Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

Cell-layer specific roles for gibberellins in nodulation and root development.

Gibberellins (GA) have a profound influence on the formation of lateral root organs. However, the precise role this hormone plays in the cell-specific events during lateral root formation, rhizobial infection and nodule organogenesis, including interactions with auxin and cytokinin (CK), is not clear. We performed epidermal- and endodermal-specific complementation of the severely GA-deficient na pea (Pisum sativum) mutant with Agrobacterium rhizogenes. Gibberellin mutants were used to examine the spatial expression pattern of CK (TCSn)- and auxin (DR5)-responsive promoters and hormone levels. We found that GA produced in the endodermis promote lateral root and nodule organogenesis and can induce a mobile signal(s) that suppresses rhizobial infection. By contrast, epidermal-derived GA suppress infection but have little influence on root or nodule development. GA suppress the CK-responsive TCSn promoter in the cortex and are required for normal auxin activation during nodule primordia formation. Our findings indicate that GA regulate the checkpoints between infection thread (IT) penetration of the cortex and invasion of nodule primordial cells and promote the subsequent progression of nodule development. It appears that GA limit the progression and branching of IT in the cortex by restricting CK response and activate auxin response to promote nodule primordia development.

Occurrence and diversity of stem nodulation in Aeschynomene and Sesbania legumes from wetlands of Madagascar.

Legumes have the ability to establish a nitrogen-fixing symbiosis with soil rhizobia that they house in specific organs, the nodules. In most rhizobium-legume interactions, nodulation occurs on the root. However, certain tropical legumes growing in wetlands possess a unique trait: the capacity to form rhizobia-harbouring nodules on the stem. Despite the originality of the stem nodulation process, its occurrence and diversity in waterlogging-tolerant legumes remains underexplored, impeding a comprehensive analysis of its genetics and biology. Here, we aimed at filling this gap by surveying stem nodulation in legume species-rich wetlands of Madagascar. Stem nodulation was readily observed in eight hydrophytic species of the legume genera, Aeschynomene and Sesbania, for which significant variations in stem nodule density and morphology was documented. Among these species, A. evenia, which is used as genetic model to study the rhizobial symbiosis, was found to be frequently stem-nodulated. Two other Aeschynomene species, A. cristata and A. uniflora, were evidenced to display a profuse stem-nodulation as occurs in S. rostrata. These findings extend our knowledge on legumes species that are endowed with stem nodulation and further indicate that A. evenia, A. cristata, A. uniflora and S. rostrata are of special interest for the study of stem nodulation. As such, these legume species represent opportunities to investigate different modalities of the nitrogen-fixing symbiosis and this knowledge could provide cues for the engineering of nitrogen-fixation in non-legume crops.

Unregulated GmAGL82 due to Phosphorus Deficiency Positively Regulates Root Nodule Growth in Soybean.

Nitrogen fixation, occurring through the symbiotic relationship between legumes and rhizobia in root nodules, is crucial in sustainable agriculture. Nodulation and soybean production are influenced by low levels of phosphorus stress. In this study, we discovered a MADS transcription factor, GmAGL82, which is preferentially expressed in nodules and displays significantly increased expression under conditions of phosphate (Pi) deficiency. The overexpression of GmAGL82 in composite transgenic plants resulted in an increased number of nodules, higher fresh weight, and enhanced soluble Pi concentration, which subsequently increased the nitrogen content, phosphorus content, and overall growth of soybean plants. Additionally, transcriptome analysis revealed that the overexpression of GmAGL82 significantly upregulated the expression of genes associated with nodule growth, such as GmENOD100, GmHSP17.1, GmHSP17.9, GmSPX5, and GmPIN9d. Based on these findings, we concluded that GmAGL82 likely participates in the phosphorus signaling pathway and positively regulates nodulation in soybeans. The findings of this research may lay the theoretical groundwork for further studies and candidate gene resources for the genetic improvement of nutrient-efficient soybean varieties in acidic soils.

Phosphorylation of the alpha-I motif in SYMRK drives root nodule organogenesis.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

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.

Molecular module GmPTF1a/b-GmNPLa regulates rhizobia infection and nodule formation in soybean.

