GABA Accumulation Research Articles

RNA Interference of OsGABA-T1 Gene Expression Induced GABA Accumula-tion in Rice Grain

γ-氨基丁酸（GABA）是一种四碳非蛋白质氨基酸,具有降血压等功能。为提高稻米中GABA含量,利用RNA干扰技术,构建水稻中GABA代谢关键酶GABA转氨酶1基因（Os GABA-T1）的干扰载体,通过农杆菌介导法,将其转化至粳稻品种宁粳1号中。实时荧光定量PCR检测结果表明导入的RNA干扰结构成功地降低了目的基因Os GABA-T1的表达,且干扰家系中Os GABA-T2基因表达也随之下降。对转基因T3代稻米GABA含量测定发现,糙米中GABA含量相对于对照增加了13倍以上,精米中GABA含量也显著增加,而其他主要氨基酸含量则没有明显变化。测定储藏4个月的转基因稻米发现,GABA含量仍具有较高水平。所以,利用RNA干扰技术可有效提高稻米γ-氨基丁酸（GABA）含量,为培育富含GABA的降血压功能性水稻品种提供基础。

‘눈큰흑찰’의 가바(GABA) 함량 증진을 위한 이화학적 처리 효과

거대쌀눈의 특성과 검정쌀 그리고 찹쌀의 특성을 보유한 눈큰흑찰의 침종과 발아 그리고 기질로 글루탐산을 처리하였을 경우의 가바를 포함한 주요 성분의 분석 결과는 다음과 같다. 1. 싹 발아 길이를 기준으로 눈큰흑찰의 가바 함량은 싹 길이가 5~10 mm인 stage-3에 93.9 mg/100 g으로 최고를 보였고, 대량 제조 조건에서는 최고 126.9 mg의 가바를 함유하고 있음을 확인하였다. 2. 침종에 의한 성분 분석에서는 72시간 침종에서 폴리페놀, 플라보노이드, 아미노산 그리고 가바 함량이 가장 많이 축적됨을 확인하였다. 3. 글루탐산 탈탄산효소의 작용에 의한 가바 함량 증가는 현미와 쌀겨 모두에서 급격한 증가가 관찰되었고, 눈큰흑찰 현미와 쌀겨의 경우 가바 함량이 각각 354.6 mg/100 g과 726.4 mg으로 현미와 쌀겨에 비해 각각 14배와 3배 함량이 증가하였다. 따라서 본 연구에서 눈큰흑찰의 발아와 침종에 따른 품질특성을 분석한 결과, 뇌의 대사전달 물질의 조정 작용과 고혈압 그리고 스트레스 조정 기능이 있는 가바의 함량이 침종과 발아에 의해 일반 쌀에 비해 급격히 증가함을 확인하였다. 특히나 글루탐산탈탄산 효소의 작용에 의한 눈큰흑찰의 현미와 쌀겨의 가바 생성은 현재까지 보고된 그 어떤 쌀품종에서 분석된 가바 함량보다도 가장 우수함을 확인할 수 있어 향후 기능성 발아현미 가공을 위한 원료로 활용이 가능하며, 침종 조건은 가바 함량이 증진된 밥의 제조에 활용이 가능하고, 일본에서 시판되는 가바 건강식품의 용량과 맞먹는 함량을 지닌 눈큰흑찰을 이용한 가바쌀 생산과 가바의 효능에 의한 혈압강하, 체중조절, 알코올 관련 질환 등에 효능이 있는 건강기능성 식품 원료로 활용이 가능할 것으로 사료된다. We assessed the GABA accumulation and other components after the 'Nunkeunhukchal (BGE)', 'Josanghukchal (BR)', and 'Ilmibyeo (IB)' grain was soaked in water for 24, 36, 48, 60, 72 and 96 hr. The results showed a continuous accumulation of GABA in soaking treated brown rice of BGE and IB. Among the treated hours, 72 hours of soaking had the maximal accumulation of GABA (51.4 mg/100 g), amino acid, polyphenol and other components. The activities of glutamate decarboxylase (GAD) in brown rice and rice-bran were the same in BGE rice. However, the formation of GABA treated with L-glutamate as substrate showed dramatic increase of 354.6 (fourteen times higher than normal extraction) and 726.4 mg/100 g in BGE rice and rice-bran, respectively. These results suggested that the soaking and extraction with L-glutamate buffer could be better methods for the harvest of increased GABA.

Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys.

In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid-contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL-TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys.

Synaptic recruitment of gephyrin regulates surface GABAA receptor dynamics for the expression of inhibitory LTP.

Postsynaptic long-term potentiation of inhibition (iLTP) can rely on increased GABAA receptors (GABAARs) at synapses by promoted exocytosis. However, the molecular mechanisms that enhance the clustering of postsynaptic GABAARs during iLTP remain obscure. Here we demonstrate that during chemically induced iLTP (chem-iLTP), GABAARs are immobilized and confined at synapses, as revealed by single-particle tracking of individual GABAARs in cultured hippocampal neurons. Chem-iLTP expression requires synaptic recruitment of the scaffold protein gephyrin from extrasynaptic areas, which in turn is promoted by CaMKII-dependent phosphorylation of GABAAR-β3-Ser383. Impairment of gephyrin assembly prevents chem-iLTP and, in parallel, blocks the accumulation and immobilization of GABAARs at synapses. Importantly, an increase of gephyrin and GABAAR similar to those observed during chem-iLTP in cultures were found in the rat visual cortex following an experience-dependent plasticity protocol that potentiates inhibitory transmission in vivo. Thus, phospho-GABAAR-β3-dependent accumulation of gephyrin at synapses and receptor immobilization are crucial for iLTP expression and are likely to modulate network excitability.

Impact of 1-methylcyclopropene and controlled atmosphere storage on polyamine and 4-aminobutyrate levels in "Empire" apple fruit.

1-Methylcyclopropene (1-MCP) delays ethylene-meditated ripening of apple (Malus domestica Borkh.) fruit during controlled atmosphere (CA) storage. Here, we tested the hypothesis that 1-MCP and CA storage enhances the levels of polyamines (PAs) and 4-aminobutyrate (GABA) in apple fruit. A 46-week experiment was conducted with “Empire” apple using a split-plot design with four treatment replicates and 3°C, 2.5 kPa O2, and 0.03 or 2.5 kPa CO2 with or without 1 μL L-1 1-MCP. Total PA levels were not elevated by the 1-MCP treatment. Examination of the individual PAs revealed that: (i) total putrescine levels tended to be lower with 1-MCP regardless of the CO2 level, and while this was mostly at the expense of free putrescine, large transient increases in soluble conjugated putrescine were also evident; (ii) total spermidine levels tended to be lower with 1-MCP, particularly at 2.5 kPa CO2, and this was mostly at the expense of soluble conjugated spermidine; (iii) total spermine levels at 2.5 kPa CO2 tended to be lower with 1-MCP, and this was mostly at the expense of both soluble and insoluble conjugated spermine; and (iv) total spermidine and spermine levels at 0.03 kPa were relatively unaffected, compared to 2.5 kPa CO2, but transient increases in free spermidine and spermine were evident. These findings might be due to changes in the conversion of putrescine into higher PAs and the interconversion of free and conjugated forms in apple fruit, rather than altered S-adenosylmethionine availability. Regardless of 1-MCP and CO2 treatments, the availability of glutamate showed a transient peak initially, probably due to protein degradation, and this was followed by a steady decline over the remainder of the storage period which coincided with linear accumulation of GABA. This pattern has been attributed to the stimulation of glutamate decarboxylase activity and inhibition of GABA catabolism, rather than a contribution of PAs to GABA production.

Gamma-aminobutyric acid and glutamic acid contents, and the GAD activity in germinated brown rice (Oryza sativa L.): Effect of rice cultivars

The γ-aminobutyric acid (GABA) and glutamic acid (Glu) contents, and the glutamate decarboxylase (GAD) activity in brown rice (BR) and germinated BR (GBR) of the 5 Thai rice cultivars KDML105, PT1, CN1, SP1, and PL2 were investigated. BR was soaked at 35°C for 12 h and then germinated for 24 h to produce GBR. Amounts of GABA and Glu, and the GAD activity in samples were determined. The GABA content and GAD activity in GBR samples of all cultivars were increased 7–50× and 10–100× over levels in BR. The GABA content in BR had no effect on GABA accumulation in GBR. There was no trend in changes in the Glu content for different cultivars after soaking and germination. Variation in the GABA amount in GBR was due to both the GAD activity and the amount of Glu. These two parameters affected accumulation of GABA in all cultivars.

