Fixation and distribution of 14C in Populus deltoides during dormancy induction

Photosynthetically fixed ¹⁴C was analyzed in various chemical fractions from leaves and stems of cottonwood (Populus deltoides Bartr. ex. Marsh.) during dormancy induction. Dormancy was induced by 8-h photoperiods and 20/14°C temperature regimes. Within 4 weeks under short days, terminal buds were set and leaf expansion and stem elongation had stopped. ¹⁴C₂ was fed to a leaf at Leaf Plastochron Index 7 for 30 min. Either after this 30 min feeding period or after a 48-h translocation period the plants were sampled, freeze-dried, extracted and analyzed for¹⁴C. ¹⁴C-fixation decreased during dormancy induction from 60% to 17% of the 3.7 MBq ¹⁴C applied at 0 week and 8 weeks, respectively. Percentage distribution of ¹⁴C in chemical fractions of source leaves reflected leaf age and translocation inhibition. In rapidly growing plants, considerable ¹⁴C was incorporated into leaf protein while most of the soluble¹⁴C-sugars were either metabolized or translocated out of the leaf. After terminal bud set, the percentage of ¹⁴C in the protein and residue fractions decreased rapidly and that in the sugar fraction increased. Percent distribution in stems closely reflected changing metabolic pathways of carbon flow as influenced by dormancy induction. For example, the ¹⁴C in structural carbohydrates decreased in 5 weeks under short days from 65 to less than 10% of the ¹⁴C recovered in the chemical fractions, thus indicating cambium inhibition. At the same time the percentage of ¹⁴C in starch and sugar increased indicating storage. Short term (after 30 min) incorporation of ¹⁴C into the protein and starch fractions of leaves changed relatively little throughout the 8-week induction period. In contrast the turnover rates of these fractions (¹⁴C present after 48 h) increased considerably after active growth of the whole plant stopped.     

Metabolism of [14C]trans-zeatin and [14C]benzyladenine by letached yellow, green and variegated leaves of Schefflera

[8-¹⁴C]Benzyladenine (BA) and [8-¹⁴C] trans-zeatin (tZ) were fed through the petiole to mature, detached green, yellow and variegated leaves of Schefflera arboricola. Recovery of radioactivity from the plant material ranged between 4.2 and 22.1%. More radioactivity was recovered when tZ was applied compared to BA. Green leaves or the green parts of variegated leaves yielded more radioactivity than the yellow leaf material. BA was metabolized much faster than the endogenous cytokinin tZ. It would appear that while lower amounts of radioactivity were present in yellow leaves, as well as in yellow parts of variegated leaves, the rate of cytokinin metabolism was nevertheless faster. Metabolites that were formed to a greater extent in these yellow parts were the nucleotides of both cytokinins. Currently it is not known whether or not cytokinins influence chlorophyll and other pigment development in chimeric variegated leaves.     

Translocation of 14C-assimilates in roses

The upper shoot on decapitated rose branches ( Rosa hybrids cv. Marimba) grows faster than lower shoots on the same branch. Transport of radioactive assimilates to the upper shoot is higher than to the lower ones. Darkening of the uppermost shoot resulted in the reduction of growth and ^I4C-assimilate accumulation in the darkened shoot as well as the promotion of growth and ¹⁴C transport to the lower 2 shoots, thereby rendering dominance to the second shoot. Benzyladenine treatment to the uppermost shoot reversed the effect of darkening and restored the apical control of this shoot.     

Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain

Abstract: Production of [¹⁴C]acetylcholine and ¹⁴CO₂ was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [¹⁴C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 m m - and 31 m m -K⁺, contrasting with the lack of significant change in ¹⁴CO₂ production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13-month and senescent animals. Measurements of the release of [¹⁴C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.     

