The simultaneous use of 14CO2 and 15N2 labelling techniques to study the carbon and nitrogen economy of legumes grown under natural conditions

A method based on simultaneous short-term exposure to ¹⁴CO₂ and ¹⁵N₂ is described for studying nitrogen fixation and distribution in legumes relative to carbon assimilation and use. Equipment designed to accomodate experiments under natural conditions with very little disturbance of the N₂ fixing association is used. It permits continuous measurement and regulation of variables such as air temperature, humidity and CO₂ concentration as well as soil aeration. Measurements of distribution and use of assimilates, respiration of nodulated roots, quantitative N₂ fixation and the distribution and fate of fixed N as a function of time lead to a precise estimation of C and N budgets for each labelling period. When experiments are done at several phenological stages they give a new insight into the complex C and N interrelations in legume symbiosis.
A series of trials throughout the growth period of Glycine max (L.) Merr. cv. Hodgson demonstrated the sensitivity of the method. The development of the plants from vegetative to reproductive stages was accompanied by a complete change in the distribution patterns of current assimilates and products of nitrogen fixation. Maximum sink strength moved from the leaves to the pods and seeds which ended up receiving 70% of the incoming C and 35% of the fixed N. The fact that up to 85% of fixed N in the plants was in the reproductive organs at maturity can be accounted for by remobilisation from vegetative parts.
The respiration of nodulated roots utilized 33% of carbon translocated to below-ground plant parts before nitrogen fixation started, but as much as 50% during the period of optimal fixation. The advantages and limitations of the isotopic method described are critically discussed as a prelude to future investigations.     

Osmoregulation and cell composition in salt-adaptation of Nostoc muscorum

Adaptation to salt in the cyanobacterium Nostocmuscorum, is composed of a few mechanisms which together lead to the generation of a salt-tolerant cell. The initial mechanism combines a stimulation of photosynthetic activity with the accumulation of sucrose as an osmoregulator. The secondary mechanism involves the adaptation of N₂ fixation activity and protein biosynthesis. The adaptation is most efficient in response to NaCl-induced stress and functions only partially under stress induced by either KCl or a nonionic osmoticum such as mannitol.     

Effect of darkness and CO2 starvation on NH4+ and NO3− assimilation in the unicellular alga Cyanidium caldarium

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO₂, assimilated NH₄⁺ at high rates both in the light and in the dark. The assimilation of NO₃⁻, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO₂-free air, NH₄⁺ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO₂ completely restored the capacity of the alga to assimilate NH₄⁺. NO₃⁻ assimilation, by contrast, was 33% higher in the absence of CO₂ and was linear with time. It is suggested that NO₃⁻ and NH₄⁺ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.     

The effect of Cd2+ on photosynthetic reactions of mesophyll protoplasts

Photosynthetic CO₂-fixation of mesophyll protoplasts of lambs lettuce [Valerianella locusta (L.) Betcke] was inhibited by short time exposure to Cd⁺. Inhibition was due to uptake of the metal ion into the protoplasts and increased with increasing Cd²⁺ concentrations and the time of preincubation. A 10 min pretreatment at 2 mM Cd²⁺ reduced CO₂-fixation by 40–60%. Inhibition of photosynthesis was independent of the light intensity to which the protoplasts were exposed. Measurement of the lightinduced electrochromic pigment absorption change at 518nm and chlorophyll fluorescence studies revealed that primary photochemical reactions associated with the thylakoid membranes were not affected by the metal ion. Also, light activation of the ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was not inhibited by Cd²⁺. Under rate-limiting CO₂ concentrations, inhibition of CO₂-fixation was smaller than at V_max of CO₂ reduction indicating that the carboxylation reaction of the Calvin cycle is not susceptible to Cd²⁺. Cd²⁺ treatment of protoplasts significantly extended the lagphase of CO₂-supported O₂-evolution and partly inhibited light activation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and the ribulose-5-phosphate kinase (EC 2.7.1.19). Measurement of relative concentrations of [¹⁴C]-labeled Calvin cycle intermediates showed that Cd²⁺ caused a decrease in the 3-phosphoglycerate/triose phosphate ratio and an increase in the triose phosphate/ribulose-1,5-bisphosphate ratio. It is concluded that in protoplasts Cd²⁺ affects photosynthesis mainly at the level of dark reactions and that the site of inhibition may be localized in the regenerative phase of the Calvin cycle.     

