Changes in accumulation of seed nitrogen compounds in maize under conditions of sulphur deficiency

Maize ( Zea mays L., hybrid INRA 260) was grown in the greenhouse with mineral nutrition of different sulphate concentrations. Mature seeds from these plants were compared for their free amino acid and protein N forms. For the most S-deficient sample, the Asx (asparagine + aspartic acid) content increased by 30% as compared with control, while methionine and cysteine decreased (by 25 and 30%, respectively), as well as glycine, lysine, histidine, arginine and tryptophan. In seeds lowest in S the non-protein N to total N ratio was 77% higher than in the control. Free asparagine dominated in starved seeds (50 mol % of total free amino acids) and was ten-fold more concentrated than in the control, where proline was the predominant free amino acid. Thus the Asx of non-protein N reached 28% of the total mol Asx of the whole starved seed. Altered S nutrition had virtually no effect on the amino acid composition of the main protein fractions, but it significantly changed their ratios. Zeins, which are poor in S-containing amino acids, showed 25% higher level than in seeds supplied with normal S. As a counterbalance, two glutelin subfractions rich in S-containing amino acids, decreased by 36–71% under limiting S nutrition.
It is concluded that the plant reacts against S deficiency by modifying its N metabolism. Significant accumulation occurred of free asparagine, which is the main form of N transportation. The biosynthesis of seed storage protein occurred through the accumulation of the highest possible protein quantity allowed by the available S-containing amino acids, i.e. proteins low in S-containing amino acids were preferentially synthesized.     

A proteomic approach to studying biogenic amine producing lactic acid bacteria

All fermented foods are subject to the risk of biogenic amine contamination. Histamine and tyramine are among the most toxic amines for consumers' health, exerting undesirable effects on the central nervous and vascular systems, but putrescine and cadaverine can also compromise the organoleptic properties of contaminated foods. These compounds are produced by fermenting microbial flora that decarboxylate amino acids to amines. Little is known of the factors which induce biosynthesis of decarboxylating enzymes and/or which modulate their catalytic activity: the accumulation of amines is generally considered to be a mechanism that contrasts an acidic environment and/or that produces metabolic energy through coupling amino acid decarboxylation with electrogenic amino acid/amine antiporters. Two Lactobacillus strains, Lactobacillus sp. 30a (ATCC 33222), and a Lactobacillus sp. strain (w53) isolated from amine-contaminated wine, carrying genetic determinants for histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), were studied and the influence of some environmental and nutritional parameters on amine production and protein biosynthesis was analyzed through a proteomic approach; this is the first report of a proteomic analysis of amine-producing bacteria. HDC and ODC biosynthesis were shown to be closely dependent on the presence of high concentrations of free amino acids in the growth medium and to be modulated by the growth phase. The stationary phase and high amounts of free amino acids also strongly induced the biosynthesis of an oligopeptide transport protein belonging to the proteolytic system of Lactic Acid Bacteria. At least two isoforms of glyceraldehyde-3-phosphate dehydrogenase, with different M(r), pI and expression profiles, were identified from Lactobacillus sp. w53: the biosynthesis of one isoform, in particular, is apparently repressed by high concentrations of free amino acids. Other proteins were identified from the Lactobacillus proteome, affording a global knowledge of protein biosynthesis modulation during biogenic amine production.     

Impact of potassium,sodium, and salinity on the protein-and free amino acid content of wheat grain

A lysimeter study was conducted on Cajeme wheat (Triticum aestivum L.) to investigate the impact of salinity on protein and free amino acid content of the grain. Cross correlations were obtained between 16 different soil-plant-water based parameters and the concentration and total accumulation of amino acids. The results indicated that after 3 years of irrigation, the majority of protein bound and free amino acids increased in concentration in the grain. However, both free tryptophan and free proline revealed decreasing concentrations with increasing salinity. Free tryptophan showed a synergism between total accumulation, yield and concentration. Free proline concentrations decreased in association with increasing protein concentrations. Cross correlations of the 16 soil-plant-water based parameters with free and protein bound amino acids revealed significant correlations for free aspartic acid and glycine with total accumulation but not with concentrations. Only methionine plus cystine was lower than suggested FAO levels for essential amino acids and was lower in the third year than in the first year.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Amino acid composition of the lymph and blood in allergic transformations of the body

The time-course of changes in the content of free amino acids and total protein in lymph of the thoracic duct and peripheral blood was studied in dogs during sensitization and anaphylactic shock (AS). Allergic rearrangement of the body during AS was accompanied by considerable disorders in redistribution of free amino acids in body fluids (blood, interstitial fluid and lymph). It is assumed that changes in the quantitative ratios occurring between individual amino acids during metabolic processes under sensitization are likely to cause protein biosynthesis disorders in the body experiencing allergic rearrangement.     