Testing the branch autonomy theory: a 13C/14C double-labelling experiment on differentially shaded branches

The impact of a heterogeneous within‐crown light environment on carbon allocation was investigated on young walnut trees trained on two branches: one left in full sunlight, the other shaded until leaf fall resulting in 67% reduction in photosynthetically active radiation. In September, the two branches were separately labelled with ¹⁴CO₂ and ¹³CO₂, respectively, so that the photosynthates from each branch could be traced independently at the same time. Although some carbon movements could be detected within 5 d in both directions (including from the shaded branch to the sun branch), between‐branch carbon movements were very limited: approximately 1% of the diurnal net assimilation of a branch. At this time of the year branch autonomy was nearly total, leading to increased relative respiratory losses and a moderate growth deficit in the shaded branch. The ratio of growth to reserve storage rate was only slightly affected, indicating that reserves acted not as a mere buffer for excess C but as an active sink for assimilates. In winter, branch autonomy was more questionable, as significant amounts of carbon were imported into both branches, possibly representing up to 10% of total branch reserves. Further within‐plant carbon transfers occurred in spring, which totally abolished plant autonomy, as new shoots sprouted on each branch received significantly more C mobilized from tree‐wide reserves than from local, mother‐branch located reserves. This allowed great flexibility of tree response to environment changes at the yearly time scale. As phloem is considered not functional in winter, it is suggested that xylem is involved as the pathway for carbohydrate movements at this time of the year. This is in agreement with other results regarding sugar exchanges between the xylem vessels and the neighbouring reserve parenchyma tissues.     

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.     

Metabolic Fluxes Between [14C]2-Deoxy-D-Glucose and [14C]2-Deoxy-D-Glucose-6-Phosphate in Brain In Vivo

The rates of the phosphorylation and dephosphorylation of 2-deoxyglucose were measured in rat brain in vivo using tracer kinetic techniques. The rate constant for each reaction was estimated from two separate experiments with different protocols for tracer administration. Tracer amounts of [1-14C]2-deoxyglucose (1 microCi) were injected through the internal carotid artery (intraarterial experiment), or through the atrium (intravenous experiment). Brains were sampled by freeze-blowing at various times after the injection. In the intraarterial experiment, the rate constant for the forward reaction from 2-deoxyglucose to 2-deoxyglucose phosphate was calculated by dividing the initial rate of 2-deoxyglucose phosphate production by the 2-deoxyglucose content in brain. The rate constant for the reverse reaction from 2-deoxyglucose phosphate to 2-deoxyglucose was calculated from the decay constant of 2-deoxyglucose phosphate. The rate constants estimated were 10.1 +/- 1.4%/min (SD) and 3.00 +/- 0.01%/min (SD), respectively, for the forward and reverse reactions. In the intravenous experiment, rate constants for both reactions were estimated by compartmental analysis. By fitting data to program SAAM-27, the rate constants for the forward and reverse reactions were estimated as 11.4 +/- 0.4%/min (SD) and 5.1 +/- 0.4%/min (SD), respectively. The rate constants determined were compared to those for the reactions between glucose and glucose-6-phosphate, estimated previously from labeled glucoses. It is concluded that the rate of glucose utilization measured by the 2-deoxyglucose method reflects the rate of the hexokinase reaction and not the rate of glucose utilization or brain energy utilization.     

The fate of [14C]glutamate and [14C]malate in birch roots is strongly modified under inoculation with Paxillus involutus

The impact of inoculation with Paxillus involutus on the utilization of organic carbon compounds by birch roots was studied by feeding [¹⁴C]Glu or [¹⁴C]malate to the partners of the symbiosis, separately or in association, and by monitoring the subsequent distribution of ¹⁴C. Inoculation increased [¹⁴C]Glu and [¹⁴C]malate absorption capacities by up to eight and 17 times, respectively. Six- and 15-d-old mycorrhizal roots showed about four-fold higher [¹⁴C]Glu and [¹⁴C]malate absorption capacities compared with 60-d-old mycorrhizal roots, suggesting that the early stages of mycorrhiza formation induced higher requirements for C skeletons. Moreover, the results demonstrated that inoculation strongly modified the fate of [¹⁴C]Glu and [¹⁴C]malate. It was demonstrated that exogenously supplied Glu and malate might serve as C skeletons for amino acid synthesis in mycorrhizal birch roots and in the free-living fungus. Gln was the major ¹⁴C-sink in mycorrhizal roots and in the free-living P. involutus. In contrast, citrulline and insoluble compounds were the major ¹⁴C sinks in non-mycorrhizal roots, whatever the ¹⁴C source. It was concluded that mycorrhiza formation leads to a profound alteration of the metabolic fate of exogenously supplied C compounds. The ecological significance of amino acid and organic acid utilization by mycorrhizal plants is further discussed.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants

We assessed the effects of doubling atmospheric CO₂ concentration, [CO₂], on C and N allocation within pedunculate oak plants (Quercus robur L.) grown in containers under optimal water supply. A short-term dual ¹³CO₂ and ¹⁵NO₃^? labelling experiment was carried out when the plants had formed their third growing flush. The 22-week exposure to 700 μl l^?1 [CO₂] stimulated plant growth and biomass accumulation (+53% as compared with the 350 μl l^?1 [CO₂] treatment) but decreased the root/shoot biomass ratio (-23%) and specific leaf area (-18%). Moreover, there was an increase in net CO₂ assimilation rate (+37% on a leaf dry weight basis; +71% on a leaf area basis), and a decrease in both above- and below-ground CO₂ respiration rates (-32 and -26%, respectively, on a dry mass basis) under elevated [CO₂]. ¹³C acquisition, expressed on a plant mass basis or on a plant leaf area basis, was also markedly stimulated under elevated [CO₂] both after the 12-h ¹³CO₂ pulse phase and after the 60-h chase phase. Plant N content was increased under elevated CO₂ (+36%), but not enough to compensate for the increase in plant C content (+53%). Thus, the plant C/N ratio was increased (+13%) and plant N concentration was decreased (-11%). There was no effect of elevated [CO₂] on fine root-specific ¹⁵N uptake (amount of recently assimilated ¹⁵N per unit fine root dry mass), suggesting that modifications of plant N pools were merely linked to root size and not to root function. N concentration was decreased in the leaves of the first and second growing flushes and in the coarse roots, whereas it was unaffected by [CO₂] in the stem and in the actively growing organs (fine roots and leaves of the third growth flush). Furthermore, leaf N content per unit area was unaffected by [CO₂]. These results are consistent with the short-term optimization of N distribution within the plants with respect to growth and photosynthesis. Such an optimization might be achieved at the expense of the N pools in storage compartments (coarse roots, leaves of the first and second growth flushes). After the 60-h ¹³C chase phase, leaves of the first and second growth flushes were almost completely depleted in recent ¹³C under ambient [CO₂], whereas these leaves retained important amounts of recently assimilated ¹³C (carbohydrate reserves?) under elevated [CO₂].     

The fate of carbon during the assimilation of carbamoyl phosphate in white spruce seedlings as revealed by [14C]-carbamoyl phosphate, [14C]-cyanate, and [14C] -bicarbonate labeling patterns

The fate of carbamoyl phosphate in white spruce seedlings revolves around the production of spontaneous degradation products, cyanate, bicarbonate, and carba-mate. When [¹⁴C]-carbamoyl phosphate and [¹⁴C]-cyanate are assimilated, urea is a common early metabolic intermediate that appears in the alcohol soluble N. By contrast, urea is not detected among the products of [¹⁴C]-bicarbonate. Carbamoyl phosphate and glutamic acid are implicated as having pivotal roles in the production of amides, arginine, and biotin. Within 2-h exposures to radioactive substrates considerably more carbon from bicarbonate was diverted into amino acids Incorporated into proteins than with carbon-nitrogen substrates. Specific activities of bound amino acid residues support the view that proteins formed from these [¹⁴C]-substrates have different rates of metabolic turnover.     

