Free Amino Acid Pool Research Articles

Proteasome Inhibition in Brassica napus Roots Increases Amino Acid Synthesis to Offset Reduced Proteolysis.

Cellular homeostasis is maintained by the proteasomal degradation of regulatory and misfolded proteins, which sustains the amino acid pool. Although proteasomes alleviate stress by removing damaged proteins, mounting evidence indicates that severe stress caused by salt, metal(oids), and some pathogens can impair the proteasome. However, the consequences of proteasome inhibition in plants are not well understood and even less is known about how its malfunctioning alters metabolic activities. Lethality causes by proteasome inhibition in non-photosynthetic organisms stem from amino acid depletion, and we hypothesized that plants respond to proteasome inhibition by increasing amino acid biosynthesis. To address these questions, the short-term effects of proteasome inhibition were monitored for 3, 8 and 48 h in the roots of Brassica napus treated with the proteasome inhibitor MG132. Proteasome inhibition did not affect the pool of free amino acids after 48 h, which was attributed to elevated de novo amino acid synthesis; these observations coincided with increased levels of sulfite reductase and nitrate reductase activities at earlier time points. However, elevated amino acid synthesis failed to fully restore protein synthesis. In addition, transcriptome analysis points to perturbed abscisic acid signaling and decreased sugar metabolism after 8 h of proteasome inhibition. Proteasome inhibition increased the levels of alternative oxidase but decreased aconitase activity, most sugars and tricarboxylic acid metabolites in root tissue after 48 h. These metabolic responses occurred before we observed an accumulation of reactive oxygen species. We discuss how the metabolic response to proteasome inhibition and abiotic stress partially overlap in plants.

Omics Data Reveal Putative Regulators of Einkorn Grain Protein Composition under Sulfur Deficiency.

Understanding the molecular mechanisms controlling the accumulation of grain storage proteins in response to nitrogen (N) and sulfur (S) nutrition is essential to improve cereal grain nutritional and functional properties. Here, we studied the grain transcriptome and metabolome responses to postanthesis N and S supply for the diploid wheat einkorn (Triticum monococcum). During grain filling, 848 transcripts and 24 metabolites were differentially accumulated in response to N and S availability. The accumulation of total free amino acids per grain and the expression levels of 241 genes showed significant modifications during most of the grain filling period and were upregulated in response to S deficiency. Among them, 24 transcripts strongly responded to S deficiency and were identified in coexpression network analyses as potential coordinators of the grain response to N and S supply. Sulfate transporters and genes involved in sulfate and Met metabolism were upregulated, suggesting regulation of the pool of free amino acids and of the grain N-to-S ratio. Several genes highlighted in this study might limit the impact of S deficiency on the accumulation of grain storage proteins.

Leucine Supplementation Does Not Enhance Translation Initiation of Low Birth‐Weight Neonatal Pigs Despite Greater Sestrin2 Expression in Skeletal Muscle

