Young Nodules Research Articles

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO 3 (-) addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO 3 (-) had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO 3 (-) was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO 3 (-) and another 5 d in the presence of 18 mM NO 3 (-) resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO 3 (-) levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO 3 (-) . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.

Fixation d'azote chez les espèces ligneuses symbiotiques. I. Ultrastructure des nodules, mycorhizes à vésicules et à arbuscules et activité réductrice de C2H2 de jeunes plants de Robinia pseudoacacia cultivés au laboratoire

Black locust (Robinia pseudoacacia L.), a native species of North America, has found a specific and efficient Rhizobium strain in French soils. Ultrastructural studies of young nodules showed that infection of the host cell occurred by numerous infection threads. Rhizobia are liberated in the cell by endocytotic processes. No important differences were found between rhizobia in the infection threads and bacteroids in the host cell; the bacteroid cells were only enlarged, without any structural change. This Rhizobium strain is a "fast grower".Roots were also infected by an endomycorrhizal fungus producing numerous vesicles and arbuscules in the root cortex.Nitrogen-fixing activity, assayed by the acetylene reduction method, was measurable only 5 to 10 days after nodular development. Until the 50th day after seed germination, the activity increased slowly, then more rapidly. By the 80th day, fixing activity was about 9 μmol/h per plant.No relationships between the form, age, and fixing activity of the nodules were found.

Fluctuations of nitrogenase and cytosol glutamine synthetase activity in pea (Pisum sativum L.) nodules in the course of vegetation

Changes in glutamine synthetase activity located in the cytosol of root nodules were followed in pea (Pisum sativum L.) plants in relation to their nitrogenase activity. The highest glutamine synthetase activity was found in young nodules (15 days after inoculation) and its changes in 17-to 45-day-old plants showed a positive correlation with nitrogenase activity. In contrast to nitrogenase activity, changes in glutamine synthetase activity during the day and night period could not be unequivocally interpreted in terms of diurnal fluctuation.

15N2 fixation, H2 evolution and C2H2 reduction relationships in Phaseolus vulgaris

AbstractIn a study of N2 fixation in nodules of beans (Phaseolus vulgaris L.) cv. Venezuela‐350 of different ages and kept under controlled light and temperature conditions, three parameters ‐ 15N2 fixation, C2H2 reduction and H2 evolution—were measured.C2H2 reduction was positively related to the N fixed. A standardization of sampling is necessary to keep experimental errors to a minimum. To decrease the variation between replications, the nodules should be carefully incubated (adequate time and temperature), and the samples for chromatography should also be carefully stored. No significant difference was found in C2H2 reduction in the systems studied ‐ nodulated roots, disturbed whole plants and intact plants—during the first hour of measurement.H2 evolution was detected in nodules over 20 days of age and was positively related to N2 fixation and C2H2 reduction. This measurement was essential to account for the equilibrium between the three parameters in the electron balance ratio. Under phytotron conditions, maximum H2 evolution occurred 20–30 days after planting.Nodulated roots from different plants were used to determine the N2 fixed and C2H2 reduction, and on average the ratios C2H2:N2 varied between 7.3 and 8.3 for all ages. In vivo C2H4/(3N2+ H2) electron balance values were over 1, varying from 1.32 to 1.43. These data suggest that overestimation of actual N2 fixation might be occurring when using the C2H2 reduction technique, even if taking account for H2 evolution. In this case, this method should be used with great care to avoid an overestimation of on average 32‐43% of the actual value. Variation was smaller between the measures of the three parameters when younger nodules were used.

SCANNING ELECTRON MICROSCOPY OF THE NITROGEN‐FIXING ACTINORHIZA OF MYRICA PENSYLVANICA L. (BAYBERRY)*

SummaryThe nitrogen‐fixing root nodules of Myrica pensylvanica L. (bayberry) were shown to be inhabited by a filamentous micro‐organism typical of non‐leguminous root nodules (actinorhizal nodules) by scanning electron microscopy (s.e.m.). The micro‐organism was hyphal in young nodule areas and host cell infection was accomplished by cell wall penetration with subsequent proliferation of the micro‐organism. The penetration was often massive with hyphal entries recorded less than 1 μm apart. The smooth transition between host cell wall and endophyte hypha indicated some homology between their surfaces. The second stage of development of the endophyte was vesicular; club‐shaped vesicles arose as extensions of hyphal tips. The vesicles resembled similar structures from other non‐leguminous nitrogen‐fixing plants. X‐ray microanalysis of the nodules revealed the presence of crystalline deposits containing substantial calcium content packed within randomly distributed cortical cells. The crystals were typical of calcium oxalate, a common constituent in plant tissue.

