Glutamate Decarboxylase Activity Research Articles

Drought priming-induced stress memory improves subsequent drought or heat tolerance via activation of γ-aminobutyric acid-regulated pathways in creeping bentgrass.

Recurrent drought can induce stress memory in plants to induce tolerance to subsequent stress, such as high temperature or drought. Drought priming (DP) is an effective approach to improve tolerance to various stresses; however, the potential mechanism of DP-induced stress memory has not been fully resoved. We examined DP-regulated subsequent drought tolerance or thermotolerance associated with changes in physiological responses, GABA and NO metabolism, heat shock factor(HSF) and dehydrin (DHN) pathways in perennial creeping bentgrass. Plants can recover after two cycle of DP, and DP-treated plants had significantly higher tolerance to subsequent drought or heat stress, with higher leaf RWC, Chl content, photochemical efficiency, and cell membrane stability. DP significantly alleviated oxidative damage through enhancing total antioxidant capacity in response to subsequent drought or heat stress. Endogenous GABA was significantly increased by DP through activating glutamic acid decarboxylase activity and inhibiting GABA transaminase activity. DP also enhanced accumulation of NO, depending on NOS activity, under subsequent drought or heat stress. Transcript levels of multiple transcription factors, heat shock proteins, and DHNs in the HSF and DHN pathways were up-regulated by DP under drought or heat stress, but there were differences between DP-regulated heat tolerance and drought tolerance in these pathways. The findings indicate that under recurrent moderate drought, DP improves subsequent tolerance to drought or heat stress in relation to GABA-regulated pathways, providing new insight into understanding of the role of stress memory in plant adaptation to complex environmental stresses.

Effect of Lactiplantibacillus plantarum cell-free supernatant on the physiology, quorum sensing, transcription, and enhanced GABA production of Enterococcus faecium

A co-culture strategy has been proposed for the production of desired products. However, the interaction effect on enhancing γ-aminobutyric acid (GABA) production requires further investigation. In this study, the mechanism by which Lactiplantibacillus plantarum BC112 cell-free supernatant (CFS) enhances GABA production in Enterococcus faecium AB157 was investigated, including physiological characteristics, quorum sensing (QS), and mRNA expression. The results showed CFS can significantly enhance the production of γ-aminobutyric acid (GABA) in E. faecium AB157, with a maximum increase of 4.6 times compared to the strain without CFS. CFS promoted the growth of E. faecium AB157, enhanced the activity of glutamate decarboxylase (GAD) and ATPase, and reduced the pH. The effect of CFS on QS was demonstrated through changes in Autoinducer-2 (AI-2) activity and luxS expression. Moreover, 16 amino acids were detected in CFS, with glutamic acid having the highest content. Additionally, 974 genes of E. faecium AB157 differed significantly in cultures with added CFS, which involved glycolytic, amino acid, and GABA biosynthetic pathways. Therefore, Lb. plantarum BC112 CFS enhances GABA production in E. faecium AB157 by affecting energy metabolism, the QS system, and the GABA biosynthesis system.

Controlled atmosphere storage alleviates the browning of walnut (Juglans regia L.) fruit through enhancing GABA-mediated energy metabolism

