Whole Plant Studies using Radioactive 13-Nitrogen: I. TECHNIQUES FOR MEASURING THE UPTAKE AND TRANSPORT OF NITRATE AND AMMONIUM IONS INHYDROPONICALLY GROWN ZEA MAYS

Methods are described for studying the uptake, by hydroponicallygrown Zea mays seedlings, of ammonium and nitrate ions labelledwith radioactive nitrogen-13, which has a half-life often minutes.For nitrate only, some of the activity absorbed by the rootexchanges back out again into the root bathing solution. Theamount of this activity is about five times too large to beattributable to exchange with ions in the root cortical apoplasm.Much of it must be transferred from the root symplasm with ahalf-time of exchange of 2–5 min. After exposing the rootto the labelled solution, equilibrium rates of transport to,and distribution in the shoot were attained within 2 min, fornitrate, or 5 min, for ammonium. The pools within the root,and the transport pathway through which the label passes musttherefore rapidly attain the specific activity of the nutrientsolution. Distribution patterns through the plant are reasonablyconsistent with earlier work on nitrogen assimilation and transport. Key words: Zea mays, Nitrate uptake, Amonium uptake, ¹³N tracer     

Whole Plant Studies using Radioactive 13-Nitrogen: II. A COMPARTMENTAL MODEL FOR THE UPTAKE AND TRANSPORT OF NITRATE IONS BY Zea mays

Nitrate ion uptake by the roots of hydroponically grown maizeseedlings was measured using the short-lived isotope ¹³N. Itis shown to be described by a four compartment model, recognizablynitrogen in the root bathing solution, nitrogen which is readilyexchangeable from the root, nitrogen bound in the root, andnitrogen transported from the root. Some of the absorbed activity leaks back into the root bathingsolution with the efflux from the root, as a fraction of theinflux, increasing with concentration to be greater than 0–8at external nitrate ion concentrations above about 1.0 mol m^–3.The capacity of the exchangeable root pool increases with externalnitrate ion concentration, approaching the expected cytoplasmicnitrate ion content at the highest external nitrate ion concentrationsstudied (70 mol m^–3). The investigation has highlighted the problems of interpretinguptake profiles in experiments for which the 10 min half-lifeof ¹³N dictates experimental times that are comparable withthe times for saturation of root pools. Key words: Zea mays, ¹³N, Compartmental model, Nitrate uptake     

Whole Plant Studies Using Radioactive 13-Nitrogen: III. THE RELATIONSHIP BETWEEN NITRATE AND AMMONIUM ION UPTAKE AND WATER FLUX FOR ZEA MAYS

Nitrogen-13 labelled nitrate and ammonium ions were used tomeasure uptake and assimilation rates in non-destructive experimentsin which the water flux through hydroponically grown maize plantswas doubled by changing the ambient humidity. Although no immediateeffects on ion uptake were observed, longer term observationsshowed different patterns of coupling for the uptake parametersfor nitrate and for ammonium. The patterns were compared withpatterns predicted for different combinations of processes foruptake and for discharge to the xylem. Those which were compatiblewith the experimental patterns indicated that for nitrate theuptake into the root showed coupling to water flux whereas dischargeto the xylem did not. For ammonium there appeared to be couplingto the water flux of both uptake into the root and of dischargeto the xylem though it was less evident in the former when theexternal ammonium ion concentration was low. Key words: Ion-water coupling, Ammonium uptake, Nitrate uptake     

UPTAKE OF ORGANIC SUBSTRATES BY CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE): EFFECTS OF IONS,IONOPHORES AND METABOLIC AND TRANSPORT INHIBITORS1,2