Lean growth is impaired in low birth‐weight (LBWT) neonatal pigs and correlates with a reduction in protein synthesis rate in skeletal muscle. Translation initiation via mTOR signaling pathway is the rate limiting step in protein synthesis. Leucine binds and inactivates Sestrin2, an inhibitor of the mTORC1 complex. The objective of this study was to determine whether dietary branched‐chain amino acids (BCAA) stimulate translation initiation in skeletal muscle of LBWT pigs. LBWT and normal birth weight (NBWT) were defined when weight at birth was 2 SD below or within 0.5 of the corresponding litter average. Three day old LBWT and NBWT pigs (n=11‐13/group) were assigned to a Control or BCAA sow milk‐replacer formula in a 2×2 factorial arrangement. Formulas were isonitrogenous and contained either 1% BCAA (1:0.68:0.5 leucine:isoleucine:valine) or 0.7% alanine, and were offered at 250 mL·kg body weight−1·day−1 in 5 equal meals. Carotid and jugular catheters were surgically placed on day 21. On day 29, blood samples were taken over 4 h following a meal. Pigs were euthanized 1 h after a final meal. Data were analyzed using MIXED procedure of SAS version 9.4. BCAA supplementation increased BCAA concentration in plasma (P<0.0001) and in muscle free amino acid pool (P<0.001) compared to the Control diet, which similarly increased alanine concentration in plasma and muscle (P<0.0001). Although BCAA supplementation decreased circulating Pro, Ser, Asp, and Lys (P<0.001, 0.05, 0.05, 0.06, respectively), concentrations were not different in skeletal muscle. LBWT pigs expressed less BCAT2 mRNA in the longissimus dorsi than NBWT pigs, but expression was enhanced to normal levels with BCAA supplementation (P<0.05). BCKDHA expression was greater in the liver of LBWT compared to NBWT pigs irrespective of diet (P<0.05). Circulating branched‐chain α‐keto acids increased only in response to BCAA supplementation (P<0.0001). LBWT pigs expressed less Sestrin2 in skeletal muscle (P<0.01), suggesting enhanced sensitivity to leucine. However, despite lower expression of Sestrin2 and increased intramuscular BCAA concentrations, 4EBP1 and S6K1 phosphorylation were not affected by BCAA supplementation. These data suggest that branched‐chain amino acid supplementation did not enhance translation initiation in muscles of neonatal pigs regardless of birth weight.

Sources and Transformation Processes of Proteinaceous Matter and Free Amino Acids in PM2.5

AbstractAmino acids is an important organic nitrogen compound class in aerosol. Its origins and transformation in the atmosphere remain poorly understood. The concentrations of free amino acids (FAAs) and combined amino acids (CAAs) and δ15N values of free and combined Glycine (Gly) in PM2.5 and main emission sources (plant, soil, and aerosol from biomass burning) were investigated from five sampling sites in Nanchang area (Jiangxi Province, China). Our results showed that the composition profiles of CAAs and FAAs were quite different from each other in PM2.5. For CAAs, percentages of individual combined amino acids were different between biomass burning sources and plant or soil sources, and we found that the composition profiles of aerosol CAAs could reflect the contribution of the main emission sources. For FAAs, a predominance of free Gly was observed in aerosol FAAs pool because atmospheric photooxidative processes could increase free Gly and decrease other reactive FAAs. This implied that the composition of aerosol FAAs could not reflect their origins. A strong correlation between δ15NF‐Gly values in aerosols or sources and the δ15NC‐Gly values in their parent CAAs was observed, indicating that the isotope effect associated with Gly transformation in aerosols may be small. Additionally, we found more positive δ15NGly in aerosols emitted by biomass burning, which was likely due to 15N‐depleted amino‐N loss from Gly molecule. The results suggested that δ15NGly values in aerosols might be used to identify biomass burning sources.

Utilization of GC-MS untargeted metabolomics to assess the delayed response of glufosinate treatment of transgenic herbicide resistant (HR) buffalo grasses (Stenotaphrum secundatum L.).

