The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDA kilodalton - Lb leghemoglobin - TBST Tris-buffered saline plus Tween 20     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

Membrane lipid peroxidation, nitrogen fixation and leghemoglobin content in soybean root nodules

Membrane lipids in soybean nodules may undergo oxidative degradation resulting in the loss of membrane structural integrity and physiological activities. One of the final products of lipid peroxidation is malondialdehyde (MDA), which can react with thiobarbituric acid (TBA) in vitro to form a chromogenic adduct, a Schiff base product that can be measured spectrophotometrically. MDA formation was quantified in the nodules as well as in the adjacent root tissue. Lipid peroxidation was initially high in soybean nodules induced by Bradyrhizobium japonicum, but sharply declined following an increase in both leghemoglobin content and nitrogen fixation rate. Lipid peroxidation was 2 to 4 times higher in the nodules than in their corresponding adjoining root tissue. Malondialdehyde levels in ineffective nodules were 1.5 times higher than those in effective nodules. MDA formation was also shown to occur in the ‘leghemoglobin-free’ cytosolic fraction, the ‘leghemoglobin’ fraction, and the nodule tissue pellet. Antioxidants, such as reduced ascorbic acid, glutathione, and 8-hydroxyquinoline, caused a partial suppression of lipid peroxidation, whereas ferrous sulfate, hydrogen peroxide, iron EDTA, disodium-EDTA, and β-carotene induced MDA formation. In contrast, quenchers of oxygen free radicals such as HEPES, MES, MOPS, PIPES, phenylalanine, Tiron, thiourea, sodium azide, and sodium cyanide (uncouplers of oxidative phosphorylation) caused somewhere between a 12 to 70 percnt; reduction in MDA production. TBA-reactive products were formed despite the incorporation of superoxide dismutase, proxidase, and catalase into the reaction mixture.     

TheRhizobium-legume symbiosis Two methods to discriminate between nodules and other root-derived structures

Summary Two methods have been developed in order to discriminate between lateral roots, nodules and root-derived structures which exhibit both root and nodule histological features and which can develop on legumes inoculated with certainRhizobium mutants. The first method, known as the clearing method, allows the observation by light microscopy of cleared undissected root-structures. The second, known as the slicing method, is a complementary technique which provides a greater degree of structural information concerning such structures. The two methods have proved invaluable in defining unequivocally the nature of the interaction between a rhizobial strain and a legume host.     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

Morphological and structural adaptation of nodules of cowpea to functioning under sub- and supra-ambient oxygen pressure

Nodules of cowpea (Vigna unguiculata (L.) Walp. cv. Vita 3:Bradyrhizobium CB 756) from 28-d-old plants cultured for 23 d with their root systems maintained in O₂ levels from 1 to 80% (v/v, in N₂) in the external gas phase showed a range of structural changes which have been interpreted in relation to an over- or under-supply of O₂. A response to the partial pressure of O₂ in the gas phase (pO₂) was noted with respect to nodule size, lenticel development, the relative distributions of cortical and infected central tissue, the differentiation of cortex, especially the inner cortex, the frequency and size of infected and uninfected interstitial cells, the volume of extracellular spaces both in cortex and infected tissue, and in the frequency of bacteroids. As a consequence of these changes the surface area of inner cortex relative to the nitrogenase-containing units of fixing tissue (infected cells or bacteroids) was increased by as much as 20-fold. Effectiveness of bacteroid functioning increased from 0.10 ± 0.02 · 10^-9 μmol acetylene reduced per bacteroid in air-grown nodules to 0.9 ± 0.16 · 10^-9 (same units) per bacteroid in those cultured in 1% O₂. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian International Development Assistance Bureau postgraduate fellowship (to F.D.D.). The authors wish to thank Dr. W.F.C. Blumer for his considerable help with morphometric analysis, Dr. J. Kuo for guidance in the use of histological techniques, and to Dr. J.S. Pate for the suggestion that lenticel development might be quantified by surface staining of nodules.     