The uptake of glucose and amino acids by the euryhaline diatom Cyclotella cryptica Reimann, Lewin & Guillard does not appear to be related to proton gradients. Instead, the transport systems for these organic solutes show a strong requirement for the presence of NaCl. The relationship between uptake and NaCl concentration is hyperbolic, with optimal uptake rates being approached at 100 mM NaCl. High concentrations of KCl cause strong reductions in uptake rates. The (Na⁺, K⁺)-stimulated ATPase inhibitor ouabain has no effect on glucose uptake, whereas the diphenolic glucoside phlorizin and its aglucone phloretin are strongly inhibitory. The proton translocating uncoupler CCCP (carbonylcyanide m-chlorophenyl hydrazone) and the ATPase inhibitor DCCD (dicyclohexylcarbodiimide) both almost completely abolish glucose transport, and low concentrations of the ionophares monensin and valenomycin strongly inhibit glucose uptake by the diatom. The requirement of high external NaCl concentrations for glucose transport, and the inhibitory effect an transport of the Na⁺-specific ionophore monensin are consistent with a coupling of Na⁺ and organic substrate transport, but could also be explained by a Na⁺ requirement for glucose binding to a transport carrier, and/or a possible interference with energy producing reactions associated with a monensin-induced collapse of the normal Na⁺ gradient.     

TAXONOMY OF ZEA (GRAMINEAE). II. SUBSPECIFIC CATEGORIES IN THE ZEA MAYS COMPLEX AND A GENERIC SYNOPSIS

A compromise classification of the genus Zea, reflecting both phylogeny and practical needs, recognizes six taxa, as follows: Section Luxuriantes : Zea perennis. Zea diploperennis, Zea luxurians. Section Zea : Zea mays ssp. mexicana (Neo-volcanic Plateau), Zea mays ssp. parviglumis Iltis & Doebley ssp. n. var. parviglumis (Rio Balsas drainage, Pacific slope from Guerrero to Jalisco), Zea mexicana ssp. parviglumis var. huehuetenangensis Iltis & Doebley var. n. (Pacific slope, western Guatemala, Prov. Huehuetenango), Zea mays ssp. mays. The new subspecies is distinguished by smaller spikelets and rachis joints, the varieties by different habitats, blooming dates and their genetic behavior in relation to cultivated Zea mays. Zea mays ssp. mexicana is the ancestor of corn.     

THE COMBINATION OF SALTS AND PROTEINS : II. A METHOD FOR THE DETERMINATION OF THE CONCENTRATION OF COMBINED IONS FROM MEMBRANE POTENTIAL MEASUREMENTS.

A method is described for measuring membrane potentials of gelatin-salt solutions, and it is pointed out that such measurements, together with the analysis of the solutions, allow the calculation of the concentration of ions combined with the protein. The values for the combined ions obtained in this way for ZnCl₂, KCl, LiCl, and HCl agree quite well with those obtained by direct concentration cell measurements.     

A TEST FOR DIFFUSIBLE IONS : II. THE IONIC NATURE OF PEPSIN.

1. The distribution of pepsin between particles of gelatin or coagulated egg albumin and the outside solution has been found to be equal to the distribution of chloride or bromide ion under the same conditions. This is the case from pH 1 to 7, and in the presence of a variety of salts. 2. Pepsin is therefore probably a monovalent anion. 3. Under certain conditions the enzyme may be adsorbed on the surface of the protein particles. This reaction is irreversible and is markedly influenced by the presence of low concentrations of electrolytes.     

Studies with Radioactive Tracers in Plant Nutrition: II. THE ESTIMATION OF RADIOACTIVE TRACERS

Part I Tracer methods can yield valid results only if the behaviourof the tracer within the plant is indistinguishable from thatof the normal element. The extent to which radioactive isotopessatisfy this requirement must be considered from the followingviewpoints:

1. their chemical and physical bahaviour before disintegration;
2. the effects and substances formed by their disintegration;
3. the effects of the radiation they emit.

