Dual function of pyrimidine metabolism in potato (Solanum tuberosum) plants: pyrimidine salvage and supply of β-alanine to pantothenic acid synthesis

Uridine and cytidine are major nucleosides and are produced as catabolites of pyrimidine nucleotides. To study the metabolic fates and role of these nucleosides in plants, we have performed pulse (2 h) and chase (12 h) experiments with [2-¹⁴C]uridine and [2-¹⁴C]cytidine and determined the activities of some related enzymes using tubers and fully expanded leaves from 10-week-old potato plants ( Solanum tuberosum L.). In tubers, more than 94% of exogenously supplied [2-¹⁴C]uridine and [2-¹⁴C]cytidine was converted to pyrimidine nucleotides and RNA during 2-h pulse, and radioactivity in these salvage products still remained at 12 h after the chase. Little degradation of pyrimidine was found. A similar pyrimidine salvage was operative in leaves, although more than 20% of the radioactivity from [2-¹⁴C]uridine and [2-¹⁴C]cytidine was released as ¹⁴CO₂ during the chase. Enzyme profile data show that uridine/cytidine kinase (EC 2.7.1.48) activity is higher in tubers than in leaves, but uridine nucleosidase (EC 3.2.2.3) activity was higher in leaves. In leaves, radioactivity from [U-¹⁴C]uracil was incorporated into β-ureidopropionic acid, CO₂, β-alanine, pantothenic acid and several common amino acids. Our results suggest two functions of uridine and cytidine metabolism in leaves; these nucleosides are not only substrates for the classical pyrimidine salvage pathways but also starting materials for the biosynthesis of β-alanine. Subsequently, some β-alanine units are utilized for the synthesis of pantothenic acid in potato leaves.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

Abscisic acid metabolism in Ceratocystis coerulescens

The fungus Ceratocystis coerulescens Bakshi (strain RWD 390) has been shown to produce the plant hormone, abscisic acid (ABA). The production of ABA in defined liquid medium during a culture period of 50 days was measured by gas-liquid chromatography. A considerable accumulation of ABA occurred in the stationary phase. Maximum ABA contents were 3.5 ng ml⁻¹ in culture media and 218 ng (g dry weight)⁻¹ in mycelial extracts.
The ABA-metabolizing capability of the fungus was investigated. Dihydrophaseic acid, and phaseic acid, ABA metabolites in higher plants, were not present in cultures of Ceratocystis coerulescens . When [2-¹⁴C]-ABA was fed to the fungus, the formation of [2-¹⁴C]- 2-trans , 4- trans -ABA and a second metabolite, less polar than ABA, was observed. This suggests a different metabolic pathway of ABA in the fungus.     

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.     

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.     

On the mechanism of oleate transport across human brain microvessel endothelial cells

The blood–brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the CNS. As most fatty acids in the brain enter from the blood, we examined the mechanism of oleate (C18:1) transport across primary human brain microvessel endothelial cells (HBMEC). The permeability of [1-¹⁴C]oleate was determined using confluent cells grown on Transwell® inserts in both the absence or presence of bovine serum albumin in the basolateral media, and following inhibition of various fatty acid transporters. The passage of [1-¹⁴C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. The presence of the non-specific fatty acid uptake inhibitor phloretin significantly decreased [1-¹⁴C]oleate uptake by HBMEC and the subsequent release of [1-¹⁴C]oleate into the basolateral medium. Knockdown of fatty acid transport protein-1 or fatty acid translocase/CD36 significantly decreased [1-¹⁴C]oleate transport across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that a fatty acid acceptor is a requirement for oleate transport across HBMEC monolayers. In addition, transport of oleate across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins.     

