Diversity in Legume Root Nodules

4.3. Autoregulation of nodulation

11.4.8 Peptides in regulating nodulation

Nodulation is essential for nitrogen fixation by rhizobial bacteria. Genetic analysis of mutants of L. japonicus with a supernodulation phenotype allowed identifying the HYPERNODULATION ABERRANT ROOT FORMATION (HAR) gene that is important for regulating the nodule number in roots. HAR is homologous to CLV1, and is found to regulate nodule development systemically (Nishimura et al., 2002). Since CLV1 is demonstrated to bind the CLV3 peptide in regulating SAM maintenance, HAR is speculated to bind a CLE peptide for regulating nodulation as well. Expression of 39 LjCLE genes from L. japonicus is analyzed after inoculation with Mesorhizobium loti, leading to the identification of three LjCLE genes LjCLE-RS1, LjCLE-RS2, and LjCLE-RS3 with a significant upregulation of expression. Overexpression of LjCLE—RS1 and LjCLE—RS2 inhibits the nodulation systemically, and the nodulation suppression depends on the HAR1 receptor (Okamoto et al., 2009). With nano-LC-MS/MS analysis, LjCLE-RS2 was identified to be an arabinosylated glycopeptide with the hydroxylated proline at the seventh position that was modified further with three arabinose residues. The LjCLE-RS2 peptide synthesized in vitro binds directly to HAR1 at its arabinose chain in a sequence-dependent manner. LjCLE-RS2 produced in roots was found in xylem sap collected from shoots, implying that LjCLE-RSs may provide a long-distance mobile signal in the regulation of the initial step of nodulation (Okamoto et al., 2013).

Later, an LRR-RLK KLAVIER (KLV), which is highly homologous to the Arabidopsis RPK2 receptor kinase, was found to negatively regulate nodulation in L. japonicus. Double mutant analysis indicates that HAR and KLV act in the same signaling pathway. Biochemical analyses reveal a direct interaction between these two RLKs. Overexpression of LjCLE-RS1 and LjCLE-RS2 does not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required for LjCLE-RS1 and LjCLE-RS2 signaling, and acts downstream of LjCLE-RS1 and LjCLE-RS2 (Miyazawa et al., 2010).

Three CLE-related genes, GmRIC1, GmRIC2, and GmRIC3, were found in soybean (Glycine max), with a conserved CLE motif of 12 amino acid residues. GmRIC1, GmRIC2, and GmRIC3 regulate nodulation through the GmNARK RLK (Reid et al., 2011; Lim et al., 2011). Among 25 CLE genes identified in the Medicago truncatula genome, MtCLE12 and MtCLE13 regulate nodulation through an LRR-RLK called SUNN (Mortier et al., 2010). WOX5 was expressed during nodule organogenesis in M. truncatula. Its expression level was increased in supernodulation mutants such as har and klv, indicating that WOX5 may be involved in the CLE peptide-mediated nodulation process (Osipova et al., 2012).

4.13.2.2.1 Root nodules

Parasponia

Nodulation of the nonlegume genus Parasponia by rhizobia has many of the above ‘deviant’ properties. The rhizobia enters between epidermal cells and, as the bacteria penetrate deeper, true infection threads may form. Nitrogen fixation occurs in fixation threads. One additional property distinguishes Parasponia nodules from all known legume nodules. In the mature nodule, the infected cells, those that harbor the large fixation threads, are in the periphery (the nodule cortex) and vasculature develops in the center as it does in roots. This is the reverse of one property that appears to be true of all legume nodules: the infected nitrogen-fixing cells are central, with the vasculature being peripheral. In this respect, Parasponia nodule development is like that of the nodules of hosts of the actinorhizal bacterial genus Frankia. The presence of nod genes has been confirmed in rhizobia that infect Parasponia in all cases examined for this property. When the nod genes of one of these strains were mutated, it could not nodulate Parasponia or its legume hosts siratro and cowpea.

In all of these cases, it is the plant that determines these differences in nodule development; the differences assort with membership in different legume taxonomic groups. In contrast, a given rhizobial strain (usually a Bradyrhizobium strain) can nodulate and fix nitrogen on plants showing different modes of nodule development and infection, for example, crack-entry on peanut and root hair infection on cowpea. Whether the set of bacterial genes and components required is any different on these different hosts is for the most part undetermined.