Differential accumulation of γ‐aminobutyric acid in elicited cells of two rice cultivars showing contrasting sensitivity to the blast pathogen

Intracellular free amino acid pools were quantified in suspension cultured cells of a blast-sensitive and a blast-resistant rice genotype at increasing times after treatment with Magnaporthe oryzae cell wall hydrolysates. Besides some expected variations in free phenylalanine, a remarkable early increase of γ-aminobutyric acid (GABA) levels was evident in both cultivars. Glutamate decarboxylase activity and protein levels were unaffected. GABA homeostasis was recovered in the sensitive cultivar 48h after the treatment. In contrast, a further GABA accumulation and a general increase of most amino acids was found at this later stage in the resistant genotype, which showed a larger decrease in cell viability as a consequence of elicitor addition. Data support a recently hypothesised role of GABA metabolism in the plant response to fungal pathogens.

Role of elicitation on the health-promoting properties of kidney bean sprouts

The influence of germination of kidney beans for 4, 6 and 8 days in different elicitors (ascorbic acid, folic acid, glutamic acid, glutamic acid/chitosan and lactic acid/chitosan) on the production of health-promoting ingredients was studied. Sprouts were characterized according to their protein profile, soluble phenolic compounds (SPC) and γ-aminobutyric acid (GABA) content. Additionally, the antioxidant and angiotensin-converting enzyme (ACE) inhibition activities of aqueous extracts from kidney bean elicited sprouts were determined. Elicitation by glutamic acid for 8 days led to the highest SPC and GABA accumulation in kidney bean sprouts and their extracts were selected based on their highest phytochemical content. These extracts also showed remarkable antioxidant and ACE-inhibitory activities. The IC50 for ACE inhibition of glutamic acid-extracts increases 89-fold after the in-vitro gastrointestinal digestion. These results will contribute not only to improving the health-promoting potential of kidney bean sprouts but also to obtaining ingredients which might reduce oxidative stress and hypertension.

Sequence analysis of diamine oxidase gene from fava bean and its expression related to γ‐aminobutyric acid accumulation in seeds germinating under hypoxia‐NaCl stress

γ-Aminobutyric acid (GABA) is synthesized via the polyamine degradation pathway in plants, with diamine oxidase (DAO) being the key enzyme. In this study the cDNA of DAO in fava bean was cloned and its expression in seeds germinating under hypoxia-NaCl stress was investigated. Fava bean DAO cDNA is 2199 bp long and contains 2025 bp of open reading frame that encodes 675 amino acid peptides with a calculated molecular weight of 76.31 kDa and a pI of 5.41. Hypoxia and hypoxia-NaCl stress enhanced DAO activity and resulted in GABA accumulation in germinating fava bean. However, DAO gene expression was down-regulated under hypoxia compared with non-stress condition, while its expression in the cotyledon and shoot was up-regulated under hypoxia-NaCl. In addition, DAO expression could be promoted to enhance GABA accumulation after increasing the stress intensity using NaCl. DAO gene expression was significantly inhibited by aminoguanidine treatment under hypoxia but increased under hypoxia-NaCl. Under hypoxia, GABA accumulation due to NaCl was mainly concentrated in the cotyledon. The GABA content increase under hypoxia did not result from DAO gene expression, but DAO existing in seeds was activated under hypoxia. DAO gene expression was up-regulated to enhance GABA accumulation after increasing the stress intensity.

