Short-Term Nitrate Effects on Hydroponically-Grown Soybean cv. Bragg and its Supernodulating Mutant: I. CARBON, NITROGEN AND MINERAL ELEMENT DISTRIBUTION, RESPIRATION AND THE EFFECT OF NITRATE ON NITROGENASE ACTIVITY

A comparison between two hydroponically-grown soybean genotypes(Glycine max [L.] Merr.) cv. Bragg and the supernodulating mutantnts 1007 was made in terms of dry matter accumulation, carbon,nitrogen, and mineral element distribution, ¹⁵N natural abundanceand the effect of short-term treatment with 4·0 mol m^–3KNO₃ on nitrogenase activity and respiration. Differences weremost pronounced in nodule dry weight and plant nitrogen content,both of which were recorded to be substantially elevated inthe mutant. Mineral element concentrations in different plantparts proved to be rather similar with the exception of Ca,found to be lower in leaves of the mutant, and Mn concentrationswhich were twice as high in roots of nts 1007. The values of¹⁵N natural abundance showed that both genotypes were equallydependent on nitrogen fixation when nitrate was absent. Theresults of the acetylene reduction assays indicated similarspecific nodule activity, while on a per plant basis nitrogenaseactivity of the mutant proved to be more than twice the amountof Bragg. This effect was also reflected in higher nodule respirationwhile root respiration remained below that of Bragg. Nitrate induced a substantial reduction in nitrogenase activitynot only in Bragg, but also in nts 1007. Nodule respiratoryactivity of Bragg was reduced by nitrate from 1·27 to0·34 mg C h^–1 plant^–1. In nts 1007 correspondingvalues were 2·70 to 1·52 mg C h^–1 plant^–1.Starch concentration in nodules was decreased in both genotypes,but nevertheless remained higher in nts 1007. Values for solublesugars in nodules even increased in the mutant in response tonitrate while the same treatment caused a reduction in Bragg.The data indicate that nitrogenase activities of Bragg and nts1007 are equally sensitive to short-term application of nitrate. Key words: Glycine max, C and N distribution, nitrate, root respiration, ¹⁵N natural abundance     

Autoregulation and Nitrate Inhibition of Nodule Formation in Soybean cv. Enrei and its Nodulation Mutants

The regulation and nitrate inhibition of nodule formation insoybean, Glycine max (L.) Merr., was further examined usingthe nodulation mutants of cv. Enrei. The non-nodulating mutantsEn115, Enl282, and En1314 produced extremely few markedly-curledroot hairs which were all devoid of infection threads, and invariablyfailed to initiate sub-epidermal cell divisions (SCDs) in theroot cortex. A considerable number of arrested SCDs was foundbefore nodule emergence in Enrei, but not in En6500 which hadsignificantly more SCDs that progressively increased at moreadvanced stages of nodule ontogeny. These observations indicatethat autoregulation acts by blocking the developmental stagebefore nodule emergence. In both Enrei and En65OO, the maturationof emerged nodules was restricted by a late-acting nodulationcontrol mechanism that is apparently unrelated to autoregulation.Reciprocal wedge-grafts of plants inoculated at sowing showedthat the control of the supernodulating phenotype resides inthe shoot, while the non-nodulating phenotype is strictly root-controlled.The nodulation phenotype of the current non-nodulating mutantsresults not from an alteration of the autoregulatory mechanism,but from mutation that exerts a root-localized effect that blocksSCDs which trigger the autoregulatory mechanism. Reciprocalgrafting experiments on Enrei and En6500 seedlings grown undervarious nitrate levels suggest that nitrate inhibition of nodulation,like autoregulation, is shoot-controlled. Since these two processesare invariably expressed together, they are probably causallyrelated, acting synergistically to regulate nodule formationin soybean. These results indicate that the regulation and nitrateinhibition of nodulation in the nodulation mutants of cv. Enreiare similar to those of cv. Bragg nodulation mutants. Key words: Autoregulation, nitrate-tolerant symbosis, non-nodulating mutants, soybean, supernodulating mutant     

