GABA Formation Research Articles

Участь γ-аміномасляної кислоти у клітинних сигнальних процесах і в адаптації рослин до дії абіотичних стресорів

Adaptation of plants to stress factors occurs with the participation of stress phytohormones, signaling network and plant neurotransmitters. Among the latter, in particular, γ-aminobutyric acid (γ-aminobutyric acid – GABA) is a non-proteinogenic four-carbon amino acid found in many prokaryotic and eukaryotic organisms. Its functions in plants have been actively studied only in the past decade. During this period, a lot of information has been accumulated about the protective effect of exogenous GABA on plants of various taxonomic groups under the influence of stress factors of various nature. The first national review is devoted to the analysis and generalization of data on the mechanisms of stress-protective action of GABA in plants. The ways of synthesis and metabolism of GABA in plant cells and the mechanisms of activation of these processes under stressful conditions are described. It is noted that the main way of GABA formation in plants is decarboxylation of glutamate by means of glutamate decarboxy­lase. Possible mechanisms of GABA reception and signal transmission to the genetic apparatus are discussed. Special attention is paid to the analysis of new data on the role of calcium in the activation of GABA synthesis and the realization of its physiological effects. Possible mechanisms of GABA’s influence on the functioning of mitochondria, its role in maintaining redox homeostasis under stressful conditions are discussed. At the same time, data on the increase in the expression of genes encoding the catalytic subunit of NADPH oxidase under the influence of GABA are presented. Functional connections between GABA and nitric oxide as a signaling mediator are considered. The effect of exogenous GABA on the main protective reactions of plants is characte­rized: the state of the antioxidant system, the accumulation of multifunctional low-molecular protectors, the synthesis of dehydrins and chaperones. The data on the phenomeno­logy of the effects of GABA under the main abiotic stresses are presented: the effects of extreme temperatures, drought and salinity on plants. The prospects for the practical use of GABA as a compound that combines the functions of an energy metabolite and a signaling mediator are noted.

Culture-dependent and -independent analyses of bacterial compositions and its contributions to formation of γ-aminobutyric acid and poly-γ-glutamic acid in Cheonggukjang

This study was performed to unveil bacterial compositions and their contributions to the formation of γ-aminobutyric acid (GABA) and poly-γ-glutamic acid (γ-PGA) in Cheonggukjang. To predict possible key factors contributing to the content of the bioactive compounds in Cheonggukjang, commercial products were analyzed for various parameters. The content of GABA and γ-PGA showed a negative (R2 = 0.61 – 0.73) and positive correlation (R2 = 0.53 – 0.96) with antioxidative activity. Consistently, GABA content showed a moderate negative correlation with γ-PGA content (R2 = 0.58). Among the physicochemical and microbial parameters, only salinity showed a moderate negative correlation with γ-PGA content (R2 = 0.75), which might be due to the inhibition of bacterial growth. It was also suggested that multiple factors (including bacterial species) were involved in the formation of GABA and γ-PGA in Cheonggukjang. To reveal dominant bacterial species and further presume their contributions to the bioactive compound formation in Cheonggukjang, both culture-independent (metagenomic) and -dependent (culturomic) methods were used. Culture-independent method showed that Bacillus piscis was dominant (23.37 – 94.89 %), followed by B. hisashii (0.00 – 62.45 %) and B. coagulans (0.00 – 13.82 %). Considering the quantitative speciation data on the bioactive compound content in Cheonggukjang (and bacterial production capability) together, it was further elucidated that B. piscis contributed primarily to the bioactive compound formation. Unlike this, culture-dependent analysis revealed that B. licheniformis and B. subtilis were dominant (30.0 – 47.6 and 17.5 – 39.5 %, respectively). Based on the quantitative speciation data on the bacterial production capability of GABA and γ-PGA, B. subtilis was the primarily contributing bacterial species to the bioactive compound formation. Consequently, it was observed that the bacterial compositions and their contributions to the bioactive compound formation determined by the two methods differed considerably, i.e., B. piscis and B. subtilis were identified to be prominent bacterial contributors, respectively, depending on the method used.

