The formation of nitrogen-fixing nodules on legumes requires an integration of infection by rhizobia at the root epidermis and the initiation of cell division in the cortex, several cell layers away from the sites of infection. Several recent developments have added to our understanding of the signaling events in the epidermis associated with the perception of rhizobial nodulation factors and the role of plant hormones in the activation of cell division leading to nodule morphogenesis. This review focuses on the tissue-specific nature of the developmental processes associated with nodulation and the mechanisms by which these processes are coordinated during the formation of a nodule.

Coordinating nodule morphogenesis with rhizobial infection in legumes.

The P450 enzymes (mixed function oxidases, cytochrome P450 monooxygenases), a diverse class of enzymes found in virtually all insect tissues, fulfill many important tasks, from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of foreign chemicals of natural or synthetic origin. This diversity in function is achieved by a diversity in structure, as insect genomes probably carry about 100 P450 genes, sometimes arranged in clusters, and each coding for a different P450 enzyme. Both microsomal and mitochondrial P450s are present in insects and are best studied by heterologous expression of their cDNA and reconstitution of purified enzymes. P450 genes are under complex regulation, with induction playing a central role in the adaptation to plant chemicals and regulatory mutations playing a central role in insecticide resistance. Polymorphisms in induction or constitutive expression allow insects to scan their P450 gene repertoire for the appropriate response to chemical insults, and these evolutionary pressures in turn maintain P450 diversity.

Insect P450 enzymes.

Proanthocyanidins (PA) (condensed tannins) and hydrolyzable tannins (HT) are the two major classes of tannins. Proanthocyanidins are flavonoid polymers. Hydrolyzable tannins are polymers of gallic or ellagic acid esterified to a core molecule, commonly glucose or a polyphenol such as catechin. Proanthocyanidins are the most common type of tannin found in forage legumes. Problems in the analysis of tannins are that sample processing and drying decrease extraction and reactivity, suitable standards are unavailable, and quantitative analytical methods are poorly correlated with enzyme inhibition, protein precipitation, and nutritional effects. Hydrolyzable tannins are potentially toxic to ruminants. Pyrogallol, a hepatotoxin and nephrotoxin, is a product of HT degradation by ruminal microbes. Proanthocyanidins are considered to be non-toxic because they are not absorbed, but they are associated with lesions of the gut mucosa. Research on tannins in forage legumes has determined their effects on protein digestion and metabolism but more research on tannin structure in relation to digestion of specific proteins is needed. The widely accepted explanation for positive effects of PA on protein digestion and metabolism is that PA-protein complexes escape ruminal degradation and the protein is available in the lower tract. This proposed mechanism may be incorrect because PA also complex carbohydrates, endogenous proteins, and microbial products and the degradability of PA-protein complexes by ruminal microbes has not been adequately studied. Several alternative hypotheses (to escape protein) that explain the effect of PA on protein digestion and metabolism in ruminants are also consistent with experimental results on forage legumes. These include increased microbial protein synthesis, increased use of endogenous nitrogen in the rumen, and increased secretion of salivary glycoproteins. Research on manipulating the content and type of PA in forage legumes is justified because they are associated with non-bloating legumes, lower soluble non-protein nitrogen in silage, and improved efficiency of protein utilization. Research on the biosynthesis, molecular genetics, and cell biology of PA in forage legumes needs to be integrated with research on toxicology and nutrition.

Nutritional toxicology of tannins and related polyphenols in forage legumes

(1988). Inhibition of legume nodule formation and N2 fixation by nitrate. Critical Reviews in Plant Sciences: Vol. 7, No. 1, pp. 1-23.

Inhibition of legume nodule formation and N2 fixation by nitrate

Legumes are highly important food, feed and biofuel crops. With few exceptions, they can enter into an intricate symbiotic relationship with specific soil bacteria called rhizobia. This interaction results in the formation of a new root organ called the nodule in which the rhizobia convert atmospheric nitrogen gas into forms of nitrogen that are useable by the plant. The plant tightly controls the number of nodules it forms, via a complex root-to-shoot-to-root signaling loop called autoregulation of nodulation (AON). This regulatory process involves peptide hormones, receptor kinases and small metabolites. Using modern genetic and genomic techniques, many of the components required for nodule formation and AON have now been isolated. This review addresses these recent findings, presents detailed models of the nodulation and AON processes, and identifies gaps in our understanding of these process that have yet to be fully explained.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1744-7909.2010.00899.x

Molecular Analysis of Legume Nodule Development and Autoregulation

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.

Regulation of the Soybean-Rhizobium Nodule Symbiosis by Shoot and Root Factors

Extended target-site specificity for a hammerhead ribozyme.

We happily accepted the invitation to write this chapter because we have experienced the development of new approaches to research into the genetics, microbiology and biochemistry of symbiotic nitrogen fixation. We were confronted with a broad range of advances in the biochemical genetics of Rhizobium, especially relating to the genetic elements controlling the key symbiotic phenotypes: nitrogen fixation (the nif and fix genes) and nodulation (controlled by the nod genes). Likewise there has been a major application of plant molecular biology to the analysis of nodule-specific or nodule-enhanced plant gene products (Lee et al., 1983; Fuller et al., 1983; Govers et al., 1985; and Kaninakis and Verma, 1985). This major class of proteins or nodulins allows the molecular visualisation of the plant genome’s contribution to the symbiosis (Verma et al., 1985).

Plant Genetic Approaches to Symbiotic Nodulation and Nitrogen Fixation in Legumes

Ten of 11 supernodulating mutants of soybean [Glycine max (L.) Merr.] cv. Bragg, in which nodulation was far in excess of that in the wild type, showed pronounced tolerance of nodulation to applied nitrate. Mutant nts (nitrate-tolerant symbiosis) 1116 had an intermediate nodulation response and also showed some inhibition by nitrate. Mutant 1029, a revertant of nts382 (an extreme supernodulator), showed a wild-type nodulation pattern and was equally sensitive to nitrate as cv. Bragg. Grafting experiments with cv. Bragg and nts382 indicated that both supernodulation and tolerance of nodulation to nitrate were dependent on shoot factors. Total leaf nitrate reductase (EC 1.6.6.1 and EC 1.6.6.2) activity of the supernodulating mutants was similar to that in cv. Bragg. We conclude from these results that the inhibitory effect of nitrate on nodule initiation and development in soybean depends on an interaction between nitrate and the autoregulation singal. In the supernodulating mutants, the autoregulation signal is either altered or absent and cosequently nodulation in these mutants is not sensitive to nitrate.

Angela C. Delves

Papers

Regulation of the Soybean-Rhizobium Nodule Symbiosis by Shoot and Root Factors

Extended target-site specificity for a hammerhead ribozyme.

Plant Genetic Approaches to Symbiotic Nodulation and Nitrogen Fixation in Legumes

Relationship between autoregulation and nitrate inhibition of nodulation in soybeans

Biochemistry and physiology of esterases in organophosphate-susceptible and -resistant strains of the Australian sheep blowfly, Lucilia cuprina