Influence of red irradiation and of removal of integuments on the metabolism of (±) [2-14C]-abscisic acid by Lactuca sativa Grand Rapids achenes

Achenes of Lactuca saliva L. cv. Grand Rapids, imbibed for 6 h in water or in a 10 µ M solution of non-radioactive abscisic acid (ABA), were cultivated on (2-¹⁴C]-ABA (10 µ M ) for 40 to 90 h. Red irradiation (660 ± 2.5 nm, 5 min, 2 W m ^-2) or removal of integuments were carried out before transfer to (2-¹⁴C]-ABA. When both treatments were applied, irradiation preceded removal of integuments. Imbibition and culture took place in darkness at 24°C. Two acidic diethyl ether phases, which contained the free acids (free phase) and the acids released after mild alkaline hydrolysis, respectively, were isolated. They were analyzed by thin layer chromatography (TLC). as well as the remaining aqueous phase.
Both red irradiation and removal of integuments led to increased [2-¹⁴C|-ABA uptake. Application of ABA during imbibition partly limited the stimulating effect of red irradiation on radioactive ABA uptake. Red irradiation stimulated [2-¹⁴C|-ABA metabolism by achenes, favouring the formation of the polar compound found in the remaining aqueous phase. Removal of the integuments stimulated metabolism notably, leading to an increase of the radioactivity in the remaining aqueous phase. This treatment also induced the appearance of new metabolites in the free phase (compound believed to be 7'-hydroxy-ABA) as well as in the remaining aqueous phase. The glucose ester of ABA was the only representative compound of the ester phase. Irrespective of the experimental conditions, there was no classical oxidative metabolism indicating that oxygen was not the limiting factor.     

Time-course of uptake of dissolved inorganic carbon through willow roots in light and in darkness

Uptake of dissolved inorganic carbon (DIC) from a nutrient solution by willow roots was measured in light and darkness and the distribution in the plant of DIC taken up by the roots was determined. It was also studied whether the transport system could be activated by preincubation with dissolved inorganic carbon.
Willow plants ( Salix cv. Aquatica gigantea) grown in hydroponic culture media were preincubated for 2 days with or without 0.74 mM NaHCO₃. After preincubation, either unlabelled or [¹⁴C]-labelled NaHCO₃ was injected into the media and after 1, 5, 10 and 24 h either in light or in darkness the plants were harvested in pieces into liquid nitrogen, lyophilized and burned in a combustion chamber.
¹⁴C was transported through the roots to the shoots and leaves both in light and in darkness, although incorporation of ¹⁴C in darkness was only half of that in light at the end of the 24-h feeding period. Both in light and in darkness the amount of ¹⁴C increased in all parts of willow plants with time. In light the rate of labelling was highest into cuttings and shoots. In darkness more than half of the total label was detected in cuttings of both the non-activated and the activated treatments.
In the shoots the middle part was most strongly labelled after 5 and 10 h, but after 24 h ¹⁴C moved towards the base of the shoot. In the leaves at all feeding times most radioactivity was incorporated into the young, fully open leaves on the upper part of the shoots. Preincubation of plants with unlabelled NaHCO³ in growth media had no clear effect on the rate of DIC uptake either in light or in darkness.     

Chlorophyll-protein levels and degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants

Lemna gibba plants were incubated aseptically on medium containing labelled 10^-7 M indole-3-acetic acid (IAA-1-¹⁴C). Most of the radioactivity disappeared from the culture medium during a 24 h light period. A high percentage of the loss was due to photolysis and only a low percentage of the radioactivity was recovered in the plants. Uptake of ¹⁴C by the plants was strongly stimulated by light. The radioactivity taken up by the plants was the sum of photosynthetically taken up ¹⁴CO₂ and ¹⁴C taken up in IAA. Analyses with the indolo-α-pyrone fluorescence method revealed that the free IAA content was almost the same in plants grown in control and in IAA media for 5 h, whereas the amount of IAA which could be liberated by alkaline hydrolysis was doubled by the presence of IAA in the medium.     

