Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

(14) C]-Assimilate translocation in the light and dark in celery (Apium graveokns) leaves of different ages

The 2 major photosynthetic products and translocated carbohydrates in celery ( Apium graveolens L.) are sucrose and the sugar alcohol, mannitol. Sucrose is produced and utilized in leaves of all ages. Mannitol, however, is synthesized primarily in mature leaves, utilized in young leaves and stored in all leaves. Here we show that mannitol export was lower from young, expanding leaves than from older leaves. After a 10 min pulse of ¹⁴CO₂ and a 2 h chase in the light or dark there was more radioactivity in sucrose than in mannitol in petiole tissues from leaves of all ages. However, after a chase of 15 h in the dark or 6 h in the light followed by 9 h in the dark, mannitol was the predominant [¹⁴C]-labeled carbohydrate remaining in all leaf and petiole tissues. Thus, newly synthesized sucrose was apparently exported at a faster rate than mannitol and more mannitol was partitioned into vacuolar storage pools than was sucrose. It also appears that in the light both sucrose and mannitol were exported, but in the dark, once sucrose pools were depleted, mannitol remained as the predominant substance translocated. Both mannitol and sucrose were unloaded into petiole storage parenchyma tissue, but sucrose was hydrolyzed prior to storage.     

Effect of auxins on spermidine uptake into carrot protoplasts

The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot ( Daucus carota L. cv. Ingrid) was studied. In the presence of 1 m M Ca²⁺, IAA (10⁻⁷ to 10⁻⁴ M ) enchances [¹⁴C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca²⁺. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La³⁺, known not to penetrate the plasmalemma, exerts the same effect as Ca²⁺, but gives lower uptake values than Ca²⁺. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca²⁺ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca²⁺ -induced increase of spermidine uptake.     

Distribution and metabolism of root-applied cytokinins in Pisum sativum

When N ⁶ [8–¹⁴C] furfuryladenine was applied to the intact root system of Pisum sativum L. cv. Meteor seedlings it was almost completely metabolised to other compounds within 24 h. Of the total activity recovered from the plants 94.5% was retained in the root system itself. ¹⁴C was recovered in a number of ethanol-soluble compounds and in ribonucleic acid, deoxyribonucleic acid and protein fractions of roots, stems, leaves and axillary buds. In rapidly growing axillary buds released from apical dominance by removal of the shoot apex the combined nucleic acid fractions accounted for 63.3% of the total ¹⁴C recovered from these organs. Xylem exudate collected from decapitated plants 0 to 12 h after supplying N ⁵[8–¹⁴C]furfuryladenine to the roots consistently contained a single major ¹⁴C-labelled compound which, in three different solvent systems, had the same Rf values as a major endogenous cytokinin isolated from the xylem of unlabelled plants. The content of N ⁶ [8–¹⁴C] furfuryladenine itself in the xylem exudate was always low and in some experiments it could not be detected.
It is suggested that part of the label from N ⁶ [8- ¹⁴CJfurfuryladenine taken up by the intact root system may have become incorporated in an endogenous cylokinin before export to the shoot.     

Transport and accumulation of indole-3-acetic acid in pea cuttings under two levels of irradiance

The transport and accumulation of 2-[¹⁴C]-IAA applied to the apex of cuttings of Pisum sativum L. cv. Alaska was greater in cuttings from stock plants grown under 38 W m⁻² than 16 W m⁻². Accumulation of ^14C in the base of the cuttings from the highest level of irradiance was correspondingly more significant. The level of irradiance to the stock plants greatly affected the rate of accumulation, while the light conditions during IAA transport had a minor effect. The amount of IAA reaching the base of the cuttings increased with increasing concentration of IAA in the treatment solution, but the percentage of applied IAA reaching the base decreased.
The relative chromatographic partition of ethanol-extractable ¹⁴C showed that, after 12 h of IAA-transport, the amount of 2-[¹⁴C]-IAA was higher in the base of cuttings from 38 W m⁻² than in those from 16 W m⁻². After a further 12 h of transport the relative amounts of 2-[¹⁴C]-IAA in the two types of cuttings were reduced to the same lower level.
A possible role of an irradiance-mediated difference in the topographic distribution of IAA in the base of pea cuttings on the subsequent adventitious root formation is discussed.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Localisation of translocated 14C in roots and root exudates of field-grown maize

The roots of a mature, field-grown maize plant are dimorphic: the primary root and those from the oldest nodes are bare with a heavily lignified cortex arid sloughed epidermis; those from younger nodes, except for a bare elongation zone, have an intact epidermis surrounded by a persistent soil sheath. Sheathed roots consistently have more layers of cortical cells, but the ratio of volumes of cortex to stele (ca 4) and the cross-sectional area of phloem (ca³× 10⁻² mm²) are similar in each type. Assimilated carbon (from ¹⁴ C applied to a small area of one leaf) was translocated to all roots and actively metabolized in cortex and stele of both types. After 1 to 2 days the proportion of ¹⁴C exuded from a given length of mature root into its soil sheath, or into the adjacent unattached soil in the case of bare roots, was the same (5%) in both root types when compared with the ethanol-soluble ¹⁴C in the tissues of this length. Up to 75% of the ethanol-soluble label in the roots was in a cationic fraction (amino acids and unidentified compounds), ca 1% was in an anionic fraction and the remainder was in a neutral fraction (sugars). Approximately equal amounts of soluble ¹⁴C were found in the stele, cortex and laterals.     

