The role of light in soybean seed filling metabolism

Soybean (Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U‐¹³C₆]‐glucose, [U‐¹³C₅]‐glutamine, the combination of [U‐¹⁴C₁₂]‐sucrose + [U‐¹⁴C₆]‐glucose + [U‐¹⁴C₅]‐glutamine + [U‐¹⁴C₄]‐asparagine, or ¹⁴CO₂ labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO₂, O₂) were established by a combination of gas chromatography‐mass spectrometry, ¹³C‐ and ¹H‐NMR, scintillation counting, HPLC, gas chromatography‐flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3‐phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

Non-morphogenic metabolism during shoot induction in radiata pine cotyledon explants

Summary The effects of excision, light and cytokinin (N⁶-benzyladenine) on¹⁴C-acetate metabolism in cotyledons ofPinus radiata (D. Don) were determined.¹⁴CO₂ was released and the distribution of radioactivity into lipids, sugars, organic acids and amino acids was determined. While light and cytokinin generally caused some increase in metabolism, the effect of excision, i.e., wounding, was most pronounced. Specific metabolites examined (citrate, malate, succinate, alanine, aspartate, glutamate and glutamine) were at least 50% greater in¹⁴C-labeling in excised cotyledons as compared to intact seedlings. This enhancement of wound metabolism would mask possible morphogenically-related changes occurring at that time. This research was supported by the Natural Sciences and Engineering Research Council of Canada Grant A-6467 to T.A. Thorpe.     

Studies on the biochemistry and fine structure of silica-shell formation in diatoms

Summary The distribution of radioactivity in ethanol-water-soluble compounds after short periods of photosynthetic incorporation of ¹⁴CO₂ is consistent with the operation of the photosynthetic carbon reduction (PCR) cycle in the fresh water diatom Navicula pelliculosa. Incorporation of ¹⁴CO₂ for extended time periods established the presence of the intermediates of the PCR and tricarboxylic acid (TCA) cycles, amino acids, and organic acids; free sugars were not observed. The main labelled soluble carbohydrate was a glucan. Hydrolysis of the radioactive insoluble material indicated the presence of carbohydrates containing several distinct sugars, and proteins with the usual amino-acid composition. During silicon starvation of exponentially growing cultures, rates of incorporation of both ³²P _i and ¹⁴CO₂ decreased. Incorporation into the lipid increased, with a corresponding decrease into protein and carbohydrate. Reintroduction of Si to staryed cells led to an increased rate of incorporation of both isotopes, and transient changes in the radioactivity in most metabolic intermediates investigated. After 30 min the radioactivity in all PCR cycle intermediates, except phosphoglyceric acid, had increased by about 300%. The radioactivity of citrate and -keto-glutarate increased, whereas that of other TCA-cycle intermediates decreased. An initial decrease in the levels of glutamate, aspartate and glutamine was apparently reversed by cleavage of glutamate-aspartate peptides, as radioactivity of other amino acids increased. Incorporation into the soluble glucan and into protein increased markedly although the rate of incorporation into insoluble carbohydrates remained constant.     

Photosynthetic Activity in the Flower Buds of ;Valencia' Orange (Citrus sinensis [L.] Osbeck)

Flower buds of `Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix ¹⁴CO₂ into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total ¹⁴C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the ¹⁴C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO₂ per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO₂ into different compounds suggests that this CO₂ assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

Abstract: CO₂ fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with ¹⁴CO₂ and the incorporation of ¹⁴C into medium or cell extract products was determined. After chromatographic separation of ¹⁴C-labelled products, the fractions of ¹⁴C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the ¹⁴C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO₂ fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [¹⁴C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net ¹⁴CO₂ fixation, but did alter the distribution of ¹⁴C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or ¹⁴C incorporation). Low rates of conversion of α-[¹⁴C]ketoglutarate to [¹⁴C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.     

Photosynthetic products of division synchronized cultures of euglena

Rates and products of photosynthetic ¹⁴CO₂ fixation by division synchronized cultures of Euglena gracilis strain Z were determined over the cycle. Rate of ¹⁴CO₂ fixation doubled in a continuous manner throughout the light phase followed by a slight reduction of photosynthetic capacity in the dark phase. Greater ¹⁴C incorporation into the nucleic acid-polysaccharide fraction occurred with mature cells. Products of ¹⁴CO₂ fixation varied markedly over the cycle: although with mature cells ¹⁴C-labeled sucrose was not detected, with dividing cells this was the main sugar labeled; in young cells ¹⁴C maltose was formed. Cells removed at end of dark phase accumulated ¹⁴C in glycolate, whereas at other stages over the cycle less ¹⁴C was present in glycolate, and this was accompanied by a rapid incorporation of ¹⁴C into glycine and serine. Glycerate was an early and major product of photosynthesis with cells at the mature stage of the cycle.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

A study of potassium gradients in the epidermis of intact leaves of Commelina communis L. in relation to stomatal opening

Summary Activated phytochrome lowers the free amino acid pools of all plants investigated by about 15%. The action of red light is preferentially directed to Asp, Glu and Phe. Exogenously supplied Leu-U-¹⁴C is incorporated more quickly into protein of red-light-treated samples compared with dark controls. In contrast, red light decreases the amount of Asp-U-¹⁴C incorporated into protein, but increases the amount of ¹⁴CO₂ respired after feeding with Asp-U-¹⁴C. Red light has no effect on the amount of ¹⁴CO₂ respired after feeding with Leu-U-¹⁴C. Red-light-mediated stimulation of incorporation of Leu-U-¹⁴C into protein occurred within 15 min, well before the red-light-mediated increase in ¹⁴CO₂ production following feeding with Asp-U-¹⁴C could be detected.Abbreviations R red light - FR far-red light - R+FR red immediately followed by far-red light - D dark control - TCA tricarboxylic acid cycle For amino acids as stated in Biochem. J. 126, 773–780 (1972).     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

