of sodium molybdate spraying (0.2 and 0.4% solutions in water) on nodulation, nitrogen uptake and translocation in soybean plants under different levels of salt stress (0, 5 and 10 dS m−1 NaCl, respectively). Salinity reduced the nodulation, root and shoot growth and special flavonoids content in roots, which are have a key role in nodulation includes, daidzein, genistein, coumestrol and glycitein, also diminished nitrogenase, glutamine synthetase (GS), glutamate dehydrogenase (GDH), glutamine oxoglutarate aminotransferase (GOGAT) and nitrate reductase (NR) activities in nodes, nitrogen content of nodes, roots and leaves, nitrogen uptake and translocation by soybean plants. Under salt stress and nonsaline condition, sodium molybdate treatments improved the nodulation by increasing flavonoids content of roots, also these treatments enhanced the plant growth and nitrogenase, GS, GDH, GOGAT and NR activities of nodes. Furthermore, nitrogen content of nodes, roots and leaves, nitrogen uptake and translocation by soybean plants improved by sodium molybdate applications. Both of the sodium molybdate doses, exposed the similar effects on improving nodulation and nitrogen metabolism of soybean.

Genome Wide Identification and Expression Profiling of Ethylene Receptor Genes during Soybean Nodulation.

It has long been known that the gaseous plant hormone ethylene plays a key role in nodulation in legumes. The perception of ethylene by a family of five membrane-localized receptors is necessary to trigger the ethylene signaling pathway, which regulates various biological responses in Arabidopsis. However, a systematic analysis of the ethylene receptors in leguminous plants and their roles in nodule development is lacking. In this study, we performed a characterization of ethylene receptor genes based on the latest Glycine max genome sequence and a public microarray database. Eleven ethylene receptor family genes were identified in soybean through homology searches, and they were divided into two subgroups. Exon–intron analysis showed that the gene structures are highly conserved within each group. Further analysis of their expression patterns showed that these ethylene receptor genes are differentially expressed in various soybean tissues and organs, including functional nodules. Notably, the ethylene receptor genes showed different responses to rhizobial infection and Nod factors, suggesting a possible role for ethylene receptors and ethylene signaling in rhizobia–host cell interactions and nodulation in soybean. Together, these data indicate the functional divergence of ethylene receptor genes in soybean, and that some of these receptors mediate nodulation, including rhizobial infection, nodule development, and nodule functionality. These findings provide a foundation for further elucidation of the molecular mechanism by which the ethylene signaling pathway regulates nodulation in soybean, as well as other legumes.

Search for Nodulation and Nodule Development-Related Cystatin Genes in the Genome of Soybean (Glycine max).

Nodulation, nodule development and senescence directly affects nitrogen fixation efficiency, and previous studies have shown that inhibition of some cysteine proteases delay nodule senescence, so their nature inhibitors, cystatin genes, are very important in nodulation, nodule development, and senescence. Although several cystatins are actively transcribed in soybean nodules, their exact roles and functional diversities in legume have not been well explored in genome-wide survey studies. In this report, we performed a genome-wide survey of cystatin family genes to explore their relationship to nodulation and nodule development in soybean and identified 20 cystatin genes that encode peptides with 97–245 amino acid residues, different isoelectric points (pI) and structure characteristics, and various putative plant regulatory elements in 3000 bp putative promoter fragments upstream of the 20 soybean cystatins in response to different abiotic/biotic stresses, hormone signals, and symbiosis signals. The expression profiles of these cystatin genes in soybean symbiosis with rhizobium strain Bradyrhizobium japonicum strain 113-2 revealed that 7 cystatin family genes play different roles in nodulation as well as nodule development and senescence. However, these genes were not root nodule symbiosis (RNS)—specific and did not encode special clade cystatin protein with structures related to nodulation and nodule development. Besides, only two of these soybean cystatins were not upregulated in symbiosis after ABA treatment. The functional analysis showed that a candidate gene Glyma.15G227500 (GmCYS16) was likely to play a positive role in soybean nodulation. Besides, evolutionary relationships analysis divided the cystatin genes from Arabidopsis thaliana, Nicotiana tabacum, rice, barley and four legume plants into three groups. Interestingly, Group A cystatins are special in legume plants, but only include one of the above-mentioned 7 cystatin genes related to nodulation and nodule development. Overall, our results provide useful information or clues for our understanding of the functional diversity of legume cystatin family proteins in soybean nodulation and nodule development and for finding nodule-specific cysteine proteases in soybean.