Browning is the primary physiological damage that shortens the postharvest life and deteriorates the quality of stored fruits. Controlled atmosphere (CA) storage has been shown to be effective in alleviating the browning of ‘Xifu 1′ in-hull walnut; however, its alleviation mechanism is less understood. To further explore the effect and regulatory mechanism of CA on the browning of fresh in-hull walnut, we stored four varieties of in-hull walnuts under CA-treated (5% O2 + 7.5% CO2) and untreated conditions at 0 ± 0.5 °C with 70–80% relative humidity for 70 d. The indicators related to browning and γ-aminobutyric acid (GABA) metabolism were measured until 56 d of storage. The results showed that the CA significantly increased GABA levels by maintaining higher activities of glutamate decarboxylase (GAD), GABA transaminase (GABAT) and succinate dehydrogenase (SDH). This reduced the changes in the browning index (BI), color values, and respiration rate (RR) of the four walnut fruit varieties compared with controls during storage. Consistent with the active GABA metabolism, higher energy charge (EC) and adenosine triphosphate (ATP), succinic acid (SA), malic acid (MLA), fumaric acid (FA), and citric acid (CTA) contents were also observed in the CA-treated walnut hull compared to the controls. The epicarp degradation of the CA-treated ‘Xifu 1′ walnut fruit occurred later, and their mitochondria and cells maintained better integrity compared to the controls. Among the differentially expressed genes (DEGs) screened via RNA-Sequencing (RNA-Seq), 32 were annotated as part of the citrate cycle (TCA cycle) pathway, and 27 were annotated in the GABA metabolism pathway. Real-time quantitative PCR (RT-qPCR) analysis showed that eight candidate genes were closely related to the effect of CA on the synthesis, transport, and transformation of GABA. In conclusion, CA effectively controlled the browning of different varieties of fresh in-hull walnuts by enhancing GABA metabolism and maintaining energy balance.

Selenium treatment alters the accumulation of osmolytes in arsenic-stressed rice (Oryza sativa L.).

Arsenic (As), one of the major pollutants in the soil, is an important environmental concern as its consumption can cause adverse health symptoms in living organisms. Its contamination of rice grown over As-contaminated areas is a serious concern in South Asian countries. Selenium (Se) has been reported to influence various osmolytes under metal stress in plants. The present study reports the role of Se in mitigating As stress in rice by modulating osmolyte metabolism. Rice plants grown in As-amended soil (2.5-10mgkg-1) in pots were treated with sodium selenate (0.5-1.0mg Se kg-1 soil) in glass house conditions and leaf samples were collected at 60 and 90days after sowing (DAS). As-treated rice leaves displayed a reduction in relative water content (RWC) and dry weight than control with a maximum reduction of 1.68- and 2.47-fold in RWC and 1.95- and 1.69-fold in dry weight in As10 treatment at 60 and 90 DAS, respectively. Free amino acids (1.38-2.26-fold), proline (3.88-3.93-fold), glycine betaine (GB) (1.27-1.72-fold), choline (1.67-3.1-fold), total soluble sugars (1.29-1.61-fold), and reducing sugars (1.67-2.19-fold) increased in As-treated rice leaves as compared to control at both stages. As stress increased the γ-aminobutyric acid (GABA), putrescine content, and glutamate decarboxylase activity whereas diamine oxidase and polyamine oxidase activities declined by 1.69-1.88-fold and 1.52-1.86-fold, respectively. Se alone or in combination with As improved plant growth, RWC, GB, choline, putrescine, and sugars; lowered proline and GABA; and showed a reverse trend of enzyme activities related to their metabolism than respective As treatments. As stress resulted in a higher accumulation of osmolytes to combat its stress which was further modulated by the Se application. Hence, the current investigation suggested the role of osmoprotectants in Se-induced amelioration of As toxicity in rice plants.

Changes in Protein-Related Enzyme Activities, Concentrations of GABA and Nitrogen-Containing Constituents of Vigna Radiata L. Seeds Germinated under Different Circumstances

Germination is one of the most important techniques to enhance the nutrients and functionality of legume seeds. This academic work investigated the effects of pre-treatment with acidifying soaking water and germination time on changes in concentrations of gamma-aminobutyric acid (GABA) and nitrogenous constituents, and protein-related enzyme activities in mung bean seeds. Higher amounts of crude protein, non-protein nitrogenous, free amino acids, and enzyme activities, but lower protein nitrogenous concentrations were found when soaking in more acidic water and germinating at longer germination time. Steeping water of pH 5.5 and germination duration of 8 h induced mung bean seeds to manifest the highest activities of glutamate decarboxylase (GAD) (60.9 U/g powder, db) and protease (2.81 U/g powder, db), responsible for the highest values of GABA (1.60 g/kg, db), free amino acids (5.92 g/kg, db) and non-protein nitrogen (14.7 g/kg, db), and the lowest amount of protein nitrogen (30.8 g/kg, db). These findings indicate that pre-treatment with acidic soaking water before germinating was more likely to enrich the GABA and amino acid-containing compounds of the mung bean seeds.