Effects of Ultraviolet-B Irradiances on Soybean: II. INTERACTION BETWEEN ULTRAVIOLET-B AND PHOTOSYNTHETICALLY ACTIVE RADIATION ON NET PHOTOSYNTHESIS, DARK RESPIRATION, AND TRANSPIRATION

Soybean plants (cv. Hardee) were grown from seed under four ultraviolet-B radiation flux densities and four photosynthetically active radiation levels in a factorial design. Net photosynthesis, dark respiration, and transpiration were measured after 2 and 6 weeks of exposure. Effects of ultraviolet-B radiation were dependent upon photosynthetically active radiation levels. Ultraviolet-B radiation adversely affected net photosynthesis at low photosynthetically active radiation levels, but had little consequence at levels normally saturating photosynthesis in the field. Ultraviolet-B radiation affected both stomatal and nonstomatal resistances to carbon dioxide under low levels of photosynthetically active radiation. The present study demonstrates interactions between ultraviolet-B and photosynthetically active radiation.     

COMPARATIVE STUDIES ON RESPIRATION : II. THE EFFECT OF ANESTHETICS AND OTHER SUBSTANCES ON THE RESPIRATION OF ASPERGILLUS NIGER.

1. In concentrations which are high enough to produce any effect, formaldehyde, ether, and acetone cause an increase, followed by a decrease, in the rate of respiration. 2. 3.65 per cent ether, which causes an increase with certain cultures, produces only a decrease with others. 3. The reaction producing an increase in the respiration with 7.3 per cent ether is a reversible process, while the reaction producing the decrease is not reversible. 4. 0.5 per cent caffeine produces only a decrease in respiration while a saturated solution causes an increase, which is followed by a decrease.     

Carbohydrates in Soybean Nodules: II. DISTRIBUTION OF COMPOUNDS IN SEEDLINGS DURING THE ONSET OF NITROGEN FIXATION

During the first few days of nitrogen fixation activity by soybean (Glycine max (L.) Merr) root nodules, d-chiro-inositol, myo-inositol, sucrose, alpha,alpha-trehalose, and maltose accumulate rapidly and reach concentrations several fold greater than concentrations in other plant organs. Concentrations of d-pinitol in nodules (>/=1.0 milligrams per gram fresh weight) were similar to concentrations in leaf blades. The concentration of fructose in nodules was lower than concentrations in other plant organs.Comparison of nonnodulated roots, nodulated roots (after removal of nodules), and nodules indicated that nodules may compete successfully with roots for carbohydrates, especially the disaccharides sucrose, alpha,alpha-trehalose, and maltose. Based on the isolation of protoplasts and bacteroids, it was tentatively concluded that the highest concentrations of cyclitols in soybean nodules are located in the infected region and that, inside infected cells, the highest concentrations of d-pinitol and myo-inositol are outside of bacteroids.Evidence for the identification of d-chiro-inositol and maltose in soybean nodules is presented.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : II. Effects of Fatty Acids and Albumin on Respiration

Fatty acids inhibited the ability of Escherichia coli membrane-envelope fragments to catalyze the oxidation of succinate and nicotinamide adenine dinucleotide, reduced form (NADH) and also inhibited the response of the Clark oxygen electrode to nonenzymatic oxygen uptake. In all cases, unsaturated fatty acids were much more inhibitory than saturated fatty acids. Albumin afforded complete protection from inhibition in the nonenzymatic oxygen-uptake experiments but only partial protection for the respiratory activities of the membrane fragments. The succinoxidase activity was totally inhibited by bovine serum albumin at concentrations that inhibited succinate dehydrogenase only slightly and NADH oxidase not at all. The E. coli acellular preparation showed no dehydrogenase or oxidase activity for any of the fatty acids under a variety of conditions. These conditions included variations of pH, concentration of fatty acids, and the presence or absence of albumin, CoA, ATP, NAD, cysteine, succinate, and carnitine. It thus appears that E. coli grown in the absence of fatty acid can not use fatty acids as an energy source.     