Cerebral Glucose Utilization: Comparison of [14C]Deoxyglucose and [6-14C]Glucose Quantitative Autoradiography

The [14C]deoxyglucose [Sokoloff et al., J. Neurochem. 28, 897-916 (1977)] and [6-14C]glucose [Hawkins et al., Am. J. Physiol. 248, C170-C176 (1985)] quantitative autoradiographic methods were used to measure regional brain glucose utilization in awake rats. The spatial resolution and qualitative appearance of the autoradiograms were similar. In resting animals, there was no significant difference between the two methods among 18 gray and three white matter structures over a fourfold range in glucose utilization rates (coefficient of correlation = 0.97). In rats given increasing frequencies of photoflash visual stimulation, the two methods gave different results for glucose utilization within visual pathways. The linearity of the metabolic response was studied in the superior colliculus using an on-off checkerboard stimulus between 0 and 33 Hz. The greatest increment in activity occurred between 0 and 4 Hz stimulation with both methods, probably representing recruitment of neuronal elements into activity. Above 4 Hz, there was a progressive increase in labeling with [14C]deoxyglucose up to 1.7 times control at 33 Hz. With [6-14C]-glucose, there was no further increment in change above a 30% increase seen at 4 Hz. Measurement of tissue glucose revealed no drop in the visually stimulated structures compared to control. We interpret these results to indicate that, with increasing rates of physiological activity, the products of deoxyglucose metabolism accumulate progressively, but the products of glucose metabolism are removed from brain in 10 min.     

Alterations in the Production of 14CO2 and [14C]Acetylcholine from [U-14C]Glucose in Brain Subregions Following Transient Forebrain Ischemia in the Rat

Abstract: The production of ¹⁴CO₂ and [¹⁴C]acetylcholine from [U-¹⁴C]glucose was determined in vitro using tissue prisms prepared from the dorsolateral striatum (a region developing extensive neuronal loss following ischemia) and the paramedian neocortex (an ischemia-resistant region) following 30 min of forebrain ischemia and recirculation up to 24 h. Measurements were determined under basal conditions (5 mMK⁺) and following K⁺ depolarization (31 mM K⁺). The production of ¹⁴CO₂ by the dorsolateral striatum was significantly reduced following 30 min of ischemia for measurements in either 5 or 31 mM K⁺ but recovered toward preischemic control values during the first hour of recirculation. Further recirculation resulted in ¹⁴CO₂ production again being reduced relative to control values but with larger differences (20–27% reductions) detectable under depolarized conditions at recirculation times up to 6 h. Samples from the paramedian neocortex showed no significant changes from control values at all time points examined. [¹⁴C]Acetylcholine synthesis, a marker of cholinergic terminals that is sensitive to changes in glucose metabolism in these structures, was again significantly reduced only in the dorsolateral striatum. However, even in this tissue, only small (nonstatistically significant) differences were seen during the first 6 h of recirculation, a finding suggesting that changes in glucose oxidation during this period were not uniform within all tissue components. The results of this study provide evidence that in a region susceptible to ischemic damage there were specific changes during early recirculation in the metabolic response to depolarization. This apparent inability to respond appropriately to an increased need for energy production could contribute to the further deterioration of cell function in vivo and ultimately to the death of some cells.     

Translocation of 14C, carbohydrate content and activity of the enzymes of sucrose metabolism in rose petals temperatures

Both export of ¹⁴C from the source leaves of roses (Rosa × hybrida cv. Golden Times) and import of ¹⁴C to the petals were reduced by plant exposure to low night temperature. However, the import was affected to a greater extent than the export. During all stages of flower bud development the concentration of reducing sugars in petals of roses grown at reduced night temperature was lower than in petals of plants grown at higher night temperature. There was no significant difference in starch content in response to the night temperature, and the content of starch decreased toward complete flower bud opening. The concentration of sucrose in flowers at the low night temperature remained low during all stages of flower bud development, while at the high night temperature the concentration of sucrose increased during flower bud development, reaching a peak at the stage when petals start to unfold. At both temperatures the concentration of sucrose declined at complete flower opening. The activity of sucrose synthase (EC 2.4.1.14) was inhibited by low temperature in young rose shoots more than in the petals, while the activity of acid invertase (EC 3.2.1.26) was affected similarly in both tissues by the temperature treatments.     