Ca2+ UPTAKE BY SYNAPTOSOMES AND ITS EFFECT ON THE INHIBITION OF ACETYLCHOLINE RELEASE BY BOTULINUM TOXIN

Abstract— ⁴⁵Ca²⁺ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K⁺ at all times investigated. The accumulated ⁴⁵Ca²⁺ was bound within the synaptosome. ⁴⁵Ca²⁺-ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal ⁴⁵Ca²⁺ then returned to control values. A23187 also increased intra-synaptosomal Na⁺ and Cl⁻ contents. Botulinum toxin inhibits the K.⁺-stimulated release of [¹⁴C]ACh from synaptosomes but the ionophore released [¹⁴C]ACh from both normal and botulinum-treated preparations in a Ca²⁺-dependent manner. However, it also elicited Ca²⁺-dependent release of [choline. Increased extracellular Ca²⁺ (10 mM and 20 mM) released [¹⁴C]ACh (but not [¹⁴C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca²⁺ essential for the mechanism of ACh release.     

Distribution of 14C assimilates in the storage root of sugar beet plants after import from leaves of different age

In the sugar beet plant ( Beta vulgaris L. ssp. altissima ) the vascular bundles of old leaves lead to the center and those of young leaves to the periphery of the storage root. Whether the flux of assimilates follows these anatomical routes was tested by applying ¹⁴CO₂ for 4 h to either an old (10th) or a young (20th) leaf in intact sugar beet plants. Four-month-old plants, which had about 30 leaves, were used in the experiment. The ¹⁴C distribution in the storage root was measured by autoradiography and counting in about 20 cross and longitudinal sections per root.
About 37% of assimilated ¹⁴C from an old leaf and 23% from a young leaf were exported within 24 h. Although some ¹⁴C moved into younger leaves, most was exported into the storage root. During its rapid movement towards the root tip, which took place perferentially in the orthostichon belonging to the [¹⁴C]-treated leaf, the label spread laterally.
The autoradiograms indicate that the distribution of assimilates within the storage root is roughly determined by the course of the vascular bundles extending from the source leaf. The fine distribution, however, seems to be controlled by sucrose gradients between storage cells.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Nitrogenase activity decay and energy supply in Frankia after addition of ammonium to the host plant Alnus incana

A clone of Alnus incana (L.) Moench was grown in symbiosis with a local source of Frankia or with Frankia Ar14. Seven to 9-week-old plants were given 20 m M NH₄Cl (20 m M KCl = control) for 3 days. Nitrogenase activity of intact plants decreased gradually within the 3 days of treatment to about 10% of the initial rates. Hydrogen evolution in air and total nitrogenase activity responded similarly to the treatment. Relative efficiency of nitrogenase thus remained the same throughout the study period. Control plants were not affected. Measurements of nitrogenase activity in root nodule homogenates (in vitro measurements) indicated loss of active nitrogenase rather than shortage of energy for nitrogenase activity in Frankia from ammonium-treated plants. Shoots were exposed to ¹⁴CO₂ and translocation of ¹⁴C to Frankia vesicle clusters prepared from root nodules was studied. Frankia vesicle clusters from ammonium-treated plants contained about half as much ¹⁴C as those of control plants during all 3 days studied. One explanation for the observed effects is that a reduced supply of carbon to Frankia vesicles in the root nodules caused a reduced metabolic rate, including reduced protein synthesis and synthesis of nitrogenase.     

The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase

Abstract: [¹⁴C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-¹⁴C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited ≥79% by α-ketoglutaric acid (10 m m ), Cu²⁺ (1 m m )and Zn²⁺(1 m m ). [¹⁴C]Oxaloacetate, [¹⁴C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity.     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

Compound 48/80, a calmodulin antagonist, inhibits ion-porter function in plant roots

A rather specific inhibitor of calmodulin-regulated functions, compound 48/80. inhibited ³⁶Cl⁻, ⁸⁶Rb⁺ and ²²Na⁺ absorption by Hordeum vulgare, Phaseolus mungo and Zea mays roots. The compound did not inhibit plasmalemma or tonoplast Mg:²-ATPase activity; neither did it inhibit or uncouple respiration, or affect ATP content and seemed also not to cause depolarization of the membrane potential of roots cells. Compound 48/80 inhibited the ΔpH-mediated Cl⁻-H⁺ symport in ATP-depleted roots.     