Nitrogen metabolism in cultures of Tabernaemontana divaricata

A cell suspension culture from Tabernaemontana divaricata was fed with ¹⁵N-labelled ammonium or nitrate. The incorporation of label in free amino acids, protein amino acids and indole alkaloids was determined. Ammonium was found to be used more extensively than nitrate in the biosynthesis of these compounds. For tryptamine considerably lower labelling percentages were found than for the indole alkaloid O-acetylvallesamine and the amino acids. This indicates a vacuolar pool of tryptamine, formed at the beginning of the culture-period and not available for further alkaloid biosynthesis.     

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an α subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.     

Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts

A major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein-bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.     

Differential molecular responses of rice and wheat coleoptiles to anoxia reveal novel metabolic adaptations in amino acid metabolism for tissue tolerance

Rice (Oryza sativa) and wheat (Triticum aestivum) are the most important starch crops in world agriculture. While both germinate with an anatomically similar coleoptile, this tissue defines the early anoxia tolerance of rice and the anoxia intolerance of wheat seedlings. We combined protein and metabolite profiling analysis to compare the differences in response to anoxia between the rice and wheat coleoptiles. Rice coleoptiles responded to anoxia dramatically, not only at the level of protein synthesis but also at the level of altered metabolite pools, while the wheat response to anoxia was slight in comparison. We found significant increases in the abundance of proteins in rice coleoptiles related to protein translation and antioxidant defense and an accumulation of a set of enzymes involved in serine, glycine, and alanine biosynthesis from glyceraldehyde-3-phosphate or pyruvate, which correlates with an observed accumulation of these amino acids in anoxic rice. We show a positive effect on wheat root anoxia tolerance by exogenous addition of these amino acids, indicating that their synthesis could be linked to rice anoxia tolerance. The potential role of amino acid biosynthesis contributing to anoxia tolerance in cells is discussed.     

Effect of tryptophan excess in a diet on amino acid composition of skin collagen and on an initial stage of protein biosynthesis in rat liver

Protein deficiency and tryptophane load against its background lead to the acid-soluble collagen synthesis in the rat skin. The amino acid composition of the collagen differs from the norm. This is accompanied by changes in the free amino acid pool of blood serum and liver, under tryptophane load the free amino acids pool of the liver increasing twice as high. At the same time protein deficiency increases and tryptophane load decreases the level of tRNA amino acylation with tryptophane in the animal liver. Thus, protein deficiency and tryptophane load against its background cause deep changes in the protein biosynthesis.     

Penicillin biosynthesis and amino acid metabolism in Penicillium chrysogenum in experiments with washed mycelium]

The study of the amino acid metabolism in Penicillium chrysogenum with the use of washed mycelium showed that the amount of the free intracellular amino acids significantly decreased during the process of penicillin production. Still, such a decrease did not cover the nitrogen requirements of the culture for the antibiotic synthesis and mobilization of the protein nitrogen took place. By the end of the process the amount of the protein nitrogen markedly decreased. At the same time alpha-amino nitrogen was absent in the fermentation broth filtrate. About 14 amino acids (including cysteine and valine) which participate in constriuction of the penicillin molecule nucleus were found in the amino acid poll. However, the amounts of cysteine and valine were not high and probably other free intracellular amino acids participated in their synthesis. It was shown that one of the limiting factors in the process of penicillin biosynthesis was synthesis of cysteine, a sulphur-containing amino acid which is one of the precursors of the antibiotic molecule nucleus.     

Amino Acid Pools and Metabolism During the Cell Division Cycle of Arginine-Grown Candida utilis

Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed.     