Enhancement of γ-Aminobutyric Acid and the Characteristics of Nutrition and Function in White Quinoa through Ultrasound Stress at the Pre-Germination Stage.

The global production of quinoa has been increasing in recent years. In plant-based foods, ultrasound stress has received increasing attention, owing to its ability to enhance the production of primary and secondary metabolites. We studied the effects of ultrasonic stress at the pre-germination stage on the γ-aminobutyric acid (GABA) accumulation and characteristics of nutrition and function in quinoa. The results showed that ultrasonic conditions of 100 W for 4 min promoted an increase in GABA content by 9.15-fold, to 162.47 ± 6.69 mg/100 g·DW, compared to that of untreated quinoa, through promoting a 10.2% and 71.9% increase in the water absorption and glutamate decarboxylase activity of quinoa, respectively. Meanwhile, compared to untreated quinoa, ultrasonic stress at the pre-germination stage enhanced the total phenolic, total flavonoid, and total saponin contents of quinoa by 10.2%, 33.6%, and 90.7%, to 3.29 mg GA/g·DW, 104.0 mg RE/100 g·DW, and 7.13 mg/g, respectively, without decreasing its basic nutritional quality. Ultrasonic stress caused fissures on the surface of quinoa starch particles. Additionally, germination under ultrasonic stress increased the n3 polyunsaturated fatty acids by 14.4%. Furthermore, ultrasonic stress at the pre-germination stage promoted the scavenging of 2,2-diphenyl1-picrylhydrazyl radicals and inhibitions of α-amylase, α-glucosidase, and pancreatic lipase by 14.4%, 14.9%, 24.6%, and 20.0% in vitro, compared to untreated quinoa. The results indicated that the quinoa sprouted via ultrasonic stress could represent a promising method through which to develop nutritionally balanced whole grains rich in GABA, with hypoglycemic and hypolipidemic activities, which could provide theoretical support for the development of functional whole-grain foods based on quinoa.

Physiology of γ-aminobutyric acid production by Akkermansia muciniphila.

Gut bacteria hold the potential to produce a broad range of metabolites that can modulate human functions, including molecules with neuroactive potential. One such molecule is γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system in animals. Metagenomic analyses suggest that the genomes of many gut bacteria encode glutamate decarboxylase (GAD), the enzyme that catalyzes GABA production. The genome of Akkermansia muciniphila, a mucin specialist and potential next-generation probiotic from the human gut, is predicted to encode GAD, suggesting a contributing role in GABA production in the human gut. In this study, A. muciniphila was grown in batch cultures with and without pH control. In both experiments, A. muciniphila was found to produce GABA as a response to acid (pH <5.5), although only when GABA precursors, either glutamate or glutamine, were present in the medium. Proteomic analysis comparing A. muciniphila grown with and without precursors at pH 4 did not show a difference in GAD expression, suggesting that it is expressed regardless of the presence of GABA precursors. To further investigate the function of A. muciniphila GAD, we heterologously expressed the gad gene (encoded by locus tag Amuc_0372) with a His tag in Escherichia coli and purified the GAD protein. Enzyme assays showed GAD activity in a pH range between 4 and 6, with the highest specific activity at pH 5 of 144 ± 16 µM GABA/min/mg. Overall, our results demonstrate the ability of A. muciniphila to produce GABA as an acid response and unravel the conditions under which GABA production in A. muciniphila occurs.IMPORTANCEAkkermansia muciniphila is considered to be a beneficial bacterium from the human gut, but the exact mechanisms by which A. muciniphila influences its host are not yet fully understood. To this end, it is important to identify which metabolites are produced and consumed by A. muciniphila that may contribute to a healthy gut. In the present study, we demonstrate the ability of A. muciniphila to produce γ-aminobutyric acid (GABA) when grown in an acidic environment, which often occurs in the gut. GABA is the major inhibitory neurotransmitter in the central nervous system and is present in the human gut. For this reason, it is considered an important bacterial metabolite. Our finding that A. muciniphila produces GABA in acidic environments adds to the growing body of understanding of its relationship with host health and provides an explanation on how it can survive acid stress in the human gut.

