Infection of legume root cells by rhizobia requires plant cell-wall remodeling. This must involve localized degradation of some of the plant wall components accompanied by the synthesis of new cell wall material. This remodeling can occur in root hairs, but also occurs at each new cell wall that must be bridged as the infection thread (IT) grows down through cells into the root. Even in those legumes in which root-hair infection does not occur, localized degradation and cell-wall remodeling can enable intercellular rhizobia to initiate cell infection. This review considers some of the issues that must occur during cell-wall remodeling including the degradation and modification of pectin and other cell wall carbohydrates. It is now clear that nodulation signaling by rhizobia induces the expression of a root-infection-specific form of a legume pectate lyase. It seems likely that this enzyme must be locally targeted to the sites of rhizobial infection foci and sites where growing ITs traverse plant cell walls. There is evidence that pectin methylesterases and polygalacturonases may be induced along with pectate lyase.
Nitrogenase carries at its active site a unique iron–molybdenum cofactor (FeMo-co) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex pathway involving proteins acting as molecular scaffolds, as metallocluster carriers, or enzymes to provide substrates in appropriate chemical forms. Insertion of completed FeMo-co into a P-cluster containing but FeMo-co-deficient NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for iron–sulfur clusters in biology.
Chapter 9 Regulation of nif Gene Expression in Azotobacter vinelandii César Poza-Carrión, César Poza-Carrión Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, SpainSearch for more papers by this authorCarlos Echavarri-Erasun, Carlos Echavarri-Erasun Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, SpainSearch for more papers by this authorLuis M. Rubio, Luis M. Rubio Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, SpainSearch for more papers by this author César Poza-Carrión, César Poza-Carrión Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, SpainSearch for more papers by this authorCarlos Echavarri-Erasun, Carlos Echavarri-Erasun Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, SpainSearch for more papers by this authorLuis M. Rubio, Luis M. Rubio Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, SpainSearch for more papers by this author Book Editor(s):Frans J. de Bruijn, Frans J. de BruijnSearch for more papers by this author First published: 14 July 2015 https://doi.org/10.1002/9781119053095.ch9Citations: 4 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary Nitrogen fixation is a highly regulated trait. In the free-living diazotrophic bacterium Azotobacter vinelandii the nitrogen fixation (nif) regulon consists of at least 50 genes distributed in two chromosomal regions. Analysis of polar mutations, Northern blots, lacZ transcriptional fusions and, more recently, quantitative real-time PCR and transcriptomics, has rendered a detailed picture of nif operon structures and expression patterns. Specific regulatory elements NifA and NifL, in concert with nitrogen control proteins GlnD and GlnK, comprise a system that integrates cellular redox, energy, and nitrogen signals to modulate initiation of nif gene expression by a σ54-containing RNA polymerase. Citing Literature Biological Nitrogen Fixation RelatedInformation
The ability of legumes to acquire sufficient nitrogen from the symbiosis with Rhizobium relies on the intimate contact between the endosymbiotic, intracellular rhizobia, called bacteroids, and their host cells, the symbiotic nodule cells. Nodules contain several thousand symbiotic cells, each harboring thousands of bacteroids. Bacteroids are differentiated bacteria that have a specific metabolism, and in many legumes their formation is accompanied with a morphological transformation involving strong cell enlargement and genome amplification. We have shown that in Medicago and related legumes, the symbiotic nodule cells produce large amounts of highly diverse nodule-specific effector peptides called NCR peptides. These control the bacteroid differentiation by inducing their elongation and DNA amplification. The NCR peptides are similar to antimicrobial peptides (AMPs) of innate immunity in animals and plants and can kill rhizobia in vitro. The BacA protein of Sinorhizobium meliloti provides protection against the antimicrobial activity of NCR peptides and other AMPs with similar mode of action, and intracellular rhizobia require the BacA protein to survive the challenge of the NCR peptides in the nodule cells. BacA is conserved among many bacteria, and several animal pathogens also require BacA or BacA-like proteins for pathogenesis and chronic infection of their host, most likely by providing protection against host-produced innate immunity AMPs. BacA and BacA-like proteins belong to the ABC transporter family. However, what is transported and how this protein provides protection against AMPs remain unknown. Several hypotheses are discussed.
Actinorhizal plants can establish an ecologically important symbiosis that leads to the development of nitrogen-fixing nodules in response to the soil actinobacteria Frankia. Actinorhizal root nodulation following intracellular infection by Frankia is a multistep process involving both plant and Frankia specific signals that activate a symbiotic signaling pathway. Flavonoids have been shown to improve the nodulation process and to be necessary for root hair infection, and ongoing research efforts focus on the biochemical characterization of Frankia signals. With the development of RNA interference to knock down candidate symbiotic genes in the actinorhizal plants Casuarina glauca and Datisca glomerata, plant genes essential for both infection, nodule formation and endomycorrhization have been characterized. Data provide evidence that there are some similarities in the molecular mechanisms underlying the nodulation process by Frankia and rhizobia.
