The combination of mutation-based genetics and functional genomics has allowed a detailed dissection of the nodulation-induction and autoregulation of nodulation(AON) pathways of soybean. Applicable to all legumes, nodulation is induced by Rhizobium/Bradyrhizobium-produced lipopolysaccharides(Nod factors), perceived by Nod factor receptors(NFR1/NFR5 dimers), leading to cortical and pericycle cell divisions. These induce the production of CLAVATA3-like(CLE) peptides, which travel in the xylem to the shoot, where they are perceived by a receptor complex including a leucine-rich repeat(LRR) receptor kinase, encoded by Gm NARK, Lj HAR1, Mt SUNN and closely related receptors in other legumes like Phaseolus vulgaris(common bean), Pisum sativum(pea), and Glycine soja. The activated receptor complex negatively regulates by phosphorylation of the constitutive synthesis of mi R2111 in the shoot. This is normally is translocated via the phloem to the entire plant body, initiating suppression of a rootexpressed Kelch repeat-containing F-box protein“Too Much Love(TML),” which in turn suppresses the nodule initiation cascade. Nodulation is therefore permitted during a developmental window between the induction and progress of the nodulation/cell division/infection cascade during the first few days after inoculation and the functional“readiness” of the AON cascade, delayed by the root–shoot–root loop. Loss-of-function mutations in Gm NARK and Lj TML result in excessive nodulation(supernodulation/hypernodulation/supernummary nodulation) as well as localized tolerance to externally applied nitrate. Recent analyses have indicated an interaction of the AON with lateral root formation as well as with the autoregulation of mycorrhization(AOM). Further details of the parallel functions of key points in this regulatory loop remain to be elucidated.

Introduction Crop rotation, a crucial agricultural practice that enhances soil health and crop productivity, is widely used in agriculture worldwide. Soybeans play a crucial role in crop rotation owing to their nitrogen-fixing ability, which is facilitated by symbiotic bacteria in their root systems. The soybean-rapeseed rotation is an effective agricultural practice in the Yangtze River Basin of China. However, the mechanism underlying the effectiveness of this system remains unknown. Objectives The aim of this study was to decipher the mechanisms by which previous soybean cultivation enhances the growth of subsequent rapeseed. Methods Soybeans with three distinct nodulation genotypes were rotated with rapeseed, and the impact of previous soybean cultivation on subsequent rapeseed growth was evaluated by examining the soybean root secretome and soil rhizosphere microbiome. Results Soybean-rapeseed rotation significantly enhanced subsequent rapeseed growth and yield, especially when supernodulating soybean plants were used, which released the most nitrogen into the soil rhizosphere. The differences in soybean nodulation capability led to variations in root exudation, which in turn influenced the bacterial communities in the rhizosphere. Notably, the supernodulating soybean plants promoted Sphingomonadaceae family of bacteria growth by secreting oleic acid and cis-4-hydroxy-D-proline, and further attracted them through cis-4-hydroxy-D-proline. Furthermore, the exogenous application of Sphingomonadaceae bacteria, either alone or in combination with rhizobia, significantly enhanced the growth of rapeseed. Conclusion Our data definitively demonstrated the crucial role of previous soybean cultivation in enhancing the yield of rapeseed, with the assistance of Sphingomonadaceae bacteria and rhizobia. This study elucidates the role of soybean nodulation in rhizosphere bacterial dynamics, highlighting its importance in sustainable agricultural practices.