GmFW1 expression decreased in GmSymRK knockdown transgenic soybean roots

ABSTRACTSymRK and GmFWL1 both play important roles in nodulation. However, during symbiotic development, the details of Nod factor signaling association with the regulation of cell division in nodules are unknown. SymRK, the immediately downstream component of these Nod factor receptors, is central to the Nod factor signaling cascade. In this study, specified RNAi plasmid of GmSymRK was constructed and transformed into soybean roots by agrobacterium rhizogenes-mediated hairy root transformation. We found that the nodule number decreased substantially in GmSymRK knockdown soybean transgenic roots. Further to study the relationship between GmFWL1 and Nod factor signaling, we analyzed the GmFWL1 expression levels in the GmSymRK RNAi soybean transgenic roots and found that rhizobia inoculation led to substantially reduced GmFWL1 expression in GmSymRK RNAi soybean transgenic roots. Our studies showed that the regulation of cell division was affected by Nod factor signaling during nodule development in soybean, which provides important information toward understanding the functions of GmSymRK and GmFWL1 in symbiotic signaling and nodule development.

GmACP expression is decreased in GmNORK knockdown transgenic soybean roots

NORK and soybean acyl carrier protein (ACP) both play important roles in nodulation. However, the relationship between Nod factor signaling and fatty acid (FA) biosynthesis during symbiotic development is unknown. In this study, an RNAi plasmid of GmNORK was constructed and transformed into soybean roots by Agrobacterium rhizogene-mediated hairy-root transformation. The nodule number decreased substantially in GmNORK knockdown soybean transgenic roots. To investigate the relationship between GmACP and Nod factor signaling, we measured GmACP expression levels in GmNORK RNAi soybean transgenic roots and found that rhizobia inoculation led to substantially reduced GmACP expression. Thus, FA biosynthesis was affected by Nod factor signaling during nodule development in soybean, a finding that provides valuable information that improves our understanding of the functions of GmNORK and GmACP in symbiotic signaling and nodule development.

The LysM receptor-like kinase SlLYK10 regulates the arbuscular mycorrhizal symbiosis in tomato.

Most plants have the ability to establish a symbiosis with arbuscular mycorrhizal (AM) fungi, which allows better plant nutrition. A plant signaling pathway, called the common symbiosis signaling pathway (CSSP), is essential for the establishment of both AM and root nodule symbioses. The CSSP is activated by microbial signals. Plant receptor(s) for AM fungal signals required for the activation of the CSSP and initial fungal penetration are currently unknown. We set up conditions to use virus-induced gene silencing (VIGS) in Solanum lycopersicum to study the genes potentially involved in AM. We show that the lysin motif receptor-like kinase SlLYK10, whose orthologs in legumes are essential for nodulation, but not for AM, and SlCCaMK, a component of the CSSP, are required for penetration of the AM fungus Rhizophagus irregularis into the roots of young tomato plants. Our results support the hypothesis that the SILYK10 ancestral gene originally played a role in AM and underwent duplication and neofunctionalization for a role in nodulation in legumes. Moreover, we conclude that VIGS is an efficient method for fast screening of genes playing major roles in AM.

Differential response of kabuli and desi chickpea genotypes toward inoculation with PGPR in different soils.

Pakistan is among top three chickpea producing countries but the crop is usually grown on marginal lands without irrigation and fertilizer application which significantly hampers its yield. Soil fertility and inoculation with beneficial rhizobacteria play a key role in nodulation and yield of legumes. Four kabuli and six desi chickpea genotypes were, therefore, evaluated for inoculation response with IAA-producing Ochrobactrum ciceri Ca-34T and nitrogen fixing Mesorhizobium ciceri TAL-1148 in single and co-inoculation in two soils. The soil type 1 was previously unplanted marginal soil having low organic matter, P and N contents compared to soil type 2 which was a fertile routinely legume-cultivated soil. The effect of soil fertility status was pronounced and fertile soil on average, produced 31% more nodules, 62% more biomass and 111% grain yield than marginal soil. Inoculation either with O. ciceri alone or its co-inoculation with M. ciceri produced on average higher nodules (42%), biomass (31%), grains yield (64%) and harvest index (72%) in both chickpea genotypes over non-inoculated controls in both soils. Soil 1 showed maximum relative effectiveness of Ca-34T inoculation for kabuli genotypes while soil 2 showed for desi genotypes except B8/02. Desi genotype B8/02 in soil type 1 and Pb-2008 in soil type 2 showed significant yield increase as compared to respective un-inoculated controls. Across bacterial inoculation treatments, grain yield was positively correlated to growth and yield contributing parameters (r = 0.294* to 0.838*** for desi and r = 0.388* to 0.857** for kabuli). PCA and CAT-PCA analyses clearly showed a site-specific response of genotype x bacterial inoculation. Furthermore, the inoculated bacterial strains were able to persist in the rhizosphere showing colonization on root and within nodules. Present study shows that plant growth promoting rhizobacteria (PGPR) inoculation should be integrated with national chickpea breading program in Pakistan especially for marginal soils. Furthermore, the study shows the potential of phytohormone producing strain Ca-34T as promising candidate for development of biofertilizer alongwith nodulating strains to get sustainable yield of kabuli and desi chickpea with minimum inputs at marginal land.