Behaviour of Nif− mutants of Rhodobacter capsulatus in photosynthetic, ammonia-limited chemostat cultures

Abstract Nif⁻ mutants of Rhodobacter capsulatus carrying mutations either in the nifR4 regulatory gene or in the nifH structural gene both outgrew the wild-type strain B10 in mixed chemostat cultures under conditions favouring nitrogenase-mediated H₂ production by the wild-type (ammonia as limiting nutrient, inert argon atmosphere, light as energy source), whereas under aerobic conditions in the dark, or in batch culture, the growth of Nif⁻ mutants was not favoured. Nitrogenase-mediated H₂ production therefore appears to be detrimental to the growth of R. capsulatus in nitrogen-limited continuous culture, as may also be the case for other nitrogen fixers.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

Soybean (Glycine max) modulation and N2-fixation as affected by exposure to a low root-zone temperature

Low root-zone temperatures (RZTs) are known to reduce soybean N₂-fixation. However, the relative sensitivity of the various stages of symbiosis establishment and function (N₂-fixation) to suboptimal RZTs is unresolved. We conducted experiments to examine the effect of exposure to a RZT of 15°C on nodulation. The control RZT was 25°C. Root temperatures were controlled by circulating cooled water around pots on a growth bench. Soybean seedlings [ Glycine max (L.) Merr. cv. Maple Arrow] were inoculated with 1 ml of a log-phase culture (approximately 10⁻⁸ cells) of Bradyhizobium japonicum strain 532C. They were then (1) maintained continuously at RZTs of 15 or 25°C, transferred to 15 or 25°C from the alternate temperature 7 days after inoculation (DAI), or transferred to 15 or 25°C at 14 DAI, and (2) maintained at 15 or 25°C, or transferred at either 1, 4 or 7 DAI. When seedlings were maintained at a RZT of 25°C nodule primordia (<1 mm) were visible at 7 DAI and N₂-fixation commenced at 14 DAI. Nodule function (N₂-fixation) appeared to be relatively insensitive to low RZTs since exposure of plants to 15°C following the onset of N₂-fixation (14 DAI) resulted in 68% of the N fixed and 78% of the dry weight of the 25°C RZT, although N partitioning to shoot tissues was reduced. In contrast, exposure to the low RZT shortly after inoculation declayed the onset of N₂-fixation for 4 to 6 weeks, primarily by inhibiting the early stages of nodulation. This resulted in fixed N and dry weight levels of 9% and 22% of controls, respectively.     

Differential effects of Na+, Mg2+, K+, Ca2+ and osmotic stress on the wild type and the NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii

In order to identify physiological components that contribute to salinity tolerance, we compared the effects of Na⁺, Mg²⁺ and K⁺ salts (NaCl, Na₂SO₄, MgCl₂, MgSO₄, KCl and K₂SO₄), Ca₂₊ (CaSO₄), mannitol and melibiose on the wild type and the single-gene NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii. Compared with gametophytic growth of the wild type, stl2 showed a low level of tolerance that was restricted to Na⁺ salts and osmotic stress. stl2 exhibited high tolerance to both Na⁺ and Mg²⁺ salts, as well as to osmotic stress. In response to short-term exposure (3 d) to NaCl, accumulation of K⁺ and Na⁺ was similar in the wild type and stl1. In contrast, stl2 accumulated higher levels of K⁺ and lower levels of Na⁺. Ca²⁺ supplementation (1.0 mol m^?3) ameliorated growth inhibition by Na⁺ and Mg²⁺ stress in wild type and stll, but not in stl2. In addition, under Na⁺ stress (175 mol m^?3) wild-type, stll and stl2 gametopbytes maintained higher tissue levels of K⁺ and lower levels of Na⁺ when supplemented with Ca²⁺ (1.0 mol m^?3). stl2 gametophytes were extremely sensitive to K⁺ supplementation. Growth of stl2 was greater than or equal to that of the wild type at trace concentrations of K⁺ but decreased substantially with increasing K⁺ concentration. Supplementation with K⁺ from 0 to 1.85 mol m^?3 alleviated some of the inhibition by 75 mol m^?3 NaCl in the wild type and in stl1. In stl2, growth at 75 mol m^?3 NaCl was similar at 0 and 1.85 mol m^?3 K⁺ supplementation. Although K⁺ supplementation above 1.85 mol m^?3 did not alleviate inhibition of growth by Na⁺ in any genotype, stl2 maintained greater relative tolerance to NaCl at all K⁺ concentrations tested.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

The Family of Na+/Cl− Neurotransmitter Transporters

Abstract: The termination of neurotransmission is achieved by rapid uptake of the released neurotransmitter by specific high-affinity neurotransmitter transporters. Most of these transporters are encoded by a family of genes (Na⁺/Cl⁻ transporters) having a similar membrane topography of 12 transmembrane helices. An evolutionary tree revealed five distinct subfamilies: γ-aminobutyric acid transporters, monoamine transporters, amino acid transporters, "orphan" transporters, and the recently discovered bacterial transporters. The bacterial transporters that belong to this family may help to develop heterologous expression systems with the aim of solving the three-dimensional structure of these membrane proteins. Some of the neurotransmitter transporters have been implicated as important sites for drug action. Monoamine transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs. Localization of individual transporters in specific cells and brain areas is pertinent to understanding their contribution to neurotransmission and their potential as targets for drugs. The most important questions in the field include resolving the mechanism of neurotransmitter transport, the structure of the transporters, and the interaction of each transporter in complex neurological activities.     

Binding of Cd2+, Ni2+, Cu2+ and Zn2+ by isolated envelopes of Pseudomonas fluorescens

Abstract Cell envelopes of Pseudomonas fluorescens , cytoplasmic membrane, peptidoglycan and outer membrane were obtained from a fractionation procedure and tested for their metal binding capacity. Isolated envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) were chemically modified and functional carboxyl groups transformed to electropositive amine groups, using carbodiimide ethylenediamine. Transformation of carboxyl groups was evaluated by measuring total amine groups in all fractions (modified or not). Using equilibrium dialysis and Scatchard plots for the data, we have established that isolated unmodified cell envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) possess at least two types of metal binding sites with different association constants ( K _a and K '_a). Introduction of positive charges into the bacterial envelopes resulted in the disappearance of one type of metal binding site which had the highest association constant value for Ni²⁺, Cu²⁺ and Zn²⁺. All fractions, modified or not, always presented at least two types of binding sites with different association constants for Cd²⁺.     

Effects of atmospheric SO2 on Azolla and Anabaena symbiosis

The water fern Azolla pinnata R. Br. was fumigated for 1 week with either 25, 50 or 100 nl 1⁻¹ SO₂. The symbiosis of Azolla with Anabaena azollae (spp.) was severely damaged by atmospheric SO₂ even at concentrations as low as 25 nl 1⁻¹, with significant reductions in growth, reduction of C₂H₂, NH₃ assimilation, protein synthesis, and heterocyst development. These disturbances appear to be mainly responsible for the extreme sensitivity of this fern to atmospheric SO₂. Changes in violaxanthin/antheraxanthin and epoxy-lutein/lutein ratios also indicate that free radical products are induced by atmospheric SO₂. These results suggest that the Azolla-Anabaena symbiotic system is a very responsive and reliable lower plant model to study the detailed effects of total sulphur deposition upon the balances between various important plant metabolic processes.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.