Gut production of GABA by a probiotic formula: an in vitro study.

The objective of this study was to evaluate the influence of a probiotic formula on the production of neuroactive compounds in different parts of the colon in batch culture and in vitro gut simulator experiments. Thirteen lactic acid bacterial strains, belonging to the species Levilactobacillus brevis, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Ligilactobacillus salivarius, Streptococcus thermophilus, were characterised for their in vitro ability to produce neurotransmitters. L. brevis P30021 and L. plantarum P30025 were selected based on their capability to produce γ-aminobutyric acid (GABA) and acetylcholine in vitro. A probiotic formulation with potential psychobiotic activity was prepared and tested in a batch culture of human microbiota monitoring the formation of GABA and acetylcholine. Samples of the three colonic tracts were taken from the Simulator of the Human Intestinal Microbiota (SHIME®) evaluating the production of GABA and other neurotransmitters by LC-MS. Short chain fatty acids (SCFA) by GC and microbiota composition by 16S rRNA gene sequencing were also determined. Probiotic supplementation led to the formation of GABA and acetylcholine with a decrease in glutamate concentrations in the in vitro batch fermentation. Production of GABA after the treatment with probiotics was confirmed in the SHIME® short-term experiment. No differences in short-chain fatty acids were observed up to 72h of fermentation. Different microbiota composition was found in the three different parts of the colon, with a higher abundance of Veillonellaceae in the ascending colon vessels. The probiotic-exposed microbiota showed higher levels of Bacteroides, a gut microbe associated with anti-inflammatory activities and a potential GABA producer. Results demonstrate the impact of the tested probiotic formula on gut microbiota structure and GABA production. In conclusion, the probiotic treatment changed the microbiota composition and increased neuroactive metabolites production, indicating promising potential as psychobiotics, even if further clinical evidence is needed to confirm the effectiveness of these probiotics in improving mental health.

Polymorphism and Multi-Component Crystal Formation of GABA and Gabapentin.

This study exploits the polymorphism and multi-component crystal formation of γ-amino butanoic acid (GABA) and its pharmaceutically active derivative, gabapentin. Two polymorphs of GABA and both polymorphs of gabapentin are structurally revisited, together with gabapentin monohydrate. Hereby, GABA form II is only accessible under special conditions using additives, whereas gabapentin converts to the monohydrate even in the presence of trace amounts of water. Different accessibilities and phase stabilities of these phases are still not fully clarified. Thus, indicators of phase stability are discussed involving intermolecular interactions, molecular conformations, and crystallization environment. Calculated lattice energy differences for polymorphs reveal their similar stability. Quantification of the hydrogen bond strengths with the atoms-in-molecules (AIM) model in conjunction with non-covalent interaction (NCI) plots also shows similar hydrogen bond binding energy values for all polymorphs. We demonstrate that differences in the interacting modes, in an interplay with the intermolecular repulsion, allow the formation of the desired phase under different crystallization environments. Salts and co-crystals of GABA and gabapentin with fumaric as well as succinic acid further serve as models to highlight how strongly HBs act as the motif-directing force in the solid-phase GABA-analogs. Six novel multi-component entities were synthesized, and structural and computational analysis was performed: GABA fumarate (2:1); two gabapentin fumarates (2:1) and (1:1); two GABA succinates (2:1) and (1:1); and a gabapentin:succinic acid co-crystal. Energetically highly attractive carboxyl/carboxylate interaction overcomes other factors and dominates the multi-component phase formation. Decisive commonalities in the crystallization behavior of zwitterionic GABA-derivatives are discussed, which show how they can and should be understood as a whole for possible related future products.