BIOCHEMICAL AND PHYSIOLOGICAL ASPECTS OF LEAF DEVELOPMENT IN COCOA (THEOBROMA CACAO)

The dormant phase of the flush cycle of leaf growth in cocoa is known to be correlated with high abscisic acid (ABA) levels in the mature leaves of the new flush (NF) and previous flush (PF) leaves. Defoliation of either the NF leaves or PF leaves of cocoa seedlings reduced the length of the dormant phase of the flush cycle, thus showing that the mature leaves were a source of growth inhibitors which could affect shoot apical activity. The application of ABA to the NF and PF leaves led to an extension of the dormant phase, whereas application of zeatin or gibberellic acid decreased it. The distribution of [¹⁴C]ABA following its application to NF and PF leaves at different stages throughout the growth cycle showed that [¹⁴C] ABA was accumulated by the bud in relatively larger amounts during the final stages of bud dormancy (I-1 and I-2) than in the earlier stage (F-2). The results suggest that internal competition for nutrients may be responsible for the inhibition of growth at the F-2 stage but that ABA translocated from the mature leaves causes the buds to remain dormant during the subsequent stages of I-1 and I-2.     

Regulation of abscisic acid translocation during embryo maturation of Phaseolus vulgaris

When R, S [2-¹⁴C] abscisic acid (ABA) was applied to the leaf of Phaseolus vulgaris , a part of the radioactivity was always found 24 h later in the only pod left on the plant. In early podfill, a large part of the labeled material was found in the maternal tissues while in late podfill, most had migrated to the embryos. During embryogenesis, embryo cells became more and more alkaline with respect to the seed coat cells. These results suggest that the distribution of ABA within the tissue is regulated by the pH differential between the two compartments.
The decrease of endogenous ABA level observed in situ during the second part of the embryo development therefore cannot be explained only in terms of the passive diffusion of the undissociated species (ABA-H). The empty-ovules technique revealed that ABA was only partly affected by an inversion of the pH gradient and that metabolic inhibitors influenced the release of ABA. These results indicate that in addition to a diffusive path, an energy-dependent component was involved.     

Cation fluxes in the saps of Sinapis alba during the floral transition

Plants of Sinapis alba L. were induced to flower by either a single long day or a single displaced short day. The levels of three cations. Ca²⁺, Mg²⁺ and K⁺, were measured by atomic absorption spectrophotometry in exudates from roots, leaves and apical stem tips. The export of all three cations out of the root system (root exudate) was increased in induced as compared to non-induced plants. No changes were observed in cation export out of the mature leaves (leaf exudate). The supply of cations to the apical bud (apical exudate) did not originate from the phloem and, so, should mainly be of apoplastic origin. Only the supply of Ca²⁺ to the apical bud was increased, not the supply of Mg²⁺ or K⁺. The increase in Ca²⁺ supply was transient and occurred at about the same time as a conspicuous stimulation of cell division, previously detected in the apical bud.     

AXONAL TRANSPORT OF LIPIDS IN THE RABBIT OPTIC SYSTEM

Abstract— Axonal transport of lipids was demonstrated in the rabbit optic system using [2-³H]glycerol and [3-¹⁴C]serine. Following intraocular injection of these precursors, radioactive lipids were detected in the optic tract, superior colliculus and lateral geniculate body over a 31 day period. The bulk of lipid appeared to migrate at a rate equivalent to that of rapidly transported protein which, when combined with a prolonged period of release into the axon, led to a peak of transported radioactivity at 6-10 days for the 3 tissues. The suggestion of a second peak at 17 days indicated the possibility of a smaller slow component, although another interpretation is suggested. Analysis of individual transported lipids revealed [2-³H]glycerol to label phosphoglycerides preferentially and [3-¹⁴C]serine to be an effective precursor for sphingolipids and certain of the phosphoglycerides. [3-¹⁴C]Serine labeled axonally transported proteins to an even greater extent than lipids, revealing the same fast and slow components previously shown with other amino acids.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

Patterns of adenine metabolism and caffeine biosynthesis in different parts of tea seedlings