MANAGING CHILLING INJURY IN FRUITS

Most tropical and subtropical fruits are susceptible to chilling injury, mainly manifested as pitting, surface or internal browning, failure to ripening, development of a woolly or leathery texture, and decay. The development of these chilling injury symptoms negatively affects fruit quality and thus shortens storage life. Chilling injury of fruits can be alleviated by physical techniques such as low temperature conditioning, heat treatment, controlled or modified atmosphere storage, waxing, and UV-C irradiation, by chemical treatments with methyl jasmonate (MeJA), methyl salicylate (MeSA), γ-aminobutyric acid (GABA), 1-methylcyclopropene (1-MCP), nitric oxide, oxalic acid, or other natural compounds and genetic engineering. Treatment with MeJA enhances antioxidant activity, increases endogenous GABA and proline content and maintains a higher degree of unsaturation of membrane lipid in loquat fruit. GABA treatment enhances the accumulation of endogenous GABA and proline, induces higher antioxidant activity, and maintains higher adenosine triphosphate (ATP) content and energy charge in peaches. The combination of heat with MeJA or MeSA treatment has a synergistic effect on alleviating chilling injury and inducing higher antioxidant activity in peach fruit. For loquat, avocado, pineapple, and persimmon fruits whose chilling injury is enhanced by increased ethylene production, 1-MCP can be used to reduce its development. Low temperature conditioning activates the expression of membrane lipid modification enzymes such as fatty acid desaturase (FAD) and lipid transfer protein (LTP), whereas heat treatment induces the expression of various stress-related genes, including heat shock proteins (HSPs), dehydrin and universal stress protein (USP), increases antioxidant activity and maintains membrane stability in citrus fruits. Genetic engineering has the potential to enhance chilling tolerance by generation of genetically-modified fruits with overexpressed genes that encode HSPs and antioxidant enzymes.

The effects of volatile anesthetics on the extracellular accumulation of [(3)H]GABA in rat brain cortical slices.

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096mM) with an EC50 of 0.26mM and 35μM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23mM sevoflurane and 0.048mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63%, respectively. Incubation of brain cortical slices at low temperature (17°C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75%, respectively, relative to that at normal temperature (37°C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because β-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.

Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in apple fruit

BackgroundThe ubiquitous, non-proteinaceous amino acid GABA (γ-aminobutyrate) accumulates in plants subjected to abiotic stresses such as chilling, O2 deficiency and elevated CO2. Recent evidence indicates that controlled atmosphere storage causes the accumulation of GABA in apple (Malus x domestica Borkh.) fruit, and now there is increasing interest in the biochemical mechanisms responsible for this phenomenon. Here, we investigated whether this phenomenon could be mediated via Ca2+/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity.ResultsGAD activity in cell-free extracts of apple fruit was stimulated by Ca2+/CaM at physiological pH, but not at the acidic pH optimum. Based on bioinformatics analysis of the apple genome, three apple GAD genes were identified and their expression determined in various apple organs, including fruit. Like recombinant Arabidopsis GAD1, the activity and spectral properties of recombinant MdGAD1 and MdGAD2 were regulated by Ca2+/CaM at physiological pH and both enzymes possessed a highly conserved CaM-binding domain that was autoinhibitory. In contrast, the activity and spectral properties of recombinant MdGAD3 were not affected by Ca2+/CaM and they were much less sensitive to pH than MdGAD1, MdGAD2 and Arabidopsis GAD1; furthermore, the C-terminal region neither bound CaM nor functioned as an autoinhibitory domain.ConclusionsPlant GADs typically differ from microbial and animal GAD enzymes in possessing a C-terminal 30–50 amino acid residue CaM-binding domain. To date, rice GAD2 is the only exception to this generalization; notably, the C-terminal region of this enzyme still functions as an autoinhibitory domain. In the present study, apple fruit were found to contain two CaM-dependent GADs, as well as a novel CaM-independent GAD that does not possess a C-terminal autoinhibitory domain.

γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii

γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.

Is GABA-shunt functional in endodormant grapevine buds under respiratory stress?

It has been suggested that a respiratory stress is part of the mechanism through which the dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, induce the release of buds from the endodormancy (ED) in grapevines. The accumulation of metabolites like succinate, alanine (Ala) and γ-amino butyric acid (GABA), together with the activation of the GABA-shunt pathway, is a general feature of plants in response to oxygen deprivation and to respiratory stress. Unexpectedly, in a previous study, we found that GABA applied exogenously to grapevine buds, down-regulated the expression of most genes encoding for antioxidant enzymes, suggesting that its accumulation under respiratory stress conditions could be deleterious for the bud. In order to analyze whether GABA accumulates under respiratory stress conditions in grapevine buds, we analysed in this study, the effect of hypoxia, the respiration inhibitor KCN and the dormancy breaker compound HC, on the level of GABA, and on the expression levels of the GABA-shunt genes (VvGAD, VvGABA-T, VvSSADH). Additionally, genes from the Ala fermentative pathway (VvAlaAT, VvAspAT) were also analysed. The results revealed that although the three treatments mentioned above, up-regulated the expression of VvGAD1, the content of GABA remained constant, while Ala content increased. The lack of GABA accumulation under respiratory stress is an important physiological fact in grapevine buds, since it avoids the down-regulation of antioxidant genes, and promotes the incorporation of succinate into the TCA cycle, a fact that would be important in the release of buds from the ED.