Biochemical and Autoradiographic Study of Cerebral Protein Synthesis with [18F]- and [14C]Fluorophenylalanine

Fluorine-18-labeled ortho or para isomers of L-fluorophenylalanine were used in double-label experiments together with L-[3H]phenylalanine for amino acid incorporation into cerebral proteins of Mongolian gerbil brain. It was demonstrated by qualitative regional comparison of the 18F and 3H autoradiographic images that L-p-[18F]fluorophenylalanine is incorporated into proteins and exhibits a regional cerebral protein synthesis pattern. To a minor extent, L-p-fluorophenyl[3-14C]alanine and L-o-[18F]fluorophenylalanine are hydroxylated in vivo to form labeled tyrosine or tyrosine analogues that are incorporated into cerebral proteins as well. The advantage and validity of the application of L-p-[18F]fluorophenylalanine with positron emission tomography for noninvasive studies of cerebral protein synthesis in humans are evaluated on the basis of an experimental animal approach.     

Interaction between K-Deficiency and Light in 14C-Sucrose Translocation in Bean Plants

The effect of K deficiency on sucrose transport was studied in bean plants from which the cotyledons had been removed soon after germination and which were subsequently grown in culture solution. Labelled sucrose wassupplied to the primary leaf. Mild Kdeficiency depressed translocation. A statistically significant interaction between the presence of K and light was observed. K deficiency had no effect on translocation if the plants had been placed in the dark before the experiment and remained in the dark during the translocationperiod. Maximum correction for differences in the specific activity of the sugar being translocated, as between control and K-deficient plants (based on sugar analyses in various plant parts), failed to abolish the K effect when the amount of sugar applied was relatively low (high specific activity). On the other hand no effect of K-deficiency was observed on translocation when the amount of applied sugar was high (low specific activity). In this case ¹⁴CO₂ fixation was greatly reduced in the controls, but not in the K-deficient plants. Significant interaction existed between added sugar and K-deficiency on ¹⁴CO₂ fixation. All results reported here can be explained on the basis of a scheme whereby K plays a rôle in reversible intermediate steps between sugar formation in the chloroplast and the entry of this photosynthesised sugar into the “translocation pool” where it mixes with exogenous sugar.     

The trans-tissue pathway and chemical fate of 14C photoassimilate in carrot taproot

Axial and radial transport and the accumulation of photoassimilates in carrot taproot were studied using ¹⁴C labelling and autoradiography. Axial transport of the ¹⁴C labelled assimilates inside the taproot was rapid and occurred mainly in the young phloem found in rows radiating from the cambium. The radial transport of the assimilate inward (to cambium, xylem zone and pith) and outward (to phloem zone and periderm) from the conducting phloem was an order of magnitude slower than the longitudinal transport and was probably mainly diffusive. The cambial zone of the taproot presented a partial barrier in the inward path of the assimilate to the xylem zone. We suggest that this is due to the cambium comprising a strong sink for the assimilate on the basis that our previous work has shown that it contains very low concentrations of free sucrose. By contrast, a high accumulation of nonsoluble ¹⁴C was found in the cambium region in good agreement with the active growth of this zone. Autoradiography following the feeding of ¹⁴C labelled sugars to excised sections of taproot indicated that only a ring of cells at and/or just within the cambium take up sugars from the apoplast. This indicates that radial movement in the phloem and pith must be symplastic. An apoplastic step between phloem and xylem is possible. The rapid uptake of sugars from the apoplast at this point might represent a mechanism for keeping photoassimilates away from the transpiration stream and re-location back to the leaves.     

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.     

The translocation of 14C photosynthate from leaves and pods in Phaseolus vulgaris

Field experiments were undertaken to study the pattern of distribution of photosynthate produced by the leaves and the pods of Phaseolus vulgaris (cv. Purley King) by means of the ¹⁴C technique. It was found that the ^UC photosynthate produced by a trifoliate leaf (38 days after anthesis) was shared almost equally between the leaf and the pod at its axil with 33–50% of the fixed ¹⁴C finding its way to the seeds in that pod. However, during the early stages of pod development (10 days after anthesis) some 13–14% of the fixed ¹⁴C was detected in the stem, indicating the inadequacy of the pod as a sink at that stage. When the pod was treated, virtually no ¹⁴C was detected in other parts of the plant. Of the ¹⁴C fixed by pod photosynthesis in the later stages (38 days after anthesis), 55–60% was translocated to the seeds within the same pod. These results indicate the importance of current photosynthesis during the pod fill stage in P. vulgaris as has been suggested in other grain legume crops.