Contents of purine alkaloids in different parts of tea ( Camellia sinensis L. cv. Yabukita ) seedlings, seeds and tissue cultures were determined with high-performance liquid chromatography. More than 99% of the caffeine detected was in the leaves of the 4-month-old seedlings. The amount expressed per g fresh weight was higher in older leaves. Theobromine, a precursor of caffeine biosynthesis, was found only in younger leaves. Zero or only trace amounts of theophylline, a degradation product of caffeine, were found in the seedlings. Almost all the caffeine in tea seeds was found in the seed coats. Theobromine and theophilline could not be detected in any part of the seeds.
Tracer experiments using [8-¹⁴C]-adenine indicate that (i) caffeine biosynthesis from [8-¹⁴C]-adenine occurs only in younger leaves,(ii) "salvage" of [8-¹⁴C]-adenine for nucleic acid synthesis takes place in all parts of the seedlings, (iii) considerable degradation of [8-¹⁴C]-adenine by conventional purine degradation pathway via uric acid takes place in roots and lower parts of stem tissue.
The results strongly suggest that caffeine is synthesized in younger leaves and accumulated within the leaves. Both caffeine contents and its synthetic activity from adenine were extremely low in tissue culture of tea.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Distribution of 14C-sucrose applied to leaves at different nodes of okra (Abelmoschus esculentum) during flowering and early pod growth

The distribution pattern of ¹⁴C-sucrose from ¹⁴C-sucrose applied to vegetative okra plants and leaves 1–9 on separate plants during the green pod development stage were investigated in relation to duration and leaf position. Results indicated bi-directional transport of assimilates to both apical and basal portions of the stem. Within 48 h ¹⁴C moved to all plant parts; stem and leaves appeared to be strong sinks. In plants fed at the vegetative stage, 48 h after feeding, 66% of the fed activity was exported from the fed leaf. At the pod development stage, about 35% of the activity exported from the fed leaf was present in green pods and 65% in vegetative parts. In plants where leaf 1–9 was fed, irrespective of the position of the fed leaf, the subtending fruit was the strongest sink among the reproductive parts. Leaves and stems were the principal sinks.     

Translocation of labelled assimilates following photosynthesis of 14CO2 by the field bean, Vicia faba

When whole plants were exposed to ¹⁴CO₂, almost the same amount of radioactivity was taken up initially by each leaf regardless of its position on the stem and of the presence of beans at that node. Thus, although developing beans are a powerful sink for assimilated carbon, they do not increase the CO₂ uptake by adjoining leaves.
The distribution of labelled assimilates 6 hours after feeding ¹⁴CO₂ to a single leaf for 1 hour varied with both the position of the treated leaf and the stage of development of the plant. Before any flowers were set most of the radioactivity from all expanded leaves moved downwards to the roots and the stem below the treated leaf (lower stem). Later, during pod-fill, the upper leaves maintained this supply to the roots and lower stem, whilst most of the carbon translocated from the lower and mid-stem leaves went to the beans. However, we found no exclusive relationship between a leaf and the supply to beans developing on the same node.
The amount of radioactivity moving out of a source leaf at a fruiting node increased over successive samplings up to 48 h; the pattern of distribution of the ¹⁴CO₂ however remained virtually unchanged.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

TL-DNA transformation decreases ABA level

The endogenous levels of ABA were measured in Agrobacterium rhizogenes A₄ T_l-DNA transformed oilseed rape (Brassica napus L. var. oleifera cv . Brutor and cv. Drakkar), cabbage (Brassica oleracea) . A₄ transformed tobacco ( Nicotiana tabacum cv. Xanthi) and their normal counterparts, using high performance liquid chromatography and enzyme-liked immunosorbent assay. Measurements were made on different plant tissues (i. e. floral stem, terminal bud, young leaf, mature leaf, root and root tips) and ABA levels were compared in unstressed and osmotically stressed oilseed rape plants (cv. Brutor). In unstressed Plants. in each of the 5 independent transformation events studied, a significant reduction (about 65% of control) in ABA concentration was observed in all transformed plants. When subjected to an osmotic stress, T_L transformed Brutor plants showed a higher ABA accumulation than untransformed plants. The change in ABA content as a consequence of T_L-DNA transformation is discussed with regard to phenotype, drought resistance and adaptability.