Herbicide resistant (HR) buffalo grasses were genetically engineered to resist the non-selective herbicide, glufosinate in order to facilitate a modern, 'weeding program' which is highly effective in terms of minimizing costs and labor. The resistant trait was conferred by an insertion of the pat gene to allow for the production of the enzyme phosphinothricin acetyltransferase (PAT) to detoxify the glufosinate inhibitive effect. To date, there are only a few reports using metabolomics as well as molecular characterizations published for glufosinate-resistant crops with no reports on HR turfgrass. Therefore, for the first time, this study examines the metabolome of glufosinate-resistant buffalo grasses which not only will be useful to future growers but also the scientific community. A major aim of this present work is to characterize and evaluate the metabolic alterations which may arise from a genetic transformation of HR buffalo grasses by comprehensively using gas chromatography-mass spectrometry (GC-MS) based untargeted metabolomics. Eight-week old plants of 4 HR buffalo grasses, (93-1A, 93-2B, 93-3C and 93-5A) and 3 wild type varieties (WT 8-4A, WT 9-1B and WT 9-1B) were selected for physiological, molecular and metabolomics experiments. Plants were either sprayed with 1, 5, 10 and 15% v/v of glufosinate to evaluate the visual injuries or submerged in 5% v/v of glufosinate 3days prior to a GC-MS based untargeted metabolomics analysis. In contrast, the control group was treated with distilled water. Leaves were extracted in 1:1 methanol:water and then analysed, using an in-house GC-MS untargeted workflow. Results identified 199 metabolites with only 6 of them (cis-aconitic acid, allantoin, cellobiose, glyceric acid, maltose and octadecanoic acid) found to be statistically significant (p < 0.05) between the HR and wild type buffalo grass varieties compared to the control experiment. Among these metabolites, unusual accumulation of allantoin was prominent and was an unanticipated effect of the pat gene insertion. As expected, glufosinate treatment caused significant metabolic alterations in the sensitive wild type, with the up-regulation of several amino acids (e.g. phenylalanine and isoleucine) which was likely due to glufosinate-induced senescence. The aminoacyl-tRNA biosynthetic pathway was identified as the most significant enriched pathway as a result of glufosinate effects because a number of its intermediates were amino acids. HR buffalo grasses were very similar to its wild type comparator based on a comprehensive GC-MS based untargeted metabolomics and therefore, should guarantee the safe use of these HR buffalo grasses. The current metabolomics analyses not only confirmed the effects of glufosinate to up-regulate free amino acid pools in the sensitive wild type but also several alterations in sugar, sugar phosphate and organic acid metabolism have been reported.

Abstract C50: Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state

Abstract Tumors contain a heterogeneous mixture of live cells that are either undergoing proliferation or reside in a nonproliferative state. Cells that do not divide are often associated with increased chemoresistance, challenging therapies that target rapidly proliferating cells. In pancreatic cancer, paucity of blood vessels frequently generates hypoperfused, “arid” regions that constrain tumor cell metabolism and proliferation. Nonetheless, experimental conditions for studying tumor cells in vitro typically mimic well-nourished, “fertile” environments optimized for supporting proliferating cells rather than the more prevalent quiescent state. Characterization of the poorly vascularized KPC model confirmed that the quiescent phenotype is common and is strongly correlated to reduced tissue perfusion. To formulate culture conditions that will emulate the arid microenvironment associated with the nonproliferative state in vitro, we systematically deprived various nutrients from tumor cells and established the necessary components for attaining cell quiescence. Deprivation of amino acids on top of glucose, oxygen, and serum levels was essential for obtaining a complete and reversible cell cycle arrest without loss of viability. Cell cycle arrest was accompanied by a number of physiologic adaptations. Metabolomic analysis indicated reduction in the levels of TCA cycle and mevalonate pathway intermediates indicative of increased reliance on nonglycolytic metabolism. In addition, free amino acid pools were diminished and tumor cells exhibited a significant increase in autophagic flux and micropinocytosis, suggesting that amino acid availability is limited under arid conditions. Concomitant with reduced proliferation and metabolic rewiring, tumor cells under arid conditions exhibited remarkable resistance to gemcitabine. To explore alternative strategies to target these nonproliferating pancreatic tumor cells, we performed a comprehensive genome-wide CRISPR/Cas9-based genetic screen and compared vulnerabilities under ”fertile” and “arid” conditions. Functional annotation of sgRNAs depleted under arid conditions indicated a striking dependence on oxidative phosphorylation for survival. Remarkably, our analysis also revealed that a number of targetable genetic and epigenetic programs driving cell cycle progression were not essential and, in some instances, even hazardous for cells under arid conditions. These results suggest that pancreatic tumor cells that cease proliferation in a nutrient-depleted environment are physiologically rewired and may exhibit a distinct drug response profile from rapidly proliferating cells. The experimental model we developed allows investigation of this dominant yet understudied cell population and may serve as a platform for identification of agents targeting the quiescent fraction of tumors. Citation Format: Yogev Sela, Jinyang Li, Miriam Doepner, Shivahamy Maheswaran, Clementina Mesaros, Ian Blair, Ophir Shalem, Ben Stanger. Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C50.