Nodulation begins with the initiation of infection threads (ITs) in root hairs. Though mutual recognition and early symbiotic signaling cascades in legumes are well understood, molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis remain largely unexplored. We functionally investigated a novel pectate lyase enzyme, GmNPLa, and its transcriptional regulator GmPTF1a/b in soybean (Glycine max), where their regulatory roles in IT development and nodule formation were elucidated through investigation of gene expression patterns, bioinformatics analysis, biochemical verification of genetic interactions, and observation of phenotypic impacts in transgenic soybean plants. GmNPLa was specifically induced by rhizobium inoculation in root hairs. Manipulation of GmNPLa produced remarkable effects on IT and nodule formation. GmPTF1a/b displayed similar expression patterns as GmNPLa, and manipulation of GmPTF1a/b also severely influenced nodulation traits. LI soybeans with low nodulation phenotypes were nearly restored to HI nodulation level by complementation of GmNPLa and/or GmPTF1a. Further genetic and biochemical analysis demonstrated that GmPTF1a can bind to the E-box motif to activate transcription of GmNPLa, and thereby facilitate nodulation. Taken together, our findings potentially reveal novel mediation of cell wall gene expression involving the basic helix-loop-helix transcription factor GmPTF1a/b acts as a key early regulator of nodulation in soybean.

Shoot-to-root communication via GmUVR8-GmSTF3 photosignaling and flavonoid biosynthesis fine-tunes soybean nodulation under UV-B light.

Legume nodulation requires light perception by plant shoots and precise long-distance communication between shoot and root. Recent studies have revealed that TGACG-motif binding factors (GmSTFs) integrate light signals to promote root nodulation; however, the regulatory mechanisms underlying nodule formation in changing light conditions remain elusive. Here, we applied genetic engineering, metabolite measurement, and transcriptional analysis to study soybean (Glycine max) nodules. We clarify a fine-tuning mechanism in response to ultraviolet B (UV-B) irradiation and rhizobia infection, involving GmUVR8-dependent UV-B perception and GmSTF3/4-GmMYB12-GmCHS-mediated (iso)flavonoid biosynthesis for soybean nodule formation. GmUVR8 receptor-perceived UV-B signal triggered R2R3-MYB transcription factors GmMYB12-dependent flavonoid biosynthesis separately in shoot and root. In shoot, UV-B-triggered flavonoid biosynthesis relied on GmUVR8a, b, c receptor-dependent activation of GmMYB12L-GmCHS8 (chalcone synthase) module. In root, UV-B signaling distinctly promotes the accumulation of the isoflavones, daidzein, and its derivative coumestrol, via GmMYB12B2-GmCHS9 module, resulting in hypernodulation. The mobile transcription factors, GmSTF3/4, bind to cis-regulatory elements in the GmMYB12L, GmMYB12B2, and GmCHS9 promoters, to coordinate UV-B light perception in shoot and (iso)flavonoid biosynthesis in root. Our findings establish a novel shoot-to-root communication module involved in soybean nodulation and reveal an adaptive strategy employed by soybean roots in response to UV-B light.

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.

CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions.

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume-rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

NopAA and NopD Signaling Association-Related Gene GmNAC27 Promotes Nodulation in Soybean (Glycine max).

Rhizobia secrete effectors that are essential for the effective establishment of their symbiotic interactions with leguminous host plants. However, the signaling pathways governing rhizobial type III effectors have yet to be sufficiently characterized. In the present study, the type III effectors, NopAA and NopD, which perhaps have signaling pathway crosstalk in the regulation of plant defense responses, have been studied together for the first time during nodulation. Initial qRT-PCR experiments were used to explore the impact of NopAA and NopD on marker genes associated with symbiosis and defense responses. The effects of these effectors on nodulation were then assessed by generating bacteria in which both NopAA and NopD were mutated. RNA-sequencing analyses of soybean roots were further utilized to assess signaling crosstalk between NopAA and NopD. NopAA mutant and NopD mutant were both found to repress GmPR1, GmPR2, and GmPR5 expression in these roots. The two mutants also significantly reduced nodules dry weight and the number of nodules and infection threads, although these changes were not significantly different from those observed following inoculation with double-mutant (HH103ΩNopAA&NopD). NopAA and NopD co-mutant inoculation was primarily found to impact the plant-pathogen interaction pathway. Common differentially expressed genes (DEGs) associated with both NopAA and NopD were enriched in the plant-pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways, and no further changes in these common DEGs were noted in response to inoculation with HH103ΩNopAA&NopD. Glyma.13G279900 (GmNAC27) was ultimately identified as being significantly upregulated in the context of HH103ΩNopAA&NopD inoculation, serving as a positive regulator of nodulation. These results provide new insight into the synergistic impact that specific effectors can have on the establishment of symbiosis and the responses of host plant proteins.

