Inhibition by isonicotinyl hydrazide of pigment formation in higher plants

The inhibition of greening of illuminated etiolated maize seedlings by isonicotinyl hydrazide can be alleviated by serine or pyruvate. The similar inhibition in barley can be reversed only by pyruvate. In both plants earlier intermediates in the glycollate pathway and other related compounds were ineffective in overcoming the inhibition of greening produced by isonicotinyl hydrazide. In maize seedlings radioactivity from l-serine-[3-¹⁴C] is poorly incorporated into β-carotene, a typical chloroplast terpenoid, unless glycine and formate or, more effectively, glycine together with isonicotinyl hydrazide are supplied. These supplementations may minimize interconversion of serine and glycine, and hence dilution of radioactivity at C-3 of l-serine by unlabelled C-1 units, before incorporation into terpenoids. The results support the view that in young greening tissue the C2-3 fragment of l-serine can give rise to acetyl-CoA, an obligatory precursor of chloroplast terpenoids.     

The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO₂ concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with ¹⁴C showed that the barley mutants incorporated [¹⁴C]formate and [2-¹⁴C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway. Received: 14 August 1998 / Accepted: 30 September 1998     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

The interconversion of glycine and serine by plant tissue extracts

1. Extracts prepared from a variety of higher-plant tissues by ammonium sulphate fractionation were shown to catalyse the interconversion of glycine and serine. This interconversion had an absolute requirement for tetrahydrofolate and appeared to favour serine formation. 2. The biosynthesis of serine from glycine was studied in more detail with protein fractionated from 15-day-old wheat leaves. Synthesis of [¹⁴C]serine from [¹⁴C]glycine was not accompanied by labelling of glyoxylate, glycollate or formate. 3. The synthesis of serine from glycine was stimulated by additions of formaldehyde, and [¹⁴C]formaldehyde was readily incorporated into C-3 of serine in the presence of tetrahydrofolate. 4. The results are interpreted as indicating that serine biosynthesis involves a direct cleavage of glycine whereby the α-carbon is transferred via N⁵N¹⁰-methylenetetrahydrofolate to become the β-carbon of serine.     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Metabolism of [5-h]proline by barley leaves and its use in measuring the effects of water stress on proline oxidation

The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H₂O as a measure of proline oxidation. When [5-³H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H₂O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and γ-aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the ³H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring ³H₂O recovery from [5-³H]proline were only slightly lower than rates estimated by incorporation of ¹⁴C into oxidized products and loss of ¹⁴C from total proline. Therefore, ³H₂O recovery from [5-³H]proline is useful in assessing the effects of stress on proline metabolism.

Water stress inhibited proline oxidation, as reported previously. In addition, a reconversion of proline oxidation products to proline occurred in stressed leaves. This observation probably indicates a breakdown in cellular compartmentation of proline synthesis and proline oxidation.

     

Oxidation of one-carbon compounds to formate and carbon dioxide in rat liver mitochondria.

In rat liver mitochondria, swollen with phosphate and supplemented with NAD⁺, the oxidation of the methyl carbon of sarcosine to formate is enhanced by the addition of NADP⁺. No carbon dioxide is formed. Formaldehyde and serine, which are the only oxidation products of the methyl group in the absence of the pyridine nucleotides, are decreased by an amount equal to the formate produced. Carbon dioxide, as well as formate, is produced when the mitochondria are treated with EDTA, even without the addition of the pyridine nucleotides. When the mitochondria are exposed to pyrophosphate without added NAD⁺ and/or NADP⁺, all of the oxidized sarcosine-methyl can be recovered as formate, [3-C]serine, and carbon dioxide. Formaldehyde accumulates only if the system is supplemented with Mg²⁺. In the presence of NADP⁺ or the combined pyridine nucleotides, serine accumulation is depressed by an amount equal to the increase in carbon dioxide production. Both carbons of glycine and the 3-C of serine can also be oxidized to carbon dioxide in the pyrophosphate-treated mitochondria. The oxidation of the methyl carbon of S-adenosylmethionine to formaldehyde, [3-C]serine, formate, and carbon dioxide requires a whole homogenate supplemented with glycine. Neither exogenous formaldehyde nor formate is oxidized to carbon dioxide in any of the mitochondrial systems capable of converting sarcosine-methyl to carbon dioxide. Under conditions in which [N⁵,N¹⁰-¹⁴C-methylene]- and [N¹⁰-¹⁴C-formyl]tetrahydrofolate can be isolated as intermediate products of [¹⁴CH₃]sarcosine, exogenous [N⁵,N¹⁰-¹⁴C-methylene]tetrahydrofolate can also be converted to [3-¹⁴C]serine, [¹⁴C]formate, and [¹⁴C]carbon dioxide.     