Whereas, chemically and physically, isotopes with the exceptionof H¹ and H³ satisfy this requirement, they may cause significantradiation damage even when supplied in low concentration ifthey are accumulated metabolically. The investigation of possibleradiation effects is therefore an essential preliminary to allphysiological investigations. The value of tracer methods is considered. Part II Equipment and laboratory procedures for the electronic assayof P³² are described. For the assay of samples containing P³²a wet ashing method and the subsequent use of a liquid countingtube is preferred. Improvements of equipment to suit the requirementsof plant nutritional studies are considered. Sources of experimentalerror are examined. On account of the decay of radioactive isotopes,the time factor may be of special importance in tracer work.Its effect on both experimental design and on laboratory proceduresis considered. It is stressed that the scope and replicationof large experiments, particularly if they are of long duration,may be subject to serious limitations. The importance of simplifyingthe equipment used in tracer assay and increasing its reliabilityis emphasized.     

A TEST FOR DIFFUSIBLE IONS : I. THE IONIC NATURE OF TRYPSIN.

1. The Donnan equilibrium furnishes a test for the ionic nature of any diffusible substance, since the ratio of the concentration of any ion on the two sides of a membrane must be equal to the ratio of the concentrations of any other ion of the same sign and valence, whereas a non-ionic substance would be equally distributed on both sides. 2. The distribution of trypsin inside and outside of gelatin particles has been compared to the distribution of hydrogen and chloride ions under the same conditions. 3. The ratio of the trypsin concentration in the gelatin to the concentration in the outside liquid is equal to the ratio of the hydrogen ion under the same conditions and to the reciprocal of the chloride ion ratio. 4. This result was obtained between pH 2.0 and 10.2. At pH 10.2 the trypsin is equally distributed and on the akaline side of 10.2 the ratio is directly equal to the chloride ratio. 5. Trypsin is therefore a positive monovalent ion in solutions of pH 10 to 2. It is probably isoelectric at 10.2 and a monovalent negative ion on the alkaline side of 10.2 6. Trypsin must also be a strong base since there is no evidence of any undissociated form on the acid side of pH 10.2.     

HIGH AFFINITY UPTAKE SYSTEMS FOR TAURINE IN TISSUE SLICES AND SYNAPTOSOMAL FRACTIONS PREPARED FROM VARIOUS REGIONS OF THE RAT CENTRAL NERVOUS SYSTEM. CORRECTION OF TRANSPORT DATA BY DIFFERENT EXPERIMENTAL PROCEDURES

Abstract— —The uptake of taurine into tissue slices of specific regions of the rat central nervous system (CNS) was compared with the uptake of taurine into synaptosomal fractions prepared from the corresponding regions. Two different techniques for performing control experiments were also compared: procedure I, correction for the uptake of taurine obtained from duplicate incubations but at 2°c and procedure II, correction of taurine uptake into extracellular or extrasynaptosomal space measured by inulin uptake experiments plus correction for diffusion (non-saturable) processes.
Kinetic analyses of the uptake data in tissue slices utilizing the procedure I correction technique indicate that six regions of the rat CNS (spinal cord, diencephalon, cortex, striatum, hippocampus, and midbrain) possess high affinity uptake systems (K_m values approx 60 μM or less). The K_m value for the cerebellum (105.4 ± 15.7 μM) is intermediate between a high and low affinity uptake system while the K_m value for the pons-medulla (210.0 12.4 μM) is considered to be low affinity. When procedure II techniques were utilized for correcting the uptake data all eight regions demonstrated high affinity uptake systems (11.8–73.2μM).
Synaptosomal fractions prepared from the spinal cord, pons-medulla, diencephalon, and midbrain demonstrate high affinity uptake systems (procedure I) for taurine (10.3–47.2 μM) while the hippocampus, cortex, striatum, and cerebellum have intermediate (but still high affinity) values (59.4–96.4 μM). High affinity uptake systems (8.2–79.8 μM) were obtained for all eight regions of the rat CNS when procedure II was utilized for correction of the data.     