Preface. DNA Amplification Fingerprinting and its Potential Application for Genome Analysis (Brant J. Bassam, Gustavo Caetano-Anolles, and Peter M. Gresshoff). The Plant Molybdenum Cofactor (Moco) (Ralf R. Mendel). Motility and Chemotaxis in the Life of Rhizobia (Wolfgang D. Bauer). Distinct Entities between Soybean Agglutinin Receptor and Soybean Root Hair Binding Site on Bradyrhizobium japonicum Cell Surface (Siu-Cheong Ho). Rhizobium Lipopolysaccharides Their Structures and Evidence for Their Importance in the Nitrogen-Fixing Symbiotic Infection of Their Host Legumes (Russell W. Carlson, U. Ramadas Bhat, and Brad Reuhs). Molecular Signaling in the Bradyrhizobium japonicum-Soybean Symbiosis (Gary Stacey). Cytokinins and Legume Nodulation (Barbara J. Taller). Nodulation in the Absence of Rhizobium (Gustavo Caetano-Anolles, Priyavadan A. Joshi, and Peter M. Gresshoff). Altered Tryptophan Biosynthesis in Bradyrhizobium japonicum Gives Enhanced Nodulation and Nitrogen Fixation (L. David Kuykendall and William J. Hunter). Alfalfa Nodule Development Ribonucleotide Pools and Ribonucleotide Reductase Activity in Cultured and Symbiotic Rhizobium meliloti (J.R. Cowles and S.F. Yet). Systemic Regulation of Nodulation in Legumes (Peter M. Gresshoff and Gustavo Caetano-Anolles). RFLP Linkage Analysis of Symbiotic Mutants of Soybean (Deborah Landau-Ellis, Randy Shoemaker, Sieglinde Angermuller, and Peter M. Gresshoff). Physical Mapping of the nts Region of the Soybean Genome Using Pulse Field Gel Electrophoresis (PFGE) (Roel P. Funke and Peter M. Gresshoff). Gene Transfer to Barley (R.R. Mendell, E. Clauss, J. Schulze, H.H. Steinbiss, and A. Nerlich). Application of Molecular Analyses to Questions Relating to the Genetics, Ecology and Evolution of Actinorhizal Symbioses (Beth C. Mullin, Susan M. Swenson, Paul Twigg, and Paula Goetting-Minesky). Intracellular Receptor Proteins for Calcium Signals in Plants (Daniel M. Roberts, C. David Weaver, and Suk-Heung Oh). Pathogen and Pest Resistance in Endophyte-Infected Tall Fescue (Kimberly D. Gwinn, Ernest C. Bernard, and Charles D. Pless). The Commercial Pathway for Agricultural Biotechnology (Zachary S. Wochok).

Symbiotic nitrogen fixation boosts legume growth and production in nitrogen-poor soils. It has long been assumed that fixed nitrogen increases reproductive success, but until now, the regulatory mechanism was unknown. Here, we report a symbiotic flowering pathway that couples symbiotic and nutrient signals to the flowering induction pathway in legumes. We show that the symbiotic microRNA–microRNA172c (miR172c) and fixed nitrogen systemically and synergistically convey symbiotic and nutritional cues from roots to leaves to promote soybean ( Glycine max ) flowering. The combinations of symbiotic miR172c and local miR172c elicited by fixed nitrogen and development in leaves activate florigen-encoding FLOWERING LOCUS T ( FT ) homologs ( GmFT2a/5a ) by repressing TARGET OF EAT1-like 4a ( GmTOE4a ). Thus, FTs trigger reproductive development, which allows legumes to survive and reproduce under low-nitrogen conditions.

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.

Plant roots are constantly exposed to a diverse microbiota of pathogens and mutualistic partners. The host’s immune system is an essential component for its survival, enabling it to monitor nearby microbes for potential threats and respond with a defence response when required. Current research suggests that the plant immune system has also been employed in the legume-rhizobia symbiosis as a means of monitoring different rhizobia strains and that successful rhizobia have evolved to overcome this system to infect the roots and initiate nodulation. With clear implications for host-specificity, the immune system has the potential to be an important target for engineering versatile crops for effective nodulation in the field. However, current knowledge of the interacting components governing this pathway is limited, and further research is required to build on what is currently known to improve our understanding. This review provides a general overview of the plant immune system’s role in nodulation. With a focus on the cycles of microbe-associated molecular pattern-triggered immunity (MTI) and effector-triggered immunity (ETI), we highlight key molecular players and recent findings while addressing the current knowledge gaps in this area.

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 Forster 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 GA(3) 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.

Plants coordinate development through complex signaling networks involving many different types of molecules, genes, proteins, and hormones. CLAVATA3-Endosperm Surrounding Region-related (CLE) peptides are one such family of signaling molecules that have roles in general plant development as well during interactions with abiotic and biotic factors. Members of the CLE peptide family have been well studied in the model species, Arabidopsis thaliona; with extensive CLV3, CLE40 and TDIF signaling pathways having been identified. These CLE peptide pathways tend to have three core elements including a CLE peptide, which is perceived by a Receptor-Kinase that regulates a WUSCHEL-like homeobox transcription factor. Such elements are also present in the Autoregulation of Nodulation pathway that controls nodule number during the legume-rhizobia symbiosis. In the soybean genome, 84 CLE peptide-encoding genes are present, although little research outside of nodulation has been undertaken to understand how they modulate growth in the model legume. Understanding CLE peptides may provide novel targets to improve soybean growth and productivity. Moreover, it also represents a unique opportunity to study CLE peptides in the context of beneficial symbiosis with rhizobia and mycorrhizae of which A. thaliana cannot undergo. This chapter reviews the current understanding of CLE peptide signaling in plants, what is currently known about soybean orthologues, and avenues that we can target to improve soybean development using the CLE peptides and their associated molecular networks.

Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture.

Autoregulation of nodulation (AON) plays a central role in nodulation by inhibiting the formation of excess number of legume root nodules. In this study, the effect of hydroxymethylglutaryl-coenzyme A reductase 1 (GmHMGR1) gene expression on nodulation and the AON system in Glycine max (L.) Merr was investigated. Wild-type soybean (cultivar Bragg) and its near-isogenic supernodulating mutant (nitrate tolerant symbiotic) nts1007 were selected to identify the expression pattern of this gene in rootlets after inoculation by its microsymbiont Bradyrhizobium. For further analysis, the full length of GmHMGR1 and its promoter were cloned after amplification by inverse-PCR and BAC library screening. Also, we constructed an intron hairpin RNA interference (ihpRNAi) and a GmHMGR1 promoter: β-glucuronidase fusion constructs, consequently for suppression of GmHMGR1 and histochemical analysis in transgenic soybean hairy roots induced by Agrobacterium rhizogenes strain K599. The GmHMGR1 gene was functional during the early stages of nodulation with the AON system having a negative effect on GmHMGR1 expression and nodule formation in wild-type rootlets. GmHMGR1 was particularly expressed in the developing phloem within the root, nodules and nodule lenticels. Expression of GmHMGR1 in transgenic hairy roots was suppressed by RNAi silencing approximately 85% as compared to empty vector controls. This suggests that the GmHMGR1 gene has an important role in triggering nodule formation as its suppression caused a reduction of nodule formation in nts mutant lines with a deficient AON system.

Legumes form a symbiosis with atmospheric nitrogen (N-2)-fixing soil rhizobia, resulting in new root organs called nodules that enable N-2-fixation. Nodulation is a costly process that is tightly regulated by the host through autoregulation of nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been investigated in Medicago truncatula, for which only rhizobia-induced MtCLE12 and MtCLE13 have been characterised. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. MtCLE35 was found to inhibit nodulation in a SUNN- and RDN1-dependent manner via overexpression analysis. Together, our findings indicate that MtCLE12 and MtCLE13 have a specific role in AON, while MtCLE35 regulates nodule numbers in response to both rhizobia and nitrate. MtCLE34 likely had a similar role to MtCLE35, but its function was lost due to a premature nonsense mutation.

Legume plants form a symbiosis with N2-fixing soil rhizobia, resulting in new root organs called nodules that enable N2-fixation. Nodulation is a costly process that is tightly regulated by the host through Autoregulation of Nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been characterised in Medicago truncatula, with only rhizobia-induced MtCLE12 and MtCLE13 identified. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control pathways. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. Overexpression of MtCLE12, MtCLE13 and MtCLE35 inhibits nodulation. Together, our findings indicate that MtCLE12 and MtCLE13 have a distinct role in AON, while MtCLE35 regulates nodule numbers in a rhizobia- and nitrate-dependent manner. MtCLE34 likely had a similar role to MtCLE35 but its function was lost due to a nonsense mutation resulting in the loss of the mature peptide.

Genome Mapping and Agriculture (R.C. Shoemaker, L.L. Lorenzen, B.W. Diers, and T.C. Olson). Usefulness of Plant Genome Mapping to Plant Breeding (F.L. Allen). Technology for Molecular Breeding: RAPD Markers, Microsatellites, and Machines (J.A. Rafalski, M.K. Hanafey, S.V. Tingey, and J.G.K. Williams). Multiple Arbitrary Amplicon Profiling Using Short Oligonucleotide Primers (G. Caetano-Anolles, B.J. Bassam, and P.M. Gresshoff). Length Polymorphisms of Simple Sequence Repeat (SSR) DNA as Molecular Markers in Plants (P.B. Cregan, M.S. Akkaya, A.A. Bhagwat, U. Lavi, and J. Rongwen). Approaches to Mapping in Horticultural Crops (N. Weeden). FISH, DNA Amplification Markers and Conifers (J.E. Carlson, Y.-P. Hong, G.R. Brown, and J.C. Glaubitz). Molecular Exploitation of Soybean Genetic Resources (L.O. Vodkin). Molecular Mapping of Soybean Nodulation Genes (P.M. Gresshoff and D. Landau-Ellis). Plant Telomeres as Molecular Markers (A.M. Kolchinsky and P.M. Gresshoff). Plant Yeast Artificial Chromosome Libraries and Their Use: Status and Some Strategic Considerations (R.P. Funke and A.M. Kolchinsky). Duplicate Loci in Soybean: Characterization of the sle and A071 Loci (K.M. Polzin, E.S. Calvo, T.C. Olson, and R.C. Shoemaker). Antisense RNA Inhibition of Photosynthetic Gene Expression (S. Rodermel, C-Z. Jiang, D. Kliebenstein, and J. Qian). Organization and Expression of Mitochondrial DNA Sequences Associated with Cytoplasmic Male Sterility in Phaseolus vulgaris L. (C.D. Chase and M.J. Bassett). Cell Cycle Genes and Their Plant Homologues (J. Deckert, N. Taranenko, and P.M. Gresshoff). The Basics of the Patent Process (L. Terlizzi). Recent Cases and Legislative Initiatives Pertaining to Biotechnology (R.H. Kjeldgaard). Patenting DNA: Is There a Bull in the China Shop? (L.L. Greenlee). Glossary. Index.

