Fine structure of nitrogen-fixing leguminous root nodules from the Canadian Arctic

Effective (nitrogen-fixing) root nodules of Oxytropis maydelliana Trautv., O. arctobia Bunge and Astragulus alpinus L. were collected in the high Arctic tundra and subsequently processed for structural studies. The cylindrically-shaped perennial nodules consisted of the following tissues: nodule cortex, nodule meristem, nodular vascular bundles, an active central region with uninfected and infected cells at various stages of development, and a proximal region of senescent cells. The active central region was dark red-coloured due to the presence of the pigment leghemoglobin. The host cells became infected by the growth of infection threads into cells recently derived from the nodule meristem and the subsequent endocytotic release of rhizobia from unwalled membrane-bound regions of the infection thread. The host plasma membrane adjacent to the unwalled regions of infection thread gave rise to the peribacteroid membrane which surrounded the released bacteria. Thus, nodule development and the basic tissue arrangement of the arctic nodules was similar to that of cylindrically-shaped nodules formed on temperate species of legumes.
The arctic legume nodules are unique in having large numbers of lipid droplets present in the cytoplasm of the nodule cortex and uninfected cells of the central active region. Newly infected cells also have lipid droplets. More developed infected cells lack lipid droplets but often contain amyloplasts. Mature differentiated bacteria were spherically-shaped and contained electron-dense inclusions. Electron-dense material was also present in vesicles formed from dilated endoplasmic reticulum and in the peribacteroid space. The lipid droplets present in the host cytoplasm of the nodule cortex and uninfected cells of the central tissue may be storage products which are used to support nitrogen-fixation in nodules growing under cool temperatures of this harsh environment.     

The occurrence of nitrogen-fixing root nodules on non-leguminous plants

At present it is known that some 13 genera of non-leguminous Angiosperms include at least some species which bear root nodules with the property of nitrogen fixation. Alnus is the best known example. To assist in the assessment of the ecological importance of these plants, the author has surveyed the relevant literature to discover how many species in each of the genera have been recorded to bear noduleS. He also provides evidence of this feature in several species not hitherto reported. Of an estimated total complement of 342 species in the 13 genera, 118 species haye so far been recorded to bear nodules. The remaining species do not appear to have been examined for the presence of nodules.     

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia , the nitrogen-fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O₂ transport.     

Observations on the fine structure of the endophyte of the root nodules of alnus glutinosa (L.) gaertn.

Summary Electron micrographs have been prepared showing different stages in the development of the endophyte in Alnus glutinosa root nodules. Although the complete life cycle of the organism has not yet been worked out it is concluded that it is an Actinomycete. The organism can exist as a septate, branching filament of diameter 0.7–1.0 . However, under certain conditions within the nodule, the endophyte can form spherical sub-divided vesicles 4–5 in diameter and it is suggested that these vesicles are analogous to sporangia. Yet again under other conditions the organism fragments to form bacteroids. It would appear that the different forms of the organism are associated with different seasons of the year and with different metabolic patterns, of particular interest in this connection is the fixation of nitrogen which is associated with the nodule symbiosis.     

Sterols of roots and nitrogen-fixing root nodules of some non-leguminous species

Sterols of both roots and nodules of three species of Alnus were found to consist only of sitosterol, whereas Casuarina cunninghamiana contained substantial amounts of campesterol, stigmasterol and sitosterol. In all four cases more sterol was extracted from nodules than from roots.     

Ammonium effects on function and structure of nitrogen-fixing root nodules of Alnus incana (L.) Moench

Cloned plants of Alnus incana (L.) Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammonium on the function and structure of the root nodules were studied by adding 20 mM NH₄Cl (20 mM KCl=control) for four days. Nitrogenase activity decreased to ca. 50% after one day and to less than 10% after two days in ammonium treated plants, but was unaffected in control plants. The results were similar at photon flux densities of 200 and 50 mol m^-2 s^-1. At the higher light level the effect was concentration dependent between 2 and 20 mM NH₄Cl. The recovery was slow, and more than 11 d were needed for plants treated with 20 mM ammonium to reach initial activity. The distribution of ¹⁴C to the root nodules after assimilation of ¹⁴CO₂ by the plants was not changed by the ammonium treatment. Microscopical studies of root nodules showed high frequencies of endophyte vesicles being visually damaged in nodules from ammonium-treated plants, but not in nodules from control plants. When nitrogenase activity was restored, visually damaged vesicles were again few, whereas young developing vesicles were numerous. The slow recovery, the ¹⁴C-translocation pattern, and the structural changes of the endophyte indicate a more complex mechanism of ammonium influence than simply a short-term reduction in supply of carbon compounds to the nodules.     