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.Abbreviations FH₄ tetrahydrofolic acid - PRPP 5-phospho--D-ribose 1-pyrophosphate - PRPP synthetase ribosephosphate pyrophosphokinase (phosphoribosylpyrophosphate synthetase)     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules

Methods are reported for the preparation from soybean (Glycine max (L.) Merr.) root nodules, of well-washed, intact membrane envelopes containing bacteroids. The intact envelopes are of much lower density than the bacteroids within and therefore only low speed centrifugation (approx. 150 g) may be used. The optimum osmotic strength is 600 mOsm/kg H₂O. The envelope contents were recovered following mild osmotic shock and-or hard centrifugal packing at >10,000 g. Extracts prepared in this way contained leghaemoglobin (identified spectrophotometrically), low-molecular-weight fluorescent materials and other components which are yet to be identified. Envelope leghaemoglobin did not react with specific antibody until the envelopes were ruptured. ¹³¹I-Labelled leghaemoglobin or bovine serum albumin, added during initial breakage of nodule cells, was not released when envelopes were ruptured to release leghaemoglobin. It is therefore concluded that this leghaemoglobin is located within the envelope space and did not arise from adhering or occluded cytosol leghaemoglobin. Based on the number and dimensions of microscopically intact envelopes in these preparations, the concentration within that space was in the range 178–523 M. Based on these estimates, leghaemoglobin within envelopes represented about one third of the total amount present in the nodule cells. Flat-bed isoelectric focusing of partially-purified envelope leghaemoglobin demonstrated that the latter contained all of the leghaemoglobin components previously reported for soybean nodules and an additional minor component focusing between leghaemoglobins a and b.     

Possible involvement of nodule superoxide dismutase and catalase in leghemoglobin protection

Superoxide anion is able to oxidize oxyleghemoglobin prepared from soybean nodules. Furthermore, ferrileghemoglobin is oxidized to leghemoglobin (IV) by hydrogen peroxide and this irreversible reaction leads to a complete inactivation of the hemoprotein. In scavenging O ₂ ^- and H₂O₂, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) are able to limit these oxidations. The occurrence of these enzymes within soybean nodules and their main characteristics are reported here. A general scheme taking into account their roles in leghemoglobin protection in vivo is proposed.Abbreviations Lb leghemoglobin - SOD superoxide dismutase     

Ultrastructure of chickpea nodules

Summary Developing and senescing chickpea (Cicer arietinum L.) nodules formed byRhizobium sp. (Cicer) CC 1192 have been shown by light and electron microscopy to have general morphological and ultrastructural features that are characteristic of indeterminate nodules. These features included the presence of persistent meristematic tissue at the distal ends of the multi-lobed nodules, and a gradient of cells at different stages of development towards the proximal point of attachment of the nodules to the parent root. The cytoplasm of infected cells in the nitrogen-fixing region of the nodules was densely packed with symbiosomes, most of which contained a single bacteroid. Infection threads containing bacteria were noted in invaded cells from the nitrogen-fixing region of the nodules. Other features that were observed in chickpea nodules included the presence of electron-dense occlusions in intercellular spaces in the nitrogen-fixing region, and plasmodesmata that connected infected cells with other infected cells and with uninfected cells. No poly--hydroxybutyrate granules were noted in the bacteroids. In later stages of development, infected cells became enlarged and highly vacuolated, and eventually lost their contents. Uninfected cells in the central region were smaller than infected cells and were also highly vacuolated. Some of the degenerative processes that take place in senescing bacteroids were noted.     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

Phosphoenolpyruvate carboxylase in soybean root nodules: An immunochemical study

The activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) strongly increased during the maturation of soybean (Glycine max L. Weber) root-nodules. By using a specific immune serum it was shown that this increase was the consequence of an elevated population of enzyme molecules whose appearance preceded the emergence of nitrogen fixing capacity. Whether or not the phenomenon could be ascribed to the formation of a specific isoenzyme is not known. The location of the enzyme was also investigated. Immunocyto-fluorescence experiments established that phosphoenolpyruvate carboxylase was present in the cytoplasmic compartment of both infected and uninfected cells of nodules.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Arabinogalactan proteins during the development of soybean root nodules