CmCML11 interacts with CmCAMTA5 to enhance γ-aminobutyric acid (GABA) accumulation by regulating GABA shunt in fresh-cut cantaloupe

The effect of calcium chloride (CaCl2) treatment on γ-aminobutyric acid (GABA) accumulation in fresh-cut cantaloupe and the involved mechanisms were investigated. The result showed that 1% (w/v) CaCl2 treatment increased GABA content and activities of glutamate decarboxylase (GAD) and succinate semialdehyde dehydrogenase (SSADH), while decreased glutamate (Glu) content and GABA transaminase (GABA-T) activities in fresh-cut cantaloupe. CmCML11 and CmCAMTA5 expressions of CaCl2-treated fruit increased by 187.4% and 165.6% than control fruit in the initial 6 h. Besides, expressions of GABA shunt genes, including CmGAD1, CmGAD2, CmGABA-T and CmSSADH were also up-regulated by CaCl2 treatment during early storage. Moreover, acting as a transcriptional activator, CmCAMTA5 could bind to the CG-box in promoters of CmGAD1, CmGABA-T and CmSSADH and activate their transcription. Furthermore, the interaction between CmCML11 and CmCAMTA5 could enhance the transcriptional activation on GABA shunt genes which were regulated by CmCAMTA5. Collectively, our findings revealed that CaCl2 treatment promoted GABA accumulation in fresh-cut cantaloupe via the combined effect of CmCML11 and CmCAMTA5 in the regulation of expressions of CmGAD1, CmGABA-T, and CmSSADH in GABA shunt.

γ-Aminobutyric acid is involved in overlapping pathways against chilling injury by modulating glutamate decarboxylase and defense responses in papaya fruit.

The effect of γ-aminobutyric acid (GABA) treatment at two concentrations (1 mM or 5 mM) on papaya fruit stored at 4°C and 80%-90% relative humidity for 5 weeks was investigated. The application of GABA at 5 mM apparently inhibited chilling injury, internal browning, electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H2O2), polyphenol oxidase (PPO), phospholipase D (PLD), and lipoxygenase (LOX) activities of papaya fruit. Fruit treated with 5 mM GABA enhanced the activities of ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutamate decarboxylase (GAD), and phenylalanine ammonia-lyase (PAL). In addition, GABA treatment significantly displayed higher levels of proline, endogenous GABA accumulation, phenolic contents, and total antioxidant activity than the nontreated papaya. The results suggested that GABA treatment may be a useful approach to improving the chilling tolerance of papaya fruit by reducing oxidative stress and enhancing the defense system.

Lactococcus lactis CNCM I-5388 versus NCDO2118 by its GABA hyperproduction ability, counteracts faster stress-induced intestinal hypersensitivity in rats.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and invitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, invivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients.

A Novel Method for γ-Aminobutyric Acid Biosynthesis Using Glutamate Decarboxylase Entrapped in Polyvinyl Alcohol-Sodium Alginate Capsules.

γ-aminobutyric acid (GABA) has essential physiological functions in the human body. A novel method using glutamate decarboxylase (GAD) entrapped in polyvinyl alcohol (PVA)-sodium alginate (SA) capsules provides a green biological strategy for GABA synthesis. In this investigation, the stability range of immobilized GAD was effectively broadened, and immobilized GAD could be repeatedly used as a batch and fixed-bed column catalyst. The immobilized enzymes were stable and retained 89% of their activity in a pH range of 4.0-5.6, while there was an approximately 50% decrease in free GAD activity in the pH range of 4.8 ± 0.4. The immobilized GAD affinity to the substrate improved, and this was evidenced by the apparent decrease in Km to 13.3 mmol/L from the 30.9 mmol/L for free GAD. The immobilized GAD retained >90.6% activity after eight cycles and a near-100% enzyme activity retention after 120 h of a continuous fixed-bed column catalyst operation. This study has thus presented an effective PVA-SA-GAD immobilization method that could be used to continuously scale-up GABA biosynthesis.

Ameliorative Potential of (-) Pseudosemiglabrin in Mice with Pilocarpine-Induced Epilepsy: Antioxidant, Anti-Inflammatory, Anti-Apoptotic, and Neurotransmission Modulation

One prevalent neurological disorder is epilepsy. Modulating GABAergic/glutamatergic neurotransmission, Nrf2/HO-1, PI3K/Akt, and TLR-4/NF-B pathways might be a therapeutic strategy for epilepsy. Eight-week-old BALB/c mice were administered 12.5, 25, or 50 mg/kg (-) pseudosemiglabrin orally one hour before inducing epilepsy with an i.p. injection of 360 mg/kg pilocarpine. (-) Pseudosemiglabrin dose-dependently alleviated pilocarpine-induced epilepsy, as revealed by the complete repression of pilocarpine-induced convulsions and 100% survival rate in mice. Furthermore, (-) pseudosemiglabrin significantly enhanced mice’s locomotor activities, brain GABA, SLC1A2, GABARα1 levels, glutamate decarboxylase activity, and SLC1A2 and GABARα1mRNA expression while decreasing brain glutamate, SLC6A1, GRIN1 levels, GABA transaminase activity, and SLC6A1 and GRIN1 mRNA expression. These potentials can be due to the suppression of the TLR-4/NF-κB and the enhancement of the Nrf2/HO-1 and PI3K/Akt pathways, as demonstrated by the reduction in TLR-4, NF-κB, IL-1β, TNF-α mRNA expression, MDA, NO, caspase-3, Bax levels, and Bax/Bcl-2 ratio, and the enhancement of Nrf2, HO-1, PI3K, Akt mRNA expression, GSH, Bcl-2 levels, and SOD activity. Additionally, (-) pseudosemiglabrin abrogated the pilocarpine-induced histopathological changes. Interestingly, the (-) pseudosemiglabrin intervention showed a comparable effect to the standard medication, diazepam. Therefore, (-) pseudosemiglabrin can be a promising medication for the management of epilepsy.

Sex hormone dose escalation for treating abnormal sleep in ovariectomized rats: in vitro GABA synthesis in sleep-related brain areas.

No data in the literature have evaluated sex hormone dose escalation for treating abnormal sleep of ovariectomized rats-nor studies on the role of sex hormones in GABA synthesis of rats' sleep-related areas. The main aim of this study was to determine the maximum tolerated dose (MTD) of estradiol (ET), progesterone (PT), and the mixture of both (EPT) to restore normal sleep in a model of menopause in rats. The second purpose was to describe the in vitro activity of glutamate decarboxylase (GAD) in sleep-related brain areas in the presence or absence of sex hormones. A weekly dose-escalation design of ET, PT, or EPT was implemented in ovariectomized rats (six per group). Dose escalation continued until the dose at which 100% of the rats exhibited a state of "complete somnolence." Doses that were not toxic or did not show side effects were considered. For in vitro experiments, sleep-related brain areas were separated and incubated with radiolabeled glutamate. Estradiol (17β-E2), progesterone (P), and pyridoxal phosphate (PLP) were added to this assay, and GAD activity was determined. Under the same conditions, a second test was carried out, but the P antagonist RU486 was added to assess the role of P in GAD activity. Ovariectomy increased periodic awakenings compared to those determined for the SHAM group. The EPT for ovariectomized rats was very effective by the fifth week in decreasing arousal and achieving a similar sleep behavior to the SHAM-control group. Rats tolerated the ET, PT, and EPT well to the maximum planned dose (0.66mg/kg and 4.4mg/kg, respectively). No lethal events occurred; the MTD was reached. The in vitro studies indicated that the presence of 17β-E2 plus P in the assay triggered the activity of isotype 65 GAD in all the studied brain areas. RU486 in the incubation medium blocked such activity; however, the action of isotype 67 GAD was not blocked by RU486. A dose-escalation model was determined; the MTD coincided with the maximum dose of ET and PT used. However, the EPT combination restored normal sleep in the menopause model compared to the SHAMs without toxic effects. The in vitro model demonstrated that 17β-E2 plus P presence in the assay increased the activity of GAD65 in the studied brain tissues.

FaGAPC2/FaPKc2.2 and FaPEPCK reveal differential citric acid metabolism regulation in late development of strawberry fruit.

Citric acid is the primary organic acid that affects the taste of strawberry fruit. Glycolysis supplies key substrates for the tricarboxylic acid cycle (TCA cycle). However, little is known about the regulatory mechanisms of glycolytic genes on citric acid metabolism in strawberry fruits. In this study, the citric acid content of strawberry fruit displayed a trend of rising and decreasing from the initial red stage to the full red stage and then dark red stage. Thus, a difference in citric acid metabolic regulation was suspected during strawberry fruit development. In addition, overexpression of either cytoplasm glyceraldehyde-3-phosphate dehydrogenase (FxaC_14g13400, namely FaGAPC2) or pyruvate kinase (FxaC_15g00080, namely FaPKc2.2) inhibited strawberry fruit ripening and the accumulation of citric acid, leading to a range of maturity stages from partial red to full red stage. The combined transcriptome and metabolome analysis revealed that overexpression of FaGAPC2 and FaPKc2.2 significantly suppressed the expression of phosphoenolpyruvate carboxykinase (FxaC_1g21491, namely FaPEPCK) but enhanced the content of glutamine and aspartic acid. Meanwhile, the activities of PEPCK and glutamate decarboxylase (GAD) were inhibited, but the activities of glutamine synthase (GS) were increased in FaGAPC2/FaPKc2.2-overexpressed fruit. Further, functional verification demonstrated that overexpression of FaPEPCK can promote strawberry fruit ripening, resulting in a range of maturity stage from full red to dark red stage, while the citric acid synthase (CS) activities and citric acid content were significantly decreased. Overall, this study revealed that FaGAPC2/FaPKc2.2 and FaPEPCK perform an important role in reducing citric acid content in strawberry fruit, and FaGAPC2/FaPKc2.2 mainly by promoting the GS degradation pathway and FaPEPCK mainly by inhibiting the CS synthesis pathway.

Spatiotemporally resolved metabolomics and isotope tracing reveal CNS drug targets.

Deconvolution of potential drug targets of the central nervous system (CNS) is particularly challenging because of the complicated structure and function of the brain. Here, a spatiotemporally resolved metabolomics and isotope tracing strategy was proposed and demonstrated to be powerful for deconvoluting and localizing potential targets of CNS drugs by using ambient mass spectrometry imaging. This strategy can map various substances including exogenous drugs, isotopically labeled metabolites, and various types of endogenous metabolites in the brain tissue sections to illustrate their microregional distribution pattern in the brain and locate drug action-related metabolic nodes and pathways. The strategy revealed that the sedative-hypnotic drug candidate YZG-331 was prominently distributed in the pineal gland and entered the thalamus and hypothalamus in relatively small amounts, and can increase glutamate decarboxylase activity to elevate γ-aminobutyric acid (GABA) levels in the hypothalamus, agonize organic cation transporter 3 to release extracellular histamine into peripheral circulation. These findings emphasize the promising capability of spatiotemporally resolved metabolomics and isotope tracing to help elucidate the multiple targets and the mechanisms of action of CNS drugs.

