Content of indoleacetic acid and its metabolism in root nodules ofMelilotus alba

The root nodules ofMelilotus alba, a leguminous fodder herb, contain a high amount of indoleacetic acid (IAA). The tryptophan pool present in the nodule might serve as a source for the IAA production. Metabolism of IAA in the nodules was evidenced by the presence of IAA-metabolizing enzymes, IAA oxidase and peroxidase. A high amount of IAA was produced by the symbiont isolated from the nodules in culture, when supplemented with tryptophan. For IAA production, the bacteria preferred thel-isomer over thedl- ord-isomer of tryptophan. The possible role of nodular IAA production on the legume-Rhizobium symbiosis is discussed.     

Integrated effects of abiotic stresses on inoculant performance, legume growth and symbiotic dependence estimated by 15N dilution

Temperature, water, salinity, sodicity, acidity and nutrient disorders are major abiotic stresses that can affect legume growth or the establishment and function of the legume-Rhizobium symbiosis. We have examined the literature where the application of the ¹⁵N isotope dilution methodology permits the effect of individual abiotic stresses to be independently and quantitatively separated into plant growth-mediated and BNF (biological N₂ fixation)-mediated components. The response of the symbiosis to a particular stress depends on a host of factors, including legume genotype, cultivar, Rhizobium inoculant, climatic conditions, and the duration, timing and severity of the stress. Published data are analysed in terms of the above variables and their interactions. As a general rule, severe stress inhibits both legume dry matter (DM) and the proportional dependence of the legume on BNF as a source of N. The symbiosis is resilient to low to moderate stress, but there may still be a penalty on legume DM. Gaps in knowledge are identified, and general guidelines on the identification and amelioration of abiotic stresses are provided.     

Pathogenic and mutualistic plant-bacteria interactions: ever increasing similarities

Plant-interacting bacteria can establish either mutualistic or pathogenic interactions that cause beneficial or detrimental effects respectively, to their hosts. In spite of the completely different outcomes, accumulating evidence indicates that similar molecular bases underlie the establishment of these two contrasting plant-bacteria associations. Recent findings observed in the mutualistic nitrogen-fixing Rhizobium-legume symbiosis add new elements to the increasing list of similarities. Amongst these, in this review we describe the role of plant resistance proteins in determining host specificity in the Rhizobium-legume symbiosis that resemble the gene-for-gene resistance of plant-pathogen interactions, and the production of antimicrobial peptides by certain legumes to control rhizobial proliferation within nodules. Amongst common bacterial strategies, cyclic diguanylate (c-di-GMP) appears to be a second messenger used by both pathogenic and mutualistic bacteria to regulate key features for interaction with their plant hosts.     

Suitability of rhizobia-inoculated wild legumes Argyrolobium flaccidum,Astragalus graveolens,Indigofera gangetica and Lespedeza stenocarpa in providing a vegetational cover in an unreclaimed limestone quarry

Several legume-Rhizobium associations were evaluated by sowing and broadcast of seeds encapsulated with polyacrylamide-entrapped rhizobia (PER) in an opencast limestone quarry in the outer ranges of the Himalaya. Four wild legumes, Lespedeza stenocarpa, Astragalus graveolens, Argyrolobium flaccidum and Indigofera gangetica, with various rhizobial strains, showed higher seedling establishment and survival as well as higher biomass than controls (uninoculated treatments). All legumes established without aftercare. Both A. flaccidum and L. stenocarpa flowered and fruited and the self sown seeds of the experimental stands of the latter species also germinated. Within four years following seeding, many non-legumes colonized the experimental site indicating the amelioration of the derelict habitat. These results give evidence that novel, suitable wild legume-Rhizobium associations are useful in providing a vegetational cover in degraded lands, and that the ecological restoration of limestone-mined sites are possible to some extent by artificial reconstruction. Nodulation in inoculated treatments only suggests that revegetation programmes involving legumes should also include their microsymbionts. Results also suggest that the encapsulation of seeds with PER is a suitable inoculation technology for the revegetation programmes.Abbreviations ARA acetylene reduction activity - CFU colony forming unit(s) - PER polyacrylamide-entrapped rhizobia - YM yeast extract mannitol (medium)     

Methods for measuring biological nitrogen fixation in grain legumes

To assure proper management and fully realize the benefits of the legume-Rhizobium symbiosis it is necessary to be able to quantify the amount of nitrogen fixed. Having measured the effectiveness of atmospheric N₂ fixation the macro- or micro-symbionts as well as agronomic factors can be manipulated with the objective to maximize biological nitrogen fixation. A suitable method to quantify nitrogen fixation is therefore necessary in any programme aiming at increasing N₂ fixation, like the one being reported in this volume. There are several methods available to quantify nitrogen fixation and most of the commonly used ones are described in the present paper listing their advantages and disadvantages.     