Effect of blockade of synthesis of nitric oxide by L-NAME on the level of free amino acids and biogenic amines in the brain cortex of rats with subtotal cerebral ischemia

Mechanisms of development of ischemic stroke are complex and have not been fully established. The aim of this study was to estimate the changes in the pool of free amino acids and biogenic animes in the brain cortex of rats with subtotal cerebral ischemia (SCI) and treated with L-NAME. Experiment was made on 18 rats: 12 animals were undergoing bilateral filament occlusion of arteries carotid, 6 of them were treated with L-NAME. The analyses of free amino acids levels in the blood plasma extracts were carried out by reversed phase HPLC.Concentrations of several amino acids were elevated during SCI including aspartate, b-alanine, valine and leucine. In contrast, the levels of glutamate, asparagine, treonine, gamma-aminobutiric acid, tyrosine and 5-hydroxiindolylacetate were decreased. The administration of L-NAME partially prevented the imbalance of the amino acids pool due to SCI by normalizing the levels of aspartate, glutamate, asparagine, methionine, gamma-aminobutiric acid, b-alanine, 5-hydroxiindolylacetate. However, the administration of L-NAME has induced an additional imbalance in the amino acids pool in the brain cortex (decrease in the levels of glutamine, histidine, taurine, tryptophan, phenylalanine, tyrosine (in comparison to SCI) and decrease in the levels of treonine and arginine. The imbalance of the amino acids pool induced by the administration of L-NAME during SCI is more severe than the imbalance caused by SCI.

Pool of free amino acids in the blood plasma of rats undergoing subtotal cerebral ischemia after L-name administration

A stroke is one of the leading causes of morbidity, disability and mortality in many countries. Mechanisms of development of ischemic stroke are complex and have not been fully established. The aim of this study was to estimate the changes in the pool of free amino acids and their derivatives in the plasma of rats undergoing subtotal cerebral ischemia (SCI) and treated with L-NAME. Experiment was made on 18 rats: 12 animals were undergoing bilateral flament occlusion of arteries carotid, 6 of them were treated with L-NAME. The analyses of free amino acids levels in the blood plasma extracts were carried out by reversedphase HPLC. Concentrations of several amino acids were elevated after 1 hour of ischemia, including aspartate, asparagine, glutamine, glycin, alanine, taurine, phenylalanine, gistidine, 3-methilgistidine, treonine, citrulline, ornitine, as well as branched-chain amino acids. Administration of L-NAME partially prevented the imbalance of the amino acids pool caused by SCI. Preventive injection of L-NAME alleviated the imbalance in the pool of free amino acids of blood plasma caused by SCI.

Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self-sufficient for weeks.

During the life cycle of plants, seedlings are considered vulnerable because they are at the interface between the highly stress tolerant seed embryos and the established plant, and must develop rapidly, often in a challenging environment, with limited access to nutrients and light. Using a simple experimental system, whereby the seedling stage of Arabidopsis is considerably prolonged by nutrient starvation, we analysed the physiology and metabolism of seedlings maintained in such conditions up to 4weeks. Although development was arrested at the cotyledon stage, there was no sign of senescence and seedlings remained viable for weeks, yielding normal plants after transplantation. Photosynthetic activity compensated for respiratory carbon losses, and energy dissipation by photorespiration and alternative oxidase appeared important. Photosynthates were essentially stored as organic acids, while the pool of free amino acids remained stable. Seedlings lost the capacity to store lipids in cytosolic lipid droplets, but developed large plastoglobuli. Arabidopsis seedlings arrested in their development because of mineral starvation displayed therefore a remarkable resilience, using their metabolic and physiological plasticity to maintain a steady state for weeks, allowing resumption of development when favourable conditions ensue.