A novel symbiotic nitrogen-fixing member of the Ochrobactrum clade isolated from root nodules of Acacia mangium

Ten strains of root nodule bacteria were isolated from the nodules of Acacia mangium grown in the Philippines and Thailand. Partial sequences (approx. 300 bp) of the 16S rRNA gene of each isolate were analyzed. The nucleotide sequences of strain DASA 35030 indicated high homology (>99%) with members of the genus Ochrobactrum in Brucellaceae, although the sequences of other isolates were homologous to those of two distinct genera Bradyrhizobium and Rhizobium. The strain DASA 35030 was strongly suggested to be a strain of Ochrobactrum by full length sequences of the 16S rRNA gene, fatty acids composition, G+C contents of the DNA, and other physiological characteristics. Strain DASA 35030 induced root nodules on A. mangium, A. albida and Paraserianthes falcataria. The nodules formed by strain DASA 35030 fixed nitrogen and the morphology of the nodules is the same as those of nodules formed by the other isolates. This is the first report that the strain of Ochrobactrum possesses complete symbiotic ability with Acacia.     

Nocardia casuarinae sp. nov., an actinobacterial endophyte isolated from root nodules of Casuarina glauca

An actinobacterium strain BMG51109a was isolated from surface sterilized root nodules of Casuarina glauca collected in Tunisia. The 16S rRNA gene sequence of strain BMG51109a showed most similarity (96.53–96.55 %) to the type strains of Nocardia transvalensis, N. aobensis and N. elegans. Chemotaxonomic analysis supported the assignment of the strain to Nocardia genus. The major menaquinone was MK-8(H₄c) while the polar lipid profile contained diphosphatidylglycerol, phosphatidylmonomethylethanolamine, glycophospholipid, phosphatidylinositol, one uncharacterized phospholipid and three glycolipids. Whole-cell sugar analysis revealed the presence of meso-diaminopimelic acid, arabinose and galactose as diagnostic sugars, complemented by glucose, mannose and ribose. The major cellular fatty acids were tuberculostearic, oleic, palmitoleic and stearic acids. Physiological and biochemical tests showed that strain BMG51109a could be clearly distinguished from its closest phylogenetic neighbours. On the basis of these results, strain BMG51109a^T (= DSM 45978^T = CECT 8469^T) is proposed as the type strain of the novel species Nocardia casuarinae sp. nov.     

Evidence from inhibitor studies that the endophyte synthesises nitrogenase in the root nodules of Alnus glutinosa L. Gaertn.

Summary Viable spheroplasts of a marine dinoflagellate, Gonyaulax polyedra, have been prepared for the first time. This simple and rapid procedure results in a yield of over 95% intact spheroplasts. Utilizing this technique, many studies of the cell-wall-free form of this dinoflagellate are now possible.     

Cell wall (outer membrane) of bacteroids in nitrogen-fixing peanut nodules

Rhizobia in peanut nodules are transformed from rod-shaped cells to extremely large spherical bacteroids. The invading rhizobia shed their outer membranes soon after their release into the host cells. The outer membranes are seen to be peeled off and replaced by new outer membranes before rhizobia are differentiated into nitrogen-fixing bacteroids. Evidence is presented that peanut bacteroids, in spite of their spheroplast-or protoplast-like appearance, do possess both the inner and the outer membrane.     

Localization of H+-ATPases in soybean root nodules

The localization of H⁺-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H⁺-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H⁺-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids. Received: 25 November 1998 / Accepted: 6 January 1999     

Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata.

Morphogenesis of stem nodules in Sesbania rostrata was studied over a period of 6 days after inoculation with an appropriate species of Rhizobium. Nodulation sites were initially slightly raised, circular areas 0.3 to 0.6 mm in diameter and 4 to 5 mm apart in vertical rows along the length of the stem. Each site was underlaid by an adventitious root primordium. A site became susceptible to infection by a specific Rhizobium sp. when the root primordium broke through the epidermis, leaving a fissure. Rhizobia multiplied within this fissure and colonized the exposed intercellular spaces. The infection extended inward as narrow, branched intercellular threads moved into a cortical meristematic zone, where cell division was initiated, and invagination of infection thread branches into adjacent plant cells followed. Rhizobia were released into the plant cells and surrounded immediately by plant membrane. Intracellular rhizobia divided actively, leading to bacteroid-filled cells. Infected areas enlarged and coalesced as the nodule matured.     