N-α-Lauroyl-L-arginine ethyl ester hydrochloride combined with hot water treatment alleviates chilling injury of postharvest cucumber fruit

Postharvest cucumber fruit is prone to chilling injury (CI) under cold stress, leading to severe quality deterioration. In this study, the effects of N-α-Lauroyl-l-arginine ethyl ester hydrochloride (LAE), hot water (HW), and LAE combined with HW treatments on CI in cucumber fruit stored at 4 ± 1 ℃ were investigated. The response surface methodology (RSM) confirmed that the LAE concentration of 1.00 g L−1, HW temperature of 44 ℃, and immersion time of 10 min were the optimal condition for cucumber fruit preservation during cold storage. Further analysis revealed that LAE, HW, and LAE combined with HW treatments, all significantly mitigated chilling injury symptoms in cucumber fruit in comparison with the control, but the application of LAE combined with HW was the most effective method for enhancing cold tolerance in cucumber fruit. Moreover, LAE combined with HW treatment could reduce reactive oxygen species (ROS) accumulation, and enhance superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) activities in cold-stored cucumber fruit. Meanwhile, the activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), Δ1-pyrroline-5-carboxylate synthetase (P5CS), ornithine δ-aminotransferase (OAT), and glutamate decarboxylase (GAD) were also increased by LAE combined with HW treatment, while the activities of proline dehydrogenase (PDH) and γ-aminobutyric acid transaminase (GABA-T) were decreased by LAE combined with HW treatment, which contributed to maintaining higher polyamines, proline, and GABA contents. These findings suggested that LAE combined with HW treatment can reduce chilling injury in cucumber fruit via enhancing antioxidant enzymes activities, PA, proline, and GABA contents to maintain ROS homeostasis during cold storage.

Accelerated Accumulation of γ-Aminobutyric Acid and Modifications on Its Metabolic Pathways in Black Rice Grains by Germination under Cold Stress

Germination can increase γ-aminobutyric acid (GABA) accumulation in grains, but the combined effects of germination and other external stress on rice grains have been little studied. In this investigation, enhanced accumulation of GABA and modification of its metabolic pathways in black rice grains were investigated during germination under cold stress. The combination of cold stress and germination resulted in a greater accumulation of GABA than germination alone. The treatment of cold stress at 0 °C for 1 h and germination for 72 h induced a maximum GABA content of 195.64 mg/100 g, 51.54% higher compared to the control, which was superior to any other treatment. We modified the metabolism of the GABA shunt to the orientation of GABA synthesis, in which the activity of glutamic acid decarboxylase and protease were stimulated. The total content of free amino acid indicated an upward trend as germination prolonged. The degradation of polyamines was partly promoted due to elevated diamine oxidase and polyamine oxidase activity, but the activity of amino-aldehyde dehydrogenase for the direct synthesis of GABA in the pathway was suppressed. The result implied that the GABA shunt might play a major role in enhancing GABA accumulation induced by cold stress and germination rather than the polyamines degradation pathway. This investigation provides a practical reference for GABA accumulation by germination under cold stress and a theoretical basis for the possible mechanism underlying the accelerating action.

Exogenous γ-aminobutyric acid (GABA) improves salt-inhibited nitrogen metabolism and the anaplerotic reaction of the tricarboxylic acid cycle by regulating GABA-shunt metabolism in maize seedlings