Effect of a co-fermentation system with high-GABA-yielding strains on soymilk properties: microbiological, physicochemical, and aromatic characterisations

Biosynthesis is the safest method for preparing GABA; however, there are not enough GABA-producing strains to provide an effective resource. The purpose of this study was to determine the feasibility of using Lactobacillus fermentum SMN10-3(A) and Lactococcus lactis SMN15-6(B) to study the effects of strain complex pairing on the GABA formation, flavour, and metabolic pathways of fermented soymilk. It was found that group A2B1 had the highest acid production rate, GABA yield (1.76 ± 0.01 mg/mL), and flavour compound content. A total of 55 differential metabolites were produced after fermentation, of which 28 dominated by hexanal were significantly downregulated and 26 dominated by alcohols were significantly upregulated. The significant metabolic pathways involved were d-alanine, taurine and hypotaurine, and selenocompound metabolism. Finally, the components contributing to the aroma of fermented soymilk were identified, which included 2-pentylfuran and 2-butyl-2-octenal. These results provide a theoretical basis for future research on GABA-rich fermented foods.

Foliar application of procyanidins enhanced the biosynthesis of 2-acetyl-1-pyrroline in aromatic rice (Oryza sativa L.)

BackgroundProcyanidins is a polyphenolic compound with multiple properties. However, the application of exogenous procyanidins in crops has not been reported. Aromatic rice is a high-quality rice with a special aroma and popular with consumers. The 2-acetyl-1-pyrroline (2-AP) is a key compound of aromatic rice aroma. In the current study, aromatic rice plants were sprayed with procyanidins solutions at 0.25 (Pr0.25), 0.50 (Pr0.50), 1.00 (Pr1.00), 2.00 (Pr2.00) g L−1, respectively and treatment sprayed with distilled water was taken as control (CK). The effects of exogenous procyanidins on growth and 2-AP biosynthesis of aromatic rice plants were explored.ResultsCompared with CK, Pr1.00 and Pr2.00 treatments significantly increased 2-AP content by 16.67% and 37.68%, respectively. Higher proline, 1-pyrroline-5-carboxylic acid (P5C), 1-pyrroline, methylglyoxal contents, and lower γ- aminobutyric acid (GABA) content were recorded in Pr1.00 and Pr2.00 treatments than CK. Compared with CK, Pr1.00 and Pr2.00 treatments significantly improved the activities of P5CS and OAT and diminished the activity of BADH. Furthermore, compared with CK, Pr1.00 and Pr2.00 treatments significantly up-regulated the transcript levels of P5CS2, P5CR, OAT, DAO4 and down-regulated the transcript levels of BADH2. Exogenous procyanidins had no substantial effects on plant height, stem diameter, fresh weight, and dry weight of aromatic rice plants.ConclusionsIn conclusion, our findings reported the increment of 2-AP content in aromatic rice under exogenous procyanidins. Our results indicated that the application of exogenous procyanidins enhanced 2-AP biosynthesis by improving proline biosynthesis and inhibiting GABA formation.

Production of plant-based fermented beverages possessing functional ingredients antioxidant, γ-aminobutyric acid and antimicrobials using a probiotic Lactiplantibacillus plantarum strain L42g as an efficient starter culture

Plant-based probiotic beverages have gained increasing interest due to demand from health-conscious consumers. In this study, we aimed to isolate and screen lactic acid bacteria possessing functional properties for use as a starter culture of fermented almond and coix beverages. Lactiplantibacillus plantarum L42g isolated from fermented beef was selected. Both intact cells and cell free supernatant of this strain exhibited high antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging at 38.2% and 44.9%, respectively. L.plantarum L42g grown in MRS broth supplemented with 1% (w v-1) monosodium glutamate (MSG) produced a large amount of γ-aminobutyric acid (GABA) at 496.7μgmL-1. Moreover, strain L42g displayed remarkable antibacterial activity against several potential foodborne bacterial pathogens, including Bacillus cereus, Listeria monocytogenes, Listeria inocua, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, Shigella sp., Vibrio cholerae and Vibrio parahaemolyticus. Strain L42g also possessed additional probiotic properties including abilities to tolerate gastrointestinal conditions, adhere to gut mucosa, co-aggregate with pathogens, be susceptible to antibiotics, and produce protease. Probiotic strain L42g was subsequently employed in fermenting almond and coix juices containing MSG (1%) supplementation. Levels of antioxidant, GABA and antibacterial formation along with cell growth were clearly higher in fermented almond juice than in fermented coix juice. Nonetheless, both fermented almond and coix juices meet the standards required for the consumption of fermented beverages. Therefore, L.plantarum strain L42g represents a promising starter culture for producing functional plant-based probiotic beverages.

