Investigation of proteomic responses of Streptomyces lydicus to pitching ratios for improving streptolydigin production

Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.     

Insights into the roles of exogenous glutamate and proline in improving streptolydigin production of Streptomyces lydicus with metabolomic analysis

The addition of precursors was one strategy to improve antibiotic production. The exogenous proline and glutamate, as precursors of streptolydigin, could significantly improve the streptolydigin production, but their underlying molecular mechanisms remain unknown. Herein, metabolomic analysis was carried out to explore the metabolic responses of Streptomyces lydicus to the additions of proline and glutamine. The significant differences in the quantified 53 metabolites after adding the exogenous proline and glutamate were enunciated by gas chromatography coupled to time-of-flight mass spectrometry. Among them, the levels of some fatty acids (e.g., dodecanoic acid, octadecanoic acid, hexadecanoic acid) were significantly decreased after adding glutamate and proline, indicating that the inhibition of fatty acid synthesis might be benefit for the accumulation of streptolydigin. Particularly, the dramatic changes of the identified metabolites, which are involved in glycolysis, the tricarboxylic acid cycle, and the amino acid and fatty acid metabolism, revealed that the additions of glutamate and proline possibly caused the metabolic cross-talk in S. lydicus. Additionally, the level of intracellular glutamate dramatically enhanced at 12 h after adding proline, showing that exogenous proline may be firstly convert into glutamate and consequently result in crease of the streptolydigin production. The high levels of streptolydigin at 12 and 24 h after adding glutamate unveiled that part glutamate were rapidly used to synthesize the streptolydigin. Furthermore, there is the significant difference in metabolomic characteristics of S. lydicus after adding glutamate and proline, uncovering that multiple regulatory pathways are involved in responses to the additions of exogenous glutamate and proline. Taken together, exogenous glutamate and proline not only directly provided the precursors of streptolydigin biosynthesis, but also might alter the metabolic homeostasis of S. lydicus E9 during improving the production of streptolydigin.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Insight into the surfactin production of <Emphasis Type="Italic">Bacillus velezensis</Emphasis> B006 through metabolomics analysis

Bacillus velezensis B006 is a biocontrol agent which functions through effective colonization and surfactin production. To reveal the surfactin-producing mechanism, gas chromatography–mass spectrometry based untargeted metabolomics was performed to compare the metabolite profiles of strain B006 grown in industrial media M3 and M4. Based on the statistical and pathway topology analyses, a total of 31 metabolites with a fold change of less than ??1.0 were screened as the significantly altered metabolites, which distributed in 15 metabolic pathways. Fourteen amino acids involving in the metabolisms of alanine/aspartate/glutamate, glycine/serine/threonine, arginine/proline, glutathione/cysteine/methionine and valine/leucine/isoleucine as well as succinic acid in TCA cycle were identified to be the hub metabolites. Aminoacyl-tRNA biosynthesis, glycerolipid metabolism, and pantothenate/CoA biosynthesis also contributed to surfactin production. To the best of our knowledge, this study is the first to investigate the metabolic pathways of B. velezensis on surfactin production, and will benefit the optimization of commercial fermentation for higher surfactin yield.     

Metabolic changes associated with adaptation of plant cells to water stress

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from ¹⁵N labeling kinetics after substituting [¹⁵N]ammonium and [¹⁵N]nitrate for the ¹⁴N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and γ-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and γ-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and γ-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low ¹⁵N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.     