BACKGROUND AND AIMS:Although hypernodulating phenotype mutants of legumes, such as soybean, possess a high leaf N content, the large number of root nodules decreases carbohydrate availability for plant growth and seed yield. In addition, under conditions of high air vapour pressure deficit (VPD), hypernodulating plants show a limited capacity to replace water losses through transpiration, resulting in stomatal closure, and therefore decreased net photosynthetic rates. Here, we used hypernodulating (nod4) (282.33 ± 28.56 nodules per plant) and non-nodulating (nod139) (0 nodules per plant) soybean mutant lines to determine explicitly whether a large number of nodules reduces root hydraulic capacity, resulting in decreased stomatal conductance and net photosynthetic rates under high air VPD conditions.METHODS:Plants were either inoculated or not inoculated with Bradyrhizobium diazoefficiens (strain BR 85, SEMIA 5080) to induce nitrogen-fixing root nodules (where possible). Absolute root conductance and root conductivity, plant growth, leaf water potential, gas exchange, chlorophyll a fluorescence, leaf 'greenness' [Soil Plant Analysis Development (SPAD) reading] and nitrogen content were measured 37 days after sowing.KEY RESULTS:Besides the reduced growth of hypernodulating soybean mutant nod4, such plants showed decreased root capacity to supply leaf water demand as a consequence of their reduced root dry mass and root volume, which resulted in limited absolute root conductance and root conductivity normalized by leaf area. Thereby, reduced leaf water potential at 1300 h was observed, which contributed to depression of photosynthesis at midday associated with both stomatal and non-stomatal limitations.CONCLUSIONS:Hypernodulated plants were more vulnerable to VPD increases due to their limited root-to-shoot water transport capacity. However, greater CO2 uptake caused by the high N content can be partly compensated by the stomatal limitation imposed by increased VPD conditions.

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen-fixing rhizobia bacteria is central to their advantage. This plant-microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.

Pongamia pinnata has been established as a biofuel legume tree. Different methods such as acetylene reduction assay, ureide analysis, isotopic techniques, nitrogen difference method and isotope techniques (N-15 natural abundance and N-15 enrichment technique) were employed to analyze, assess and estimate symbiotic nitrogen fixation in 16-wk-old pongamia seedlings. In the acetylene reduction assay, the uninoculated control had negligible ethylene produced while inoculated plants were shown to have increasing ethylene production from 0-min to 60-min incubation with a range of 0.03 to 2.76 mL. Pongamia was found to produce 1.19 x 10-6 mole per plant with a Rhizobum (PR-UQ-05) inoculation. Ureide analysis was also done, not only to estimate fixed nitrogen, but also to determine the presence of allantoin in the xylem of pongamia. The result showed that allantoin was present in pongamia at low levels of 143-150 nmole, which means pongamia utilizes ureides in the form of allantoin to transport its fixed nitrogen to other plant parts to support growth and reproduction. The nitrogen difference method and isotope techniques quantified the fixed nitrogen of pongamia inoculated with PR-UQ-05 which was estimated to be 100 mg/plant (based on the difference of total N yield between the nodulated and non -nodulated pongamia seedlings) and from 20.4 mg/plant (natural abundance) to 47.4 mg/plant (enriched). The different methods used in this study showed different results based on the amount of fixed nitrogen calculated for each method. However, all of the methods employed in this study demonstrated that Pongamia inoculated with PR-UQ-05 fixed more nitrogen than the uninoculated control. The symbiotic nitrogen fixation of Pongamia demonstrated in this study is very relevant to the biofuel industries.