Separation and determination of the relative concentrations of the homogeneous components of soybean leghemoglobin by isoelectric focusing

The multiple components of soybean ferric leghemoglobin are readily separated by analytical and preparative flat bed isoelectric focusing in both the presence and also the absence of the ligand nicotinate. In the presence of nicotinate the separation by isoelectric focusing is more rapid and results in sharper bands of the very stable ferric leghemoglobin nicotinate complexes. The separation is sensitive enough to permit analytical experiments on leghemoglobin from single nodules. Leghemoglobins a and c 1 prepared by ion exchange chromatography are homogeneous by isoelectric focusing criteria. Leghemoglobin c 2 prepared by ion exchange chromatography is an approximately 1 : 2 mixture of leghemoglobins c 2 and c 3. Leghemoglobin d consists of three components. The ratio of leghemoglobin a to leghemoglobin c 3 content increases dramatically as very young nodules mature. The increase in relative leghemoglobin a content suggests that leghemoglobin a might be required for regulation of nodule O 2 concentration only when the nodule structure is complex. The ratio of leghemoglobin c 1 content to leghemoglobin c 3 content increases somewhat during the early period of nodule development, while the ratio of leghemoglobin c 2 content to leghemoglobin c 3 content increases slowly throughout nodule development. Ratios of leghemoglobin b content to leghemoglobin a content and of total leghemoglobin d content to total leghemoglobin c content were almost independent of nodule age. Leghemoglobins a and b might be related biosynthetically, as might leghemoglobins c and d.

A cooperative action of plant and Rhizobium to dissolvethe host cell wall during development of root nodule symbiosis

Summary Development of infection by Rhizobium on legume roots appears to involve two cell wall hydrolysing enzymes, cellulase and pectinase. The activity of these enzymes increases several fold in young nodules and is compartmentalized, i.e. pectinase is present in the bacteroids while cellulase is in the host cell cytoplasm. Cellulase activity was localized in the cell wall region of the infection threads. These data suggest that both host and Rhizobium may participate in the infection process resulting in the dissolution of the host cell wall and establishment of symbiosis.

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two- to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads. Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34--45% sucrose (mean density of 1.14 g/cm3). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining. ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix.

THE ONSET OF NITROGEN FIXATION IN YOUNG ALDER PLANTS AND ITS RELATION TO DIFFERENTIATION IN THE NODULAR ENDOPHYTE

SUMMARYThe acetylene assay was used to find how soon nitrogenase activity could be detected in young, nodulating alder plants. Samples taken at intervals initially of 2–3 days were assayed, starting as soon as external signs of nodulation were seen, and continuing for a further 33 days. Squash preparations made from the nodules of the assayed plants were examined microscopically to detect whether there was any relation between nitrogenase activity and the stage of differentiation of the nodule endophyte.No nitrogenase activity was detected on the first two assay occasions, on which the endophyte was confined to the hyphal stage in all the nodules present. Activity was detected on the subsequent occasions, its onset coinciding with the production of vesicles by the endophyte. Activity rose as the vesicles became more abundant, and in agreement with the limited previous evidence it was concluded that the vesicles, but not the hyphae, are nitrogen‐fixing. The third type of structure produced by the alder endophyte, namely the bacteroid stage, was not detected in the young nodules studied here.The onset of nitrogenase activity in the nodules preceded by 16 days the increased greening of the leaves signifying the arrival in the shoot of products of nitrogen fixation. Ethylene production per plant increased in exponential manner over the period of the experiment, while ethylene production per unit weight of nodule tissue rose ten‐fold

Preparation and Fractionation of Rhizobium Bacteroids by Zone Sedimentation through Sucrose Gradients

Zone sedimentation through sucrose gradients was used for preparing Rhizobium bacteroids from lupin nodules and for separating them into slowly and rapidly sedimenting fractions.The purity of the bacteroids was established by electron microscopy and by enzyme assays, and they were shown to contain a CO-insensitive cytochrome c oxidase.Bacteroids sedimented more rapidly than broth-cultured Rhizobium bacteria, and bacteroids from old nodules sedimented more rapidly than bacteroids from young nodules.THERE APPEAR TO BE AT LEAST TWO FORMS OF BACTEROID IN OLD NODULES: slowly sedimenting bacteroids with moderate colony-forming ability resembling the bacteroids found in young nodules, and rapidly sedimenting bacteroids with much lower colony-forming ability.