In soybean (Glycine max (L.) Merr.) root nodules the level of hydroxyproline-containing molecules is developmentally regulated. Hydroxyproline accumulates in both nodule cortex and medulla. In the cortex, the hydroxyproline is mainly localized in the cell wall, presumably as extensin, but in the medulla it is mainly in the soluble fraction as an arabinogalactan protein (AGP). Nodule-specific AGPs are present at early nodulation. The highest concentration of AGP is in the nodule medulla, followed by nodule cortex, uninfected roots, leaves, flowers, pods and seeds. Root nodules and all organs of the soybean plant that were tested were found to express a tissue-specific set of arabinogalactan proteins.Abbreviation AGP Arabinogalactan protein     

Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts

Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.     

Differentiation of nodules of Glycine max

Plants of Glycine max var. Caloria, infected as 14 d old seedlings with a defined titre of Rhizobium japonicum 3Il b85 in a 10 min inoculation test, develop a sharp maximum of nitrogenase activity between 17 and 25 d after infection. This maximum (14±3 nmol C₂H₄ h^-1 mg nodule fresh weight^-1), expressed as per mg nodule or per plant is followed by a 15 d period of reduced nitrogen fixation (20–30% of peak activity). 11 d after infection the first bacteroids develop as single cells inside infection vacuoles in the plant cells, close to the cell wall and infection threads. As a cytological marker for peak multiplication of bacteroids and for peak N₂-fixation a few days later the association of a special type of nodule mitochondria with amyloplasts is described. 20 d after inoculation, more than 80% of the volume of infected plant cells is occupied by infection vacuoles, mostly containing only one bacteroid. The storage of poly--hydroxybutyrate starts to accumulate at both ends of the bacteroids. Non infected plant cells are squeezed between infected cells (25d), with infection vacuoles containing now more than two (up to five) bacteroids per section. Bacteroid development including a membrane envelope is also observed in the intercellular space between plant cells. 35 d after infection, more than 50% of the bacteroid volume is occupied by poly--hydroxybutyrate. The ultrastructural differentiation is discussed in relation to some enzymatic data in bacteroids and plant cell cytoplasm during nodule development.     

Ultrastructural specialization for ureide production in uninfected cells of soybean root nodules

Summary Ultrastructural studies were conducted on root nodules of soybean (Glycine max) inoculated as seeds withRhizobium japonicum. The development of the large peroxisomes and abundant tubular endoplasmic reticulum (ER) characteristic of the uninfected interstitial cells was followed during nodule growth and maturation. Quantitative data on differences between the uninfected and infected cells in volumes and numbers of peroxisomes, plastids and mitochondria were analyzed statistically. The peroxisomes are 60 times greater in volume per unit cytoplasm in the uninfected cells than the small presumptive peroxisomes in the infected cells. Plastids are about equal in volume in the two types of cells. Mitochondria have 4 × the volume and 3 × the number of profiles per unit cytoplasm in the infected cells than in the uninfected. The observations are discussed in relation to published evidence that several enzymes involved in ureide production are localized in organelles of the uninfected cells. The uninfected cells are viewed as essential components in the symbiotic relationship between host and bacterium.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Immunochemical studies on xanthine dehydrogenase of soybean root nodules

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was purified from root nodules of soybean (Glycine max) and used to prepare a polyclonal rabbit antiserum. Monospecificity of this antiserum was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipate. During root nodule development of soybean, only one form of XDH was detected on an immunological basis. Titration of XDH by immunoelectrophoresis showed that a remarkable increase in the amount of XDH occurred between two and four weeks after inoculation, in parallel with the increase in enzyme activity. Localization of XDH by immunofluorescence indicated that the enzyme was present exclusively in uninfected cells where it appeared to be associated with discrete organellelsAbbreviations IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis - XDH xanthine dehydrogenase