Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O₂ (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O₂ showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O₂ treatments up to 16 d after sowing. Thereafter the Ar: O₂ treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO ₃ ^- (0.1–0.2 mM) to Ar: O₂-grown seedlings overcame the abovementioned consequences of N₂ deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O₂ treatment on nodule structure and senescence. Treatment of Ar: O₂-grown seedlings with NO ₃ ^- greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO ₃ ^- treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO ₃ ^- . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from ¹⁵N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase     

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O₂ (pO₂; 1–80%, v/v, in N₂) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO₂ tested. In sub-ambient pO₂ (1 and 2.5%) nodules contained specific activities of uricase (urate: O₂ oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O₂ contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O₂, the activities of other enzymes of ureide synthesis were relatively unaffected by pO₂. Short-term effects of pO₂ on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO₂ (1, 2.5 or 5%, v/v in N₂) and¹⁵N₂. Despite a fall in total¹⁵N₂ fixation, ureide synthesis and export was maintained at a high level except in 1% O₂ where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O₂ supply and demand to be maintained over a wide range of pO₂, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian Development Assistance Bureau postgraduate fellowship (to F.D.D.).     

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.     

The relative efficiency of nitrogen fixation in pea root nodules

Summary Experiments were performed to investigate the causes of low relative efficiency, RE, in legume root nodules. Nitrogen fixing activity and RE varied with time of incubation of nodules and with different temperatures and oxygen concentrations. The effects of nitrogen concentration and carbon dioxide concentration were also examined. In each case the RE was inversely related to nitrogen fixing activity; measured by acetylene reduction. Increasing the nitrogen concentration had no effect on either nitrogen fixing activity or RE. Experiments with isolated bacteroids gave higher RE values than the whole nodules from which they were isolated. All the results were consistent with hydrogen inhibition of nitrogenase within the nodule being the cause of low RE.     

Nodulation and symbiotic nitrogen fixation of cowpea (Vigna unguiculata (L.) Walp)

Summary Rhizobium strains CIAT 301, CIAT 79 and SLM 602 were tested and found effective in the nodulation and nitrogen fixation of cowpea cv. MI-35 (Vigna unguiculata (L.) Walp) plants in growth chamber experiments. Fresh weight of nodules increased with plant age initially and stabilized in 20–30 days from planting, followed by a secondary flush of nodule growth after 30 days. Apparent nitrogen fixation per gram nodule fresh weight reached a maximum in 20–30 days after planting and then decreased, even though a flush of new nodules was produced.     

Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

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.     

Nitrogen fixation by the woody legumeLeucaena leucocephala in Tanzania

Summary The nitrogen fixation rate in a 4-year-old stand of the woody legumeLeucaena leucocephala (Lam.) de Wit. was estimated in the field at a rather dry site in Tanzania by use of an acetylene reduction technique. The diurnal mean value during April–May was 35 nmol C₂H₄ mg^–1 (dry weight) nodules h^–1, with a variation between 22±8 and 48±12 nmol C₂H₄ mg^–1 (dry weight) nodules h^–1 in early morning and at midday, respectively. The nodule biomass was determined by auger sampling to be 51±16 kg (dry weight) ha^–1. Most of the nodules were found at the 10–30 cm soil depth level. A rough calculation of the amount of nitrogen fixed annually arrived at 110±30 kg ha^–1. The results give strong support for the use ofL. leucocephala for soil enrichment in less humid areas of tropical Africa.     

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     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.Abbreviations EF exoplasmic fracture - ER endoplasmic reticulum - ESR electron-spin resonance - IMP(s) intramembranous particle(s) - PF protoplasmic fracture     

Pathways of assimilation and transfer of fixed nitrogen in coralloid roots of cycad-Nostoc symbioses