A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation.

BACKGROUND: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. RESULTS: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. CONCLUSION: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

An emerging role of heterotrimeric G-proteins in nodulation and nitrogen sensing.

MAIN CONCLUSION: A comprehensive understanding of nitrogen signaling cascades involving heterotrimeric G-proteins and their putative receptors can assist in the production of nitrogen-efficient plants. Plants are immobile in nature, so they must endure abiotic stresses including nutrient stress. Plant development and agricultural productivity are frequently constrained by the restricted availability of nitrogen in the soil. Non-legume plants acquire nitrogen from the soil through root membrane-bound transporters. In depleted soil nitrogen conditions, legumes are naturally conditioned to fix atmospheric nitrogen with the aid of nodulation elicited by nitrogen-fixing bacteria. Moreover, apart from the symbiotic nitrogen fixation process, nitrogen uptake from the soil can also be a significant secondary source to satisfy the nitrogen requirements of legumes. Heterotrimeric G-proteins function as molecular switches to help plant cells relay diverse stimuli emanating from external stress conditions. They are comprised of Gα, Gß and Gγ subunits, which cooperate with several downstream effectors to regulate multiple plant signaling events. In the present review, we concentrate on signaling mechanisms that regulate plant nitrogen nutrition. Our review highlights the potential of heterotrimeric G-proteins, together with their putative receptors, to assist the legume root nodule symbiosis (RNS) cascade, particularly during calcium spiking and nodulation. Additionally, the functions of heterotrimeric G-proteins in nitrogen acquisition by plant roots as well as in improving nitrogen use efficiency (NUE) have also been discussed. Future research oriented towards heterotrimeric G-proteins through genome editing tools can be a game changer in the enhancement of the nitrogen fixation process. This will foster the precise manipulation and production of plants to ensure global food security in an era of climate change by enhancing crop productivity and minimizing reliance on external inputs.

MicroRNA 4407 modulates nodulation in soybean by repressing a root-specific ISOPENTENYLTRANSFERASE (GmIPT3).

MicroRNAs (miRNAs) are important regulators of plant biological processes, including soybean nodulation. One miRNA, miR4407, was identified in soybean roots and nodules. However, the function of miR4407 in soybean is still unknown. MiR4407, unique to soybean, positively regulates lateral root emergence and root structures and represses a root-specific ISOPENTENYLTRANSFERASE (GmIPT3). By altering the expression of miR4407 and GmIPT3, we investigated the role of miR4407 in lateral root and nodule development. Both miR4407 and GmIPT3 are expressed in the inner root cortex and nodule primordia. Upon rhizobial inoculation, miR4407 was downregulated while GmIPT3 was upregulated. Overexpressing miR4407 reduced the number of nodules in transgenic soybean hairy roots while overexpressing the wild-type GmIPT3 or a miR4407-resistant GmIPT3 mutant (mGmIPT3) significantly increased the nodule number. The mechanism of miR4407 and GmIPT3 functions was also linked to autoregulation of nodulation (AON), where miR4407 overexpression repressed miR172c and activated its target, GmNNC1, turning on AON. Exogenous CK mimicked the effects of GmIPT3 overexpression on miR172c, supporting the notion that GmIPT3 regulates nodulation by enhancing root-derived CK. Overall, our data revealed a new miRNA-mediated regulatory mechanism of nodulation in soybean. MiR4407 showed a dual role in lateral root and nodule development.

Laser Capture Microdissection Transcriptome Reveals Spatiotemporal Tissue Gene Expression Patterns of Medicago truncatula Roots Responding to Rhizobia.

We report a public resource for examining the spatiotemporal RNA expression of 54,893 Medicago truncatula genes during the first 72 h of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of more than 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole-root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response dataset generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of 1-aminocyclopropane-1-carboxylate synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Peter and the Beanstalk: tackling the giant questions of soybean nodulation.

The following is an edited interview, carried out by the Journal Development Editor of BioTechniques, Ashling Cannon, with Peter Gresshoff (University of Queensland, UQ; Brisbane, Australia). Peter is a plant developmental geneticist, using molecular and genetic tools to understand the complexities of gene networks during the control of nodule formation in legumes. He was the Director of the Center of Excellence for Integrative Legume Research and is now an Emeritus Professor at UQ.