Effects of gibberellic acid on patterns of carbohydrate distribution and acid invertase activity in Phaseolus vulgaris

The application of gibberellic acid (GA₃,10 μ M ) as a root drench to 16-day-old plants of Phaseolus vulgaris L. cv. Masterpiece stimulated growth of the stem internodes and reduced root growth. GA₃ treatment did not affect the export of ¹⁴C from a primary leaf to which [¹⁴C]-sucrose was applied, but greatly increased upward translocation to the elongation region of the stem at the expense of transport to the hypocotyl and root system. The observed changes in the patterns of growth and [¹⁴C]-labelled assimilate distribution were correlated with an increase in the specific activity of acid invertase in the elongating stem internodes and a decrease in invertase activity in the hypocotyl and root. Sucrose concentration in the elongating internodes fell substantially after treatment with GA₃ while the concentration of hexose sugars increased. We suggest that by stimulating acid invertase synthesis in the elongating internodes, GA₃ acts to establish a more favourable sucrose gradient between these sinks and source leaves. Under source-limiting conditions this, in turn, will lead to a reduced rate of assimilate translocation to competing sinks in the root system.     

Uptake kinetics of iron-phytosiderophores in two maize genotypes differing in iron efficiency

Iron inefficiency in the maize ( Zea mays L.) mutant ysl is caused by a defect in the uptake system for Fe-phytosiderophores. To characterize this defect further, the uptake kinetics of Fe-phytosiderophores in ysl was compared to the Fe-efficient maize cultivar Alice. Short-term uptake of ⁵⁹Fe-labeled Fe-deoxymugineic acid (Fe-DMA) was measured over a concentration range of 0.03 to 300 μM. Iron uptake in Fe-deficient plants followed Michaelis-Menten kinetics up to about 30 μM and was linear at higher concentrations, indicating two kinetically distinct components in the uptake of Fe-phytosiderophores. The saturable component had similar K_m (∼ 10 μM) in both genotypes. In contrast. V_max was 5.5 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in Alice, but only 0.6 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in _ysl. Uptake experiments with double-labeled ⁵⁹Fe-[¹⁴C]DMA suggest that in both cultivars Fe-DMA was taken up by the roots as the intact chelate. The results indicate the existence of a high-affinity and a low-affinity uptake system mediating Fe-phytosiderophore transport across the root plasma membrane in maize. Apparently, the mutation responsible for Fe inefficiency in ysl affected high-affected uptake and led to a decrease in activity and/or number of Fe-phytosiderophore transporters.     

Effects of ABA on 86Rb+ fluxes at plasmalemma and tonoplast of stomatal guard cells

Abscisic acid (ABA) induces a transient stimulation of ⁸⁶Rb⁺ from isolated guard cells of Commelina communis L. When ABA is added after 30–50 min of wash-out in the absence of ABA, when tracer is almost entirely vacuolar, its effects on vacuolar release are measured. When ABA is added early in the wash-out (at 2–4 min), when both cytoplasm and vacuole are labelled, the resulting efflux includes both vacuolar and cytoplasmic contributions. Detailed comparison of rates of efflux in the absence of ABA, and in the presence of ABA added early and late in the wash-out, allows the effects of ABA on plasmalemma and tonoplast fluxes to be assessed. Three effects of ABA can be distinguished: these are stimulation of the ⁸⁶Rb⁺ flux from vacuole to cytoplasm (by twofold to 6.7-fold); stimulation of the plasmalemma efflux, by up to twofold, a smaller factor than that of the tonoplast effect and variable between experiments; and a doubling of the half-time for cytoplasmic exchange in ABA, taken to reflect an increase in cytoplasmic ion content as ions flood out of the vacuole. Concentrations of ABA of 0.1–0.2 µM and 1–10 µM are equally effective in the stimulation of plasmalemma efflux, but the effects on tonoplast fluxes are both delayed and reduced at low external concentrations of ABA. It is argued that the delay reflects the need for a threshold internal ABA to be reached before the initiation of vacuolar release, and the reduction reflects the sensitivity of the extent of activation of tonoplast ion channels to concentration of internal ABA. It is likely that the plasmalemma change is mediated by external ABA, and could be the result of the modulation of the stretch-activated channel suggested previously.     

Effect of Chronic Lead Ingestion by Rats on Glucose Metabolism and Acetylcholine Synthesis in Cerebral Cortex Slices

Abstract: The effect of chronic low-level lead (Pb²⁺) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb²⁺-exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs-Ringer bicarbonate buffer with 10 m M glucose and tracer amounts of [6-³H]glucose and either [6-¹⁴C]glucose or [3-¹⁴C] β -hydroxybutyrate. Altering the concentration of Pb²⁺ in the drinking water produced a dose-related increase in blood and brain lead levels. When tissue from Pb²⁺-exposed rats was incubated with mixed-labeled glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the ³H/¹⁴C ratios. Similar effects of Pb²⁺ were found when ¹⁴C-labeled β -hydroxy-butyrate was substituted for the [¹⁴C]glucose. It appears from these data that Pb²⁺ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb²⁺-induced changes observed in cholinergic function.