Freshly detached coralloid roots of several cycad species were found to bleed spontaneously from xylem, permitting identification of products of nitrogen transfer from symbiotic organ to host. Structural features relevant to the export of fixed N were described for Macrozamia riedlei (Fisch. ex Gaud.) Gardn. the principal species studied. Citrulline (Cit), glutamine (Gln) and glutamic acid (Glu), the latter usually in a lesser amount, were the principal translocated solutes in Macrozamia (5 spp.), Encephalartos (4 spp.) and Lepidozamia (1 sp.), while Gln and a smaller amount of Glu, but no Cit were present in xylem sap of Bowenia (1 sp.),and Cycas (2 spp.). Time-course studies of ¹⁵N enrichment of the different tissue zones and the xylem sap of ¹⁵N₂-pulse-fed coralloid roots of M. riedlei showed earlier ¹⁵N incorporation into Gln than into Cit, and a subsequent net decline in the ¹⁵N of Gln of the coralloid-root tissues, whereas Cit labeling continued to increase in inner cortex and stele and in the xylem sap. Hydrolysis of the ¹⁵N-labeled Cit and Gln consistently demonstrated much more intense labeling of the respective carbamyl and amide groups than of the other N-atoms. Coralloid roots of M. riedlei pulse-fed ¹⁴CO₂ in darkness showed ¹⁴C labeling of aspartic acid (Asp) and Cit in all tissue zones and of Cit of xylem bleeding sap. Lateral roots and uninfected apogeotropic roots of M. riedlei and M. moorei also incorporated ¹⁴CO₂ into Cit. The ¹⁴C of Cit was restricted to the carbamyl-C. Comparable ¹⁵N₂ and CO₂-feeding studies on corallid roots of Cycas revoluta showed Gln to be the dominant product of N₂ fixation, with Asp and alanine as other major ¹⁴C-labeled amino compounds, but a total absence of Cit in labeled or unlabeled form.Abbreviations Ala alanine - Asp aspartic acid - Cit citrulline - Gln glutamine - Glu glutamic acid - Orn ornithine     

Nitrogen from senescing lower leaves of common bean is re-translocated to nodules and might be involved in a N-feedback regulation of nitrogen fixation

The objective of the present study was to elucidate whether remobilized N from lower leaves is involved in causing the drop in N(2) fixation during pod-filling in common bean (Phaseolus vulgaris L). Moreover, we addressed the question of whether remobilized N from lower leaves would reach the nodules. Nodulated common bean plants were grown in a growth chamber in quartz sand. During a 2-week period, at vegetative and at reproductive growth, 50% of the leaves (lower part) were either excised or individually darkened, thereby removing the same photosynthetic capacity yet allowing N to be remobilized from the darkened leaves. Moreover, at the vegetative growth period, three lower leaves per plants were (15)N labelled by applying (15)NH(4)NO(3) prior to imposing the darkening treatment. Leaf darkening at vegetative growth induced N remobilization as well as reduced N(2)-fixation rates and growth. Leaf excision at reproductive growth enhanced N(2) fixation. Changes in N(2)-fixation rates were in all cases the result of altered growth rates, while the % N in the whole plant and in various plant parts remained conserved. Directly after leaf labelling, but also at the end of the vegetative growth period, substantial amounts of (15)N from the leaves could be recovered in nodules in the control, and in higher amounts in the leaf-darkening treatment. It is proposed that nitrogen from leaves circulates within the plant via nodules, and that the strength or composition of this circular flow may be the signal for a putative N-feedback effect.     

Nitrogen fixation associated with a hotspring green alga

A unicellular alga which can grow in the light without a combined nitrogen source was isolated from a hot spring. The cells were almost spherical, usually 5–10 m in diameter. Absorption spectra of the watersoluble pigments and of the acetone-extracted ones revealed the existence of chlorophyll a and b and the absence of phycobilins. Thin sections examined by electron microscopy revealed an eukaryotic organization with features typical of the coccoid green algae (the Chlorococcales). Cells divided by internal cytokinesis and subsequent liberation of daughter cells from the parental wall, in a way similar to Chlorella. The alga reduced acetylene to ethylene and incorporated ¹⁵N₂ into cell protoplasm when incubated in a low oxygen atmosphere. Nitrogenase activity was light-dependent, microaerophilic and thermophilic. Although the association of symbiotic nitrogen fixing prokaryotes with the cells may still be possible, any such organisms have not so far been detected.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Chl chlorophyll - MBM modified Bristol medium - TLC thin layer chromatography     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

Use of isotope dilution to measure nitrogen fixation associated with the roots of sorghum and millet genotypes

Summary Genotypes of sorghum and millet have previously been found to have different amounts of root-associated acetylene reduction activity. Isotope dilution experiments using¹⁵N have been carried out to evaluate the amount of nitrogen fixed by bacteria which is incorporated into the different genotypes when grown in vermiculite in the glasshouse. Isotope dilution result indicated that the content of shoot nitrogen derived from biological nitrogen fixation varied by up to 27% between sorghum genotypes and 17% between millet genotypes. Considerable isotope dilution also resulted from uptake of non-exchangeable-N (as NH ₄ ⁺ ) in the vermiculite. It is possible that the genotypic differences in isotope dilution may reflect differences in the ability of plants to take up non-exchangeable-N, and that vermicultite is therefore an unsuitable growth medium for such studies.Published as Journal Article No. 493 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru P.O., Andhra Pradesh 502 324, India.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.