THE POTENTIAL AND RESPIRATION OF FROG SKIN : I. THE EFFECT OF THE HOMOLOGOUS CARBAMATES. II. THE EFFECT OF CERTAIN LYSINS

Measurements of the O₂ consumption and of the potential of frog skin, made under comparable conditions, show that the homologous carbamates (ethyl, propyl, butyl, and amyl) reduce both the O₂ consumption and the potential, but not in a similar manner. In this respect, the effect of the carbamates is like the effect of reduction in O₂ tension. The simple lysins (saponin and the bile salts), on the other hand, abolish the potential without reducing the O₂ consumption at all. Irrespective of whether one considers the concentration of carbamate in the entire system or the amount of carbamate adsorbed by the frog skin, Traube''s rule relating the effect of a carbamate to its position in the homologous series does not seem to apply.     

White Clover N2-Fixation in Response to Root Temperature and Nitrate: II. N2-FIXATION, RESPIRATION, AND NITRATE REDUCTASE ACTIVITY

Established, nodulated white clover plants were transferredto eight tanks of a flowing culture apparatus with solutiontemperatures of 5, 11, 17, and 25 ?C (two tanks per temperature).Shoot temperature and light environment were common to all plants.After 7 d, (10 mmol m^–3) was continuouslysupplied to one tank at each temperature while in the remainingfour tanks (one at each temperature) the plants were completelydependent on nodule N₂-fixation. Plants were randomly selected at intervals during the following14 d period in order to measure root and nodule respirationand acetylene reduction activity (ARA) in a flow-through systemset at the adapted root temperature. Additional plants wereassayed for in vitro nitrate reductase activity in leaves, roots,and nodules. Apparent nitrogenase activity (ARA) and respiration associatedwith it were each markedly affected by temperature in two ways;(1) Activity per unit weight of nodule was reduced at lowertemperatures; (2) Development of the plant, and thus also nodulemass, was restricted at lower temperatures which, in turn, restrictedtotal nodule activity per plant. The presence of nitrate significantly reduced ARA of nodules,particularly at higher temperatures. However, significant discrepancieswere found when N₂-fixation rates, estimated from the acetylenereduction assay, were compared with N₂-fixation rates calculatedfrom curves fitted to N accumulation data (minus the rate of uptake in the case of nitrate-treated plants). Carbon use efficiency (CO² respired per C₂H₄ produced) was notsignificantly affected by temperature or the presence of nitrate. Nitrate reductase activity (NRA) developed in all plant partsat the three highest temperatures, but not at 5 ?C. We calculatethat leaf NRA may account for 82, 75, and 68% of total nitratereduction at 11, 17, and 25 ?C respectively. Key words: Trifolium repens, white clover, N₂ fixation, root temperature, acetylene reduction assay, nitrate, nitrate reductase     

THE EFFECTS OF RADIATIONS ON BIOLOGICAL SYSTEMS : II. IMMEDIATE AND SUBSEQUENT EFFECTS OF X-RAY IRRADIATION UPON RESPIRATION OF DROSOPHILA LARVAE

By means of measurement of CO₂ respiratory rates it has been possible to observe immediate and latent effects of x-ray irradiations upon Drosophila larvae. The observations were extended over a period of several days, the duration of the prepupal period being observed also. In every instance a significant decrease in the rate of CO₂ respiration was observed immediately after irradiation. This decrease was univariant with the period of irradiation within the experimental limits.     

ON GUAIACOL SOLUTIONS : II. THE DISTRIBUTION OF SODIUM AND POTASSIUM GUAIACOLATES BETWEEN GUAIACOL AND WATER

1. Measurements are reported on the distribution of sodium and potassium guaiacolates between guaiacol and water at 25°C. 2. The variation of the partition coefficients with the concentration is explained with the aid of the Debye-Hückel interionic attraction theory and the assumption that the salts are strong electrolytes in water and weak electrolytes in guaiacol. 3. The dissociation constants of sodium and potassium guaiacolates in guaiacol previously computed from electrical conductivity determinations are shown to be in agreement with the corresponding values obtained from the distribution measurements. 4. From theoretical considerations an equation is derived with which it is possible to predict the magnitude of the limiting partition coefficients from the dielectric constants of the solvents, the size of the solute ions, and the temperature.     