Microbial growth on C1 compounds. Synthesis of cell constituents by methane- and methanol-grown Pseudomonas methanica

1. A study has been made of the incorporation of carbon from [¹⁴C]methane, [¹⁴C]methanol and [¹⁴C]bicarbonate by cultures of Pseudomonas methanica growing on methane, and [¹⁴C]methanol by cultures of the same organism growing on methanol. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compound for periods up to 3min., has been analysed by chromatography and radioautography. 3. Over 90% of the radioactivity fixed from [¹⁴C]methane or [¹⁴C]methanol at the earliest times of sampling appeared in phosphorylated compounds. Glucose phosphate and fructose phosphate together constituted the largest part of the radioactive phosphates (70–90%); phosphoglycerate was a relatively minor component (2–17%). Other compounds becoming labelled during the incubation included glycine, serine, glutamate, aspartate, malate, citrate and alanine. 4. The first stable products of [¹⁴C]bicarbonate fixation were malate and aspartate (containing between them over 90% of the total radioactivity fixed at the earliest times of sampling). 5. The percentage of the total radioactivity fixed that was contained in each of the radioactive compounds has been plotted against time. The slopes of the curves obtained show that hexose phosphates are primary stable products of [¹⁴C]methane and [¹⁴C]methanol incorporation and that aspartate and malate are primary stable products of [¹⁴C]bicarbonate incorporation. 6. No carboxydismutase activity has been found in cell-free extracts of the organism. This fact, together with the other findings, shows that an autotrophic metabolism involving the ribulose diphosphate cycle of carbon dioxide fixation cannot be operating.     

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.     

Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isozymes are central to single-carbon metabolism and are responsible for interconversion of the THF derivatives in the respective compartments. In the present work, we have used 13C NMR to study folate-mediated single-carbon metabolism in these two compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metabolism. When yeast are incubated with [13C]formate, 13C NMR spectra establish that production of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isozyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Additional supporting evidence of reversibility of GCV in vivo is the production of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metabolism is independent of C1-THF synthase since these products were observed in strains lacking both the cytoplasmic and mitochondrial isozymes. These results suggest that when formate is the one-carbon donor, assimilation is primarily cytoplasmic, whereas when serine serves as one-carbon donor, considerable metabolism occurs via mitochondrial pathways.     

Relationships between glycollate and formate metabolism in greening barley leaves

The activities of enzymes catalysing glycollate oxidation, formate production and folate-dependent formate utilization were examined in the primary leaves of Hordeum vulgare cv Galt. Seedlings were grown for 6 days in darkness and then transferred to continuous light (500 μinsteins/m² per sec) for up to 5 days. Cell-free extracts of the primary leaves contained glycollate oxidase (EC 1.1.3.1), 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5, 10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and ability to enzymically decarboxylate glyoxylate. These activities increased during greening and at the end of the light treatment were 70–450% higher than etiolated controls. Greened primary leaves also incorporated [¹⁴C]formate at rates that were three- to four-fold higher than shown by etiolated leaves. The specific activity of 10-formyltetrahydrofolate synthetase was decreased by 20–35% when the leaves were greened in the presence of 10 mM hydroxysulphonate. This inhibitor also reduced the incorporation of [¹⁴C]formate by up to 45%. A potential flow of carbon from glycollate to 10-formyltetrahydrofolate via glyoxylate and formate was suggested by the data.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Investigation of the Mechanism of Action of a Chlorosis-Inducing Toxin Produced by Pseudomonas phaseolicola

A toxin that induced chlorotic haloes (typifying haloblight disease) on primary leaves of Phaseolus vulgaris L. (var. Canadian Wonder) was partially purified from culture filtrates of the causative agent Pseudomonas phaseolicola (Burkh.) Dowson. This material was used to investigate chlorosis induction. Haloes could only be induced in those bean leaves that were expanding and synthesizing chlorophyll (Chl); the toxin, therefore, does not promote Chl breakdown. Chl, carotene, and xanthophyll synthesis were inhibited in sections of greening barley (Hordeum vulgare L.) leaves, irrespective of the irradiance level. In parallel experiments, the toxin decreased the level of 5-aminolevulinic acid by amounts sufficient to account for toxin-inhibition of Chl synthesis. Electron microscopy revealed no difference between the transformation of etioplasts into chloroplasts in toxin-treated and control tissue, despite a 60% reduction in Chl in the former. The incorporation of [¹⁴C]acetate into lipid by greening barley leaf sections and by isolated Pisum sativum chloroplasts in the light and the dark was inhibited about 60% by the toxin. The distribution of radioactivity among the spectra of acyl residues was the same in the control and toxin-treated material. It is suggested that the toxin interferes with an early process common to the synthesis of different lipids, including Chl.     

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     

The biosynthesis of δ-aminolevulinic acid in greening maize leaves

In greening maize leaves δ-aminolevulinic acid (ALA) was not formed from succinyl-CoA and glycine as shown by the incorporation of [¹⁴C]-labeled     

Oxidation of proline by plant mitochondria

Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O₂ uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O₂, but not NAD. The major ninhydrin-positive product formed via Δ¹-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO₂, organic acids, and aspartate were recovered after [¹⁴C]proline feeding experiments. These results demonstrate the mitochondrial association and O₂ dependence of plant proline metabolism.     

The importance of glyoxylate in amino acid biosynthesis in plants

1. [¹⁴C₂]Glyoxylate was rapidly metabolized by carrot storage tissues, pea leaves, pea cotyledons, sunflower cotyledons, corn coleoptiles, corn roots and pea roots. In many tissues over 70% of the supplied [¹⁴C₂]glyoxylate was utilized during the 6hr. experimental periods. 2. In all tissues, the chief products of [¹⁴C₂]-glyoxylate metabolism were carbon dioxide, glycine and serine. In several of the tissues, there was also a considerable incorporation of the label into the organic acids, particularly into glycollate. 3. Degradations of the labelled serine produced during [¹⁴C₂]glyoxylate metabolism showed that glyoxylate carbon was incorporated into all three positions of the serine molecule. 4. The results are interpreted as indicating that glyoxylate is utilized by the tissues by pathways involving transamination, transmethylation, reduction and oxidative decarboxylation of the supplied glyoxylate.