<Emphasis Type="Italic">Frankia inefficax</Emphasis> sp. nov., an actinobacterial endophyte inducing ineffective,non nitrogen-fixing,root nodules on its actinorhizal host plants

Strain EuI1c^T is the first actinobacterial endophyte isolated from Elaeagnus umbellata that was shown to be infective on members of Elaeagnaceae and Morella but lacking the ability to form effective root nodules on its hosts. The strain can be easily distinguished from strains of other Frankia species based on its inability to produce vesicles, the specialized thick-walled structures where nitrogen fixation occurs. Chemotaxonomically, strain EuI1c^T contains phosphatidylinositol, diphosphatidylglycerol, two glycophospholipids and phosphatidylglycerol as phospholipids. The whole cell sugars were composed of glucose, galactose, mannose, ribose, rhamnose and fucose as diagnostic sugars of the species. Major fatty acids were iso-C_16:0, C_17:1 ω8c and C_15:0 and C_17:0 and the predominant menaquinones were MK-9(H₆), MK-9(H₈) and MK-9(H₄). Analysis of the 16S rRNA gene sequence of strain EuI1c^T showed 97, 97.4 and 97.9% identity with Frankia elaeagni DSM 46783^T, Frankia casuarinae DSM 45818^T and Frankia alni DSM 45986^T, respectively. Digital DNA:DNA hybridizations with type strains of the three Frankia species with validly/effectively published names are significantly below 70%. These results warrant distinction of EuI1c^T (= DSM 45817^T = CECT 9037^T) as the type strain of a novel species designated Frankia inefficax sp. nov.     

Hydrogenase in actinorhizal root nodules and root nodule homogenates.

Hydrogenases were measured in intact actinorhizal root nodules and from disrupted nodules of Alnus glutinosa, Alnus rhombifolia, Alnus rubra, and Myrica pensylvanica. Whole nodules took up H2 in an O2-dependent reaction. Endophyte preparations oxidized H2 through the oxyhydrogen reaction, but rates were enhanced when hydrogen uptake was coupled to artificial electron acceptors. Oxygen inhibited artifical acceptor-dependent H2 uptake. The hydrogenase system from M. pensylvanica had a different pattern of coupling to various electron acceptors than the hydrogenase systems from the alders; only the bayberry system evolved H2 from reduced viologen dyes.     

Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules

Summary Nitrogen-fixing peanut root nodules are characterized by their unique structural organization, distinct from other legume nodules. The focus of this study has been in and around the hostsymbiont interface, where the bacterioid and the host cell surface (peribacteroid membrane envelope) interact during symbiosis. The infected nodule cells have revealed the presence of lipid bodies (oleosomes) in intimate association with the peribacteroid membrane, which encloses the large spherical bacteroids with a relatively narrow peribacteroid space. Electron dense structures, referred to as dense bodies have been found attached to the bacteroid outer membranes at the host-symbiont interface. The dense bodies are osmiophilic, amorphous and 3,3-diaminobenzidine positive. The isolated intact bacteroids with dense bodies attached to their cell wall showed significant catalase activity. Many microbodies showing DAB-positive reaction have been found in the host cytoplasm, associated closely with the peribacteroid membrane. These ultrastructural and cytochemical characteristics of peanut root nodules suggest that lipids are utilized during symbiosis and the dense bodies and microbodies may be involved in the catabolic process.Abbreviation DAB 3,3-diaminobenzidine     

白车轴草和紫花苜蓿根瘤的显微及超微结构

通过对白车轴草(Trifolium repens Linn.)和紫花苜蓿(Medicago sativa Linn.)根瘤的显微及超微结构进行观察,发现其根瘤显微结构都由4部分组成,由外向内依次为：保护层、皮层、鞘细胞层和中心组织(侵染组织)。在中心组织的侵染细胞中,分布有大量的线粒体、高尔基体、核糖体及内质网,白车轴草根瘤侵染细胞中的细菌圆形或椭圆形,有明显的周膜、细菌细胞壁和质膜,在细菌发育过程中,周膜活动旺盛,有时相邻细菌的周膜发生融合,在周膜附近常分布有大量的内质网、高尔基体以及高尔基体小泡,似与周膜融合有关。紫花苜蓿细菌椭圆形、长棒形,甚至有的细菌呈分枝状。二者细菌细胞质中分布着大量的核糖体和纤维状的核物质。在白车轴草中还有染色很深的多聚磷酸盐颗粒。