Studies in the Physiology of the Onion Plant: IV. THE INFLUENCE OF DAY-LENGTH AND TEMPERATURE ON THE FLOWERING OF THE ONION PLANT

At temperatures above about 17° C. inflorescence initiationin growing onion plants, as in stored sets, is suppressed whetherthe plants are kept in long or short days. Independently ofcurrent day-length and of previous day-length treatment, ifthe plants are sufficiently large initiation begins very shortlyafter the temperature falls below c. 15° C. Emerged infiorescencesappear some ten or so weeks later. Small plants are unable toinitiate inflores cences under any of the conditions tested,and actual size (perhaps leaf area) rather than leaf or nodenumber seems to be the important factor. Inflorescence emergenceis suppressed at high temperatures in short days or long days;in long days bulb formation also suppresses emergence at lowertemperatures. In long days at temperatures sufficiently lowfor bulbing to be delayed, however, emergence is accelerated.Plants which have produced bulbs in long days in the summershow a delay of inflorescence emergence in the following winter.     

CRYSTALLINE TRYPSIN : IV. REVERSIBILITY OF THE INACTIVATION AND DENATURATION OF TRYPSIN BY HEAT

1. If dilute solutions of purified trypsin of low salt concentration at pH from 1 to 7 are heated to 100°C. for 1 to 5 minutes and then cooled to 20°C. there is no loss of activity or formation of denatured protein. If the hot trypsin solution is added directly to cold salt solution, on the other hand, all the protein precipitates and the supernatant solution is inactive. 2. The per cent of the total protein and activity present in the soluble form decreases from 100 per cent to zero as the temperature is raised from 20°C. to 60°C. and increases again from zero to 100 per cent as the solution is cooled from 60°C. to 20°C. The per cent of the total protein present in the soluble (native) form at any one temperature is nearly the same whether the temperature is reached from above or below. 3. If trypsin solutions at pH 7 are heated for increasing lengths of time at various temperatures and analyzed for total activity and total protein nitrogen after cooling, and for soluble activity and soluble (native) protein nitrogen, it is found that the soluble activity and soluble protein nitrogen decrease more and more rapidly as the temperature is raised, in agreement with the usual effects of temperature on the denaturation of protein. The total protein and total activity, on the other hand, decrease more and more rapidly up to about 70°C. but as the temperature is raised above this there is less rapid change in the total protein or total activity and at 92°C. the solutions are much more stable than at 42°C. 4. Casein and peptone are not digested by trypsin at 100°C. but when this digestion mixture is cooled to 35°C. rapid digestion occurs. A solution of trypsin at 100°C. added to peptone solution at zero degree digests the peptone much less rapidly than it does if the trypsin solution is allowed to cool slowly before adding it to the peptone solution. 5. The precipitate of insoluble protein obtained from adding hot trypsin solutions to cold salt solutions contains the S-S groups in free form as is usual for denatured protein. 6. The results show that there is an equilibrium between native and denatured trypsin protein the extent of which is determined by the temperature. Above 60°C. the protein is in the denatured and inactive form and below 20°C. it is in the native and active form. The equilibrium is attained rapidly. The results also show that the formation of denatured protein is proportional to the loss in activity and that the re-formation of native protein is proportional to the recovery of activity of the enzyme. This is strong evidence for the conclusion that the proteolytic activity of the preparation is a property of the native protein molecule.     

Studies with Radioactive Tracers in Plant Nutrition: III. THE EFFECT OF RADIATION FROM PHOSPHORUS-32 ON THE GROWTH AND PHOSPHATE UTILIZATION OF BARLEY SEEDLINGS

The effect of radiation from P³² on the growth and phosphateabsorption of barley seedlings was studied in water culture.Concentrations of carrier phosphate ranging from less than 001to 31 p.p.m. P were employed for varying periods. Between experiments wide variations occurred in the nature andextent of the effects of radiation. An increase in the proportionof the absorbed phosphate translocated to the shoots, and thereduction of root weight, were the most consistent symptoms.While severe injury was characterized by a reduction in bothgrowth and phosphate absorption, intermediate levels of P³²on occasion caused increases in dry weight, in nutrient absorption,and in the development of lateral meristems. The interrelation of the observed effects of radiation is discussed;the reduced metabolic activity of root meristems is consideredto be the primary effect in the syndrome of radiation injury.Although injury is due to the accumulation of P³² in meristematicregions, no close correlation can be shown between its extentand the level of radiation received by the meristems. The inaccuracyof present methods for estimating the levels of radiation towhich tissues are exposed has complicated the investigationsof this subject. It appears, however, that the injurious levelof radiation may vary from under 10 to over 40 rep. per daywhen the treatment period is 5 to 7 days. If active accumulationof phosphate occurs, the meristems of plant roots may receivethis dosage when treated with more than 5 µ C. of P³²per litre for periods of 6 days. The extent to which the effects of radiation may complicatethe interpretation of the results of plant physiological investigationsis considered.     

