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     

Exogeneous energy supply and excitability of cells in embryonic atypical epidermis of Cynops cultured in vitro

Cells of in vitro cultured epidermis explants of ectoderm isolated at early gastrula stage,showed only weak excitability or even non-excitable at 6V when examined electrophysiologically.If non-excitable explants were treated with 100 mM glucose,the action potential (AP) appeared and within 1 hr reached its maximum.At the same time,their stimulus threshold became lowered gradually.And,if the glucose was washed out,AP gradually disappeared.If explants were treated with glucose of different concentrations,the percentage of explants which displayed AP increased with the increase of glucose concentration.When explants with approximately the same original stimulus threshold were treated with glucose of different concentrations,the stimulus threshold became lowered more in the more concentrated solution.If explants with different original stimulus thresholds were treated with glucose of the same concentration,the lowering of stimulus threshold was more obvious in those with higher original stimulus threshold.Other energy supplying substances used showed similar effect.     

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

Botryococcus braunii carbon/nitrogen metabolism as affected by ammonia addition

Carbon metabolism in photosynthesizing and respiring cells of Botryococcus braunii was radically changed by the presence of 1 mM NH₄Cl in the medium, when the so-called resting state previously had been subjected to a nitrogen-deficient medium. Ammonia addition to the algae photosynthesizing with ¹⁴C-labelled HCO ₃ ^- almost completely inhibited the synthesis of ¹⁴C-labelled botryococcenes and other hexane-extractable compounds, and also inhibited the formation of insoluble compounds; however, it resulted in a large increase in the synthesis of alanine, glutamine, other amino acids, and especially of 5-aminolevulinic acid. Total CO₂ fixation decreased about 60% and O₂ evolution decreased more than 50%.CO₂ fixation in the dark with ammonia present led to labelled products derived from phosphoenolpyruvate carboxylation, such as glutamine, glutamate, and malate. Respiratory uptake of O₂ increased by about 70%.The inhibition of terpenoid synthesis and increased synthesis of C₅ amino acids by Botryococcus upon ammonia addition indicates 1) a diversion of acetyl coenzyme A from synthetic pathways leading to terpenoids and 2) increased operation of pathways leading to the synthesis of amino acids, especially 5-aminolevulinic acid, a precursor to chlorophyll biosynthesis.This work was supported in part by the Office of Energy Research, Office of Basic Energy Sciences, Biological Energy Research Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098, in part by a grant from SOHIO, and, in part, by a grant from the Japan-U.S. Cooperative Science Program (The Japan Society for the Promotion of Science, National Science Foundation, Division of International Programs)     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.     

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.Abbreviations GS glutamine synthetase - NR nitrate reductase - NiR nitrite reductase     

Nitrogen nutrition and the accumulation of free and sinapoyl-bound malic acid in Raphanus sativus cotyledons

Seedlings of red radish (Raphanus sativus L. var. sativus) accumulated high amounts of free malic acid and sinapoylmalate, when grown on nitrate as the sole N-source. In the presence of ammonium (NO ₃ ^– : NH ₄ ⁺ , 1:2) both metabolites failed to accumulate, and the levels of arginine, asparagine, glutamine, histidine, and serine were greatly increased. The extractable activity of 1-sinapoylglucose: l-malate sinapoyltransferase, an enzyme which plays a key role in channelling malic acid into the sinapic-acid metabolism of this plant, was positively correlated with the malic-acid level in cotyledons. The possibility is discussed that free malic acid might be the likely candidate for regulating the activity of 1-sinapoylglucose: l-malate sinapoyltransferase.Abbreviation SMT sinapoylglucose: L-malate sinapoyltransferase     

Rhizobium inoculation trials designed to support a tropical forage legume selection programme

Summary Three phases of Rhizobium inoculation trials were carried out as part of a programme to select forage legume germplasm adapted to acid, infertile Oxisols of tropical America. Firstly, a range of tropical forage legumes were evaluated for their response to N fertilization or inoculation with strains previously shown to be effective in Leonard jars, using cores of undisturbed soil or in the field at Carimagua, Meta, Colombia. In pure legume stands onlyCentrosema macrocarpum andC. pubescens showed increases in N yield due to both inoculation and N fertilization;C. brasilianum responded only to N fertilization;Zornia latifolia, Z. brasiliensis andStylosanthes capitata responded to neither treatment. Trials in cores and in grass-legume mixtures showed responses ofDesmodium ovalifolium, Pueraria phaseoloides andS. capitata to N fertilization but not to inoculation. In the second phase of experiments strains were screened in soil cores with 16 ecotypes ofDesmodium, Centrosema, Stylosanthes andPueraria spp. Significant increases in N yield due to inoculation occurred with at least one strain in all the legumes exceptS. guianensis tardio, and in some trials withS. capitata. In the third phase of trials the most effective strains were tested in the field. Significant response ofP. phaseoloides andC. macrocarpum to inoculation at two sites and in the second year after establishment were shown. Further screening trials and field trials at different sites are needed in order to provide better recommendations for inoculation of grazing trials being set up in the region under study.     

Improving nitrogen fixation in legumes by plant breeding; the relevance of host selection experiments in red clover (Trifolium pratense L.) and subterranean clover (T. subterraneum L.)

Summary This paper reviews (i) basic studies on the genetics of symbiosis in red clover (a self-sterile species) and subterranean clover (cleistogamous) and (ii) work on selection and plant breeding to increase nitrogen fixation in these hosts.Symbiotic effectiveness in red clover is influenced by many major and minor genes. The highly effective phenotype is inherited in a complex manner associated with early nodulation and the formation of large amounts of persistent bacteroid-containing tissue. Lines bred to fix more nitrogen with one strain ofRhizobium trifolii do so with most but not all other strains examined. They also show slightly increased vigour when grown on nitrate. The highly effective response is correlated with abundant nodulation and an early flowering habit, the evidence from breeding studies indicating that this correlation is not absolute. Normally effective and highly effective nodules have the same specific nitrogenase activities. The expression of the highly effective response is relatively little affected by environmental factors (temperature, light intensity, day length, supplementary carbon-di-oxide). Inbreeding substantially degrades the symbiotic response.Heterosis is shown in crosses between cultivars of subterranean clover but otherwise selection to increase effectiveness in this host was unsuccessful.The relevance of these results (and their physiological aspects) for the improvement of grain legumes is discussed.