CsCuAOs and CsAMADH1 Are Required for Putrescine-Derived γ-Aminobutyric Acid Accumulation in Tea.

Polyamines are a potential source of γ-aminobutyric acid (GABA) in plants under abiotic stress. However, studies on GABA enrichment in tea mostly focus on the GABA shunt, while the correlation between polyamine degradation and GABA formation in tea is largely unknown. In this study, tea plants responded to exogenous putrescine, resulting in a significant increase in GABA content, while the glutamate level did not change. At the same time, five copper-containing amine oxidase (CuAO) and eight aminoaldehyde dehydrogenase (AMADH) genes involved in the putrescine-derived GABA pathway were identified from the Tea Plant Information Archive. Expression analysis indicated that CsCuAO1, CsCuAO3 as well as CsAMADH1 were induced to play an important function in response to exogenous putrescine. Thus, the three genes were cloned and the catalytic efficiency of soluble recombinant proteins was determined. CsCuAOs and CsAMADH1 exhibited indispensable functions in the GABA production from putrescine in vitro. Subcellular localization assays indicated that CsAMADH1 was localized in plastid, while both CsCuAO1 and CsCuAO3 were localized in peroxisome. In addition, the synergistic effects of CsCuAOs and CsAMADH1 were investigated by a transient co-expression system in Nicotiana benthamiana. Our data suggest that these three genes regulate the accumulation of GABA in tea by participating in the polyamine degradation pathway and improve the content of GABA in tea to a certain extent. The results will greatly contribute to the production of GABA tea.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Formation of γ-aminobutyric acid (GABA) during the natural lactic acid fermentation of cucumber

This study investigated γ-aminobutyric acid (GABA) formation during natural lactic acid fermentation of cucumbers. Two lots of cucumbers were fermented or acidified in duplicate in 4 % sodium chloride (NaCl) brine and analyzed for GABA content over 6 months storage. GABA was present in fresh and acidified pickling cucumbers at 0.83 ± 0.16 mM and 0.56 ± 0.30 mM, respectively. Additional GABA was generated in fermented cucumbers (1.21 ± 0.35 mM) and remained stable over time and during further processing. Brine salt content (2 % - 6 %) affected GABA formation with greatest GABA formation in cucumber brined in 2 % NaCl (1.38 ± 0.31 mM). Commercially available, fermented cucumber pickles packed in their original fermentation brines contained more GABA (1.32 ± 0.25 mM) than acidified (0.46 ± 0.26 mM) or fermented, desalted and re-packed products (0.49 ± 0.32 mM). This work demonstrated that low-salt fermentation enhances GABA content in fermented cucumber products prepared for direct consumption.

Chronic hyperammonemia causes a hypoglutamatergic and hyperGABAergic metabolic state associated with neurobehavioral abnormalities in zebrafish larvae

Chronic hyperammonemia is a common condition affecting individuals with inherited urea cycle disorders resulting in progressive cognitive impairment and behavioral abnormalities. Altered neurotransmission has been proposed as major source of neuronal dysfunction during chronic hyperammonemia, but the molecular pathomechanism has remained incompletely understood. Here we show that chronic exposure to ammonium acetate induces locomotor dysfunction and abnormal feeding behavior in zebrafish larvae, indicative for an impairment of higher brain functions. Biochemically, chronically elevated ammonium concentrations cause enhanced activity of glutamate decarboxylase isoforms GAD1 and GAD2 with increased formation of GABA and concomitant depletion of glutamate, ultimately leading to a dysfunctional hypoglutamatergic and hyperGABAergic metabolic state. Moreover, elevated GABA concentrations are accompanied by increased expression of GABAA receptor subunits alpha-1, gamma-2 and delta, supporting the notion of an increased GABA tone in chronic hyperammonemia. Propionate oxidation as major anaplerotic reaction sufficiently compensates for the transamination-dependent withdrawal of 2-oxoglutarate, thereby preventing bioenergetic dysfunction under chronic hyperammonemic conditions. Thus, our study extends the hypothesis of alterations in the glutamatergic and GABAergic system being an important pathophysiological factor causing neurobehavioral impairment in chronic hyperammonemia. Given that zebrafish larvae have already been successfully used for high-throughput identification of novel compounds to treat inherited neurological diseases, the reported zebrafish model should be considered an important tool for systematic drug screening targeting altered glutamatergic and GABAergic metabolism under chronic hyperammonemic conditions in the future.