PECULIARITIES OF METABOLISM OF FREE PLASMA AMINO ACIDS IN THE BLOOD OF RATS UNDER THE EXPOSURE TO SODIUM FLUORIDE

To assess and substantiate the structural and metabolic mechanisms underlying the formation of intoxication, we studied the state of the pool of free plasma amino acids under the influence of sodium fluoride in the context of chronic fluoride intoxication in white rats of the Wistar population, which were orally administered sodium fluoride for 1.5 months at a dose of 20 mg / kg of body mass. Evaluation of the state of protein metabolism was conducted according to the spectrum of free plasma amino acids by ion exchange chromatography on ionites. Our studies have shown a decrease in plasma concentrations of free amino acids such as alanine, a-aminobutyric acid, threonine, serine, proline, valine, lysine, and an increase in taurine, aspartic, glutamic acid, glycine, and cysteine. The dynamics of cystine, methionine, leucine, isoleucine, cystianine, tyrosine, phenylalanine, ornithine, histidine, arginine, ammonia were not disrupted as compared with the control observation group. The decrease in the protein synthesizing function and the prevalence of catabolic processes over anabolic processes was also confirmed by the inhibition of the incorporation processes of 3H-thymidine, 3H-uridine and 14C-leucine in hepatocytes and splenocytes, which once again indicates a slowdown of synthetic processes.

Annurca Apple Polyphenols Protect Murine Hair Follicles from Taxane Induced Dystrophy and Hijacks Polyunsaturated Fatty Acid Metabolism toward β-Oxidation.

Chemotherapy-induced alopecia (CIA) is a common side effect of conventional chemotherapy and represents a major problem in clinical oncology. Even months after the end of chemotherapy, many cancer patients complain of hair loss, a condition that is psychologically difficult to manage. CIA disturbs social and sexual interactions and causes anxiety and depression. Synthetic drugs protecting from CIA and endowed with hair growth stimulatory properties are prescribed with caution by oncologists. Hormones, growth factors, morphogens could unwontedly protect tumour cells or induce cancer cell proliferation and are thus considered incompatible with many chemotherapy regimens. Nutraceuticals, on the contrary, have been shown to be safe and effective treatment options for hair loss. We here show that polyphenols from Malus Pumila Miller cv Annurca are endowed with hair growth promoting activity and can be considered a safe alternative to avoid CIA. In vitro, Annurca Apple Polyphenolic Extract (AAE) protects murine Hair Follicles (HF) from taxanes induced dystrophy. Moreover, in virtue of its mechanism of action, AAE is herein proven to be compatible with chemotherapy regimens. AAE forces HFs to produce ATP using mitochondrial β-oxidation, reducing Pentose Phosphate Pathway (PPP) rate and nucleotides production. As consequence, DNA replication and mitosis are not stimulated, while a pool of free amino acids usually involved in catabolic reactions are spared for keratin production. Moreover, measuring the effect exerted on Poly Unsaturated Fatty Acid (PUFA) metabolism, we prove that AAE promotes hair-growth by increasing the intracellular levels of Prostaglandins F2α (PGF2α) and by hijacking PUFA catabolites toward β-oxidation.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5–15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

Metabolic Alternations of Amino Acids, γ-Aminobutyric Acid, and Salicylic Acid in Solanum lycopersicum (L.) Following Preplanting Seedling Spray with Salicylic Acid.

Preplanting foliar spray of salicylic acid (SA) (0.0, 5.0, and 10.0 μg/mL) to Solanum lycopersicum (L.) altered the metabolite profile of amino acids, γ-aminobutyric acid (GABA), and SA in leaf, root, and fruits. Free amino acid pools increased; bound amino acid pools reduced. In vegetative tissues, amino acid biosyntheses linked to osmo-compatibility (Pro, Leu, Val and GABA); N (Arg, Asn, Asp, Gln, and Glu); C (Pro, Ser, and Tyr); S (Cys) assimilation; stress tolerance (Ala, Gly, Hyp, His, Lys, Met, and Thr); and central metabolism (Phe, Trp, and Tyr) enhanced for 60-120 days. Concentrations of Ala, Arg, Gln, Gly, Leu, and Ser in leaf and of Asp, Cys, Glu, His, Hyp, Lys, Met, Pro, and Val in root predominated. In planta SA and GABA biosynthesis increased concurrently. SA affected GABA biosynthesis via Glu, Pro, and Arg metabolism. SA, GABA, Glu, and Pro were key canonical variables. This study first reported SA-induced metabolites promoting health (SA/GABA; Cys/Met) and palatability (Glu/Asp; Gln) in table tomato.

Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response.

During abiotic stress low abundant amino acids are not synthesized but they accumulate due to increased protein turnover under conditions inducing carbohydrate starvation (dehydration, salt stress, darkness) and are degraded. Metabolic adaptation is crucial for abiotic stress resistance in plants, and accumulation of specific amino acids as well as secondary metabolites derived from amino acid metabolism has been implicated in increased tolerance to adverse environmental conditions. The role of proline, which is synthesized during Arabidopsis stress response to act as a compatible osmolyte, has been well established. However, conclusions drawn about potential functions of other amino acids such as leucine, valine, and isoleucine are not entirely consistent. This study reevaluates published datasets with a special emphasis on changes in the free amino acid pool and transcriptional regulation of the associated anabolic and catabolic pathways. In order to gain a comprehensive overview about the general direction of amino acid metabolism under abiotic stress conditions a complete map of all currently known enzymatic steps involved in amino acid synthesis and degradation was assembled including also the initial steps leading to the synthesis of secondary metabolites. Microarray datasets and amino acid profiles of Arabidopsis plants exposed to dehydration, high salinity, extended darkness, cold, and heat were systematically analyzed to identify trends in fluxes of amino acid metabolism. Some high abundant amino acids such as proline, arginine, asparagine, glutamine, and GABA are synthesized during abiotic stress to act as compatible osmolytes, precursors for secondary metabolites, or storage forms of organic nitrogen. In contrast, most of the low abundant amino acids are not synthesized but they accumulate due to increased protein turnover under conditions inducing carbohydrate starvation (dehydration, salt stress, extended darkness) and are degraded.

Title: Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants

Gamma-Amino Butyric Acid (GABA) is a substantial component of the free amino acid pool with low concentration in plant tissues. Enhanced GABA content occurs during plant growth and developmental processes like seed germination. GABA level, basically, alters in response to many endogenous and exogenous stimuli. In the current study, GABA effects were studied on germination, photosynthetic performance and oxidative damages in salt-exposed lettuce plants. Three NaCl (0, 40 and 80 mM) and two GABA (0 and 25 μM) concentrations were applied on lettuce during two different developmental (seed germination and seedlings growth) stages. Negative effects of salinity on germination and plant growth were removed by GABA application. GABA significantly reduced mean germination time (MGT) in salt-exposed lettuce seeds. Although, salinity caused a significant decline in maximum quantum yield of photosystem II (Fv/Fm) during distinct steps of plant growth, GABA application improved Fv/Fm particularly on high salinity level. GABA decreased specific energy fluxes per reaction center (RC) for energy absorption and dissipation, while enhanced-electron transport flux in photosynthetic apparatus of lettuce plants was observed in GABA-supplemented plants. Moreover, decline in non-photochemical quenching (NPQ) and quenching coefficients (qP, qL, qN) by salt stress were recovered by GABA application. Elevated electrolyte leakage considerably decreased by GABA exposure on salt-treated plants. Although, proline level increased by NaCl treatments in a concentration dependent manner, combined application of salt with GABA caused a significant reduction in proline content. Catalase; EC 1.11.1.6 (CAT), l-ascorbate peroxidase; EC 1.11.1.11 (APX), and superoxide dismutase; EC 1.15.1.1 (SOD) activities were increased by GABA exposure in salt-supplemented plants that resulted in regulated hydrogen peroxide level. In conclusion, a multifaceted role for GABA is suggested for minimizing detrimental effects of salinity on lettuce through improvement of photosynthetic functionality and regulation of oxidative stress.