Hormonal Control of Lateral Root and Nodule Development in Legumes.

Many plants can establish symbioses with nitrogen-fixing bacteria, some of which lead to nodulation, including legumes. Indeed, in the rhizobium/legume symbiosis, new root organs, called nodules, are formed by the plant in order to host the rhizobia in protective conditions, optimized for nitrogen fixation. In this way, these plants can benefit from the reduction of atmospheric dinitrogen into ammonia by the hosted bacteria, and in exchange the plant provides the rhizobia with a carbon source. Since this symbiosis is costly for the plant it is highly regulated. Both legume nodule and lateral root organogenesis involve divisions of the root inner tissues, and both developmental programs are tightly controlled by plant hormones. In fact, most of the major plant hormones, such as auxin, cytokinins, abscisic acid, and strigolactones, control both lateral root formation and nodule organogenesis, but often in an opposite manner. This suggests that the sensitivity of legume plants to some phytohormones could be linked to the antagonism that exists between the processes of nodulation and lateral root formation. Here, we will review the implication of some major phytohormones in lateral root formation in legumes, compare them with their roles in nodulation, and discuss specificities and divergences from non-legume eudicot plants such as Arabidopsis thaliana.

Role of Plant Genotype and Soil Conditions in Symbiotic Plant-Microbe Interactions for Adaptation of Plants to Cadmium-Polluted Soils

We highlighted some of the key problems associated with the use of beneficial microorganisms for improving adaptation of plants to soils, polluted with heavy metals (HMs), especially Cd. Inoculation of pea line SGE and its Cd-tolerant mutant SGECdt with nodule bacteria Rhizobium leguminosarum bv. viciae demonstrated that nodulation process may be disturbed at Cd concentrations below threshold toxicity levels for each partner and the plant genotype plays a major role in nodulation under Cd stress. A comparative mathematical analysis of available information about Cd tolerance, accumulation of HMs (Cd, Cr, Cu, Ni, Pb, Sr and Zn), response to mycorrhizal fungus Glomus sp. and 15 phenotypic traits of 99 pea varieties revealed that (1) the Cd-sensitive varieties were more efficient in exploring the protective potential of symbiosis to compensate their deficit in Cd tolerance and (2) correlations between the studied traits exist and can be helpful for selection of plant-microbe systems adapted to polluted soils. In pot experiment with 11 varieties of Indian mustard, the plant growth-promoting effect of rhizobacterium Variovorax paradoxus 5C-2 negatively correlated with Cd tolerance and shoot Cd concentration of the plants grown in Cd-supplemented soil. In an outdoor pot experiment, inoculation of willow with the ectomycorrhizal fungus Pisolithus tinctorius and a cocktail of rhizobacteria stimulated root exudation, decreased soil pH and increased Cd mobilization in soil and Cd uptake by plants, but decreased plant growth at a moderate contamination level (25 mg Cd kg−1). Opposite effects were observed in highly contaminated soil (77 mg Cd kg−1). We propose a preliminary systematic framework of interactions between these factors that determine the success of microbial inoculation aimed at improving crop performance on HM-polluted soils or enhancing phytoremediation.

A Lotus japonicus Cochaperone Protein Interacts With the Ubiquitin-Like Domain Protein CIP73 and Plays a Negative Regulatory Role in Nodulation.

The calcium/calmodulin-dependent protein kinase CCaMK forms a complex with its phosphorylation target CIP73 (CCaMK-interacting protein of 73 kDa). In this work, a homolog of the animal HSC/HSP70 interacting protein (HIP) was identified as an interacting partner of CIP73 in Lotus japonicus. L. japonicus HIP contains all functional domains characteristic of animal HIP proteins. The C-terminal STI1-like domain of L. japonicus HIP was found to be necessary and sufficient for interaction with CIP73. The interaction between CIP73 and HIP occurred in both the nuclei and cytoplasm in Nicotiana benthamiana leaf cells. The interactions between CIP73 and HIP and between CIP73 and CCaMK could take place simultaneously in the same nuclei. HIP transcripts were detected in all plant tissues tested. As nodule primordia developed into young nodules, the expression of HIP was down-regulated and the HIP transcript level became very low in mature nodules. More nodules were formed in transgenic hairy roots of L. japonicus expressing HIP RNA interference at 16 days postinoculation as compared with the control hairy roots expressing the empty vector. It appears that HIP may play a role as a negative regulator for nodulation.