Studies on Orobanche hederae Physiology: Pigments and CO2 Fixation

In order to investigate some aspects of Orobanche hederae physiology in relation to its parasitism, the pigment composition and the ¹⁴CO₂ incorporation, both in light and in dark, were studied. By means of various chromatographic techniques, it was shown that chlorophyll is probably quite absent and that the carotenoids are very largely distrihuted and consist almost exclusively of xanthophylls. Flavochrome, flavoxanthin, and a pigment resembling neoxanthin were found to be the major components; lutein-5,6-epoxide, taraxanthin, and traces of β-carotene and α-carotene-5,6-epoxide were also present. CO₂ incorporation is completely of heterotrophic type, being in no way stimulated by the light; the major radioactivity was detected in the fractions containing amino acids and organic acids.     

Inorganic carbon fixation and metabolism in maize roots as affected by nitrate and ammonium nutrition

The effects of NO^?₃ and NH⁺₄ nutrition on the rates of dark incorporation of inorganic carbon by roots of hydroponically grown Zea mays L. cv. 712 and on the metabolic products of this incorporation, were determined in plants supplied with NaH¹⁴CO₃ in the nutrient solution. The shoots and roots of the plants supplied with NaH¹⁴CO₃ in the root medium for 30 min were extracted with 80%; (v/v) ethanol and fractionated into soluble and insoluble fractions. The soluble fraction was further separated into the neutral, organic acid, amino acid and non-polar fractions. The amino acid fraction was then analyzed to determine quantities and the ¹⁴C content of its individual components. The rates of dark incorporation of inorganic carbon calculated from H¹⁴CO^?₃ fixation and attributable to the activity of phosphoenolpyuvate carboxylase (EC 4.1.1.31), were 5-fold higher in ammonium-fed plants than in nitrate-fed plants after a 30-min pulse of ¹⁴C. This activity forms a small, but significant component of the carbon budget of the root. The proportion of ¹⁴C located in the shoots was also significantly higher in ammonium-fed plants than in nitrate-fed plants, indicating more rapid translocation of the products of dark fixation to the shoots in plants receiving NH⁺/sp₄ nutrition. Ammonium-fed plants favoured incorporation of ¹⁴C into amino acids, while nitrate-fed plants allocated relatively more ¹⁴C into organic acids. The amino acid composition was also dependent on the type of nitrogen supplied, and asparagine was found to accumulate in ammonium-fed plants. The ¹⁴C labelling of the amino acids was consistent with the diversion of ¹⁴C-oxaloacetate derived from carboxlyation of phosphoenolpyruvate into the formation of both asparatate and glutamate. The results support the conclusion that inorganic carbon fixation in the roots of maize plants provides an important anaplerotic source of carbon for NH⁺₄ assimilation.     

Light quality effects on subsequent dark 14CO2-fixation in Fucus serratus

The intensity and fate of ¹⁴CO₂-fixation in the dark are studied on Fucus serratus apices previously maintained under low illumination conditions using white, blue, red or yellow isoquantic lights.In the case of a 180 s pulse, light quality affected dark carbon-fixation, with a higher level of incorporation into ethanol-soluble organic matter in the case of yellow light cultivated apices. After a 30 s pulse ¹⁴C was mainly fixed into glycerate and aspartate-malate pools whatever the pre-treatment light conditions, with a higher level into glycerate when apices were pre-illuminated with blue or yellow light. After a 180 s pulse, ¹⁴C was mainly transferred into amino acids (glutamate and alanine) at the expense of aspartate and malate in red and yellow pre-illumination conditions, as found in the white light reference experiment, and only at the expense of glycerate in blue light pre-illumination conditions.The metabolic pathway of glycerate formation, principally enhanced by blue light preillumination, remains unexplained under these non-photosynthetic conditions. Results are discussed with reference to CO₂-fixation via phosphoenolpyruvate carboxykinase and light quality effects on its in vitro activity.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

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.     

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-¹⁴C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO₂), hypoxic hypoxia (15 or 10% O₂), and hypoxic hypoxia plus hypercarbia (15 or 10% O₂ with 5% CO₂). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO₂) decreased the incorporation of [U-¹⁴C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO₂) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O₂) or hypoxic hypoxia with 5% CO₂ were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.     

Effect of Oxygen Concentration on C-Photoassimilate Transport from Leaves of Salvia splendens L

Partitioning and transport of recently fixed photosynthate was examined following ¹⁴CO₂ pulse-labeling of intact, attached leaves of Salvia splendens L. maintained in an atmosphere of 300 microliters per liter CO₂ and 20, 210, or 500 milliliters per liter O₂. Under conditions of increasing O₂ (210, 500 milliliters per liter), a smaller percentage of the recently fixed ¹⁴C in the leaf was allocated to starch, whereas a greater percentage of the fixed ¹⁴C appeared in amino acids, particularly serine. The increase in ¹⁴C in amino acids was reflected in material exported from source leaves. A higher percentage of ¹⁴C in serine, glycine, and glutamate was recovered in petiole extracts when source leaves were maintained under elevated O₂ levels. Although pool sizes of these amino acids were increased in both the leaves and petioles with increasing photorespiratory activity, no significant changes in either ¹⁴C distribution or concentration of transport sugars (i.e. stachyose, sucrose, verbascose) were observed. The data indicate that, in addition to being recycled intracellularly into Calvin cycle intermediates, amino acids produced during photorespiration may also serve as transport metabolites, allowing the mobilization of both carbon and nitrogen from the leaf under conditions of limited photosynthesis.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.