ADAM17 on glutamatergic neurons inhibits GABAergic signaling in a salt‐sensitive hypertension model

The brain plays an important role in modulating sympathetic drive to individual organs, leading to changes in cardiac output, vascular resistance and blood pressure regulation. Within the central nervous system, A Disintegrin And Metalloprotease 17 (ADAM17) impairs the compensatory activity of angiotensin converting enzyme type 2 (ACE2) through ectodomain shedding during hypertension. We previously observed that activation of neuronal ADAM17, especially in excitatory neurons, contributes to the development of neurogenic hypertension through promoting sympatho‐excitation. It was also revealed that ADAM17 located on glutamatergic neurons plays a supportive role in Ang‐II‐induced neuronal activation. To investigate the contributing mechanism, deoxycorticosterone (DOCA)‐salt (1mg/g body weight sc + 1% saline po for 3 weeks) or sham treatment was given to mice harboring ADAM17 knockdown in glutamatergic neurons (A17G) and their control littermates (C57Bl6/J male mice, 14–16 week‐old, n=10). At the end of the treatment, mice were sacrificed and their brains were collected. Expression levels of NMDA receptors (Grin1), AMPA receptors (GluR1), as well as GABAa receptors and Gad67 was assessed by QRT‐PCR. Among these genes, mRNA levels for Grin1, GluR1, and GABAa receptors were shown to be unaffected in the hypothalamus of either A17G mice or controls. However, mRNA level of Gad67, a critical enzyme that catalyzes the formation of GABA, was found to be significantly reduced in DOCA‐salt‐treated control mice, while it was significantly upregulated in the hypothalamus of DOCA‐salt‐treated A17G mice, implying that activation of ADAM17 in glutamatergic neurons might have a negative impact on GABAergic signaling. It was reported previously that neuro‐inflammation, especially microglia activation, can increase glutamate activity and decrease GABA synthesis as well as its release. Through CD11b labeling, we observed that less microglia was activated by DOCA‐salt treatment in the hypothalamus of A17G mice, compared to controls. Meanwhile, the markedly reduced interleukin‐6 (IL‐6) level in the hypothalamus also suggested that DOCA‐salt‐induced neuro‐inflammation was blunted in mice lacking ADAM17 in glutamatergic neurons. In addition, the distribution of ADAM17 was also studied. Beyond glutamatergic neurons, assessment of ADAM17’s presence in other neuronal populations and brain regions revealed its expression in GABAergic neurons within the MnPO (median preoptic nucleus), PVN and BNST (bed nucleus of the stria terminalis). Although the mechanism still needs to be verified, ADAM17‐mediated shedding in glutamatergic neurons seems to blunt GABAergic inhibitory activity. This may support the increase of glutamatergic neuronal excitability and sympatho‐excitation during DOCA‐salt hypertension.Support or Funding InformationVeterans Affairs Merit Award (BX004294), National Institutes of Health (HL093178 and COBRE P30GM106392) and LSUHSC‐NO Research Enhancement Program.

Effects of aminoguanidine on γ‐aminobutyric acid accumulation pathways in fresh tea leaves under hypoxic conditions

SummaryAminoguanidine (AG), a specific inhibitor of diamine oxidase (DAO), can be used to investigate the γ‐aminobutyric acid (GABA) accumulation pathways. The effect of AG on GABA accumulation pathways of tea fresh leaves remains to be explored. Our results indicated AG concentration did not significantly affect GAD activity, but high concentration solution made GABA‐T activity increase. AG (1 mm) maximally, but not entirely, blocked the polyamine degradation pathway, but had no effect on GAD and GABA‐T. The polyamine degradation pathway was responsible for 26.8% and 17.0% of GABA formation under hypoxic conditions involving immersion and immersion–vacuum, respectively, because DAO and PAO activity was not completely inhibited.