Cell dysfunction in root nodules of soybeans grown in the presence of Rhizoctonia solani

Intercellular and intracellular penetration of hyphae of Rhizoctonia solani into young, functional root nodules of soybean Glycine max inoculated with Rhizobium japonicum was restricted to the nodule's outer cortex. Penetration of the nodule central tissue may have been impeded by a layer of thick-walled sclerenchymatous cells. A remote cytopathogenic effect on Rhizobium-containing cells of the central tissue of young nodules was nuclear breakdown characterized by absence of stainable matrix material within the nucleus, dissolution or rupture of the nuclear membrane, extrusion of the nucleolus, and eventually lack of identifiable nucleus. A weak reactivity of diseased nodule tissue to benzidine-H 2O 2 at pH 4·5 indicated low leghemoglobin content. The degenerative effects did not occur in cells of young nodules grown in absence of the fungus. Normal cell degeneration in senescent nodules was primarily characterized by cytoplasmic and nuclear shrinkage without rupture of the nuclear envelope and thus differed from the cytopathogenic effect. These results support the concept that cell dysfunction in young nodules grown in the presence of R. solani may have been caused by toxic fungal metabolites that diffuse throughout the nodule. Fungal-induced nodule cell dysfunction would interfere with nitrogenase and symbiotic N 2 fixation activities and, where the N supply is sub-optimal, would reduce the yield of the soybean plant.

LA FORMATION DES NODULES FIXATEURS D'AZOTE CHEZ ALNUS GLUTINOSA (L.) VILL.

In the present study a verification is undertaken of the research published so far on the formation of root nodules in alder plants (Alnus glutinosa (L.) Vill.) as a result of a symbiosis between the host plant and an endophyte, probably an actinomycete. After a brief discussion of the growth of the endophyte within the cortex of the host root and of the morphogenesis of root nodules, the hypothesis to the effect that a root nodule is a modified lateral root was tested. This possibility had to be rejected and the conclusion was drawn that root nodules appear adventitiously in-between the normal points of lateral root production. As, from an anatomical point of view, a nodule shows a great deal of resemblance to a lateral root, the hypothesis was formulated that root nodules are adventitious lateral roots – induced by an endophyte – which develop into nodules following the penetration of their cortex by the endophyte. In order to imitate and study the reactions in the host plant set in motion by the endophyte, phytohormones were administered to whole plants as well as to explants of roots. Use was made of kinetin and the following auxins: indole-3-acetic acid (IAA), indole-butyric acid (IBA), naphtalene-acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D). The auxins and the mixture of 2,4-D and kinetin produced an induction of primordia; by means of IBA, moreover, a slight increase in the number of nodules in the inoculation experiments was obtained. Finally, some experiments with tetrazolium salts were undertaken in order to ascertain whether the reducing conditions which are essential for the reduction of atmospheric nitrogen are already present in young nodules. Seventeen days after the inoculation a reduction of tetrazolium could be observed on the vesicles.

Evidence for the Transfer of Rhizobial Metabolites to the Polyploid Nuclei of Young Clover Nodules

AbstractAutoradiography was used to provide evidence for the transfer of Rhizobium produced moieties to the host nuclei of young clover nodule cells. Cells of Rhizobium trifolii Dangeard were labeled with 3H‐adenine or 4,5‐3H‐l‐leucine and 3,4‐3H‐l.‐proline, washed free of external label, and allowed to nodulate young seedlings of white clover (Trifolium repens L. cv. White Dutch). Sections (0.5–1.0 μm) of young nodules up to four days old were autoradiographed using the dipping technique. Grain counts indicated movement of tritium from the leucine‐proline labeled rhizobia to the polyploid nuclei of two day old clover nodule cells. Acetylene reduction was not detected until approximately 24 hours after the transfer of tritium was observed. No transfer of tritium was observed with 3H‐adenine labeled rhizobia. It is hypothesized that nodulating rhizobia may induce clover nodule cells to initiate leghemoglobin synthesis by the transfer of a bacterially produced inducer.

Esterase isoenzymes from legume root nodules

Multiple forms of esterases were separated by starch gel electrophoresis from root nodules of eight different legume species. Although nodules from each legume species had characteristic esterase-zymogram patterns, the reactivity of the various esterases with different substrates and inhibitors was quite similar. Most of the esterase activity was present in the supernatant fraction of the nodule extract. Zymogram patterns from young nodules were similar to those prepared from roots of the same plant but changes occurred in the nodule zymograms during the growth of the plant. Zymograms of esterases, proteases and dehydrogenases from the same nodule extract were also compared.

Cytological Studies on Soybean Root Nodule by Means of Electron Microscopy

Ultra-thin sections of root nodule of Glycine max infected with Rhizobium Japonicum were investigated by means of an electron microscope. Materials were fixed in buffered glutaraldehyde and post fixation was done by osmium tetroxide. In the initial phase of infection, bacteria were embedded in low dense material and were surrounded with plant cell wall. Cellulose layers infected by bacteria were thicker than the original plant cell wall, and the cellulose layer became gradually thin toward the tip of the infection thread. Bacteria after released from infection thread were surrounded by the membrane which might be derived from the host cell membrane, and this enclosing membrane remaind around the bacterial bodies after the host cytoplasm was broken down. Compact cytoplasm, nuclei, Golgi bodies and mitochondria were found in young nodule cells, and peripheral tissues in nodule contained many amyloplasts. Needle-like structures were obseved in vacuole of nodule cells, although their charactors were not yet clearly understood. Bacteria in nodule cell were rod-shaped, and had nuclear region with microfibrils. Electron-dense bodies were observed in nuclear region. In both ends of nodule bacteria, electron translucent parts were recognized.

A Quantitative Study of Fixation and Transfer of Nitrogen inAlnus

SUMMARY A quantitative study of the fixation of nitrogen and of its transfer from the nodules to the remainder of the plant has been made in Alnus plants during their first season of growth. Fixation per plant reached a maximum in late August but fell rapidly with the onset of autumn, while fixation per unit dry weight of nodule tissues was greatest in young nodules and was of the same order as in nodulated legumes. Throughout the growth season there was a steady transfer from the nodules of some 90 per cent, of the nitrogen fixed. The rate of fixation relative to the growth of the endophyte is much higher than in free-living nitrogen-fixing organisms and is clearly governed by the nitrogen requirements of the entire symbiotic system. These findings are compatible with the view that the fixation process is extracellular to the endophyte.

Studies on the root nodules of Lupin, Lupinus luteus L.

The root nodules of lupine have a number of separated bacteroid tissue with a varying amount of non-infected tissue between them. Very often, infected threads are prominent in the young nodules. The leguminous nodules can be placed in four groups with respect to the mode of infection of the bacteroid tissue. 1. Infection is produced by aid of infection threads. a) By intracellular threads. For example, Broad bean. b) By intercellular threads. For example, Serradella. 2. Infection is extended by aid of cell divisions. c) By mitotic cell divisions. For example, Lupine. d) By amitotic cell divisions. For example, Soy bean.