Plasma-free amino acid profiling of cervical cancer and cervical intraepithelial neoplasia patients and its application for early detection

In this study, plasma-free amino acid profiles were used to investigate pre-cancerous cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) metabolic signatures in plasma. Additionally, the diagnostic potential of these profiles was assessed, as well as their ability to provide novel insight into CSCC metabolism and systemic effects. Plasma samples from CIN patients (n = 26), CSCC patients (n = 22), and a control healthy group (n = 35) were analyzed by high-performance liquid chromatography, and their spectral profiles were subjected to the t test for statistical significance. Potential metabolic biomarkers were identified using database comparisons that examine the significance of metabolites. Compared with healthy controls, patients with CIN and CSCC demonstrated lower levels of plasma amino acids; plasma levels of arginine and threonine were increased in CIN patients but were decreased in cervical cancer patients. Additionally, the levels of a larger group of amino acids (aspartate, glutamate, asparagine, serine, glycine, histidine, taurine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine) were gradually reduced from CIN to invasive cancer. These findings suggest that plasma-free amino acid profiling has great potential for improving cancer screening and diagnosis and for understanding disease pathogenesis. Plasma-free amino acid profiles may have the potential be used to determine cancer diagnoses in the early stage from a single blood sample and may enhance our understanding of its mechanisms.     

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.     

Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production

Genome-scale metabolic reconstructions are routinely used for the analysis and design of metabolic engineering strategies for production of primary metabolites. The use of such reconstructions for metabolic engineering of antibiotic production is not common due to the lack of simple design algorithms in the absence of a cellular growth objective function. Here, we present the metabolic network reconstruction for the erythromycin producer Saccharopolyspora erythraea NRRL23338. The model was manually curated for primary and secondary metabolism pathways and consists of 1,482 reactions (2,075 genes) and 1,646 metabolites. As part of the model validation, we explored the potential benefits of supplying amino acids and identified five amino acids “compatible” with erythromycin production, whereby if glucose is supplemented with this amino acid on a carbon mole basis, the in silico model predicts that high erythromycin yield is possible without lowering biomass yield. Increased erythromycin titre was confirmed for four of the five amino acids, namely valine, isoleucine, threonine and proline. In bioreactor experiments, supplementation with 2.5?% carbon mole of valine increased the growth rate by 20?% and simultaneously the erythromycin yield on biomass by 50?%. The model presented here can be used as a framework for the future integration of high-throughput biological data sets in S. erythraea and ultimately to realise strain designs capable of increasing erythromycin production closer to the theoretical yield.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(?)9,10-dihydrojasmonoyl]valine,N-[(?)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Aspects of the Drought Tolerance in Creosotebush (Larrea divaricata)

In order to understand better the physiological adaption of creosotebush (Larrea divaricata Cav.) to drought conditions, its carbohydrate and nitrogen metabolism after a 7-day desiccation period under controlled conditions were studied. Although fructose was not significantly altered in the leaves of desiccated plants, as compared to those maintained under normal moisture conditions, both glucose and sucrose were significantly reduced. Total amino acids more than doubled under moisture stress, the increase being predominantly due to proline, phenylalanine, and glutamic acid. Significant increases also occurred in alanine, arginine, histidine, isoleucine, and valine. Increases or decreases in other amino acids were not significant. These stress-induced changes in certain amino acids are considered in relationship to protein hydrolysis, to accumulation of nitrogen degradation products translocated from the roots, and to the possible function of specific amino acids (e.g., proline) in NH₃⁺ storage.     

1H, 13C, 15N assignments of the dimeric regulatory subunit (ilvN) of the E. coli AHAS I

Acetohydroxyacid synthase (AHAS) is an enzyme involved in the biosynthesis of the branched chain amino acids viz, valine, leucine and isoleucine. The activity of this enzyme is regulated through feedback inhibition by the end products of the pathway. Here we report the backbone and side-chain assignments of ilvN, the 22 kDa dimeric regulatory subunit of E. coli AHAS isoenzyme I, in the valine bound form. Detailed analysis of the structure of ilvN and its interactions with the catalytic subunit of E. coli AHAS I will help in understanding the mechanism of activation and regulation of the branched chain amino acid biosynthesis.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K_m for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.     