The role of isoflavone metabolism in plant protection depends on the rhizobacterial MAMP that triggers systemic resistance against Xanthomonas axonopodis pv. glycines in Glycine max (L.) Merr. cv. Osumi

Glycine max (L.) Merr. plays a crucial role in both the field of food and the pharmaceutical industry due to their input as plant protein and to the benefits of isoflavones (IF) for health. In addition, IF play a key role in nodulation and plant defense and therefore, an increase in IF would be desirable for better field performance. IF are secondary metabolites and therefore, inducible, so finding effective agents to increase IF contents is interesting. Among these agents, plant growth promoting rhizobacteria (PGPR) have been used to trigger systemic induction of plant's secondary metabolism through their microbe associated molecular patterns (MAMPs) that fit in the plant's receptors to start a systemic response. The aim of this study was to evaluate the ability of 4 PGPR that had a contrasted effect on IF metabolism, to protect plants against biotic stress and to establish the relation between IF profile and the systemic response triggered by the bacteria. Apparently, the response involves a lower sensitivity to ethylene and despite the decrease in effective photosynthesis, growth is only compromised in the case of M84, the most effective in protection. All strains protected soybean against Xanthomonas axonopodis pv. glycines (M84 > N5.18 > Aur9>N21.4) and only M84 and N5.18 involved IF. N5.18 stimulated accumulation of IF before pathogen challenge. M84 caused a significant increase on IF only after pathogen challenge and N21.4 caused a significant increase on IF content irrespective of pathogen challenge. Aur9 did not affect IF. These results point out that all 4 strains have MAMPs that trigger defensive metabolism in soybean. Protection induced by N21.4 and Aur9 involves other metabolites different to IF and the role of IF in defence depends on the previous metabolic status of the plant and on the bacterial MAMP.

A Soybean Acyl Carrier Protein, GmACP, Is Important for Root Nodule Symbiosis

Legumes (members of family Fabaceae) establish a symbiotic relationship with nitrogen-fixing soil bacteria (rhizobia) to overcome nitrogen source limitation. Single root hair epidermal cells serve as the entry point for bacteria to infect the host root, leading to development of a new organ, the nodule, which the bacteria colonize. In the present study, the putative role of a soybean acyl carrier protein (ACP), GmACP (Glyma18g47950), was examined in nodulation. ACP represent an essential cofactor protein in fatty acid biosynthesis. Phylogenetic analysis of plant ACP protein sequences showed that GmACP was classified in a legume-specific clade. Quantitative reverse-transcription polymerase chain reaction analysis demonstrated that GmACP was expressed in all soybean tissues but showed higher transcript accumulation in nodule tissue. RNA interference-mediated gene silencing of GmACP resulted in a significant reduction in nodule numbers on soybean transgenic roots. Fluorescent protein-labeled GmACP was localized to plastids in planta, the site of de novo fatty acid biosynthesis in plants. Analysis of the fatty acid content of root tissue silenced for GmACP expression, as determined by gas chromatography-mass spectrometry, showed an approximately 22% reduction, specifically in palmitic and stearic acid. Taken together, our data provide evidence that GmACP plays an important role in nodulation.

Soybean Ureases, but Not That of Bradyrhizobium japonicum, Are Involved in the Process of Soybean Root Nodulation

Ureases are abundant in plants, bacteria, and in the soil, but their role in signaling between soybean and soil microorganisms has not been investigated. The bacterium Bradyrhizobium japonicum forms nitrogen-fixing nodules on soybean roots. Here, we evaluated the role(s) of ureases in the process of soybean nodulation. Chemotaxis assays demonstrated that soybean and jack bean ureases were more chemotactic toward bacterial cells than the corresponding plant lectins. The eu1-a,eu4 soybean, deficient in urease isoforms, formed fewer but larger nodules than the wild-type, regardless of the bacterial urease phenotype. Leghemoglobin production in wild-type plants was higher and peaked earlier than in urease-deficient plants. Inhibition of urease activity in wild-type plants did not result in the alterations seen in mutated plants. We conclude that soybean urease(s) play(s) a role in the soybean-B. japonicum symbiosis, which is independent of its ureolytic activity. Bacterial urease does not play a role in nodulation.

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process. A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated. Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones. No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.