Biosynthesis and Metabolism of Indol-3yl-acetic acid: II. IN VIVO EXPERIMENTS WITH 14C-LABELLED PRECURSORS OF IAA IN TOMATO AND BARLEY SHOOTS

Our previous investigation of the naturally occurring indolecompounds in barley and tomato shoots suggested that the biosynthesisof indol-3yl-acetic acid (IAA) from tryptophan might proceedvia either the indol-3yl-pyruvic acid or tryptamine pathwaysin both species. The results further indicated that the indol-3yl-lacticacid pathway for IAA formation might also be operative in tomato.In the present study, tryptophan-3-¹⁴C and tryptamine-2-¹⁴Cwere fed to excised shoots of both barley and tomato, and indol-3yl-lacticacid-3-¹⁴C was also fed to shoots of tomato. All three compoundswere found to give rise to radioactive IAA with little dilutionin specific activity. Feeding tryptophan-3-¹⁴C also resultedin the labelling of indol-3yl-pyruvic acid, indol-3yl-acetaldehyde,and tryptamine, which were isolated and chemically identifiedfrom both species, and radioactive indol-3yl-lactic acid andtryptophol were also produced in tomato. Indol-3yl-acetaldehydewas found to be labelled in both species after administrationof tryptamine-2-¹⁴C, while the principal metabolite of indol-3yl-lacticacid-3-¹⁴C was radioactive tryptophan. These findings, alongwith the results from a quantitative study of the radioactivemetabolites, indicate that both the indol-3yl-pyruvic acid andtryptamine pathways can operate in both species, while the formationof IAA from indol-3yl-lactic acid in tomato probably occursindirectly, via tryptophan. These conclusions were supportedby the demonstration of the enzymes, L-tryptophan transaminase,L-trypto-phan decarboxylase, and indol-3yl-acetaldehyde dehydrogenasein cell-free extracts of both tissues, and of indol-3yl-pyruvicacid decarboxylase in the tomato extract. No indol-3yl-lacticacid decarboxylase activity was observed in the extracts fromeither tissue.     

Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance

Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean (Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO₃⁻-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO₃⁻ in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR-4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days.     

TEMPERATURE CHARACTERISTICS FOR THE METABOLISM OF CHLORELLA : II. THE RATE OF RESPIRATION OF CULTURES OF CHLORELLA PYRENOIDOSA AS A FUNCTION OF TIME AND OF TEMPERATURE

The respiration of the green alga Chlorella pyrenoidosa, suspended in Knop''s solution, has been studied in the dark as a function of time and of temperature. The rates of oxygen consumption and of carbon dioxide production (at constant temperature) decline for about 25 hours to a low, constant level. From an analysis of the curves it is suggested that two substances, A and B, are utilized, whose respiratory quotients are 1 and 0.65 respectively. The values of the temperature characteristics were found to be: for oxidation of A, 19,500 (0.6 to 11.5°C.) and 3,500 (11.5 to 28°C.); for oxidation of B, 5,600 (23.4 to 0.6°C.).     

Root Zone Temperature Effects on Growth and Nitrate Absorption in Rape (Brassica napus cv. Emerald)

Effects of root temperatures, ranging from 10–35 °C, on growth and nitrate inflow of fodder rape seedlings (cv.Emerald) were examined. These were cultured in solution, withtheir shoots held at 25 ° C. Nitrate inflow (uptake rateper unit root length) was little affected over the temperaturerange 10–30 ° C, although enhanced values were foundat 35 ° C. Nitrate absorption by roots at 10-30 ° Cdepleted solution concentrations to an apparent minimum of approximately6.0 µM NO₃^–. Relative growth rates were highestwith root temperatures of 25 ° C and 30 °C, and thesewere associated with the greatest nitrate depletion rates fromsolution. Root: shoot weight ratios were also greatest at 25°C and 30 °C. At 10 °C and 35 °C a relativelylarge shoot on a small root maintained nitrate inflow in spiteof the plants' slow growth rate. The nitrogen concentrationin the shoots was little affected by root temperature. Slowgrowth at a root temperature of 10 °C was not associatedwith a shortage of nitrogen in the shoots. The principal influenceof temperature appears to be on extension and differentiationof root tissues, possibly through effects on carbohydrate supplyto root meristems.     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: II. EFFECT OF PLANT N-STATUS ON THE KINETIC PARAMETERS OF NITRATE INFLUX