THE PENETRATION OF LUMINOUS BACTERIA BY THE AMMONIUM SALTS OF THE LOWER FATTY ACIDS : PART I. GENERAL OUTLINE OF THE PROBLEM,AND THE EFFECTS OF STRONG ACIDS AND ALKALIES.

It is shown that disappearance of the light of luminous bacteria may be used as a criterion of cell penetration; that luminous bacteria are cytolyzed by water, hypotonic solutions, and by freely penetrating solutions; that luminous bacteria are not injured by hydrogen or hydroxyl ions in the external solutions within the range of pH values employed with the ammonium salts and that therefore disappearance of the light in isotonic solutions of these salts must be due to penetration of the solute; and that there is a characteristic difference between the effects of strong and of weak acids and alkalies on luminous bacteria.     

EFFECTS OF POPULATION AND INDUSTRIALIZATION ON THE DEGRADATION OF BIOMASS AND ITS REGENERATION BY AFFORESTATION: A MATHEMATICAL MODEL

The bio-mass(Food, Fodder and Forestry, etc. ) in a habitat has beencontinuously depleting due to rapid industrialization and over-population leadingto various kinds of undesirable effects on our environment and on land masscausing deep concern to planners and management experts. In this paper, therefore, a mathematical model for degradation of bio-massis presented to study the effects of industrialization and associated growth ofpopulation, It is shown that with the increase in the levels of these factors,the bio-mass resource may not last long. It has also been pointed out mathematicallythat the level of the biomass can be maintained at desired equilibrium level bya suitable afforestation programme.     

INTERACTIONS BETWEEN IRON, LIGHT, AMMONIUM, AND NITRATE: INSIGHTS FROM THE CONSTRUCTION OF A DYNAMIC MODEL OF ALGAL PHYSIOLOGY

A dynamic mechanistic mathematical model (FeLANIM) is described, capable of simulating the major documented interactions between iron, light, ammonium, and nitrate in phytoplankton. There are various points in the model where species- or group-specific (e.g. eukaryote vs. prokaryote) details may be altered. Cellular Fe-containing processes accounted for in the model are photosynthesis, oxidative phosphorylation, and nitrate assimilation; synthesis of photosystems and nitrate and nitrite reductase (NNiR) are functions of the surplus Fe quota (i.e. total Fe quota minus Fe in functional components). Model simulations were run using a range of different physiological parameters for Fe-dependent processes and contrasting ammonium–nitrate interaction scenarios. Model output indicated the following interactions. Because the proportion of Fe in photosystems varies with irradiance, it is not possible to have a single Fe cost for nitrate assimilation. Fe stress can affect the relationship between ammonium and nitrate assimilations (the f-ratio), with a more rapid depression of nitrate use at lower concentrations of ammonium. However, depending on the degree of the repression of nitrate assimilation at high N:C, such a result may not be universal, and further experimental studies are required to clarify this issue. Fe refeeding results in a rapid recovery of growth rate accompanied by a proportionately greater increase in the amount of chl a within 24– 48 h. Estimates of enhanced production in iron fertilization experiments based only on increases in pigment may thus be exaggerated. Stimulation of NNiR activity on Fe refeeding appears indirect, through enhancement of photosynthesis rather than relief of nitrate stress. During Fe refeeding at high light, N nutrient transport increases proportionately more in nitrate-fed cells than in ammonium-fed cells. Changing the N source supplied to simulated Fe-stressed cells from nitrate to ammonium results in a rapid increase in growth rate and iron use efficiency with an increase in pigment content as NNiR content declines. When nitrate replaces ammonium, acclimation is slower because of the redirection of Fe formerly associated in photosystems to NNiR. Manipulation of the model may prove useful as an indicator for new research, by revealing elements of physiology that may be most significantin determining competitive advantage between species.     