Although the issues of infection threads and fixation threads have been addressed in recent sampling of the Caesalpinioideae, the issues of root hair infection and the requirement for Nod factor have hardly been addressed at all. It is important to do so, to assess whether or not the evolution of Nod factor was central to the early evolution of the symbiosis. Many of the variations in nodule development appear to be exclusively controlled by the plant, but Nod factor obviously required evolution involving the bacteria. Similarly, it would be revealing to investigate the requirements for bacterial polysaccharides (see ‘Rhizobial genes and components required in symbiosis’ and ‘Other properties of the rhizobia’) in the symbioses of the Caesalpinioideae. In general, the theory of nodule development would benefit from investigating mutants of the rhizobia that form symbioses with properties that deviate from those of the paradigm described above.

3.5 EICOSANOIDS IN HEMOCYTE SPREADING AND MIGRATION

Microaggregation and nodulation reactions are large-scale, visible cellular defense reactions to microbial challenge. We take these large-scale reactions to be the culmination of an unknown number of small-scale, relatively invisible reactions. Plasmatocyte spreading on surfaces may be regarded as one of the component actions that comprise the overall nodulation process. Because of the important role in nodulation, investigated the hypothesis that eicosanoids mediate cell spreading in primary hemocyte cultures prepared from tobacco hornworms.

Plasmatocytes from control hornworms elongated to about 41 μm after 60 min incubations. The most rapid cell elongation took place during the first 30 min and length did not significantly increase in longer incubation periods. The elongation reactions were severely truncated in primary hemocyte cultures prepared from hornworms that had been treated with Dex and the Dex effect was expressed in a dose-related manner. The inhibitory influence of Dex was reversed by injecting AA into Dex-treated hornworms. also found that inhibitors of COX and LOX pathways resulted in truncated elongation. Hence, the outcomes of these experiments allow the conclusion that eicosanoids mediate one of the smaller-scale steps in the overall nodulation process, namely plasmatocyte elongation.

Continuing in this line, Kwon et al. (2007) considered the influence of bacterial infection on plasmatocyte elongation. We found that hemocytes prepared from tobacco hornworms 15 and 60 min after infection were altered in size. Specifically, all hemocytes were smaller than 15 μm and none of the hemocytes exhibited cell spreading. On the idea that this change could result from an adventitious influence of S. marcescens, our standard bacterial challenge species, we performed these experiments with three additional bacterial species, Escherichia coli, Bacillus subtilis and Micrococcus luteus. The results were similar for all four species. In another set of experiments, we found that the influence of bacterial infection on cell spreading declined with time after infection. As seen before, the retarding influence of Dex was reversed by AA, PGH₂ and CM. For these experiments, we considered the possibility that CM prepared in the presence of bacteria could be contaminated with bacterial molecules that passed through the filter step. The appropriate control experiment was to prepare CM using only bacterial cells in the absence of hemocytes. We showed that CM prepared in the absence of hemocytes did not influence cell behavior. Overall, Kwon et al. (2007) demonstrated that bacterial infection exerts a strong effect on eicosanoid mediate cell spreading reactions to challenge.

Formation of root nodules in plants

The rhizobium-mediated nodulation of legumes has more consideration because of its vast varieties of applications. Nodules are various in shape and they are determinate (spherical with lenticels) noticed on different plant varieties like soybeans and other species grown in tropical and subtropical areas. Another one form of nodule is indeterminate (cylindrical and branched) found on alfalfa, peas, and clover grown in temperate regions (Allito et al., 2021; Hassen et al., 2016). Kitts and Ludwig (1994) documented the formation of in rice plant by A. caulinodans have the ability to fix the atmospheric nitrogen in the free-living state up to 3% (v/v) oxygen and without differentiation into bacteroids. The rhizobia are able to colonize the roots of nonlegume plants like barley, wheat, and maize. Gough et al. (1996) determined the plant-based chemical constituents such as flavonoid and naringenin could stimulate the colonies of Azorhizobium sp. on nonlegume root systems.