Rhizobial communities in symbiosis with legumes: genetic diversity,competition and interactions with host plants

The term ‘Rhizobium-legume symbiosis’ refers to numerous plant-bacterial interrelationships. Typically, from an evolutionary perspective, these symbioses can be considered as species-to-species interactions, however, such plant-bacterial symbiosis may also be viewed as a low-scale environmental interplay between individual plants and the local microbial population. Rhizobium-legume interactions are therefore highly important in terms of microbial diversity and environmental adaptation thereby shaping the evolution of plant-bacterial symbiotic systems. Herein, the mechanisms underlying and modulating the diversity of rhizobial populations are presented. The roles of several factors impacting successful persistence of strains in rhizobial populations are discussed, shedding light on the complexity of rhizobial-legume interactions.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Oxidative processes at initial stages of interaction of nodule bacteria (<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>) and pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): A review

A possible physiological mechanism of legume-Rhizobium symbiosis, consisting in regulation of the intensity of oxidative processes by the macrosymbiont in response to infection with Rhizobium, was analyzed using our own and published data. The results used in the analysis included data on the content of reactive oxygen species (O ₂ ^·? and H₂O₂), activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), and intensity of lipid peroxidation proceeding with the involvement of lipophilic phenolic compounds of the microsymbiont.     

Nodulins and nodulin genes of Glycine max

Summary Nodulins are organ-specific plant proteins induced during symbiotic nitrogen fixation. Nodulins play both metabolic and structural roles within infected and uninfected nodule cells. In soybean, several nodulin genes, coding for abundant nodulins, have been identified and isolated. Structural analysis of some of these genes has revealed their possible mode of regulation and the subcellar location of the protein product. Studies of ineffective symbiosis based on cultivar-strain genotype differences suggested that both partners influence the expression of nodulin genes. Concomitant with nodule organogenesis, the Rhizobium undergoes substantial differentiation leading to the accumulation of nodule-specific bacterial proteins, bacteroidins. The major structural alteration occuring in the infected cell is the formation of a membrane enclosing the bacteroid (peribacteroid membrane). A number of nodulins are specifically targetted to this membrane during endosymbiosis. The induction of nodulins and bacteroidins leads to the formation of an effective nodule. Nodulin genes can be induced in vitro by factors derived from nodules suggesting that trans-activators may be involved in derepression of the host genes necessary for Rhizobium-legume symbiosis.     

Signal Transduction Pathways in Mycorrhizal Associations: Comparisons with theRhizobium–Legume Symbiosis

A number of genera of soil fungi interact with plant roots to establish symbiotic associations whereby phosphate acquired by the fungus is exchanged for fixed carbon from the plant. Recent progress in investigating these associations, designated as mycorrhizae (sing., mycorrhiza), has led to the identification of specific steps in the establishment of the symbiosis in which the fungus and the plant interact in response to various molecular signals. Some of these signals are conserved with those of theRhizobium–legume nitrogen-fixing symbiosis, suggesting that the two plant–microbe interactions share a common signal transduction pathway. Nevertheless, only legume hosts nodulate in response toRhizobium,whereas the vast majority of flowering plants establish mycorrhizal associations. The key questions for the future are: what are the signal molecules produced by mycorrhizal fungi and how are they perceived by the plant?     

Rhizobium extracellular structures in the symbiosis

The extracellular and surface polysaccharides produced by Rhizobium species constitute a composite macromolecular interface between the bacterial cell and its environment. Several of these polysaccharides are involved in the complex series of interactions leading to the establishment of an effective Rhizobium-legume symbiosis. Extracellular heteropolysaccharides (EPSs) are found in culture supernatants, while capsular polysaccharides adhere to the cell surface. Cyclic (1–2)--d glucan is a periplasmic oligosaccharide that has also been found in the culture supernatants of some strains. The lipopolysaccharides (LPSs), which form part of the outer membrane and contain the O-somatic antigens, comprise the other major group of extracellular polysaccharides. In this review we will describe the major Rhizobium extracellular structures and their role in symbiosis with leguminous plants.The authors are with the Departamento de Microbiologia y Parasitologia, Facultad de Farmacia. Universidad de Sevilla, 41012 Sevilla, Spain     

Heterologous expression to assay for plant lectins or receptors

Heterologous expression of genes for membrane proteins can provide a useful approach to analyze ligand binding and other cell surface characteristics. We analyzed the expression and processing of a barley lectin gene in mammalian cells and demonstrated that this cytotoxic plant lectin could be expressed in a functional state using a transient expression system. The mammalian cells did not recognize all the processing signals on the lectin, and, as a result, the protein was secreted into the medium. The lectin expression studies suggest that it would be feasible to use the mammalian system for the expression and identification of plant genes encoding proteins that are able to bind the Nod factor, a bacterially-produced signal molecule required for the establishment of the legume-Rhizobium symbiosis. A Nod factor-binding assay was developed, and specific sets of transfected mammalian cells were shown to exhibit Nod factor-binding activity.     