THE BIOLOGY AND CHEMISTRY OF NITROGEN FIXATION BY LEGUME BACTERIA

SummarySterile culture systems for the examination of symbiotic nitrogen fixation are described, and emphasis is laid on their significance in preventing erroneous results.The hypothesis advanced by some previous workers of the change of poor bacterial strains to effective ones, and vice versa, by repeated plant passages is disproved.There is regular difference in colour between the effective and ineffective nodules. Effective nodules always contain red pigment, ineffective never. Another difference between effective and ineffective nodules is the occurrence of swollen bacteroids in the former and their non‐occurrence in the latter. This difference is a striking one in the experiments carried out in our laboratory with peas but in the literature some records are found which indicate that it is not general in all legumes. The problem requires further elucidation.The red pigment of effective root nodules is haemoglobin, which is able to store and transfer oxygen. For the sake of brevity it has been named leghaemoglobin. Its divalent iron is easily changed to the trivalent form, legmethaemoglobin. Young nodules chiefly contain leghaemoglobin when growing in a good light; with decreasing light the amount of brown legmethaemoglobin increases. Leghaemoglobin or legmethaemoglobin changes gradually during the ripening of plants to a green pigment or pigments. A similar change occurs in the pigments if young plants are kept in the dark. The green pigment still contains iron though it can be easily split off with acids. The green pigment may still contain protein. It is assumed that the green pigment is similar to those formed from the haemoglobin of blood in a solution containing ascorbic acid through the agency of oxygen or hydrogen peroxide.Leghaemoglobin has been isolated with a purity of about 85 %, assuming that the haemin content of the leghaemoglobin is the same as that of the haemoglobin of blood. The molecular weight was 34,000.The observations so far made show a distinct parallelism between nitrogen fixation and the occurrence of leghaemoglobin. If the pigment is not present in the nodule, or it it is decomposed, nitrogen fixation does not occur. The formation of the leghaemoglobin is evidently brought about by the host‐bacteria symbiosis, as also is the formation of bacteroiď mass in the nodules. The causes of the inability of ineffective strains to form ‘bacteroids’ and leghaemoglobin is not known. The possibility is suggested that the slimy layer surrounding the rods may isolate the bacteria and thus prevent the onset of symbiosis.Both the bacteria and the host‐plant specificity are discussed in the light of the above observations. An effective bacterial strain forming red nodules on a certain host plant may on another closely related variety form white ineffective nodules. Thus the host plant largely determines the effectiveness of bacterial strains.The chemical significance of leghaemoglobin in nitrogen fixation is discussed. It is possible that its effect is restricted to the transfer of oxygen.The excretion of organic nitrogen compounds to the substrate of inoculated legumes, about which there has been much argument, has gradually been established in many laboratories. The causes of the great differences in the rate of excretion are, however, still obscure. The problem has been treated from a new aspect: the unusually high excretion of amino‐dicarboxylic acid nitrogen, in certain cases exceeding the proportion of fixed nitrogen the plant has taken up, supports the idea that these amino‐acids are of primary importance in nitrogen fixation. The data recently obtained with Azotobacter by the isotopic method lead to the same result.The transamination of the amino group from amino‐dicarboxylic acids to aliphatic α‐keto acids has been demonstrated in green plants and in root nodules.The conception that the nitrogen fixed by the root nodules is transferred chiefly by excretion to the cytoplasm seems justifiable in view of the experimental data. Not until the last stage of nitrogen‐fixation does the central bacterial mass of the nodule decompose. Then the decomposition products are partly used by the host plant.The carbohydrate nitrogen ratio in symbiotic nitrogen fixation has a marked effect on nitrogen fixation. No explanation of this phenomenon is available, but the question has been considered from a new point of view.Investigations of the accessory growth factors of legume bacteria and of their bacteriophages have failed to throw light on the phenomenon of symbiosis.

Stimulation of Root-Hair Growth in Legumes by Sterile Secretions of Nodule Bacteria

WHEN a legume root is infected by the nodule organism, the formation of the young nodule is brought about by the multiplication and growth in size of the root-cells, principally in the cortex. In lucerne and clover, the bacteria are at first enclosed in infection threads, and it can be seen in young nodules that the stimulation to growth and division of the host cells extends beyond the cells actually entered by the bacteria. Stimulation must be due, therefore, to diffusible secretions of the bacteria. Nevertheless, scarcely any evidence appears in published work to show that the separated secretions of nodule bacteria can stimulate cell growth of the host plant. We have now obtained quantitative evidence of such stimulation in a study of the action of the bacterial secretions upon root-hair growth.

The structure and physiological significance of the root-nodules of Myrica gale

The peculiar nodule formations on the roots of Myrica gale were first described and figured by Brunchorst in 1886, who stated that they were caused by an inhibiting fungus with septate hyphæ and terminal spores. Möller in 1890 placed this fungus in the group Frankia, naming it Frankia Brunchorstii , and considered it to be closely related to a similar fungus in Alder nodules. In 1902 Shibara stated that the fungus is found exclusively in a peripheral sub-cork layer of tissue, one to three cells thick, and because of its peculiar ray-branching and club-shaped spores, it belongs to the group Actinomyces. Peklo in 1910, working on greenhouse-grown plants, supported Shibata’s view. Roots of Myrica gale were obtained for this investigation from plants growing wild in Wales, Ireland, and the North of England, and from cultivated plants growing in the Chelsea Physic Gardens. In all cases the roots were found to possess nodules of varying size. The young nodules are from 2-3 mm. long and 0·8-1 mm. broad, but these by branching form “clusters,” sometimes as large as a nutmeg, and surrounded by peculiar rootlets which grow out through the end of each nodule or branch. The branching is associated with the outgrowth of lateral roots, and is not due to dichotomy of the apical meristem of the root as is the case in the nodules of Cycas, Alder, and Elæagnus.