α-核突触蛋白基因突变小鼠脑组织中内源性代谢产物的变化

目的:明确α-核突触蛋白与帕金森病的病理生理相关性及其临床意义。方法:采用相色谱-质谱联用(UPLC-MS)检测野生型小鼠和基因突变型小鼠脑组织中内源性代谢性产物,通过mzcloud法对小鼠脑组织中内源性代谢物质进行鉴定,将相应数据进行主成分分析(PCA)和聚类分析,分析其相关差异表达代谢物,并构建通路图和互作网络图。结果:(1)基于LC/MS法的代谢组分析结果显示两组间差异代谢物以氨基酸类及磷脂类等为主,包括β-丙氨酰-L-组氨酸、L-精氨酸、L-组氨酸、L-亮氨酸、L-苯丙氨酸、L-缬氨酸、L-天门冬氨酸、L-丙氨酸、磷脂酰胆碱等;(2)构建的代谢通路主要涉及酮体的合成和降解、牛磺酸和亚牛磺酸代谢、丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、苯丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢等,从中发现18个具有标志性的代谢成分。结论:α-核突触蛋白基因突变后,酮体的合成和降解、牛磺酸和亚牛磺酸代谢、丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、苯丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢等代谢通路发生了变化,涉及β-丙氨酰-L-组氨酸、L-精氨酸、L-组氨酸、L-亮氨酸、L-苯丙氨酸、L-缬氨酸、L-天门冬氨酸、L-丙氨酸、磷脂酰胆碱等的生物学标志性代谢产物变化。     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf

Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and γ-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine.     

Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

Divergent low water potential response in Arabidopsis thaliana accessions Landsberg erecta and Shahdara

The Arabidopsis thaliana accession Shahdara (Sha) differs from Landsberg erecta (Ler) and other accessions in its responses to drought and low water potential including lower levels of proline accumulation. However, Sha maintained greater seedling root elongation at low water potential and a higher NADP/NADPH ratio than Ler. Profiling of major amino acids and organic acids found that Sha had reduced levels of all glutamate family amino acids metabolically related to proline, but increased levels of aspartate‐derived amino acids (particularly isoleucine), leucine and valine at low water potential. Although Sha is known for its different abiotic stress response, RNA sequencing and co‐expression clustering found that Sha differed most from Ler in defence/immune response and reactive oxygen‐related gene expression. HVA22B and Osmotin34 were two of the relatively few abiotic stress‐associated genes differentially expressed between Ler and Sha. Insensitivity to exogenous glutamine and a different expression profile of glutamate receptors were further factors that may underlie the differing metabolism and low water potential phenotypes of Sha. These data define the unique environmental adaptation and differing metabolism of Sha including differences in defence gene expression, and will facilitate further analysis of Sha natural variation to understand metabolic regulation and abiotic/biotic stress interaction.     

The role of proline in thidiazuron-induced somatic embryogenesis of peanut

Summary Peanut seeds germinated on media supplemented with thidiazuron [TDZ: N-phenyl-N′-(1,2,3-thiadiazol-yl)urea], formed somatic embryos at the hypocotyledonary notch region by Day 35 of the culture period. Supplementation of the culture media with proline, thioproline, or glutamine reduced the total number of embryos formed, but the resulting embryos were larger, greener and had a more synchronous development than the regenerants formed on media containing TDZ alone. Analysis of the endogenous amino acid content of the germinating seeds during the induction phase of somatic embryogenesis revealed accumulation of proline to 6% of the dry seed weight. Concurrent with the emergence of the radicle, the proline concentration remained significantly elevated throughout the expression phase of embryogenesis. Several other amino acids including alanine, aspartate, asparagine, glutamate, glutamine, γ-aminobutyrate (GABA), hydroxyproline, isoleucine, threonine and valine accumulated to peak values approximately 10-fold higher than those of the controls. These results indicate that proline plays a key role in directing the route of TDZ-induced somatic embryogenesis and that TDZ effectively stimulates a cascade of metabolic events resulting in the production of specific metabolites, including amino acids, required for the regenerative process.