Light and metabolic signals control the selective degradation of sucrose synthase in maize leaves during deetiolation

The content and activity of Suc (Suc) synthase (SUS) protein is high in sink organs but low in source organs. In this report, we examined light and metabolic signals regulating SUS protein degradation in maize (Zea mays) leaves during deetiolation. We found that SUS protein accumulated in etiolated leaves of the dark-grown seedlings but was rapidly degraded upon exposure to white, blue, or red light. This occurred concurrent with the accumulation of photosynthetic enzymes, such as Rubisco and Rubisco activase, and enzymes of Suc biosynthesis such as Suc-phosphate synthase. Deetiolation-induced SUS degradation was not inhibited by the proteasome inhibitor MG132. Moreover, neither full-length nor truncated SUS phosphorylated at the serine-170 site was found in the crude 26S proteasome fraction (150,000g postmicrosomal pellet) isolated in the presence of MG132. However, SUS degradation was strongly inhibited by feeding cycloheximide or amino acids to detached leaves, while Suc feeding had no effect. Of the amino acids tested, exogenous glutamate had the greatest effect. Collectively, these results demonstrate that SUS protein degradation during deetiolation: (1) is selective; (2) can be triggered by either blue- or red light-mediated signaling pathways; (3) does not involve the 26S proteasome; and (4) is inhibited by free amino acids. These findings suggest that SUS degradation is important to supply residues for the synthesis of other proteins required for autotrophic metabolism.     

A rapid method for determining tRNA charging levels in vivo: analysis of yeast mutants defective in the general control of amino acid biosynthesis.

We describe a simple method to quantitate the intracellular levels of charged tRNA species representing all 20 amino acids. Small RNA species are isolated from yeast cells under conditions where amino acids remain bound to their cognate tRNAs. After chromatographic removal of free amino acids, the tRNAs are discharged, and the amounts of the released amino acids are then quantitated. This method was applied to yeast cells from a wild type strain and from three mutant strains that are defective both in the general control of amino acid biosynthesis and in protein synthesis. Two of these mutant strains, previously shown to be defective in the methionine or isoleucine tRNA synthetases, respectively contain undetectable amounts of charged methionine or isoleucine although their levels of the remaining 19 amino acids are similar to a wild type strain. In contrast, a gcd1 mutant strain has normal levels of all 20 amino-acyl tRNA species. Thus, gcd1 strains are defective in general control of amino acid biosynthesis for reasons other than artifactual starvation of an amino acid due to a failure in tRNA changing.     

A Quantitative Regional Analysis of Amino Acids Involved in Rat Brain Protein Synthesis by High Performance Liquid Chromatography

A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood-brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl-labeled amino acids and terminated 2 min after infusion of 3H-labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of 14C- or 3H-labeled phenylalanine, tyrosine, leucine, isoleucine, and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl-tRNAs, and proteins. Amino acids were converted to their corresponding 5-dimethylamino-naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18 reversed-phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu greater than Tyr greater than Ile greater than Phe greater than Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl-tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane-anesthetized rat were determined to be 30 and 67-91 pmol total amino acid incorporation/min/mg tissue for white and gray matter, respectively.     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

The mitochondrial pool of free amino acids reflects the composition of mitochondrial DNA-encoded proteins: indication of a post- translational quality control for protein synthesis

Mitochondria can synthesize a limited number of proteins encoded by mtDNA (mitochondrial DNA) by using their own biosynthetic machinery, whereas most of the proteins in mitochondria are imported from the cytosol. It could be hypothesized that the mitochondrial pool of amino acids follows the frequency of amino acids in mtDNA-encoded proteins or, alternatively, that the profile is the result of the participation of amino acids in pathways other than protein synthesis (e.g. haem biosynthesis and aminotransferase reactions). These hypotheses were tested by evaluating the pool of free amino acids and derivatives in highly-coupled purified liver mitochondria obtained from rats fed on a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the amino acid composition of mtDNA-encoded proteins, suggesting that there is a post-translational control of protein synthesis. This conclusion was supported by the following findings: (i) correlation between the concentration of free amino acids in the matrix and the frequency of abundance of amino acids in mtDNA-encoded proteins; (ii) the similar ratios of essential-to-non-essential amino acids in mtDNA-encoded proteins and the mitochondrial pool of amino acids; and (iii), lack of a correlation between codon usage or tRNA levels and amino-acid concentrations. Quantitative information on the mammalian mitochondrial content of amino acids, such as that presented in the present study, along with functional studies, will help us to better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial metabolism.