While numerous isolations of diazotrophic bacteria from sugarcane in different countries and regions have led to the characterization of many new species of diazotrophic bacteria in the last decades, the question still remains whether these bacteria represent the complete community of diazotrophic bacteria in sugarcane plants, which actively fix nitrogen in planta (rhizosphere, roots, stems and leaves). Therefore, cultivation-independent molecular approaches to characterize the diazotrophic bacterial community of sugarcane, which may provide information about hitherto not cultivated bacteria contributing biologically fixed nitrogen to sugarcane plants were carried out. The experiments were conducted from 2008 to 2009 at Embrapa-Agrobiologia in Seropédica, Rio de Janeiro, Brazil, with the sugarcane cultivar RB867515, which is currently one of the most used sugarcane cultivars in Brazil. In these greenhouse and field studies, polymerase chain reaction (PCR) amplification of 16S rDNA and nifH DNA as well as 16S-cDNA and nifH-cDNA was performed using DNA and RNA extracted from roots and leave sheaths of sugarcane. A number of sequences were retrieved from genera and species that had been already characterized by the cultivation approach, although it appeared that the diversity reflected by the amplified sequences was much higher in terms of different bacterial species (which may not have been characterized in detail yet). However, many bacterial sequences that were not represented in the existing cultured strains, such as Ideonella, Bradyrhizobium or Rhizobium, also appeared. Interestingly, some of these species represented apparently active nitrogen-fixing bacteria in the sugarcane tissues, because we frequently found them within the RNA-pool (16S-cDNA- and nifH-cDNA-sequence libraries). Therefore, attempts were made to cultivate Bradyrhizobium bacteria from the same field-grown sugarcane variety, using new isolation approaches that have not yet been applied to sugarcane, such as enrichment with promiscuous leguminous trap plants, for example, Vigna unguiculata (cowpea). Indeed, a high diversity of Bradyrhizobium spp. could be isolated, which were found to fix nitrogen – at least to some extent – also in vitro. These newly isolated nitrogen-fixing bacteria resembled part of the sequences retrieved from the same plant material using cultivation-independent approaches. It remains to be investigated whether these bacteria are indeed able to contribute substantial amounts of fixed nitrogen to sugarcane, which has been repeatedly demonstrated using 15N-natural abundance and dilution techniques.
Biogenesis of the symbiosome membrane is accompanied by the synthesis and targeting of a variety of channel and transporter proteins with functions that support rhizobia–legume symbioses. Among these proteins are members of the “nodulin 26 intrinsic protein” family of aquaporin-like channels, which are a major protein component of the mature nitrogen-fixing symbiosome. Unlike aquaporins, nodulin 26 is a multifunctional channel that has a low intrinsic aquaporin activity that is regulated by phosphorylation, as well as the ability to transport a number of other metabolites, including the product of nitrogen fixation, NH3. Nodulin 26 also serves as a docking site for the interaction of the major nitrogen assimilatory enzyme in the infected cell cytosol, glutamine synthetase. The nodulin 26/glutamine synthetase interaction may serve as a metabolic channel, allowing facile assimilation of transported ammonia and lowering the potential toxicity of ammonia by preventing cytosolic accumulation.
The Bradyrhizobium–Aeschynomene system initially attracted attention for the unusual ability of Aeschynomene species to develop both root and stem nodules with photosynthetic Bradyrhizobium. The recent discovery that some of these Aeschynomene species are efficiently nodulated by Bradyrhizobium strains lacking the canonical nodABC genes has challenged the paradigm of the rhizobial Nod factors as compulsory keys to trigger the nitrogen-fixing symbiosis in legumes. The presence of a Nod-independent symbiosis, along with an intercellular infection process, in Aeschynomene contrasts with what has been described for Medicago truncatula and Lotus japonicus and highlights the need for developing an additional model legume. In this review, we present the emergence of A. evenia to fulfill this role. Its symbiotic properties, the advantageous genetic characteristics, and the development of functional tools make it a promising model for molecular and genetic analysis.
Recent developments in massive parallel sequencing opened new possibilities in the field of transcriptomic studies in rhizobia. With sequencing costs ever more affordable, it became possible to obtain close-to-complete transcript inventories from cellular pools of total RNA. This approach known as RNA-Seq was applied to the analysis of the promiscuous and fast-growing symbiotic Sinorhizobium fredii strain NGR234. In this study, we report on the comparative analysis of RNA-Seq data collected in three experimental conditions designed to stimulate the molecular responses of NGR234 to the major and consecutive symbiotic steps of nodulation and nitrogen fixation. In the first two conditions, free-living cells of NGR234 were grown in vitro in a minimal medium supplemented with a carbon source. To mimic the presence of a compatible host plant, NGR234 cells were exposed for 6 h to a flavonoid that induces transcription of nodulation genes while control cells were not. To monitor gene expression during symbiotic nitrogen fixation, total RNA was isolated from nodules collected on roots of cowpea 38 days post-inoculation with NGR234. These three RNA samples were analyzed by high throughput sequencing, and the resulting sequence reads were mapped onto the NGR234 genome. This chapter provides a description of the experimental setup and data analysis pipeline, as well as illustrates the wealth of complementary data obtained by probing the NGR234 symbiotic responses with RNA-Seq.
Rhizobiales diversity was characterized using previously published high-throughput DNA sequence data from 88 soil samples. To determine how environmental variables affect Rhizobiales diversity, soil and site characteristics were compared to Rhizobiales abundance and community assemblage patterns. On average, Rhizobiales accounted for 9.2% of soil bacteria; across all soils Rhizobiales relative abundance ranged from 3.7% to 18.2%. Six environmental variables correlated significantly with Rhizobiales relative abundance, with potential carbon mineralization rate correlating most strongly with relative abundance (R = 0.41). Over 60% of Rhizobiales phylotypes occurred in only a single soil sample, and less than 6% were detected in more than 10 soil samples. Soil pH had the strongest effect on Rhizobiales community structure (R = 0.50), with the presence or absence of particular lineages dictated by soil pH.