Salinity stress hampers the growth of most crop plants and reduces yield considerably. In addition to its role in metabolism, γ-aminobutyric acid (GABA) plays a special role in the regulation of salinity stress tolerance in plants, though the underlying physiological mechanism remains poorly understood. In order to study the physiological mechanism of GABA pathway regulated carbon and nitrogen metabolism and tis relationship with salt resistance of maize seedlings, we supplemented seedlings with exogenous GABA under salt stress. In this study, we showed that supplementation with 0.5 mmol·L−1 (0.052 mg·g−1) GABA alleviated salt toxicity in maize seedling leaves, ameliorated salt-induced oxidative stress, and increased antioxidant enzyme activity. Applying exogenous GABA maintained chloroplast structure and relieved chlorophyll degradation, thus improving the photosynthetic performance of the leaves. Due to the improvement in photosynthesis, sugar accumulation also increased. Endogenous GABA content and GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH) activity were increased, while glutamate decarboxylase (GAD) activity was decreased, via the exogenous application of GABA under salt stress. Meanwhile, nitrogen metabolism and the tricarboxylic acid (TCA) cycle were activated by the supply of GABA. In general, through the regulation of GABA-shunt metabolism, GABA activated enzymes related to nitrogen metabolism and replenished the key substrates of the TCA cycle, thereby improving the balance of carbon and nitrogen metabolism of maize and improving salt tolerance.

Structural Diversity Design, Synthesis, and Insecticidal Activity Analysis of Ester-Containing Isoxazoline Derivatives Acting on the GABA Receptor.

To explore insecticides targeting the γ-aminobutyric acid (GABA) receptor, two series of novel isoxazoline derivatives containing sulfonic and carboxylic esters were designed and synthesized. Their insecticidal activities against Plutella xylostella, Mythimna separata, and Aedes aegypti larvae and their structure-activity relationship were investigated. The sulfonate-containing isoxazoline derivatives (10k-q) exhibited promising insecticidal activities against the three insect larvae. Compound 10o displayed excellent activities with LC50 values of 8.32, 5.23, and 0.35 μg/mL at 48 h against P. xylostella, M. separata, and A. aegypti larvae, respectively, which were better than or similar to those of avermectin. Furthermore, compound 10o exhibited a faster insecticidal effect than avermectin against M. separata. The mode of action of 10o was preliminarily verified by molecular docking, theoretical calculations, and measurement of glutamate decarboxylase and glutamic pyruvic transaminase activities. Compound 10o is a novel insecticidal candidate acting on GABA receptors, which could guide the discovery of isoxazoline insecticides.

Screening of lactic acid bacteria strains isolated from Iranian traditional dairy products for GABA production and optimization by response surface methodology

A total of 50 lactic acid bacteria (LAB) isolates from Iranian traditional dairy products (Motal and Lighvan cheeses, and artisanal yogurt) were screened for gamma-aminobutyric acid (GABA) production. Firstly, a rapid colorimetric test was performed to evaluate the glutamate decarboxylase (GAD) activity among the LAB isolates examined. Thin layer chromatography (TLC) was then performed on selected strains to identify isolates with high/moderate GABA producing capacity, and a GABase micro-titer plate assay was employed to quantify GABA. Finally, two Lactococcus (Lac.) lactis strains were selected for GABA production optimization via Response Surface Methodology (RSM) following Central Composite Design (CCD). Forty-one out of the 50 isolates showed GAD activity according to the colorimetric assay. Eight isolates displayed strong GAD activity, while nine showed no activity; low to moderate GAD activity was scored for all other isolates. GABA production was confirmed by TLC in all isolates with high GAD activity and in four selected among isoaltes with moderate activity. Among the Lactococcus strains tested, Lac. lactis 311 and Lac. lactis 491 were the strongest GABA producers with amounts of 3.3 and 1.26 mM, respectively. These two strains were subjected to GABA production optimization applying RSM and CCD on three key variables: Monosodium glutamate concentration (MSG) (between 25 and 150 mM), incubation temperature (between 25 and 37 °C), and pH (between 4.0 and 5.0). Optimal conditions for GABA production by Lac. lactis 311 and Lac. lactis 491 of temperature, pH and MSG concentration were, respectively, 35.4 and 30 °C, pH 4.5 and 4.6, and MSG concentration of 89 and 147.4 mM, respectively. Under the above conditions, the amount of GABA produced by Lac. lactis 311 and Lac. lactis 491 was 0.395 and 0.179 mg/mL, respectively. These strains and the optimal culture conditions determined in this study could be used for the biotechnological production of GABA or applied in food fermentations for the development of naturally GABA-enriched foods.