Influx of nitrate into the roots of intact barley plants wasfollowed over periods of 1–15 min using nitrogen-13 asa tracer. Based on measurements taken over 15 min from a rangeof external nitrate concentrations (0·2–250 mmolm^–3), the kinetic parameters of influx, I_max and K_m, werecalculated. Compared with plants grown in the presence of nitrate throughout,plants that had been starved of N for 3 d showed a significantlygreater value ofI_max for ¹³N-nitrate influx (by a factor of1·4–1·8), but a similar value of K_m (12–14mmol m^–3). Pre-treating N-starved plants with nitratefor about 5 h further increased the subsequent rate of ¹³N-nitrateinflux, but had little effect in the unstarved controls. Allowingfor this induction of additional nitrate transport, the differencein rates of nitrate influx in control and N-starved plants wassufficient to account for the previously-observed differencein net uptake by the two groups of plants. In barley plants grown without any exposure to nitrate, butwith ammonium as N-source, both I_max and K_m for subsequent ¹³N-nitrateinflux were significantly decreased (by about one-half) comparedwith the corresponding nitrate-grown controls. The importance of changes in the rate of influx in the regulationof net uptake of nitrate is discussed. Key words: Ion transport, nitrate, influx, kinetic parameters, N-deficiency     

The Effect of Subjecting Peas to Air Enriched with Carbon Dioxide: II. RESPIRATION AND THE METABOLISM OF THE MAJOR ACIDS

Whole peas at about 75 per cent of their maximum fresh weightwere subjected to 5–30 per cent CO₂ in air for periodsof from 1–6 d, then returned to air for a further 1 d.Samples were withdrawn at intervals and organic acids, ^TCO₂and ethanol estimated as well as the rate of respiration. Slicesof cotyledons suspended in water were also subjected to highconcentrations of CO₂ in air for 3 h. The rate of respiration was inhibited progressively by increasein CO₂ content of the tissue. The high internal CO₂ contentof the intact pea causes an inhibition of its rate of respirationby about 25 per cent. Alcohol production commenced at between10 and 15 per cent CO₂ in the ambient gas and slowly increasedin rate up to 37 per cent. The CO₂-air mixtures reduced the content of malate, pyruvateand -oxoglutarate, increased that of succinate and left citrateunaffected. On return to air malate rose rapidly and succinatefell slowly to their original concentrations. During the sameperiod the concentration of PEP fell sharply and after about1 h rose again, whereas oxalacetate showed a reverse response.It is argued that the rapid re-synthesis of malate was by carboxylationof PEP to oxalacetate and that this reaction was stimulatedby a change in pH rather than by the direct effect of the changein concentration of CO₂. In one experiment ¹⁴CO₂ was supplied for 2 h before return toair and the movement of ¹⁴C followed for 6 h. The results supportthe method of re-synthesis of malate proposed.     

The Transport and Metabolism of Urea in Chara australis: II. UREASE AND THE DISTRIBUTION OF TRANSPORTED UREA

The ureolytic enzyme in Chara was investigated. This enzymewas shown to be a urease with an unusually high affinity forurea(K_m = 158 mmol m^-3). Little inhibition of urease activitywas found when intact Chara cells were exposed to the ureaseinhibitors hydroxyurea, acetohydroxamic acid and N-ethylmaleimide,although there was some inhibition of urea uptake. The distribution of radioactivity amongst the amino acid, organicacid and sugar/neutral fractions, determined by ion-exchangechromatography, was very similar whether the Chara internodeswere exposed to ¹⁴C-urea or to H¹⁴CO₃. This suggests that thefraction of the urea-carbon liberated by the urease as CO₂ andretained by the cell is used in photosynthetic carbon-fixation.During the initial 15 min of ¹⁴C-urea uptake, label appearsin the vacuole only in the form of unmetabolized urea. Afterthis time a variety of labelled compounds appear in the vacuole,presumably reflecting the gradual movement of carbon-fixationproducts from the chloroplasts to the cytoplasm and thence intothe vacuole. Key words: Urea transport, metabolism, Chara, urease