Association between high titers of glutamic acid decarboxylase antibody and epilepsy in patients with type 1 diabetes mellitus: A cross-sectional study

PurposeIndividuals with type 1 diabetes mellitus (T1D) are at higher risk of epilepsy. T1D is a progressive immune-mediated disease and the etiology of epilepsy remains unknown in most. Glutamic acid decarboxylase (GAD) catalyzes GABA formation. GABA-secreting neurons and pancreatic beta cells are the major cells expressing GAD. MethodsCross-sectional study. Patients with T1D from a multiethnic population underwent GADA measurement to investigate possible association between T1D and epilepsy of unknown etiology. ResultsT1D patients were analyzed (n = 375). Overall frequency of epilepsy was 5.9% (n = 22). Frequency of epilepsy of unknown etiology was 3.2% (n = 12). Of these, 8 (2.1%) had idiopathic generalized epilepsy (IGE) and 4 (1.1%) MRI-negative temporal lobe epilepsy (TLE). Patients with T1D and epilepsy of unknown etiology did not show differences in GADA frequency (83.3% vs 50%; p = 0.076); however, their titers were higher (106.9 ± 136.5 IU/mL; median 7; IQR 1.65–256 vs 10.2 ± 14.5 IU/ml; median 4.3; IQR 1.9–8.9; p = 0.019) compared to patients without epilepsy. Moreover, epilepsy of unknown etiology was associated with GADA titers ≥ 100 UI/mL [odds ratio (OR) 4.42, 95% CI 2.36–8.66]. ConclusionEpilepsy frequency was elevated in patients with T1D and multiethnic background. Presence of epilepsy of unknown etiology was associated with high titers of GADA in this population with long-standing T1D, which has different ethnic and genetic background compared to previous studies. Further prospective studies are required to identify if GADA presence or its persistence are directly responsible for epilepsy in individuals with T1D.

Evaluation of lactic acid bacteria and component change during fermentation of Ishizuchi‐kurocha

Ishizuchi-kurocha is a postfermented tea produced in Saijo, Ehime, Japan. Harvested tea leaves are fermented aerobically by fungi. Subsequently, the tea leaves are fermented anaerobically by lactic acid bacteria. In the current study, fermentation-related lactic acid bacteria were examined during the processing of Ishizuchi-kurocha obtained from three producers over a period of 3 years. Lactobacillus plantarum subsp. plantarum was isolated as the dominant species in Ishizuchi-kurocha. Lactobacillus brevis was also frequently isolated. In contrast, none of these species could be isolated from raw tea leaves. A component analysis of the tea extract was performed. The amount of catechins, amino acids, and γ-amino butyric acid in tea leaves changed during anaerobic fermentation. L. plantarum was selected during aerobic fermentation and grew exponentially during anaerobic fermentation. And L. brevis is important for GABA formation. Our results suggest that a controlled growth of L. plantarum is essential for the processing of the Ishizuchi-kurocha. Practical applications In the present study, we revealed dynamics of lactic acid bacteria during fermentation of Japanese postfermented tea. Optimization of fermentation process is important for stable and safety processing of the postfermented tea. Good growth of Lactobacillus plantarum subsp. plantarum and Lactobacillus brevis is important for flavor, taste, and function of the postfermented tea. Our work contributes to stable and efficient processing of the traditional postfermented tea.

Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

AMADH inhibitor optimization and its effects on GABA accumulation in soybean sprouts under NaCl-CaCl2 treatment.