The symbiotic relationship between the rhizobial isolates and legumes generates root nodules, in where the bacteria can fix the atmospheric nitrogen through the action of the nitrogenase enzyme (Peix et al., 2015). The coinoculation of rhizobial strain with phosphate solubilizing microbes induces the plant growth and nodulation. Oldroyd et al. (2011) documented the root nodule found in symbiotic legume plants involves a complex molecular signaling between the legume host and the rhizobial microsymbiont. In accordance with these, Sibponkrung et al. (2020) performed the coinoculation of Bacillus velezensis (S141) along with Bradyrhizobium sp. (USDA110) into a plant soybean showed enhanced nodulation and high N₂-fixing efficiency. Allito et al. (2021) observed the efficacy of Rhizobium sp. for nodulation on faba bean through greenhouse effect and field conditions.

4.07.4.2 Microbial Signal Compounds and Plant Growth Promotion

31.1.5 Resistance-Nodulation-Cell Division Superfamily

The efflux pumps of the RND superfamily are composed of tripartite proteins and composed of an inner membrane pump protein, a periplasmic fusion protein, and an outer membrane porin. The outer membrane porin is quite promiscuous, interacting with other RND systems as the protein that completes the cell efflux through the periplasm and outer membrane. The most well-studied member of this superfamily is AcrAB-TolC, and a homolog of this efflux pump has been identified in almost every Gram-negative organism sequenced to date (Elkins and Mullis, 2007). These efflux pumps are active transporters, driven by the PMF. As the best-studied member of this superfamily, AcrAB from E. coli has crystallographic structures both with and without substrates bound. The TolC outer membrane component is associated with AcrAB, AcrEF, MdtABC, and MdtEF in the enteric bacterial systems.

AcrAB is constitutively expressed in wild-type E. coli and provides resistances to a broad range of substrates. These substrates include antibiotics such as penicillins, macrolides, tetracyclines, tigecycline, chloramphenicol, and novobiocin, in addition to disinfectants, detergents, and organic solvents including ethidium bromide, sodium dodecyl sulfate, macrolides, and triclosan.

Soil nutrient availability

Soil nutrient status has a tremendous influence on the symbiosis as well as independent growth and survival of both partners.

Several field studies also demonstrate that nodulation and nitrogen fixation can be inhibited by high field nitrogen levels. High levels of nitrate in or near the nodules inhibits nitrogenase activity through a feedback mechanism, thereby reducing nitrogen fixation. Above a certain concentration, excess nitrogen can inhibit nodule initiation entirely. Estimates for nitrogen levels that will eliminate nodulation vary widely from lower value of 50 kg/ha to high value of 120 kg/ha. Tolerance of nitrogen fixation to high soil nitrate levels varies across species and even among genotypes of the same species (). Ammonium, the direct product of nitrogen fixation, is well known to inhibit nitrogen fixation (see sections above “” and “Nitrogen regulation”) and has been shown to inhibit nitrogenase synthesis at the genetic level through the regulation of nifA gene transcription. Expression of nitrogenase in symbiotic diazotrophs is fairly insensitive to ammonium because export of ammonium to their symbiont suppresses ammonium levels. The expression of nif genes in free-living diazotrophs is more sensitive to cellular ammonium levels.

The availability of phosphorus and micronutrients, including Fe, Mo, and V, is known to influence nitrogen fixation (). In low phosphorus-content soil, phosphorus has been shown to control the rates of heterotrophic biological nitrogen fixation and to limit biological nitrogen fixation in rhizobial symbioses. Phosphorus appears essential for both nodulation and nitrogen fixation, as phosphorus deprivation is often associated with decreased nodule tissue formation and low rates of nitrogen fixation. Legume-Rhizobia symbiosis requires micronutrients including boron, cobalt, copper, iron, manganese, molybdenum, nickel, selenium, and zinc, sometimes at higher rates than the plant or free-living bacteria require alone. Iron, sulfur, molybdenum and vanadium are essential components of nitrogenases (Nif, Vnf, and Anf) (see section above, “Biological nitrogen fixation and nitrogenase enzymes”), and the availability of these trace metals may be critical for the nitrogen cycle of terrestrial ecosystems.

The process of nodulation and its molecular mechanism