Salt tolerance mechanisms in three Irano-Turanian Brassicaceae halophytes relatives of Arabidopsis thaliana.

Salt tolerance mechanisms were studied in three Irano-Turanian halophytic species from the Brassicaceae ‎‎(Lepidium latifolium, L. perfoliatum and Schrenkiella parvula) and compared with the glycophyte Arabidopsis thaliana. According to seed germination under salt stress, L. perfoliatum was the most tolerant species, while L. latifolium and S. parvula were rather susceptible. Contrastingly, based on biomass production L. perfoliatum was more salt sensitive than the other two species. In S. parvula biomass was increased up to 2.8-fold by 100mM NaCl; no significant growth reduction was observed even when exposed to 400mM NaCl. Stable activities of antioxidative defense enzymes, nil or negligible accumulation of superoxide anion and hydrogen peroxide, as well as stable membrane integrity in the three halophytes revealed that no oxidative stress occurred in these tolerant species under salt stress. Proline levels increased in response to salt treatment. However, it contributed only by 0.3‒2.0% to the total osmolyte concentration in the three halophytes (at 400mM NaCl) and even less (0.04%) in the glycophyte, A. thaliana (at 100mM NaCl). Soluble sugars in all three halophytes and free amino acids pool in S. parvula decreased under salt treatment in contrast to the glycophyte, A. thaliana. The contribution of organic osmolytes to the total osmolyte pool increased by salt treatment in the roots, while decreased in halophyte and glycophyte, A. thaliana leaves. Interestingly, this reduction was compensated by a higher relative contribution of K in the leaves of the halophytes, but of Na in A. thaliana. Taken together, biomass data and biochemical indicators show that S. parvula is more salt tolerant than the two Lepidium species. Our data indicate that L. latifolium, as a perennial halophyte with a large biomass, is highly suitable for both restoration of saline habitats and saline agriculture.

Measurement duration affects the calculation of whole body protein turnover kinetics but not between-day variability

BackgroundAssessment of whole body protein turnover (WBPT) can provide fundamental information about protein kinetics which underpins the conservation of lean tissue. Reliability and methodology studies on the measurement of WBPT are scarce. This study aimed to assess the effects of urine collection duration (9 versus 12 h) and the reproducibility of WBPT with the end product method calculated from ammonia as the end product. MethodsWBPT was assessed in 21 healthy participants (11M, 10F) on 2 test days. WBPT was assessed using the end product method with a single dose of 15N glycine with ammonia as end product in a postprandial state with 9 and 12-h urine collections. ResultsThe CV for protein flux averaged 10% and 12% for 9 and 12-h urine collections respectively. Protein flux, synthesis and balance were significantly higher and protein breakdown significantly lower with 9-h urine collections compared to 12-h collections (P < 0.01) and there was a trend towards increasingly greater overestimation of 9-h calculated WBPT kinetics with greater overall rates of WBPT. Correlations between the 9 and 12-h values were strong (r > 0.962, P < 0.001 for all variables). ConclusionsThe reproducibility of WBPT with ammonia as the end product was similar to previously reported reproducibility of the gold standard precursor technique. The use of a 12-h urine collection is more effective to achieve full turnover of the ammonia free amino acid (AA) pool.

Effect of freezing and drying processes on the molecular traits of edible yellow mealworm

Abstract Yellow mealworm (Tenebrio molitor L.) represents a highly sustainable source of proteins for food and feed. Industrial production of mealworm meals for food and feed must count on optimized processing methods based on a deep knowledge of nutritional and quality aspects. Viable mealworm larvae (VL) were frozen at −20 °C and dried under two different thermal conditions, namely high-temperature-short-time (HTST, 90 °C for 1.5 h) and low-temperature-long-time (LTLT, 50 °C for 62 h). Proximate composition, fatty acid analyses by gas-chromatography and metabolic profiling by means of proton nuclear magnetic resonance (1H NMR) spectroscopy were carried out and 1H NMR data investigated with multivariate data analysis (MVDA). While fatty acid profiles did not indicate significant differences among treatments, 1H NMR highlighted relevant molecular alterations associated to LTLT drying (95% of detected metabolites are altered by LTLT). In particular, detrimental hydrolysis of triacylglycerols (TAG) was favored during LTLT drying (approximately 25% reduction of TAG in LTLT compared to VL), accompanied by the enrichment of the free amino acid pool. Larvae composition was only minimally affected by the freezing process, with only 15% of the metabolite pool affected. Industrial relevance Sustainable industrial production of insect-derived products (insect meal, oil, and other extracts) must count on efficient processes and high standard quality. Freezing and drying are the most critical processing operations in insect industry, since they can strongly affect the quality of final products. It is demonstrated here that low-temperature-long time drying processes negatively affect insect products quality, while freezing and high-temperature-short time drying do not have a significant impact. Molecular details of nutrient degradation processes are provided.

Effects of Cold Shock on Responses of Phosphomonoesters and Free Amino Acids in Phospholipid-Rich Organs in the Amur Sleeper Perccottus Glehni

Similarities and differences were found in the mechanisms of adaptation of the brain and liver to cold shock in the freshwater fish Perccottus glehni. The quantity of phosphoethanolamine in the brain on day 4 of cold shock at +4°C increased 84-fold from the level in controls (+20°C), accounting for 22.3% of the total pool of free amino acids and ninhydrin-positive compounds. Phosphoserine was absent from the brain at +20°C, but cold shock initiated its accumulation to 1.8% of the total pool. The taurine pool at +4°C decreased from 28.3 to 20% of the total pool, yielding first place to phosphoethanolamine. However, in the liver, phosphoethanolamine (PE) and phosphoserine (PS) were not seen in the free form either in controls or at 4°C. The dominant amino acid in the liver at both temperatures was taurine, the quantity at +4°C being significantly increased. Th glutamate pool decreased nine-fold at +4°C. It is suggested that the intense accumulation of PE and PS in the brain at low temperatures is evidence of the specific features of the adaptation of the neuron membrane, reflecting quantitative changes in phospholipids, possibly including sphingosine-1-phosphate.

Variations in free amino acid concentrations in mosses and different parts of Cinnamomum camphora along an urban-to-rural gradient

Vegetation growing in urban ecosystems is frequently exposed to an environment with high atmospheric nitrogen (N) pollution. We systematically investigated the free amino acid concentrations in moss samples and Cinnamomum camphora leaf (new, middle-aged and old leaves), branch phloem, trunk phloem, and bark samples from field sites (Guiyang City, SW China) with different N deposition levels. The responses of the free amino acids to N deposition were analysed in the abovementioned plant tissues to determine whether plant free amino acids could act as biomarkers of the external N supply; moreover, differences in the N metabolism of these tissues under varying N deposition conditions were revealed by the compositions and sizes of their free amino acid pools. In particular, we reported the significant accumulation of arginine with increased N deposition in bark samples (while the arginine concentrations in the branch phloem, trunk phloem, and leaves remained low), which may indicate a long-term or historic external environment with a high N availability; additionally, the noticeable dominance and fluctuation of γ-aminobutyric acid in response to varied N deposition levels occurred in both the branch phloem and the trunk phloem, suggesting that the γ-aminobutyric acid transported in the phloem may be used as an important signal reflecting increases in the atmospheric N input. We conclude that the free amino acid concentrations in moss and camphor leaf, phloem and bark tissues are more sensitive to N deposition compared to their N concentrations and that tissue glutamine/glutamate and arginine/γ-aminobutyric acid ratios may serve as better biomarkers reflecting the tissue N accumulation status associated with increased N deposition. Therefore, free amino acid concentration analyses of different plant parts may provide a means to gain a more in-depth understanding of the impacts of atmospheric N pollution on plant physiology and N cycles.