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was the suitable inhibitor for aminoaldehyde dehydrogenase (AMADH) compared with N-ethylmaleimide (NEE) and iodoacetamide (IAM). EDC exhibited the most obvious inhibition effect on AMADH activity, while its inhibition on glutamate decarboxylase (GAD) was insignificant. Compared with the control, AMADH activity reduced by 70.4% with 0.5mM EDC, and γ-aminobutyric acid (GABA) content declined by 44.3% in soybean sprouts at 4days of germination. AMADH activity reduced by 80.62, 67.61 and 72.02% in the 4-day sprouts with 1mM EDC under NaCl, CaCl2 and NaCl + CaCl2 treatment, respectively, and GABA content decreased by 43.56, 38.84 and 35.53%, respectively. EDC is a proper inhibitor for AMADH and it could be used to quantify the contribution of polyamine degradation pathway on GABA formation. In soybean sprouts, the presence of CaCl2 under NaCl stress decreased the contribution of polyamine degradation pathway on GABA accumulation.

γ-Aminobutyric Acid (GABA) Accumulation in Tea (Camellia sinensis L.) through the GABA Shunt and Polyamine Degradation Pathways under Anoxia.

γ-Aminobutyric acid (GABA) is an important bioactive component of tea (Camellia sinensis) providing various health benefits. We studied GABA accumulation via the GABA shunt and polyamine degradation pathways under anoxia in tea leaves. Anoxia caused a ∼20-fold increment in GABA concentration, relative to fresh tea leaves. This increment was due to the increase of glutamate decarboxylase and diamine oxidase activities. Genes involved in GABA formation, such as CsGAD1 and CsGAD2, were significantly up-regulated by anoxia. The concentrations of putrescine and spermine, two substrates for GABA production, were also increased by anoxia. Treating tea leaves with aminoguanidine completely inhibited diamine oxidase activity during anoxia, but the concentration of GABA decreased by only ∼25%. We infer that about one-fourth of GABA formed in tea leaves under anoxia comes from the polyamine degradation pathway, opening the possibility of producing GABA tea based through the regulation of metabolism.

WRKY40 and WRKY6 act downstream of the green leaf volatile E-2-hexenal in Arabidopsis.

Plants are known to be responsive to volatiles, but knowledge about the molecular players involved in transducing their perception remains scarce. We study the response of Arabidopsis thaliana to E-2-hexenal, one of the green leaf volatiles (GLV) that is produced upon wounding, herbivory or infection with pathogens. We have taken a transcriptomics approach to identify genes that are induced by E-2-hexenal, but not by defence hormones or other GLVs. Furthermore, by studying the promoters of early E-2-hexenal-induced genes we determined that the only statistically enriched cis-element was the W-box motif. Since members of the plant-specific family of WRKY transcription factors act in trans on this cis-element, we focused on WRKY6, 40 and 53 that were most strongly induced by E-2-hexenal. Root elongation of Arabidopsis seedlings of the wrky40 wrky6 double mutant was much less inhibited than in wt plants, similar to the E-2-hexenal-responsive mutant her1, which is perturbed in γ-amino butyric acid (GABA) metabolism. The induction of several of the E-2-hexenal-specific genes was much higher in the wrky40, wrky6 or wrky40 wrky6 mutants, including GAD4, a glutamate decarboxylase that catalyzes the formation of GABA from glutamate. In conclusion, WRKY6 and 40 seem to act as important players transducing E-2-hexenal perception.

Accumulation of γ‐aminobutyric acid in soybean by hypoxia germination and freeze–thawing incubation

γ-Aminobutyric acid (GABA) can be synthesised by the GABA shunt and polyamine degradation pathway in plants under hypoxia stress and lower temperature. The hypoxia germination freeze-thawing incubation was used as a new technique for accumulating more GABA in soybean. Results showed that glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content increased during germination within 24 h under hypoxia. However, the contents of dry matters and protein decreased. When the hypoxia-treated sprouts were frozen at -18 °C for 12 h and thawed at 25 °C for 6 h, GABA content increased drastically to 7.21-fold of the non-frozen sprouts. Subsequently, the freeze-thawing sprouts were ground into homogenates and incubated. GABA content was 14.20-fold of the only-soaked seeds when homogenates was incubated at 45 °C for 80 min within 400 µmol L(-1) pyridoxine (VB6) (pH 6.5). The hypoxia germination freeze-thawing incubation was an effective method for accumulating GABA in soybean. During incubation, DAO was more important for GABA formation in homogenate of germinated soybean compared with GAD.