Calcification in the Green Alga Halimeda: III. THE SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS AND CALCIFICATION AND A MODEL OF THE MECHANISM OF CALCIFICATION

Calcification and photosynthetic rates in Halimeda tuna weremeasured by the ¹⁴C method under conditions of differing pHand total inorganic carbon (CO₂) concentrations. The effectsof pH and CO₂ on photosynthesis and respiration were also monitoredwith a polarographic O₂ electrode. The results obtained indicatethat the intercellular pH and CO₂ differ from those of the externalmedium. Experiments carried out over a range of pH values show thatHalimeda can use for photosynthesis. Photosynthesis appears to stimulate calcification by removing CO₂ from theintercellular spaces. As these spaces are isolated from theexternal sea water by the layer of cell wall of the adpressedperipheral utricles, the removal of CO₂ results in a rise in[] and a rise in pH. This results in an increased rate of CaCO₃ precipitation. Respiratory CO₂ evolution has aninhibitory effect on calcification by decreasing the pH and[]. A model for calcification in Halimeda is proposed based on theresults of this and previous papers. Calcification in Halimedais seen to be a result of the anatomy of the thallus in whichthe sites of calcification are within a semi-isolated chamberwhere removal or addition of CO₂ due to photosynthesis or respirationcan effectively change [CO] thereby resulting in precipitation of CaCO₃. In the Appendix to this paper theoreticalcalculations illustrate the effects of CO₂, , and removal or addition in a closed system on the relative concentrations of the other inorganic carbonspecies.     

Studies in Plant Growth Hormones: IV. CHROMATOGRAPHY OF HORMONES AND HORMONE PRECURSORS IN CABBAGE

1. Acid and neutral ethereal fractions and the non-ether-solubleaqueous fraction of an extract of cabbage leaves were chromatographed,and the chromatc-grams assayed using oat coleoptile sections. 2. In the aqueous fraction an acidic precursor of 3-indolylacetonitrile(IAN) was found. When chromatographed in isopropanol/ammonia,the precursor travelled at the position of 3-indolylacetic acid(IAA), but in n-butanol/ammonia it was much closer to the starting-line.IAN is liberated from the precursor under conditions of alkalinehydrolysis including ammoniacal chromatography, and is alsoliberated by heat. Precursor and IAN zones promoted coleoptilegrowth, but the former when sprayed with ferric chloride/perchloricacid or nitrous/nitric acid gave a yellow colour showing thatthere was no free IAN on this part of the chromatogram. Hypothesesto account for this activity are discussed. 3. A neutral inhibitor was present in the aqueous fraction.It is volatile, ether-soluble, and is thought to have been liberatedfrom a water-soluble substance. 4. The neutral fraction, chromatographed in isopropanol/ammonia,contained IAN and a growth promoter at Rf o–o·1:thelatter stimulates cress root growth above that in water. Thispromoter could be formed from a precursor in the aqueous fractionby heat treatment followed by shaking with sodium bicarbonatesolution. It is suggested that this neutral hormone is the accelerator-of Bennet-Clark and Kefford. The data of these workers are analysedto show that this interpretation is consistent with their results. 5. The acid fraction contained IAN but no IAA. The former isthought not to have been liberated from the precursor in thisfraction but to have entered into it from the neutral fractionduring separation with sodium bicarbonate solution. AlthoughIAA may have been absent from the plant material, it is possiblethat any present was destroyed during the process of extraction. 6. Evidence is presented that there are other growth promoterspresent at low concentration in the extract in addition to thosealready mentioned.