Control of the expression of bacterial genes involved in symbiotic nitrogen fixation

Several genera of N₂-fixing bacteria establish symbiotic associations with plants. Among these, the genus Rhizobium has the most significant contribution, in terms of yield, in many important crop plants. The establishment of the Rhizobium-legume symbiosis is a very complex process involving many genes which need to be co-ordinately regulated. In the first instance, plant signal molecules, known to be flavonoids, trigger the expression of host-specific genes in the bacterial partner through the action of the regulatory NodD protein. In response to these signals, Rhizobium bacteria synthesize lipo-oligosaccharide molecules which in turn cause cell differentiation and nodule development. Once the nodule has formed, Rhizobium cells differentiate into bacteroids and another set of genes is activated. These genes, designated nif and fix, are responsible for N₂ fixation. In this system, several regulatory proteins are involved in a complex manner, the most important being NifA and a two component (FixK and FixL) regulatory system. Our knowledge about the establishment of these symbioses has advanced recently, although there are many questions yet to be solved.     

Extracellular polysaccharides and polysaccharide-containing biopolymers from Azospirillum species: properties and the possible role in interaction with plant roots

This paper reviews the results obtained in studies of the extracellular polysaccharides, lipopolysaccharide-protein complexes, polysaccharide-lipid complexes, lipopolysaccharides, and O-specific polysaccharides from bacteria of the genus Azospirillum. On the basis of present knowledge, the possible roles of the extracellular polysaccharides and polysaccharide-containing complexes of azospirilla in interaction with the roots of plants are discussed. Some pieces of evidence are considered in light of the lectin hypothesis originally proposed for the legume-Rhizobium symbiosis. In the context of these views of Azospirillum-cereal associative pairs, a key process at the early stages of the interaction is the specific reaction of cereal root lectins with the extracellular polysaccharide components, containing N-acetyl-d-glucosamine as part of their structure.     

Legume-Rhizobium Interactions: Cowpea Root Exudate Elicits Faster Nodulation Response by Rhizobium Species

Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH₄⁺ in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH₄⁺, indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.     

蚜虫与其初级内共生菌进化关系: 假说及演化机理

蚜虫是半翅目(Hemiptera)中一类取食植物韧皮部汁液的昆虫, 由于具有一些独特的生物学特征, 是研究重要适应进化问题的理想模型。蚜虫体内存在一类专性的胞内共生菌Buchnera, 对于蚜虫营养代谢和正常发育有重要贡献, 被称为蚜虫的初级内共生菌。蚜虫-Buchnera是研究共生关系的理想模型, 两者系统发育格局的研究有助于理解生物间专性共生关系的演化。本文系统综述了两者在不同分类水平（高级阶元、 低级阶元）上的系统发育关系研究。现有证据暗示： 两者在低级阶元水平上具有系统发育一致性, 而在高级阶元水平上可能没有平行演化关系, 这些对早期研究提出的平行演化假说提出了质疑。在现有研究的基础上, 本文建议从增加取样类群、 增加基因数目和数据量、 系统发育一致性检验等几个方面开展更深入的系统发育研究, 并开展Buchnera的转移实验, 从而检验Buchnera的横向转移及其基因在不同蚜虫支系中的进化速率一致性, 以便更客观地揭示蚜虫-Buchnera的进化关系。     

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata

Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to nod-box-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.     

Roots and Their Symbiotic Microbes: Strategies to Obtain Nitrogen and Phosphorus in a Nutrient-Limiting Environment

The association between Rhizobium and legumes and that between arbuscular mycorrhizal (AM) fungi and most land plants display a remarkable degree of similarity. Both events involve the recognition of, entrance into, and coexistence within the plant root, with the development of a specialized interface that always separates the two partners and at which nutrient exchange occurs. Molecules produced by rhizobia during the early stages of the symbiosis are related to fungal chitin, and the plant responds to both microbes with an increase in the production of flavonoids, which may assist in recognition and development of the symbioses. Many of the same plant genes are up-regulated in the two symbiotic pathways, and notably plants that are Nod^– are often defective in the AM association as well. However, there are a number of differences between the associations, and these are important for understanding the relationship between the two symbioses. The Rhizobium and AM symbioses will be compared and the question of whether the nitrogen-fixing association evolved from the much more ancient AM symbiosis will be discussed.