Characterization of γ-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress

The molecular response of plants to abiotic stresses has been considered a process mainly involved in the modulation of transcriptional activity of stress-related genes. Nevertheless, recent findings have suggested new layers of regulation and complexity. Upstream molecular mechanisms are involved in the plant response to abiotic stress. Plants gain resistance to abiotic stress by reprogramming metabolism and gene expression. GABA is proposed to be a signaling molecule involved in nitrogen metabolism, regulating the cytosolic pH, and protection against oxidative damage in response to various abiotic stresses. The aim of our study was to examine the role of the GABA shunt pathway-specific response in five wheat (Triticum aestivum L.) cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes and Nodsieh) to salt and osmotic stress in terms of seed germination, seedling growth, oxidative damage (malondialdehyde (MDA) accumulation), and characterization of the glutamate decarboxylse gene (GAD) m-RNA level were determined using RT-PCR techniques. Our data showed a marked increase in GABA, MDA and GAD m-RNA levels under salt and osmotic stress in the five wheat cultivars. Um Qayes cultivar showed the highest germination percentage, GABA accumulation, and MDA level under salt and osmotic stresses. The marked increase in GAD gene expression explains the high accumulation of the GABA level under both stresses. Our results indicated that the GABA shunt is a key signaling and metabolic pathway that allows wheat to adapt to salt and osmotic stress. Based on our data, the Um Qayes wheat cultivar is the cultivar most recommended to be grown in soil with high salt and osmotic contents.

Suppression of γ-Aminobutyric Acid (GABA) Transaminases Induces Prominent GABA Accumulation, Dwarfism and Infertility in the Tomato (Solanum lycopersicum L.)

Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α-ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild-type and single mutant strains. Although wild-type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non-lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild-type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild-type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N-tert-butyl-α-phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild-type. Measurement of intracellular GABA, glutamate and α-ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α-ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α-ketoglutarate to succinate during heat stress.

Salt Stress Induces Accumulation of γ–Aminobutyric Acid in Germinated Foxtail Millet (Setaria italica L.)

ABSTRACTEffects of NaCl treatments on sprout length, the contents of soluble protein, free amino acids, and γ‐aminobutyric acid (GABA) and on glutamate decarboxylase (GAD) activity in germinated foxtail millet were investigated, and the regulating effects of exogenous Ca2+, along with lanthanum chloride (LaCl3, a specific inhibitor of the Ca2+ pathway) and ethylene glycol tetraacetic acid (EGTA, a chelator of Ca2+) under salt stress, on GABA accumulation in germinated millet were examined in this paper. The results showed that NaCl treatments caused a decrease in sprout length of millet. Low concentration of NaCl treatments increased soluble protein content, but high concentration decreased soluble protein content. The level of free amino acids, GAD activity, and GABA content increased significantly under NaCl stress. Exogenous Ca2+ application under NaCl stress further increased GAD activity and GABA content; the optimal concentration of Ca2+ for GAD activity and GABA accumulation was 5.0mM under 100mM NaCl for 48 hr, at which GABA content was 31.92 mg/100 g, a 1.41‐fold increase as compared with that in seeds under NaCl stress (22.64 mg/100 g). GAD activity and GABA accumulation in germinated millet decreased when treated with LaCl3 or EGTA under NaCl stress. Our results suggest that salt stress combined with Ca2+ treatment may be used for preparation of millet with higher GABA content, which can be used as a natural resource for functional foods.

Functional γ-Aminobutyrate Shunt in Listeria monocytogenes: Role in Acid Tolerance and Succinate Biosynthesis

Listeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whether L. monocytogenes has this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis in L. monocytogenes. Using crude protein extracts from L. monocytogenes EGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities, argD (GABA-AT) and lmo0913 (SSDH). Crude protein extracts prepared from a mutant lacking a functional argD gene significantly reduced GABA-AT activity, while an lmo0913 mutant lost all detectable SSDH activity. The deletion of lmo0913 increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genus Listeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle of Listeria.

Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2-1 knockout and asn2-2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO(2) assimilation was not significantly different between lines under both 21 and 2% O(2). ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered (15) N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant.