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GABA in plants: Just a metabolite?
Abstract
For decades, GABA in plants has been treated merely as a metabolite, mostly in the context of the response to stress. Recent evidence from the exploitation of Arabidopsis functional genomic tools points towards a new possible role of GABA as a signal molecule and provides further insights into the role of the GABA metabolic pathway in response to stress and carbon:nitrogen metabolism. The challenge now is to uncouple the signaling and metabolic roles of GABA, and to identify the molecular components and their mode of action.
... Studies in Arabidopsis have found that the POP 2 gene encodes GABA transaminase, which is the primary rate-limiting enzyme in GABA metabolism [1]. Further research has revealed that there are two existing forms of GABA-T in plants [2,3]. ...
... Consequently, the stems cannot elongate properly [12]. Meanwhile, knockout of the GAD gene in Arabidopsis causes a reduction in GABA content, which is needed for normal root development [2]. Alterations in the expression level of the POP2 gene in Arabidopsis altered the normal GABA concentration in the stigma, which caused a decrease in the efficiency of ovule fertilization [1]. ...
... Intermediate products produced during plant growth, such as oxaloacetate and α-ketoglutaric acid, can be used to synthesize other products that help plants to protect themselves against different stress conditions. However, if these intermediates are employed broadly, they will affect the normal operation of the TCA cycle, thus affecting the normal growth and development of the plant [2]. Normally, these intermediates are supplemented by oxaloacetate generated by the PEP pathway, but this pathway does not proceed normally when plants are subjected to certain stress conditions. ...
aminobutyric acid (GABA) is closely related to the growth, development and stress resistance of plants. Combined with the previous study of GABA to promote the cotton against abiotic stresses, the characteristics and expression patterns of GABA branch gene family laid the foundation for further explaining its role in cotton stress mechanism. Members of GAD, GAB-T and SSADH (three gene families of GABA branch) were identified from the Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii genome. The GABA branch genes were 10 GAD genes, 4 GABA-T genes and 2 SSADH genes. The promoter sequences of genes mainly contains response-related elements such as light, hormone and environment.Phylogenetic analysis shows that GAD indicating that even in the same species, the homologous sequences in the family. The GABA-T gene of each cotton genus was in sum the family had gene loss in the process of dicotyledon evolution. SSADH families Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii were closely related to the dicot plants.GABA gene is involved in the regulation of salt stress and high temperature in Gossypium hirsutum.GABA attenuated part of the abiotic stress damage by increasing leaf protective enzyme activity and reducing reactive oxygen species production.This lays the foundation for a thorough analysis of the mechanism of GABA in cotton stress resistance.
... Under drought stress in chickpeas, the amount of tryptophan increased, which is involved in the ROS scavenging mechanism, leading to decreased damage to the photosystem. When chickpeas were exposed to drought stress, tyrosin and histidine as amino acids increased [30], and it is reported that tyrosin plays a role in biotic and abiotic tolerance [38][39][40][41][42]. Different reports indicate that arginine is an important amino acid that produces a mechanism for the transmission of nitrogen in plants during high-stress conditions [43,44]. ...
... Raffinose is a sugar that acts as an osmoprotectant that protects plant cells during most stressful situations, especially at the subsequent stages of stress treatment [21], and protects plants from oxidative damage [101]. GABA is another metabolite that plays a role in biotic and abiotic stress responses [40,102,103]. This compound protects plants against different stress situations such as protection against oxidative stress, regulation of cytosolic pH, and functions of GABA as a signaling and osmoregulation molecule [40]. ...
... GABA is another metabolite that plays a role in biotic and abiotic stress responses [40,102,103]. This compound protects plants against different stress situations such as protection against oxidative stress, regulation of cytosolic pH, and functions of GABA as a signaling and osmoregulation molecule [40]. Obata and Farnie showed that amino acids, including threonine, leucine, methionine, lysine, valine, and isoleucine, were usually induced during environmental stress conditions [21]. ...
Population growth in the world has made the production of food to feed this population a major challenge. The Food and Agriculture Organization (FAO) estimates that to meet human food needs by 2050, crop productivity must double. Legumes family plays an important role in food security, poverty alleviation, and sustainability. It is determined that plant development and stress responses, as well as processes such as growth, the integrity of cells, energy storing, cellular signaling, formation of membrane and scaffolding, cellular replenishing, and whole-plant resource assignment, are managed by plant metabolites. One of the important parts of early stress responses concerns changes in plant metabolism, which includes the accumulation of antioxidants for the protection of cellular components from oxidative damage and the accumulation of compatible solutes that retain water in the cell. Other components, such as GABA and amino acids, including threonine, leucine, methionine, lysine, valine, and isoleucine, were usually induced during environmental stress conditions. In general, it was determined that plants containing various metabolites alter their physiology to adapt to various situations, such as stress. Important metabolites that play a role in tolerance to stress in legumes can help breeding programs in developing stress-tolerant cultivars to increase food security in the world.
... GABA plays a multilayered function in plants. It has been proposed, with strong evidence, that GABA is not only a key regulator of primary and secondary metabolic pathways, particularly the TCA cycle and carbon/nitrogen metabolism, but also it acts as a key signaling molecule in plant growth and development [44,55,56]. For instance, GABA is required for pollen tube growth and guidance [57], as well as cell elongation [58]. ...
... The non-proteinogenic amino acid, GABA was reported in plants more than seven decades ago when Steward and his colleagues initially reported it as a major nitrogenous compound in the potato tubers [82]. Since this time, the physiological roles of GABA as a primary metabolite in plant growth and development, as well as stress responses have been extensively studied [44,[55][56][57][58][59][60][61]. GABA regulates numerous primary and secondary metabolic pathways, particularly the TCA cycle, and acts as a signaling molecule in plant growth and development [44,55,56]. ...
... Since this time, the physiological roles of GABA as a primary metabolite in plant growth and development, as well as stress responses have been extensively studied [44,[55][56][57][58][59][60][61]. GABA regulates numerous primary and secondary metabolic pathways, particularly the TCA cycle, and acts as a signaling molecule in plant growth and development [44,55,56]. Moreover, GABA accumulates in response to various abiotic stress [59][60][61], as well as biotic stressors such as insect herbivores [53,[78][79][80], viral diseases [71,72], fungal diseases [62][63][64][65][66][68][69][70], and bacterial pathogens [32,36,[73][74][75][76][77]. ...
The devastating citrus disease, Huanglongbing (HLB), is associated with ‘Candidatus Liberibacter sp.’ and transmitted by citrus psyllids. Unfortunately, HLB has no known sustainable cure yet. Herein, we proposed γ-aminobutyric acid (GABA) as a potential eco-friendly therapeutic solution to HLB. Herein, we used GC/MS-based targeted metabolomics combined with gene expression to investigate the role of GABA in citrus response against HLB and to better understand its relationship(s) with different phytohormones. GABA supplementation via root drench boosts the accumulation of endogenous GABA in the leaves of both healthy and ‘Ca. L. asiaticus’-infected trees. GABA accumulation benefits the activation of a multi-layered defensive system via modulating the phytohormone levels and regulating the expression of their biosynthesis genes and some pathogenesis-related proteins (PRs) in both healthy and ‘Ca. L. asiaticus’-infected plants. Moreover, our findings showed that GABA application stimulates auxin biosynthesis in ‘Ca. L. asiaticus’-infected plants via the activation of the indole-3-pyruvate (I3PA) pathway, not via the tryptamine (TAM)-dependent pathway, to enhance the growth of HLB-affected trees. Likewise, GABA accumulation was associated with the upregulation of SA biosynthesis genes, particularly the PAL-dependent route, resulting in higher SA levels that activated CsPR1, CsPR2, CsPR5, and CsWRKY70, which are prominent to activation of the SA-mediated pathway. Additionally, higher GABA levels were correlated with an enhanced JA profile and linked with both CsPR3 and CsPR4, which activates the JA-mediated pathway. Collectively, our findings suggest that exogenous GABA application might be a promising alternative and eco-friendly strategy that helps citrus trees battle HLB.
... While, the roles of GABA in improving antioxidant activities is well documented, it remains unclear whether this role is direct or indirect. Bouche and Fromm [49] suggested that GABA degradation could limit ROS production under stress conditions. Evidence for this hypothesis comes from the studies on mutant yeast, where the absence of GABA-shunt genes resulted in sensitivity to H 2 O 2 , highlighting potential role of GABA in activating the antioxidant defense system to scavenge ROS. ...
... Evidence for this hypothesis comes from the studies on mutant yeast, where the absence of GABA-shunt genes resulted in sensitivity to H 2 O 2 , highlighting potential role of GABA in activating the antioxidant defense system to scavenge ROS. Furthermore, proline-GABA transporters aid plants in accumulating various osmolytes and compatible solutes, providing protection against water stress conditions [49]. Drought stress promotes the degradation of polyamines into GABA due to the activation of GABArelated enzymes [50]. ...
Background
Changing climate is causing erratic rainfall and prolonged drought periods, thus posing serious threats to crop productivity. Owing to severity of drought events, it is imperative to take proactive measures to enhance the resilience of drought sensitive crops like rice. Therefore, the present study was carried out to improve the drought stress tolerance in rice through gamma amino butyric acid (GABA) application.
Methods
The experiment was included four GABA concentrations i.e., 0 mM as control, 1 mM, 1.5 mM, and 2 mM, two water levels i.e., 100% and 50% field capacity (referred as FC100 for well-watered and FC50 for drought conditions, respectively), and two fragrant rice cultivars i.e., Super Basmati and Basmati-515.
Results
The findings unveiled a comprehensive improvement in various parameters with GABA application in fragrant rice under both well-watered (FC100) and water-limited (FC50) conditions, compared to the control. Specifically, GABA induced enhancements were observed in plant height, root length, fresh weight, dry weight, total soluble protein content, and total free amino acid content across both cultivars. Moreover, GABA application significantly improved peroxidase (POD) and catalase (CAT) enzyme activities, alongside elevating anthocyanin levels, while concurrently reducing H2O2 contents in both FC100 and FC50 treatments. Furthermore, the positive impact of GABA extended to morphological traits, with notable increases in panicle length, total tillers and productive tillers per hill, branch and grain numbers per panicle, and 1000-grain weight for Super Basmati and Basmati 515 cultivars under both water regimes, compared to Ck. Similarly, the grain yield increased by 31.01% and 27.32% under FC100 and 36.85% and 27.71% under FC50 in Super Basmati and Basmati-515, respectively, in response to GABA application, compared to Ck. Additionally, principal component analysis (PCA) revealed significant variances attributed to Dim1 and Dim2, with 86.1% and 4.0% of the variance, respectively, across three bi-plots encompassing rice cultivars, water levels, and GABA treatments. Notably, all tested indices, except for H2O2 and non-productive tillers per hill, exhibited positive correlations amongst themselves and with rice yield, further emphasizing the beneficial effects of GABA application on fragrant rice under well-watered and drought conditions.
Conclusions
GABA significantly improved fragrant rice performance under both well-watered (FC100) and water-limited (FC50) conditions. Moreover, integrating GABA application into rice cultivation practices could not only improve the crop resilience to drought stress but also potentially benefiting the future food and nutritional security globally. However, however; further research is needed to understand the cellular and molecular mechanisms of the functionality of GABA in fragrant rice, particularly under drought conditions.
... γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid that has been found in uni-and multicellular organisms and is involved in many aspects of plant development (28)(29)(30). GABA is mainly produced by the decarboxylation of glutamate, and this process is catalyzed by l-glutamate decarboxylase (GAD). Knockout (KO) of GAD genes markedly reduces the GABA levels (31,32). ...
... Knockout (KO) of GAD genes markedly reduces the GABA levels (31,32). An increasing number of studies have suggested that GABA may be a signaling molecule in addition to a metabolite in plants, and this speculation is further strengthened by evidence that GABA application effectively mitigates leaf damage induced by various abiotic stresses in plants (28,(33)(34)(35)(36)(37)(38). GABA depletion resulting from GAD1 and GAD2 mutations affects stomata closure and drought tolerance in Arabidopsis. ...
Drought is a major global challenge in agriculture that decreases crop production. γ-Aminobutyric acid (GABA) interfaces with drought stress in plants; however, a mechanistic understanding of the interaction between GABA accumulation and drought response remains to be established. Here we showed the potassium/proton exchanger TaNHX2 functions as a positive regulator in drought resistance in wheat by mediating cross-talk between the stomatal aperture and GABA accumulation. TaNHX2 interacted with glutamate decarboxylase TaGAD1, a key enzyme that synthesizes GABA from glutamate. Furthermore, TaNHX2 targeted the C-terminal auto-inhibitory domain of TaGAD1, enhanced its activity, and promoted GABA accumulation under drought stress. Consistent with this, the tanhx2 and tagad1 mutants showed reduced drought tolerance, and transgenic wheat with enhanced TaNHX2 expression had a yield advantage under water deficit without growth penalty. These results shed light on the plant stomatal movement mechanism under drought stress and the TaNHX2-TaGAD1 module may be harnessed for amelioration of negative environmental effects in wheat as well as other crops.
... Amino acids, sugars and sugar alcohols can function as osmoprotectants/osmolytes that help stabilize membranes and proteins (Nayyar et al., 2014;Jogawat, 2019). For example, sugars (Wahid & Close, 2007;Zhang et al., 2017) and c-aminobutyric acid (GABA;Bouch e & Fromm, 2004;Suguiyama et al., 2014;Bown & Shelp, 2016;Yu et al., 2017) are well-known osmolytes produced in response to heat stress. Metabolic pathways such as the mevalonate and shikimate pathways provide intermediates of important heat stress response secondary metabolites, including phenolics (Weaver & Herrmann, 1997;Santos-S anchez et al., 2019) and volatile compounds (Niinemets et al., 2004;Sharkey, 2005;Possell & Loreto, 2013) that are proposed to protect photosynthetic machinery from oxidative damage. ...
... Chlorogenic acidinduced thermotolerance in Caenorhabditis elegans (del Valle et al., 2020) was also found to be mediated by heat shock factor HSF-1 and HSPs. A significant increase in GABA (Figs S4, S5b) was also observed, a well-known stress-induced osmoregulatory and signalling molecule (Bouch e & Fromm, 2004;Suguiyama et al., 2014;Bown & Shelp, 2016;Yu et al., 2017). Given the rapid increase in shikimic acid and its high concentration, as well as possible linkage to pigments, antioxidants, phenolic compounds and chlorogenic acid that contribute to general stability of membranes and protein complexes, our results point to the shikimate pathway being one of the most rapidly heat-responsive central metabolic pathways in this tropical-subtropical rainforest tree species. ...
Heat stress interrupts physiological thermostability and triggers biochemical responses that are essential for plant survival. However, there is limited knowledge on the speed plants adjust to heat in hours and days, and which adjustments are crucial.
Tropical–subtropical rainforest tree species (Polyscias elegans) were heated at 40°C for 5 d, before returning to 25°C for 13 d of recovery. Leaf heat tolerance was quantified using the temperature at which minimal chl a fluorescence sharply rose (Tcrit). Tcrit, metabolites, heat shock protein (HSP) abundance and membrane lipid fatty acid (FA) composition were quantified.
Tcrit increased by 4°C (48–52°C) within 2 h of 40°C exposure, along with rapid accumulation of metabolites and HSPs. By contrast, it took > 2 d for FA composition to change. At least 2 d were required for Tcrit, HSP90, HSP70 and FAs to return to prestress levels.
The results highlight the multi‐faceted response of P. elegans to heat stress, and how this response varies over the scale of hours to days, culminating in an increased level of photosynthetic heat tolerance. These responses are important for survival of plants when confronted with heat waves amidst ongoing global climate change.
... Vernalization-related cold stress may cause dysregulation of pH, oxidative stress, and metabolic disfunction [25], particularly in plants sensitive to cold stress, as already proven for MDR [18]. The increase of GABA, alanine, and proline in the pre-planting phase may alleviate the effects of cold stress by buffering the cytoplasmic acidosis and potentiating the antioxidant defense [21,26]. In fact, the synthesis of both alanine and GABA involves proton-consuming reactions, thus regulating cytosolic pH. ...
... Molina-Rueda et al. [30] reported that GABA and proline are often synthesized and accumulated at the same time in response to environmental stresses to protect plant tissues from oxidative stress. Finally, after relief from cold stress, alanine, GABA, and proline can be metabolized and/or converted to intermediates of the Krebs cycle and used to produce alpha-keto acids and ATP, thus re-activating plant metabolism and accelerating flowering [26,31]. Probably the initial higher capacity to synthesize and accumulate alanine and GABA, in addition to branched chain amino acids (BCAAs), in control conditions preserved MBO from the symptoms of cold stress determining its lower sensitivity to shorter-term cold stress (V2 treatment). ...
Ranunculus asiaticus L. is an ornamental geophyte. In commercial practice, it is mainly propagated by rehydrated tuberous roots. Vernalization before planting is a common practice to overcome the natural dormancy of tuberous roots; however, little is known about the mechanisms underlying the plant’s response to low temperatures. We investigated the influence of three preparation procedures of tuberous roots, only rehydration (control, C), and rehydration plus vernalization at 3.5 °C for 2 weeks (V2) and for 4 weeks (V4), on plant growth, leaf photosynthesis, flowering, and metabolism in plants of two hybrids, MBO (early flowering, pale orange flower) and MDR (medium earliness, bright orange flower), grown in pots in an unheated greenhouse. We reported the responses observed in the aerial part in a previous article in this journal. In this paper, we show changes in the underground organs in carbohydrate, amino acids, polyphenols, and protein levels throughout the growing cycle in the different plant stages: pre-planting, vegetative growth, and flowering. The metabolic profile revealed that the two hybrids had different responses to the root preparation procedure. In particular, MBO synthesized GABA and alanine after 2 weeks and sucrose after 4 weeks of vernalization. In contrast, MDR was more sensitive to vernalization; in fact, a higher synthesis of polyphenols was observed. However, both hybrids synthesized metabolites that could withstand exposure to low temperatures.
... GABA is transported into the mitochondria, where it is converted into succinic semialdehyde by GABA transaminases using either a-ketoglutarate (by GABA-TK) or pyruvate (by GABA-TP) as amino acid acceptors. Succinic semialdehyde is then reduced by succinic semialdehyde dehydrogenase (SSADH) to form succinate, which enters the t-carboxylic acid (TCA) cycle (Bouche and Fromm, 2004). ...
... GABA shunt metabolic pathway (Source:Bouche and Fromm, 2004) ...
The Germinated Brown Rice (GBR) market is expected to benefit from changing customer tastes and the growing number of GBR product variants. One of the main supply-side drivers supporting business growth is meeting the increasing demand for health foods. Natural ingredients in GBR, along with lower commodity penetration in developed economies, are creating prospects for large- and small-scale producers all over the world. In India GBR market is still in nascent stage and needs research on suitability of GBR processing with Indian rice varieties. There is no documented work till date on development of GBR using the PJTS Agricultural university released rice varieties. If the process to develop GBR is optimized, the rice varieties can be used in designing value added products with improved nutrient and functional properties.
... It has been found that plants often undergo metabolic imbalance and energy shortages due to enhanced respiratory pathway under stressful conditions (Heinemann and Hildebrandt 2021). GABA could be metabolized into different amino acids in plants, which is known as GABA shunt (Bouche and Fromm 2004;Ansari et al. 2021). Enhanced metabolic circulation induced by GABA priming was propitious to maintenance of osmotic adjustment (OA) and metabolites homeostasis under environmental stress (Li et al. , 2017b. ...
Main conclusion
γ-Aminobutyric acid alleviates acid-aluminum toxicity to roots associated with enhanced antioxidant metabolism as well as accumulation and transportation of citric and malic acids.
Abstract
Aluminum (Al) toxicity has become the main limiting factor for crop growth and development in acidic soils and is further being aggravated worldwide due to continuous industrial pollution. The current study was designed to examine effects of GABA priming on alleviating acid-Al toxicity in terms of root growth, antioxidant defense, citrate and malate metabolisms, and extensive metabolites remodeling in roots under acidic conditions. Thirty-seven-day-old creeping bentgrass (Agrostis stolonifera) plants were used as test materials. Roots priming with or without 0.5 mM GABA for 3 days were cultivated in standard nutrient solution for 15 days as control or subjected to nutrient solution containing 5 mM AlCl3·6H2O for 15 days as acid-Al stress treatment. Roots were sampled for determinations of root characteristics, physiological and biochemical parameters, and metabolomics. GABA priming significantly alleviated acid-Al-induced root growth inhibition and oxidative damage, despite it promoted the accumulation of Al in roots. Analysis of metabolomics showed that GABA priming significantly increased accumulations of organic acids, amino acids, carbohydrates, and other metabolites in roots under acid-Al stress. In addition, GABA priming also significantly up-regulated key genes related to accumulation and transportation of malic and citric acids in roots under acid-Al stress. GABA-regulated metabolites participated in tricarboxylic acid cycle, GABA shunt, antioxidant defense system, and lipid metabolism, which played positive roles in reactive oxygen species scavenging, energy conversion, osmotic adjustment, and Al ion chelation in roots.
... GABA, which is produced endogenously in the plant organism, is an important intermediate product of nitrogen metabolism and thereby indirectly affects amino acid biosynthesis [36]. It participates in the construction of carbon skeletons [37] and provides energy for biosynthetic processes. GABA is also involved in signaling or regulatory mechanisms [38]. ...
Citation: Decsi, K.; Ahmed, M.; Rizk, R.; Abdul-Hamid, D.; Kovács, G.P.; Tóth, Z. Emerging Trends in Non-Protein Amino Acids as Potential Priming Agents: Implications for Stress Management Strategies and Unveiling Their Regulatory Functions. Int. J. Mol. Sci. 2024, 25, 6203. https:// Abstract: Plants endure the repercussions of environmental stress. As the advancement of global climate change continues, it is increasingly crucial to protect against abiotic and biotic stress effects. Some naturally occurring plant compounds can be used effectively to protect the plants. By externally applying priming compounds, plants can be prompted to trigger their defensive mechanisms, resulting in improved immune system effectiveness. This review article examines the possibilities of utilizing exogenous alpha-, beta-, and gamma-aminobutyric acid (AABA, BABA, and GABA), which are non-protein amino acids (NPAAs) that are produced naturally in plants during instances of stress. The article additionally presents a concise overview of the studies' discoveries on this topic, assesses the particular fields in which they might be implemented, and proposes new avenues for future investigation.
... This alteration in flesh firmness is likely linked to probably associated with fruit ripening or senescence provoked induced by GA 4+7 treatment through shelf life (Seo et al., 2019). Furthermore, it was reported that GABA plays a pivotal role in controlling regulating a wide range of various physiological disturbances (Bouché and Fromm, 2004). In this study, the level of GABA was lower in GA 4+7 treated than in untreated control fruit during cold storage (Figs 1 and S5). ...
... For example, decarboxylation of glutamate can lead to the production of γ-aminobutyric acid (GABA) that in our analyses resulted to be overexpressed (p < 0.05) in the experiments under pure CO 2 (Fig. S4). GABA can in turn be oxidized to mitochondrial succinate [55][56][57] that we also found in higher levels in the algae cultivated under CO 2 . Ornithine, together with arginine is implicated in the nitrogen cycle and serves as a precursor for polyamine (PA) synthesis [53,58]. ...
... These DON catabolic pathways are possessed by a wide range of bacteria (including Clostridia and Bacilli) and generate NH 4 + as a metabolite or final product (Borek & Waelsch, 1953). The most robust prediction among DON catabolic pathways was exhibited by the 4-aminobutyrate degradation pathway (PWY-5022), a compound present in the environment as a product of plant and animal tissue decay (Bouché & Fromm, 2004). This pathway is widely carried by bacteria in phylum Firmicutes (including Clostridia and Bacilli) to produce NH 4 + and acetate. ...
Microbial communities in subterranean estuaries mediate biogeochemical reactions of coastal groundwater discharging into the oceans; however, studies on their response to abrupt environmental changes caused by climate and land use alterations are still limited. In this study, we conducted a controlled laboratory study using combined geochemical and metagenomic approaches to investigate microbial structures and their metabolic pathways under a wide range of nitrate (NO3− ${{\text{NO}}_{3}}^{-}$) inputs, saline solutions, and incubation times. These factors served as proxies for land use, salinization of the shallow aquifer, and climate changes. We found a highly reducing habitat and an amplification of genes related to denitrification, sulfate reduction, and methanogenesis processes. Core communities consisting of Clostridia, Bacilli, Alphaproteobacteria, Gammaproteobacteria, and Desulfobaccia were observed across all treatments. The metabolic prediction of plant‐derived organic matter (i.e., tannin and lignin) degradation was not affected by NO3− ${{\text{NO}}_{3}}^{-}$ inputs or salinity because of it being implemented by core communities and the abundance of electron donors and acceptors. Quantification of denitrification genes shows that they are susceptible to NO3− ${{\text{NO}}_{3}}^{-}$ inputs and seawater ions. Long‐term incubation allowed sufficient time for microbes to degrade less labile DOM, promoting the re‐release of buried solid phase organic matter into the active carbon cycle and increasing the relative abundance of biofilm or spore‐forming taxa while decreasing that of rare taxa. Our results illustrate the sensitivity of microbial assemblages to environmental changes and their capacity to altering the C and N cycles in coastal areas, further affecting coastal water quality and ecosystem‐scale biogeochemistry.
... GABA shunt enzymes have unique traits in plants that are missing in other organisms. A Ca 2+ /calmodulin (CaM)-dependent calmodulin binding domain (CaMBD) is often found at the C-terminus of plant glutamate decarboxylases (GADs), allowing plants to control the activation of GAD in response to biotic and abiotic stressors (Bouché and Fromm 2004). GABA biosynthesis commences with the decarboxylation of glutamate (Glu) into GABA by GAD in cells. ...
GABA (Gamma-aminobutyric acid) is a non-protein amino acid widely known as major inhibitory neurotransmitter. It is synthesized from glutamate via the enzyme glutamate decarboxylase (GAD). GAD is ubiquitous in all organisms, but only plant GAD has ability to bind Ca²⁺/calmodulin (CaM). This kind of binding suppresses the auto-inhibition of Ca²⁺/calmodulin binding domain (CaMBD) when the active site of GAD is unfolded resulting in stimulated GAD activity. OsGAD4 is one of the five GAD genes in rice genome. It was confirmed that OsGAD4 has ability to bind to Ca²⁺/CaM. Moreover, it exhibits strongest expression against several stress conditions among the five OsGAD genes. In this study, CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD from the OsGAD4 gene, to remove its autoinhibitory function. DNA sequence analysis of the genome edited rice plants revealed the truncation of CaMBD (216 bp). Genome edited line (#14–1) produced 11.26 mg GABA/100 g grain, which is almost nine-fold in comparison to wild type. Short deletion in the coding region for CaMBD yielded in mutant (#14–6) with lower GABA content than wild type counterpart. Abiotic stresses like salinity, flooding and drought significantly enhanced GABA accumulation in #14–1 at various time points compared to wild-type and #14–6 under the same stress conditions. Moreover, upregulated mRNA expression in vegetative tissues seems correlated with the stress-responsiveness of OsGAD4 when exposed to the above-mentioned stresses. Stress tolerance of OsGAD4 genome edited lines was evidenced by the higher survival rate indicating the gene may induce tolerance against abiotic stresses in rice. This is the first report on abiotic stress tolerance in rice modulated by endogenous GABA.
... On the other hand, GABA content decreased upon desiccation of E. uniflora seeds and was barely present in E. astringens seeds (Figure 5a). This amino acid plays a role in oxidative stress mitigation by preventing ROS accumulation (Bouché & Fromm 2003) and is supposed to facilitate early metabolic reorganization during germination after desiccation in DT seeds (Du et al., 2020;Li et al., 2021). Therefore, the absence of GABA response might also be a sign of accumulated stress in E. uniflora and E. astringens seeds (Figure 5a). ...
Myrtaceae species are abundant in tropical Atlantic rainforests, but 41% of the 5500 species of this family are of extreme conservation concern. Eugenia astringens and E. uniflora are native Brazilian Myrtaceae species that occur in the same habitats and produce desiccation-sensitive (DS) seeds. We hypothesized that their seed desiccation-sensitivity degree is associated with specific metabolic signatures. To test it, we analyzed the germination and metabolic profiles of fresh and desiccated seeds. The water content (WC) at which at least half of the seeds survived desiccation was lower in E. astringens (0.17 g H 2 O g À1 DW) than in E. uniflora (0.41 g H 2 O g À1 DW). We identified 103 annotated metabolites from 3261 peaks in both species, which differed in their relative contents between E. astringens and E. uniflora seeds. The main differences in seed metabolic profiles include several protective molecules in the group of carbohydrates and organic acids and amino acid contents. The relative contents of monosaccharides and disaccharides, malic and quinic acids, amino acids
... This finding is interesting because GABA is not just a metabolite but also acts as a signalling molecule in stressful conditions. [26] In our study, it could be possible plants grown under solar panels degraded GABA in order to limit the accumulation of reactive oxygen species. [27] If this was the case, it could indicate that tomato plants under solar panels were able to regulate stress response. ...
The emergence of semi‐transparent solar panels offers opportunities for their application in greenhouses where the radiation is a critical issue. The light passing through these panels is often affected, leading to a decrease in certain wavelengths that could potentially impact plant growth and quality. To address this concern, this study is performed to investigate the growth and yield of tomato and broccoli plants cultivated under semi‐transparent photovoltaic solar panels compared to those grown under conventional greenhouse plastic. Their physiological and metabolic changes are also examined. A decrease in the yellow/green spectrum after installation of the solar panels is observed. In both plants, slight alterations are observed by solar panels, enhancing even the height of plants and fruit yield in case of tomato. However, both plants showed changes related to their photosynthetic activity and some metabolite concentration. Specifically, there are significant reductions in An (photosynthetic rate) and lower levels of trigonelline in plants grown under solar panels. These reductions may be attributed to the different radiation conditions experienced by the plants, which do not appear to directly impact plant growth. The obtained results highlight the promising potential of solar panel‐integrated greenhouses that optimize economic and energy benefits while maintaining product quality.
... This process is associated with various responses, including cytosolic pH regulation, scavenging of ROS, and maintenance of C/N balance in the TCA cycle. Ascontaminated soil may disrupt this mechanism by increasing cytosolic acidification and inducing glutamate decarboxylase activity, ultimately leading to enhanced GAD and GABA synthesis (Bouche and Fromm 2004). The addition of exogenous GABA alone significantly and directly elevated endogenous GABA levels in leaves (43.13 ± 1.78 mM g −1 FW) during the late vegetative phase. ...
In this study, the addition of γ-aminobutyric acid (GABA), Bacillus pumilus, or both, was found to enhance rice growth and yield while significantly decreasing arsenic (As) accumulation in Oryza sativa rice tissues. GABA emerged as a regulator of iron (Fe) homeostasis, acting as a signaling modulator that influenced phytosiderophore secretions in the plant. Meanwhile, B. pumilus directly increased Fe levels through siderophore production, promoting the development of Fe-rich rice plants. Subsequently, Fe competed with As uptake at the root surface, leading to decreased As levels and translocation to the grains. Furthermore, the addition of GABA and B. pumilus optimized rice indole-3 acetic acid (IAA) contents, thereby adjusting cell metabolite balance under As stress. This adjustment results in low malondialdehyde (MDA) contents in the leaves and roots during the early and late vegetative phases, effectively reducing oxidative stress. When added to As-contaminated soil, GABA and B. pumilus effectively maintained endogenous GABA levels and exhibited low ROS generation, similar to normal soil. Concurrently, GABA and B. pumilus significantly downregulated the activity of OsLsi1, OsLsi2, and OsABCC1 in roots, reducing As uptake through roots, shoots, and grains, respectively. These findings suggest that GABA and B. pumilus additions impede As translocation through grains, ultimately enhancing rice productivity under As stress.
... GABA is produced by bacteria [3,52] fungi [53,54], plants [55,56], vertebrate animals and invertebrates [57][58][59]. Using the same terms in molecular language among various species orders means having part of the language in common. ...
In recent decades, given the important role of gamma-aminobutyric acid (GABA) in human health, scientists have paid great attention to the enrichment of this chemical compound in food using various methods, including microbial fermentation. Moreover, GABA or GABA-rich products have been successfully commercialized as food additives or functional dietary supplements. Several microorganisms can produce GABA, including bacteria, fungi, and yeasts. Among GABA-producing bacteria, lactic acid bacteria (LAB) are commonly used in the production of many fermented foods. Lactiplantibacillus plantarum (previously Lactobacillus plantarum) has a long history of natural occurrence and safe use in a variety of food products. This LAB species provides functional properties to various fermented foods by producing a variety of bioactive compounds, including GABA. The present review aims, after a preliminary excursus on the function and biosynthesis of GABA, to provide an overview on the current uses of microorganisms and in particular of L. plantarum in the production of GABA, with a detailed focus on fermented foods.
... 148 The primary role of GABA are signalling molecule, osmo-regulator, and cytosolic pH regulator. 149 The accumulation of GABA in response to water stress stimuli often shares patterns with that of the BCAAs (valine, leucine, and isoleucine) and other amino acids that share their synthesis pathways (lysine, threonine, and methionine). 150 The healthy growth of plants depends on BCAAs, which can also serve as suitable solutes, alternative electron donors for the mitochondrial electron transport chain, and substrates for secondary metabolites that are protective such as cyanogenic glycosides, glucosinolate, and acyl-sugars. ...
Water stress poses a significant risk to achieving global food security. Plants experience water stress due to variations in environmental conditions, which have been identified as the primary factor impacting agricultural production under the current dispensation of global climate change. This review aims to combine data from multiple published research studies in the literature in order to completely analyze and assess the morphological, physiobiochemical, and metabolite responses of plants to water stress. Stress-related variables disturb the regular stability of plants, leading to changes in their structure, function, chemistry, and genetics that ultimately impact their development and productivity. Water stress often leads to reduced leaf-relative water content, loss of turgor, and closure of stomata. Plant secondary metabolites serve as distinct sources for medicines, food additives, and industrially essential biochemicals. Plants exposed to water stress, such as different elicitors or signal molecules, frequently experience the build-up of these metabolites. Secondary metabolites significantly contribute to plants' ability to adapt to their environment and overcome stressful conditions. Plants exhibit diverse metabolic reactions to water stress, resulting in alterations in the composition of volatile oils in different herbs. The attempt to tackle water stress conditions by plants is inherently intricate due to the complex network of signaling. This review contributes to our understanding of plant responses to water stress conditions, to the implementation of sustainable agronomic practices, and development of varieties that can tolerate the impact of climate change.
... As underscored by Miguel-Tomé & Llinás (2021), citing Toyota et al. (2018), glutamate's presence can catalyze a surge in calcium ion concentrations, facilitating electrical signal transmission in plants, particularly post-injury. Current plant research is rethinking GABA, viewing it not just as a metabolic offshoot but also as a pivotal signaling compound (Bouché et al., 2003;Bouché & Fromm, 2004). The multifunctionality of these molecules in plant processes, from growth to stress responses (Žárský, 2015;Ramesh et al., 2017), sheds light on the evolutionary progression of neurotransmitters. ...
... GABA is a four-carbon non-protein amino acid that acts as a signaling and defense molecule in plant tissues and organs (Breitkreuz and Shelp, 1995;Wang et al., 2017;Balfagoń et al., 2021). It comprises a significant fraction of the free amino acid pool in plant cells, serving as an important neurotransmitter while being involved in alleviating abiotic stresses (Nicolas Bouche, 2004;Nayyar et al., 2014;Balusǩa et al., 2020). The exogenous activity of GABA simulates the effects of stress on growth and development and increases endogenous GABA concentrations in tissues in response to diverse abiotic influences (Michaeli and Fromm, 2015). ...
Plants experience constant exposed to diverse abiotic stresses throughout their growth and development stages. Given the burgeoning world population, abiotic stresses pose significant challenges to food and nutritional security. These stresses are complex and influenced by both genetic networks and environmental factors, often resulting in significant crop losses, which can reach as high as fifty percent. To mitigate the effects of abiotic stresses on crops, various strategies rooted in crop improvement and genomics are being explored. In particular, the utilization of biostimulants, including bio-based compounds derived from plants and beneficial microbes, has garnered considerable attention. Biostimulants offer the potential to reduce reliance on artificial chemical agents while enhancing nutritional efficiency and promoting plant growth under abiotic stress condition. Commonly used biostimulants, which are friendly to ecology and human health, encompass inorganic substances (e.g., zinc oxide and silicon) and natural substances (e.g., seaweed extracts, humic substances, chitosan, exudates, and microbes). Notably, prioritizing environmentally friendly biostimulants is crucial to prevent issues such as soil degradation, air and water pollution. In recent years, several studies have explored the biological role of biostimulants in plant production, focusing particularly on their mechanisms of effectiveness in horticulture. In this context, we conducted a comprehensive review of the existing scientific literature to analyze the current status and future research directions concerning the use of various biostimulants, such as plant-based zinc oxide, silicon, selenium and aminobutyric acid, seaweed extracts, humic acids, and chitosan for enhancing abiotic stress tolerance in crop plants. Furthermore, we correlated the molecular modifications induced by these biostimulants with different physiological pathways and assessed their impact on plant performance in response to abiotic stresses, which can provide valuable insights.
... GABA connects the carbon and nitrogen metabolic fluxes in plants through the GABA shunt (Fait et al., 2008). Several studies have shown that GABA can promote pollen tube elongation and stem growth at low concentrations (Bouchéand Fromm, 2004). In addition, IAA increases the accumulation of plant biomass. ...
Introduction
The primary metabolism of plants, which is mediated by nitrogen, is closely related to the defense response to insect herbivores.
Methods
An experimental system was established to examine how nitrogen mediated tomato resistance to an insect herbivore, the oriental fruit fly (Bactrocera dorsalis). All tomatoes were randomly assigned to the suitable nitrogen (control, CK) treatment, nitrogen excess (NE) treatment and nitrogen deficiency (ND) treatment.
Results
We found that nitrogen excess significantly increased the aboveground biomass of tomato and increased the pupal biomass of B. dorsalis. Metabolome analysis showed that nitrogen excess promoted the biosynthesis of amino acids in healthy fruits, including γ-aminobutyric acid (GABA), arginine and asparagine. GABA was not a differential metabolite induced by injury by B. dorsalis under nitrogen excess, but it was significantly induced in infested fruits at appropriate nitrogen levels. GABA supplementation not only increased the aboveground biomass of plants but also improved the defensive response of tomato.
Discussion
The biosynthesis of GABA in tomato is a resistance response to feeding by B. dorsalis in appropriate nitrogen, whereas nitrogen excess facilitates the pupal weight of B. dorsalis by inhibiting synthesis of the GABA pathway. This study concluded that excess nitrogen inhibits tomato defenses in plant-insect interactions by inhibiting GABA synthesis, answering some unresolved questions about the nitrogen-dependent GABA resistance pathway to herbivores.
... The regulation of lipid synthesis was tightly linked with photosynthesis and the transport of photosynthetic byproducts (Armarego-Marriott et al. 2019). Nitrogen deficiency not only affects the carbon sequestration of photosynthesis but also inhibits TCA cycling and fatty acid synthesis (Bouche andFromm 2004, Makino 2011). During nitrogen deficiency in plants, the expression of essential enzyme genes in the Calvin cycle, the rate of energy metabolism and the levels of ATP, NADH and NADPH were significantly reduced in both roots and leaves (Shen et al. 2022, Xia et al. 2022. ...
Nitrogen is one of the most essential macronutrients for plant growth and its availability in soil is vital for agricultural sustainability and productivity. However, excessive nitrogen application could reduce the nitrogen use efficiency and produce environmental pollution. Here, we systematically determined the response in lipidome and metabolome in rapeseed during nitrogen starvation. Plant growth was severely retarded during nitrogen deficiency, while the levels of most amino acids was significantly decreased. The levels of monogalactosyl diacyglycerol (MGDG) in leaves and roots was significantly decreased, while the level of digalactosyl diacylglycerol (DGDG) was significantly decreased in roots, resulting in significant reduction of MGDG/DGDG ratio during nitrogen starvation. Meanwhile, the levels of sulfoquinovosyl diacylglycerol, phosphatidylglycerol and glucuronosyl diacylglycerol was reduced to varying extents. Moreover, the levels of metabolites in the tricarboxylic acid cycle, Calvin cycle, and energy metabolism was changed during nitrogen deficiency. These findings show that nitrogen deprivation alters the membrane lipid metabolism and carbon metabolism, and our study provides valuable information to further understand the response of rapeseed to nitrogen deficiency at metabolism level.
... 13 hardening process, thereby retaining neural function and providing better endurance against thermal stress. Furthermore, the higher levels of glutamate in H mussels could potentially supply additional energy sources to the TCA cycle, thereby increasing GABA production through the GABA shunt (Bouche and Fromm, 2004;Singh and Roychoudhury, 2020). ...
Introduction: Temperature affects organisms’ metabolism and ecological performance. Owing to climate change, sea warming constituting a severe source of environmental stress for marine organisms, since it increases at alarming rates. Rapid warming can exceed resilience of marine organisms leading to fitness loss and mortality. However, organisms can improve their thermal tolerance when briefly exposed to sublethal thermal stress (heat hardening), thus generating heat tolerant phenotypes.
Methods: We investigated the “stress memory” effect caused by heat hardening on M. galloprovincialis metabolite profile of in order to identify the underlying biochemical mechanisms, which enhance mussels’ thermal tolerance.
Results: The heat hardening led to accumulation of amino acids (e.g., leucine, isoleucine and valine), including osmolytes and cytoprotective agents with antioxidant and anti-inflammatory properties that can contribute to thermal protection of the mussels. Moreover, proteolysis was inhibited and protein turnover regulated by the heat hardening. Heat stress alters the metabolic profile of heat stressed mussels, benefiting the heat-hardened individuals in increasing their heat tolerance compared to the non-heat-hardened ones.
Discussion: These findings provide new insights in the metabolic mechanisms that may reinforce mussels’ tolerance against thermal stress providing both natural protection and potential manipulative tools (e.g., in aquaculture) against the devastating climate change effects on marine organisms.
... The E226 genome also harboured genes that could assist the plant in the mitigation of stresses including some encoding for catalase and superoxide dismutase (Mittler, 2002). Furthermore, the bacterial genome of E226 contained genes that contribute to the biosynthesis of the microbe-inhibiting gamma-aminobutyric acid (GABA) (e.g., 4-aminobutyrate aminotransferase), and protein-protecting chaperones DnaK and GroEL (Bouché and Fromm, 2004;Wang et al., 2004). ...
... The studies in barley have shown an increment of γ-Aminobutyric acid (GABA) in soaked and germinated grains [11,12]. GABA is a four-carbon non-protein amino acid occurring in plants and animals [13], which play an important role as neurotransmitter in mammal's brain cells [14]. On the other hand, most barley phenolic compounds have also been identified in malt, which implies that natural antioxidants present in barley make a large contribution to the antioxidant activity of malt [15]. ...
This work aimed to evaluate the chemical composition, bioactive compounds (phenolics and γ-aminobutyric acid, GABA), and antioxidant properties of different barley varieties (Overture, Charles, Sinfonía, Montoya, and Andreia) and their malts to weigh up them as potential ingredients for producing new bio-functional foods. For this, five barleys and five malts obtained from them were studied. Regarding chemical composition, total starch was the main component (≈62%) of barleys followed by total dietary fiber (≈22.6%) and proteins (≈9.5%). Potassium and phosphorus were the most abundant elements, with mean values being 3746.1 and 3679.1 g 100g<sup>-1</sup>d.w., respectively. Regarding the free amino acid profile, the proportion of hydrophobic free amino acids was higher than that of branched-chain amino acids or sulfur-containing amino acids and the mean value of GABA was 8.8 mg 100g<sup>-1</sup>. Ferulic acid was the most abundant free phenolic acid detected in the different barleys, followed by coumaric acid. All barley extracts showed ABTS and DPPH inhibitory activities and ferric-reducing antioxidant power (FRAP). As expected, total starch, total dietary fiber, and crude fat contents of malts were lower than those found for barley. However, the malting process increased GABA, ferulic acid, hydrophobic free amino acids, branched-chain amino acids, and sulfur amino acid contents. Additionally, the antioxidant properties of malts were higher than those obtained for barleys. Barley flour could be successfully used as a bio-functional ingredient in many foods. Furthermore, given the high content of soluble solids (mainly carbohydrates, antioxidant compounds such as free phenolic acids and free amino acids, and GABA), malted flours would be novel ingredients for preparing beverages with bio-functional properties.
... According to recent studies, GABA may lessen heat stress in plants by boosting photosynthetic process and elevating the actions of radical quenching enzymes . In numerous plant signal transduction pathways, GABA has been discovered to act as a modulator of stress-induced stimuli (Bouche and Fromm 2004;Li et al. 2016;Yu et al. 2014). ...
Plants rely on photosynthesis to convert light energy into chemical energy. However, their photosynthetic performance can be greatly affected by changes in the abiotic environment such as temperature, light intensity, and water availability. This draft summarizes the impact of changing abiotic conditions on photosynthetic adaptation in plants. Plants have developed various adaptive mechanisms to optimize their photosynthetic efficiency under different abiotic stresses. For example, under high light intensity, plants may regulate their photosynthetic apparatus by reducing the size of their light-harvesting antenna or increasing the activity of photorespiration. Similarly, under low water availability, plants can close their stomata to prevent water loss and reduce their photosynthetic activity, or activate molecular pathways to enhance drought tolerance. Understanding the molecular mechanisms underlying these adaptations is essential for developing strategies to improve crop productivity and sustainability under changing environmental conditions. Advances in molecular biology and biotechnology have provided new tools for identifying genes and proteins involved in photosynthetic adaptation in plants. These findings can be applied to develop crop varieties that are better adapted to different environmental conditions, such as drought, high temperatures, or high salinity. Despite the progress made in understanding the impact of changing abiotic environments on photosynthetic adaptation in plants, there are still many challenges to be addressed. The complex interactions between plants and their environment, as well as the potential effects of multiple stresses, require further investigation. In addition, there is a need to develop sustainable agricultural practices that can mitigate the negative impacts of climate change on crop productivity.
... A Scifinder-n search (November 2022) of the terms 'Glycine AND crystal' compared to 'GABA AND crystal' led to 12,331 and 858 results, respectively. GABA, an important nonessential amino acid with a variety of GABA receptors named after it, acts as a neurotransmitter inhibitor and is linked to sleep and stress relief [36][37][38][39]. ...
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.
... The activity of this pathway is crucial in preserving the quality of fruits and vegetables during postharvest cold storage . The pathway can reduce sensitivity to environmental stress in some mutant plants (Bouché et al. 2003;Bouché and Fromm 2004). Melatonin can increase the activity of the GABA transaminase (GABA-T) enzyme, enabling fruits and vegetables to produce more ATP used in the removal of excess ROS (Carvajal et al. 2015). ...
The physiological process of ripening occurs rapidly when fruits and vegetables become mature, and beyond a specific stage after the harvest, they to undergo rapid deterioration in quality. Melatonin, a nontoxic biological molecule with significant antioxidant capacity, plays several roles, including delaying senescence, alleviating chilling injury, enhancing resistance to diseases, and tolerance to stress conditions during postharvest preservation of fruits and vegetables. Interestingly, the application of exogenous melatonin to prolong the shelf life of fruits and vegetables augment the endogenous molecule, thereby promoting these functions. There is crosstalk among different physiological and biochemical processes involved in melatonin action, which remain largely elusive. This chapter provides insights into those mechanisms and discusses several case studies demonstrating the promising effects of melatonin treatment on the postharvest preservation of fruits and vegetables.KeywordsMelatoninPostharvest biologyFruits and vegetablesCrosstalk, melatonin augmentation
The importance of gamma-aminobutyric acid (GABA) in plants has been highlighted due to its critical role in mitigating metal toxicity, specifically countering the inhibitory effects of copper stress on rice plants. This study involved pre-treating rice plants with 1 mM GABA for one week, followed by exposure to varying concentrations of copper at 50 μM, 100 μM, and 200 μM. Under copper stress, particularly at 100 μM and 200 μM, plant height, biomass, chlorophyll content, relative water content, mineral content, and antioxidant activity decreased significantly compared to control conditions. However, GABA treatment significantly alleviated the adverse effects of copper stress. It increased plant height by 13%, 18%, and 32%; plant biomass by 28%, 52%, and 60%; chlorophyll content by 12%, 30%, and 24%; and relative water content by 10%, 24%, and 26% in comparison to the C50, C100, and C200 treatments. Furthermore, GABA treatment effectively reduced electrolyte leakage by 11%, 34%, and 39%, and the concentration of reactive oxygen species, such as malondialdehyde (MDA), by 9%, 22%, and 27%, hydrogen peroxide (H2O2) by 12%, 38%, and 30%, and superoxide anion content by 8%, 33, and 39% in comparison to C50, C100, and C200 treatments. Additionally, GABA supplementation led to elevated levels of glutathione by 69% and 80%, superoxide dismutase by 22% and 125%, ascorbate peroxidase by 12% and 125%, and catalase by 75% and 100% in the C100+G and C200+G groups as compared to the C100 and C200 treatments. Similarly, GABA application upregulated the expression of GABA shunt pathway-related genes, including gamma-aminobutyric transaminase (OsGABA-T) by 38% and 80% and succinic semialdehyde dehydrogenase (OsSSADH) by 60% and 94% in the C100+G and C200+G groups, respectively, as compared to the C100 and C200 treatments. Conversely, the expression of gamma-aminobutyric acid dehydrogenase (OsGAD) was downregulated. GABA application reduced the absorption of Cu2+ by 54% and 47% in C100+G and C200+G groups as compared to C100, and C200 treatments. Moreover, GABA treatment enhanced the uptake of Ca2+ by 26% and 82%, Mg2+ by 12% and 67%, and K+ by 28% and 128% in the C100+G and C200+G groups as compared to C100, and C200 treatments. These findings underscore the pivotal role of GABA-induced enhancements in various physiological and molecular processes, such as plant growth, chlorophyll content, water content, antioxidant capacity, gene regulation, mineral uptake, and copper sequestration, in enhancing plant tolerance to copper stress. Such mechanistic insights offer promising implications for the advancement of safe and sustainable food production practices.
The regulation of glutamate dehydrogenase, an enzyme that is involved in both nitrogen and carbon metabolism, and also links between the tricarboxylic acid cycle and the -aminobutyric acid shunt, has been studied. It was found that oxygen deficiency-induced changes in glutamate dehydrogenase activity in maize leaves (Zea mays L.) are to increase its catalytic activity by more than twice. Differential expression of genes was studied by real-time PCR in GDH1 and GDH2, which encode the - and -subunits of glutamate dehydrogenase, respectively, in the maize genome. Decreased relative level of gene transcripts GDH2 was accompanied by an increase in the expression activity of the gene GDH1. This, in turn, presumably promoted the amination reaction of 2-oxoglutarate. In the promoter of the gene GDH2, the presence of two CpG islands 404 and 383 bp in size was found. Gene promoter GDH1 does not contain a single CpG island; however, 38% of the CpNpG and CpNpN sites of the total number of studied dinucleotides in its composition were found. To assess the influence of the degree of methylation of individual CpG dinucleotides that are part of the promoter regions of genes GDH1 and GDH2 on their expression under hypoxic conditions, a comparative analysis of the dynamics of the transcriptional activity of the genes of - and -subunits of glutamate dehydrogenase from the methyl status of their promoters was carried out. Inversely proportional superposition of changes in the methylation profile of gene promoters GDH1 and GDH2 and transformation of the level of expression of these genes shows their correlation. The data obtained as a result of methyl-specific PCR indicate that an increase in the proportion of methylated CpG dinucleotides leads to a decrease in the amount of mRNA of the gene GDH2, while a decrease in this value for the gene GDH1 causes the induction of its functioning. Methylation of promoter regions of glutamate dehydrogenase genes regulates their transcriptional activity in maize leaves in vivo under conditions of oxygen deficiency. Thus, the little data on the molecular mechanisms of regulation of the synthesis of glutamate dehydrogenase isoenzymes were supplemented by new results on the role of the degree of methylation of gene promoters GDH1 and GDH2 glutamate dehydrogenases in their differential expression during maizes adaptation to hypoxia.
Soil salinity limits plant growth, affects crop production and nutrition, and endangers food security globally. The use of salt-tolerant rootstocks has proven to be an efficient strategy to alleviate the adverse effects of salinity in different crop species including Vitis vinferae. The long-term response to salinity of three grapevine rootstock varieties, Paulsen 1103 (P1103), Richter 110 (R110), and Sélection Oppenheim 4 (SO4), was tested in pots with drip irrigation at different NaCl concentrations (0, 10 mM, and 30 mM) for 82 days. During the experiment, plant morphology, physiology, and central metabolism were monitored. The results revealed that shoot growth, including stem diameter and fresh and dry weight of stems and leaves, was significantly restricted by salinity in P1103 and SO4, less in R110. Roots were more sensitive than shoots to salinity, showing significant reductions in biomass already at 10 mM NaCl. The high salinity level markedly reduced most of the photosynthetic traits measured in P1103 and R110. In contrast, SO4 was exceptionally stable from a physiological standpoint, while having the lowest chloride contents in its leaves compared to the other two varieties when exposed to salinity. Elemental analysis and GC-MS based metabolite profiling of the leaves exposed to a salt stress revealed a shift in carbon (C) and nitrogen (N) metabolism, reflected by a decreased C: N ratio and by a lower content of organic acids (e.g. succinate, malate, and citrate) likely supporting the measured accumulation of amino acids (e.g. GABA, glutamate, and proline) in P1103 and R110. In addition, correlation-based network analysis (CNA) of the metabolic data revealed increased coordination of metabolic processes under salinity than under control conditions. CNA also showed higher alteration in the metabolic network in P1103 and R110 than in SO4. Taken together, the results reveal that SO4 showed milder alteration in its photosynthetic and metabolic response to saline conditions, in association with lower Cl−accumulation in its leaves.
Aim
This study examined the gamma-aminobutyric acid (GABA) shunt pathway in response to salt and osmotic stresses in three barley (Hordeum vulgare L.) genotypes (Acsad176, Athroh, and Rum) in terms of seed germination, seedlings growth, oxidative damage through malondialdehyde (MDA) accumulation as an indicator for reactive oxygen species (ROS), GABA metabolite accumulation, chlorophyll level, total proteins, total carbohydrates and the expression of glutamate decarboxylase gene (GAD) analysis.
Background
GABA is a secondary metabolite that modulates nitrogen metabolism, protects against oxidative damage, and cytosolic pH in response to various abiotic and biotic stress in plants.
Methods
The effects of salt and osmotic stresses imposed by different concentrations of mannitol, sorbitol, and NaCl on the three barley genotypes were studied. Seed germination, seedling length, fresh weight, and dry mass were recorded. The physiological and biochemical responses as per GABA and MDA accumulation, total chlorophyll, proteins and carbohydrates, and the level of GAD expression were also characterized and determined.
Results
Mannitol, sorbitol, and NaCl treatments decreased seed germination and seedling growth for the three barely genotypes used in this study. MDA concentration was increased in seedlings of all genotypes with increasing NaCl, mannitol, and sorbitol concentrations. Acsad 176 showed high GABA accumulation under NaCl treatment. Mannitol treatment significantly increased GABA accumulation in the Rum genotype. All salt and osmotic treatments decreased chlorophyll a and b and carbohydrate content and significantly increased GAD transcription in all barley genotypes. Salt and osmotic stresses affected the total protein content in all genotypes.
Conclusion
Acsad 176 genotype may adapt to NaCl stress by accumulating carbohydrates more than Athroh and Rum. GABA shunt is a crucial signaling and metabolic pathway facilitating barley's adaptation to salt and osmotic stress. In soil with high salt and osmotic contents, the Acsad 176 genotype is the recommended genotype for cultivation.
Heat stress poses a significant challenge to global rice production, affecting yield and grain quality. Elevated temperatures during the flowering and grain-filling stages, both day and night, lead to reduced yield and compromised grain quality. This impact is more pronounced during nighttime high-temperature stress, seriously threatening rice productivity. With global temperatures rising, there is a looming threat to rice production. Aromatic rice, prized for superior aroma and grain quality, is particularly vulnerable to heat. Therefore, the present work has been carried out to investigate how high temperature affects the aromatic metabolites in rice grains among the 15 rice genotypes (fourteen aromatic and one non-aromatic rice i.e., Nagina 22). Results from the present study indicated that the inactive (mutated) BADH2 gene expression was down-regulated under high-temperature stress conditions and no 2-acetyl-1-pyrroline (2-AP) accumulation was detected in the selected rice genotypes. However, the increase in levels of L-proline (precursor molecule for 2-AP) was detected, and due to the down-regulation of inactive BADH2, the oxidation of L-proline into 2-AP was affected. Proline amino acid significantly increased under high temperatures, impacting aroma quality. Metabolome studies revealed variations in compound detection among scented rice genotypes. Understanding these metabolites aids in addressing the loss of aroma in fragrant rice genotypes, offering insights into developing stable aromatic rice varieties under elevated temperature conditions. The study aims to identify metabolites causing aroma loss in aromatic rice. Results will aid in understanding aroma depletion mechanisms in scented rice under high-temperature stress, guiding the development of a stable aromatic rice variety in elevated temperatures.
Drought is one of the major and growing threats to agriculture productivity and food security. Metabolites are involved in the regulation of plant responses to various environmental stresses, including drought stress. The complex drought tolerance can be ascribed to several simple metabolic traits. These traits could then be used for detecting the genetic architecture of drought tolerance. Plant metabolomes show dynamic differences when drought occurs during different developmental stages or upon different levels of drought stress. Here, we reviewed the major and most recent findings regarding the metabolite‐mediated plant drought response. Recent progress in the development of drought‐tolerant agents is also discussed. We provide an updated schematic overview of metabolome‐driven solutions for increasing crop drought tolerance and thereby addressing an impending agricultural challenge.
Gamma‐aminobutyric acid (GABA), a ubiquitously present non‐proteinogenic amino acid, has recently emerged as a key regulator of growth and development in plants during normal as well as challenging environmental conditions. GABA biosynthesis has been reported at multiple stages of plant development, particularly during vegetative and reproductive stages and in response to stress conditions. Accumulating evidence has highlighted the crucial roles of various cell cycle regulators such as type‐D cyclins and CDK;A1, transcription factors such as E2Fa, as well as Ca 2+/ Calmodulin proteins in GABA biosynthesis in plants. GABA is known to improve stress tolerance by improving photosynthetic activity, C/N metabolism, stomatal conductance, and stress‐induced reactive oxygen species (ROS) detoxification. Here, we have reviewed recent studies that have explored the novel roles of GABA in plants with a focus on plant development and stress resilience.
In recent decades, given the important role of gamma-aminobutyric acid (GABA) in human health, scientists have paid great attention to the enrichment of this chemical compound in food using various methods, including microbial fermentation. Moreover, GABA or GABA-rich products have been successfully commercialized as food additives or functional dietary supplements. Several microorganisms can produce GABA, including bacteria, fungi, and yeasts. Among GABA-producing microorganisms, lactic acid bacteria (LAB) are commonly used in the production of many fermented foods. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a LAB species that has a long history of natural occurrence and safe use in a wide variety of fermented foods and beverages. Within this species, some strains possess not only good pro-technological properties but also the ability to produce various bioactive compounds, including GABA. The present review aims, after a preliminary excursus on the function and biosynthesis of GABA, to provide an overview of the current uses of microorganisms and, in particular, of L. plantarum in the production of GABA, with a detailed focus on fermented foods. The results of the studies reported in this review highlight that the selection of new probiotic strains of L. plantarum with the ability to synthesize GABA may offer concrete opportunities for the design of new functional foods.
Amino acid metabolism is an important factor in regulating nitrogen source assimilation and source/sink transport in soybean. Melatonin can improve plant stress resistance, but whether it affects amino acid metabolism is not known. Therefore, this study investigated whether exogenous melatonin had an effect on amino acid metabolism of soybean under drought conditions and explored its relationship with yield. The treatments were normal water supply treatment (WW), drought stress treatment (D), drought stress and melatonin treatment group (D + M), sprayed with 100 μmol/L melatonin. The effects of melatonin on amino acid metabolism and grain filling were studied by physiological and omics experiments using Kangxian 9 (drought‐sensitive variety) and Suinong 26 (drought‐resistant variety) soybean cultivars. The results showed that drought stress decreased the activity of carbon and nitrogen metabolizing enzymes, which inhibited the accumulation of dry matter and protein, and decreased the yield. In the drought‐sensitive soybean variety, glycoenzymes and amino acid synthetases synthetic genes were upregulated in melatonin‐treated soybeans, hence carbon and nitrogen metabolism enzyme activity increased, increasing the carbohydrate and amino acid contents simultaneously. This resulted in higher dry matter and yield than drought‐stressed soybean not treated with melatonin. In the drought‐resistant variety, the grain weight per plant increased by 7.98% and 6.57% in 2020 and 2021, respectively, while it increased by 23.20% and 14.07% in the drought‐sensitive variety during the respective years. In conclusion, melatonin treatment can enhance the activity of nitrogen and carbon metabolism and amino acid content by upregulating the expression of soybean metabolic pathway and related genes, thus increasing the yield of soybean under drought stress.
Drought (D) and heat (H) are the two major abiotic stresses hindering cereal crop growth and productivity, either singly or in combination (D/+H), by imposing various negative impacts on plant physiological and biochemical processes. Consequently, this decreases overall cereal crop production and impacts global food availability and human nutrition. To achieve global food and nutrition security vis-a-vis global climate change, deployment of new strategies for enhancing crop D/+H stress tolerance and higher nutritive value in cereals is imperative. This depends on first gaining a mechanistic understanding of the mechanisms underlying D/+H stress response. Meanwhile, functional genomics has revealed several stress-related genes that have been successfully used in target-gene approach to generate stress-tolerant cultivars and sustain crop productivity over the past decades. However, the fast-changing climate, coupled with the complexity and multigenic nature of D/+H tolerance suggest that single-gene/trait targeting may not suffice in improving such traits. Hence, in this review-cum-perspective, we advance that targeted multiple-gene or metabolic pathway manipulation could represent the most effective approach for improving D/+H stress tolerance. First, we highlight the impact of D/+H stress on cereal crops, and the elaborate plant physiological and molecular responses. We then discuss how key primary metabolism- and secondary metabolism-related metabolic pathways, including carbon metabolism, starch metabolism, phenylpropanoid biosynthesis, γ-aminobutyric acid (GABA) biosynthesis, and phytohormone biosynthesis and signaling can be modified using modern molecular biotechnology approaches such as CRISPR-Cas9 system and synthetic biology (Synbio) to enhance D/+H tolerance in cereal crops. Understandably, several bottlenecks hinder metabolic pathway modification, including those related to feedback regulation, gene functional annotation, complex crosstalk between pathways, and metabolomics data and spatiotemporal gene expressions analyses. Nonetheless, recent advances in molecular biotechnology, genome-editing, single-cell metabolomics, and data annotation and analysis approaches, when integrated, offer unprecedented opportunities for pathway engineering for enhancing crop D/+H stress tolerance and improved yield. Especially, Synbio-based strategies will accelerate the development of climate resilient and nutrient-dense cereals, critical for achieving global food security and combating malnutrition.
The pretreatment of rice roots for 1 h in aerobic conditions with the Ca²⁺-channel blockers ruthenium red (RR) and verapamil and the calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-1-naphtylenesulfonamide (W-7) and trifluoperazine, induced during 3 h of anoxia: (i) inhibition of amino acid and γ-aminobutyric acid (Gaba) accumulation; (ii) a decline in the protein content; (iii) a release of amino acids and K⁺ into the growth media. The calcium ionophore A23187 reversed these effects in RR-treated roots. Moreover, the aerobic pretreatment of rice roots with A23187 alone or CaCl2 increased the accumulation of amino acids and Gaba. These data indicate that the Ca²⁺/CaM complex is involved in the transduction of an anaerobic signal by inhibiting proteolysis and solute release, and activating the Ca²⁺/CaM-dependent glutamate decarboxylase.
The pretreatment of rice roots for 1 h in aerobic conditions with the Ca2+-channel blockers ruthenium red (RR) and verapamil and the calmodulin (CaM) antagonists N -(6-aminohexyl)-5-chloro-1-naphtylenesulfonamide (W-7) and trifluoperazine, induced during 3 h of anoxia: (i) inhibition of amino acid and γ-aminobutyric acid (Gaba) accumulation; (ii) a decline in the protein content; (iii) a release of amino acids and K+ into the growth media. The calcium ionophore A23187 reversed these effects in RR-treated roots. Moreover, the aerobic pretreatment of rice roots with A23187 alone or CaCl 2 increased the accumulation of amino acids and Gaba. These data indicate that the Ca2+/CaM complex is involved in the transduction of an anaerobic signal by inhibiting proteolysis and solute release, and activating the Ca2+/CaM-dependent glutamate decarboxylase.
4-aminobutyrate (GABA) is a non-protein amino acid that is widely distributed throughout the biological world. In animals, GABA functions as the predominant inhibitory neurotransmitter in the central nervous system by acting through the GABA receptors. The neuromuscular system enables animals to escape from environmental stresses. Being nonmotile, plants have evolved chemical responses to mitigate stress. Mechanisms by which GABA may facilitate these responses are discussed in this review. Environmental stresses increase GABA accumulation through two different mechanisms. Stresses causing metabolic and/or mechanical disruptions, resulting in cytosolic acidification, induce an acidic pH-dependent activation of glutamate decarboxylase and GABA synthesis. Extremely marked declines in cytosolic pH occur under oxygen deprivation, which is the primary stress factor in flooded soils, and this stress induces the greatest accumulation of GABA. Other stresses, including cold, heat, salt, and mild or transient environmental factors, such as touch, wind, rain, etc. rapidly increase cellular levels of Ca. Increased cytosolic Ca stimulates calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. A review of the kinetics of GABA accumulation in plants reveals a stress-specific pattern of accumulation that is consistent with a physiological role for GABA in stress mitigation. Recent physiological and genetic evidence indicates that plants may possess GAB A-like receptors that have features in common with the animal receptors. The mechanism of action of animal GABA receptors suggests a model for rapid amplification of ion-mediated signals and GABA accumulation in response to stress. Metabolic pathways that link GABA to stress-related metabolism and plant hormones are identified. The survival value of stress-related metabolism is dependent on metabolic changes occurring before stress causes irreversible damage to plant tissue. Rapid accumulation of GABA in stressed tissue may provide a critical link in the chain of events leading from perception of environmental stresses to timely physiological responses.
Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.
Molecular procedures have been applied to isolate plant calmodulin-binding proteins. A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated. The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E. coli GAD does not bind calmodulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns. The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E. coli GAD. Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD. Moreover, the plant protein binds calmodulin in vitro. This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism. Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.
To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.
Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.
A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters. For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library. Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3. However, genes encoding eight different amino acid transporters belonging to three different transporter families were isolated. Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes. A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family. ProT1 was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers. Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline. ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAP gene family whose expression was repressed under the same conditions. These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased.
In plants, gamma-aminobutyric acid (GABA), a major transmitter in the central nervous system in animals, is synthesized by glutamate decarboxylase (GAD), the activity of which is tightly modulated by Ca2+/calmodulin. To study the molecular mechanism of GAD regulation and examine the physiological role of GABA in plants, we isolated and characterized a 1771 bp tobacco cDNA clone, pNtGAD2. The 496 amino acid sequence deduced from pNtGAD2 showed 97, 92, and 81% identity to NtGAD1, petunia, and tomato GAD, respectively. The 26 amino acid sequence within the putative calmodulin binding domain at the carboxy terminus showed a typical alpha-helical structure with hydrophobic and charged amino acid clusters. The pNtGAD2-encoded 56 kDa protein interacted strongly with a monoclonal antibody against the petunia GAD and its GAD activity was stimulated markedly by the addition of exogenous calcium and calmodulin. The molecular sequence of pNtGAD2 and biochemical characteristics of the pNtGAD2-encoded protein confirmed that pNtGAD2 is a clone encoding a functional calmodulin-binding and Ca2+/calmodulin-dependent tobacco glutamate decarboxylase. This result indicates that tobacco plants also have Ca2+/calmodulin-dependent GADs.
Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mM NH4Cl, 5 mM NH4NO3, 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.
During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.
The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.
Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.
Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the gamma-aminobutyric acid shunt. We have cloned the cDNA for SSADH from Arabidopsis, which we designated SSADH1. SSADH1 cDNA encodes a protein of 528 amino acids (56 kD) with high similarity to SSADH from Escherichia coli and human (>59% identity). A sequence similar to a mitochondrial protease cleavage site is present 33 amino acids from the N terminus, indicating that the mature mitochondrial protein may contain 495 amino acids (53 kD). The native recombinant enzyme and the plant mitochondrial protein have a tetrameric molecular mass of 197 kD. Fractionation of plant mitochondria revealed its localization in the matrix. The purified recombinant enzyme showed maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K(0.5) = 15 microM), and exclusively used NAD+ as a cofactor (Km = 130 +/- 77 microM). NADH was a competitive inhibitor with respect to NAD+ (Ki = 122 +/- 86 microM). AMP, ADP, and ATP inhibited the activity of SSADH (Ki = 2.5-8 mM). The mechanism of inhibition was competitive for AMP, noncompetitive for ATP, and mixed competitive for ADP with respect to NAD+. Plant SSADH may be responsive to mitochondrial energy charge and reducing potential in controlling metabolism of gamma-aminobutyric acid.
Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.
Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.
The action of γ-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of
this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance.
A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H2O2 and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed
against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium
regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate
reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H2O2 exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to
buffer redox changes in the cell.
In this study we addressed the function of the Krebs cycle to determine which enzyme(s) limits the availability of reduced nicotinamide adenine dinucleotide (NADH) for the respiratory chain under H2O2-induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H2O2 proved to be aconitase, being completely blocked at 50 μM H2O2. α-Ketoglutarate dehydrogenase (α-KGDH) was also inhibited but only at higher H2O2 concentrations (≥100 μM), and only partial inactivation was achieved. The rotenone-induced increase in reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] fluorescence reflecting the amount of NADH available for the respiratory chain was also diminished by H2O2, and the effect exerted at small concentrations (≤50 μM) of the oxidant was completely prevented by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNUinsensitive decline by H2O2 in the rotenone-induced NAD(P)H fluorescence correlated with inhibition of a-ketoglutarate dehydrogenase. Decrease in the glutamate content of nerve terminals was induced by H2O2 at concentrations inhibiting aconitase. It is concluded that (1) aconitase is the most sensitive enzyme in the Krebs cycle to inhibition by H2O2, (2) at small H2O2 concentrations (≤50 μM) when aconitase is inactivated, glutamate fuels the Krebs cycle and NADH generation is unaltered, (3) at higher H2O2 concentrations (≥100 μM) inhibition of α-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H2O2-induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of a-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.
Plants, in common with all organisms, have evolved mechanisms to cope with the problems caused by high temperatures. We examined specifically the involvement of calcium, abscisic acid (ABA), ethylene, and salicylic acid (SA) in the protection against heat-induced oxidative damage in Arabidopsis. Heat caused increased thiobarbituric acid reactive substance levels (an indicator of oxidative damage to membranes) and reduced survival. Both effects required light and were reduced in plants that had acquired thermotolerance through a mild heat pretreatment. Calcium channel blockers and calmodulin inhibitors increased these effects of heating and added calcium reversed them, implying that protection against heat-induced oxidative damage in Arabidopsis requires calcium and calmodulin. Similar to calcium, SA, 1-aminocyclopropane-1-carboxylic acid (a precursor to ethylene), and ABA added to plants protected them from heat-induced oxidative damage. In addition, the ethylene-insensitive mutant etr-1, the ABA-insensitive mutant abi-1, and a transgenic line expressing nahG (consequently inhibited in SA production) showed increased susceptibility to heat. These data suggest that protection against heat-induced oxidative damage in Arabidopsis also involves ethylene, ABA, and SA. Real time measurements of cytosolic calcium levels during heating in Arabidopsis detected no increases in response to heat per se, but showed transient elevations in response to recovery from heating. The magnitude of these calcium peaks was greater in thermotolerant plants, implying that these calcium signals might play a role in mediating the effects of acquired thermotolerance. Calcium channel blockers and calmodulin inhibitors added solely during the recovery phase suggest that this role for calcium is in protecting against oxidative damage specifically during/after recovery.
During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.
4-aminobutyrate (GABA) is a non-protein amino acid that is widely distributed throughout the biological world. In animals, GABA functions as the predominant inhibitory neurotransmitter in the central nervous system by acting through the GABA receptors. The neuromuscular system enables animals to escape from environmental stresses. Being nonmotile, plants have evolved chemical responses to mitigate stress. Mechanisms by which GABA may facilitate these responses are discussed in this review. Environmental stresses increase GABA accumulation through two different mechanisms. Stresses causing metabolic and/or mechanical disruptions, resulting in cytosolic acidification, induce an acidic pH-dependent activation of glutamate decarboxylase and GABA synthesis. Extremely marked declines in cytosolic pH occur under oxygen deprivation, which is the primary stress factor in flooded soils, and this stress induces the greatest accumulation of GABA. Other stresses, including cold, heat, salt, and mild or transient envir...
The four carbon, non-protein amino acid γ-aminobutyrate (GABA) accumulates rapidly in response to diverse stresses. Its synthesis is stimulated by increases in intracellular Ca2+ or H+ levels. The pathogen-induced oxidative burst is also associated with increases in Ca2+ and H+ levels. This study investigated the relationship between GABA synthesis and the oxidative burst. A Mas-7-induced consumption of oxygen in isolated Asparagus sprengeri Regel mesophyll cells was accompanied by rapid GABA synthesis. At pH 5.0, a 300% increase occurred within 16 min from 6.6 to 26.3 nmol GABA·106 cells-1. At pH 6.0, the increase was from 8.5 to 18.1 nmol GABA·106 cells -1. Mas-7 also stimulated rapid external alkalinization and intracellular acidification. Intracellular pH decreased 0.44 pH units at pH 5.0, and 0.21 pH units at pH 6.0. The Mas-7-induced oxidative burst, GABA synthesis, extracellular alkalinization, and intracellular acidification were all eliminated when lanthanum, a Ca2+ channel blocker, replaced Ca2+ in the incubation medium. The data demonstrate that GABA accumulation is associated with the oxidative burst, and results from the fluxes of H+ and Ca2+, which are known to accompany the oxidative burst. They are discussed in light of emerging data that indicate a role for GABA in plant cell to cell signaling.
Changes in metabolites (organic acids, sugars and amino acids) and subcellular
pH were studied during fruit development of cherry tomato
(Lycopersicon esculentum Mill. var.
cerasiformae). Fructose and glucose were the major
sugars, whereas citrate and malate the two major organic acids. At different
stages of fruit development, vacuolar and cytoplasmic pH changes were followed
by in vivo 13C and
31P NMR spectroscopy. Fruit compartments had a
cytoplasmic pH around 7.1 as early as the cell-divi-sion and -expansion
stages. The vacuolar pH measured by in vivo
13C NMR spectroscopy decreased from 4.5 to 3.6.
Concomitantly, strong accumulation of γ-aminobutyric acid (GABA) was
observed during the first 15 days after anthesis and glutamate decarboxylase
(GAD) activity increased 10-fold during the first 8 days of development. The
relationships between organic acid biosynthesis and storage, GABA produc-tion,
and subcellular pH changes during development of cherry tomato fruit are
discussed.
Synthesis of the nonprotein amino acid γ-aminobutyric acid is stimulated within minutes by diverse environmental factors. Synthesis (L-Glu + H+ – γ-aminobutyric acid + CO2) is catalysed by L-Glu decarboxylase, a cytosolic enzyme having an acidic pH optimum. This study uses isolated Asparagus sprengeri (Regel) mesophyll cells to investigate the possible role of Ca2+ in stimulated γ-aminobutyric acid synthesis. Abrupt cold shock (20 °C to 1 °C) stimulated γ-aminobutyric acid levels from 2.7 to 5.6 nmol/106 cells within 15 min. This 100% increase was reduced to 28% in the presence of the Ca2+ channel blocker lanthanum, and was significantly reduced by incubation with 1 mM of the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide. Incubation at 20 °C with 25 μM calcimycin, a Ca2+ ionophore, increased levels by 61% within 15 min. A fluorescent Ca2+ indicator demonstrated that cytosolic Ca2+ increased within 2 s of cold shock, followed by a return to initial levels within 25 s. In contrast, comparable experiments indicate a rapid and prolonged decrease in cytosolic H+. L-Glu decarboxylase isolated from asparagus cladophylls was stimulated 100% by addition of 500 μM Ca2+ and 200 nM calmodulin. This activity was reduced to control values by the calmodulin antagonist. Collectively, the data indicate that cold shock initiates a signal transduction pathway in which increased cytosolic Ca2+ stimulates calmodulin-dependent L-Glu decarboxylase activity and γ-aminobutyric acid synthesis. This mechanism appears independent of increased H+. Key words: cold shock, GABA, Ca2+, H+.
A novel cDNA encoding glutamate dehydrogenase (GDH) from tobacco(Nicotiana tabacum), named gdh1, was characterized.The gdh1 mRNA was detected in roots, stems and source/senescentleaves. In order to investigate diurnal regulation of gdh1 inleaves, the content in gdh1 mRNA was measured every 3 h overa 48 h period and compared to nia and gs2 mRNAlevels, encoding, respectively, nitrate reductase (NR) and chloroplasticglutamine synthetase (GS2). In source leaves, gdh1 mRNA levelsexhibit diurnal fluctuations. A 12 h shift was observedbetween the day–night rhythms of gdh1 and nia expression.Metabolite contents were also measured and a shift in the day–nightfluctuations of both glutamate (GLU) and -aminobutyricacid (GABA) was observed between sink and source leaves, whereasthe diurnal rhythm of -ketoglutarate showed no change.A possible role of GDH in the shift of GLU and GABA contents isdiscussed. Leaf disc experiments showed that gdh1 expressionis enhanced in conditions of continuous darkness. This trend isinhibited by sucrose feeding. The opposite was observed for nia expression.An important outcome of this work is the reverse regulation of gdh1 and nia genes.A possible role of sugars and amino acids in the co-regulation of gdh1 and nia genesis suggested.
-Hydroxybutyric acid (GHB) is a naturally occurring metabolite of GABA that has been postulated to exert ubiquitous neuropharmacological effects through GABAB receptor (GABABR)-mediated mechanisms. The alternative hypothesis that GHB acts via a GHB-specific, G protein-coupled presynaptic receptor that is different from the GABABR was tested. The effect of GHB on regional and subcellular brain adenylyl cyclase in adult and developing rats was determined and compared with that of the GABABR agonist (-)-baclofen. Also, using guanosine 5′-O-(3-[35S]thiotriphosphate) ([35S]GTPS) binding and low-Km GTPase activity as markers the effects of GHB and (-)-baclofen on G protein activity in the brain were determined. Neither GHB nor baclofen had an effect on basal cyclic AMP (cAMP) levels. GHB significantly decreased forskolin-stimulated cAMP levels by 40-50% in cortex and hippocampus but not thalamus or cerebellum, whereas (-)-baclofen had an effect throughout the brain. The effect of GHB on adenylyl cyclase was observed in presynaptic and not postsynaptic subcellular tissue preparations, but the effect of baclofen was observed in both subcellular preparations. The GHB-induced alteration in forskolin-induced cAMP formation was blocked by a specific GHB antagonist but not a specific GABABR antagonist. The (-)-baclofen-induced alteration in forskolin-induced cAMP formation was blocked by a specific GABABR antagonist but not a specific GHB antagonist. The negative coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a developmental time point that is concordant with the developmental appearance of [3H]GHB binding in cerebral cortex, but the effects of (-)-baclofen were present by postnatal day 14. GHB and baclofen both stimulated [35S]GTPS binding and low-Km GTPase activity by 40-50%. The GHB-induced effect was blocked by GHB antagonists but not by GABABR antagonists and was seen only in cortex and hippocampus. The (-)-baclofen-induced effect was blocked by GABABR antagonists but not by GHB antagonists and was observed throughout the brain. These data support the hypothesis that GHB induces a G protein-mediated decrease in adenylyl cyclase via a GHB-specific G protein-coupled presynaptic receptor that is different from the GABABR.
GABA (4-aminobutyric acid) is a ubiquitousnon-protein amino acid that accumulates rapidly inplants in response to stress. GABA was firstidentified in plants (potato tubers) and animals(brain tissue) 50 years ago. Although GABA is nowrecognized as the most important inhibitoryneurotransmitter in the mammalian central nervoussystem (CNS), the role of GABA in plants remainsunclear. Studies were performed using Lemna toinvestigate the possibility that GABA elicits aresponse in plants that may be related to that of asignaling molecule as described for GABA effects onthe CNS. Lemna growth was increased 2 to 3-foldby 5 mM GABA, but growth was strongly inhibited by 0.5mM of the isomers 3-aminobutyric acid and2-aminobutyric acid. Growth promotion by GABA wasrapidly terminated by addition of 2-aminobutyric acidto the culture medium, but inhibitory effects of2-aminobutyric acid were not reversed by GABAregardless of amounts added. Promotion of Lemnagrowth by GABA was associated with an increase inmineral content of treated plants in a dose dependentmanner. Results support the hypothesis that GABAactivity in plants involves an effect on ion transportand an interaction with a receptor. Evidence for GABAreceptors in Lemna was obtained from experimentswith pharmacological agents that have been used toidentify GABA receptors in animals. GABA mediatedpromotion of Lemna growth was inhibited bybicuculline and picrotoxin, which are respectivelycompetitive and non-competitive antagonists of GABAreceptors in the CNS. Growth inhibition bybicuculline was not relieved by increasing the amountsof GABA in the medium, indicating that the alkaloid isnot acting, as in the CNS, by competitive antagonismof GABA at GABA receptor sites. Baclofen, a GABAagonist that promotes GABA activity in animalssignificantly increased GABA mediated promotion ofLemna growth. These findings and the knownaction of GABA in regulating ion channels in animalssuggests a way that GABA could amplify the stressresponse in plants.
The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans— the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.
Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca2+/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. However, more information is needed concerning the control of the catabolic mitochondrial enzymes (GABA transaminase and succinic semialdehyde dehydrogenase) and the intracellular and intercellular transport of GABA. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization. There is a need to identify the genes of enzymes involved in GABA metabolism, and to generate mutants with which to elucidate the physiological function(s) of GABA in plants.
SSADH deficiency, a rare inborn error of human metabolism, disrupts the normal metabolism of the inhibitory neurotransmitter GABA. In response to the defect, physiologic fluids from patients accumulate GHB, a compound with numerous neuromodulatory properties. Clinical and bio-chemical findings in patients are contrasted with existing neuropharmacologic data on GHB in animals and men. We conclude that GHB contributes to the pathogenesis of SSADH deficiency; whether this effect is mediated by GHB, by GABA following metabolic interconversion, or via synergistic mechanisms by both compounds, remains to be determined. An animal model of SSADH deficiency should further define the role of GHB in the pathogenesis of SSADH deficiency, and provide a useful vehicle for the evaluation of new therapeutic intervention.
We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.
The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway.
In flowering plants, a series of cell-cell interactions govern the delivery of sperm to the ovules through precise guidance of pollen tubes. Two Arabidopsis genes, POP2 and POP3, were found that mediate pollen tube guidance and are critical for self-fertility in diploid reproductive cells. The pop2 and pop3 mutations exhibited genetic redundancy: Self-sterility occurred only when male and female tissues were defective in both genes. This phenotype resembles that found in many self-incompatible species.
gamma-Hydroxybutyrate is a metabolite of GABA which is synthesized and accumulated by neurons in brain. This substance is present in micromolar quantities in all brain regions investigated as well as in several peripheral organs. Neuronal depolarization releases gamma-hydroxybutyrate into the extracellular space in a Ca(2+)-dependent manner. Gamma-hydroxybutyrate high-affinity receptors are present only in neurons, with a restricted specific distribution in the hippocampus, cortex and dopaminergic structures of rat brain (the striatum in general, olfactory bulbs and tubercles, frontal cortex, dopaminergic nuclei A9, A10 and A12). Stimulation of these receptors with low amounts of gamma-hydroxybutyrate induces in general hyperpolarizations in dopaminergic structures with a reduction of dopamine release. However, in the hippocampus and the frontal cortex, it seems that gamma-hydroxybutyrate induces depolarization with an accumulation of cGMP and an increase in inositol phosphate turnover. Some of the electrophysiological effects of GHB are blocked by NCS-382, a gamma-hydroxybutyrate receptor antagonist while some others are strongly attenuated by GABAB receptors antagonists. Gamma-hydroxybutyrate penetrates freely into the brain when administered intravenously or intraperitoneally. This is a unique situation for a molecule with signalling properties in the brain. Thus, the gamma-hydroxybutyrate concentration in brain easily can be increased more than 100 times. Under these conditions, gamma-hydroxybutyrate receptors are saturated and probably desensitized and down-regulated. It is unlikely that GABAB receptors could be stimulated directly by GHB. Most probably, GABA is released in part under the control of GHB receptors in specific pathways expressing GABAB receptors. Alternatively, GABAB receptors might be specifically stimulated by the GABA formed via the metabolism of gamma-hydroxybutyrate in brain. In animals and man, these GHBergic and GABAergic potentiations induce dopaminergic hyperactivity (which follows the first phase of dopaminergic terminal hyperpolarization), a strong sedation with anaesthesia and some EEG changes with epileptic spikes. It is presumed that, under pathological conditions (hepatic failure, alcoholic intoxication, succinic semialdehyde dehydrogenase defects), the rate of GHB synthesis or degradation in the peripheral organ is modified and induces increased GHB levels which could interfere with the normal brain mechanisms. This pathological status could benefit from treatments with gamma-hydroxybutyric and/or GABAB receptors antagonists. Nevertheless, the regulating properties of the endogenous gamma-hydroxybutyrate system on the dopaminergic pathways are a cause for the recent interest in synthetic ligands acting specifically at gamma-hydroxybutyrate receptors and devoid of any role as metabolic precursor of GABA in brain.
Succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare metabolic disorder of 4-aminobutyric acid degradation, has been identified in approximately 150 patients. Affected individuals accumulate large quantities of 4-hydroxybutyric acid, a compound with a wide range of neuropharmacological activities, in physiological fluids. As a first step in beginning an investigation of the molecular genetics of SSADH deficiency, we have utilized SSADH cDNA and genomic sequences to identify two point mutations in the SSADH genes derived from four patients. These mutations, identified by standard methods of reverse transcription, PCR, dideoxy-chain termination, and cycle sequencing, alter highly conserved sequences at intron/exon boundaries and prevent the RNA-splicing apparatus from properly recognizing the normal splice junction. Each family segregated a mutation in a different splice site, resulting in exon skipping and, in one case, a frameshift and premature termination and, in the other case, an in-frame deletion in the resulting protein. Family members, including parents and siblings of these patients, were shown to be heterozygotes for the splicing abnormality, providing additional evidence for autosomal recessive inheritance. Our results provide the first evidence that 4-hydroxybutyric aciduria, resulting from SSADH deficiency, is the result of genetic defects in the human SSADH gene.
The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.
Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9 +/- 1.7 and 1.7 +/- 0.3 mM at pH 4, respectively. The simultaneous transport of [1-14C]GABA and [2,3-3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-14C]GABA transport was inhibited by proline and quaternary ammonium compounds.
Ionotropic glutamate receptors (iGluRs) bind agonists in a domain that has been crystallized and shown to have a bilobed structure. Eukaryotic iGluRs also possess a second extracellular N-terminal domain related to the bacterial periplasmic binding protein LIVBP. In NMDA receptors, the high-affinity Zn inhibition is eliminated by mutations in the LIVBP-like domain of the NR2A subunit. Using LIVBP structure, we have modeled this domain as two lobes connected by a hinge and show that six residues controlling Zn inhibition form two clusters facing each other across a central cleft. Upon Zn binding the two lobes close tightly around the divalent cation. Thus, the extracellular region of NR2A consists of a tandem of Venus flytrap domains, one binding the agonist and the other a modulatory ligand. Such a functional organization may apply to other eukaryotic iGluRs.
Plants, like other organisms, have developed mechanisms that allow them to sense and respond to changes in levels of carbon and nitrogen metabolites. These mechanisms, in turn, regulate the expression of genes and the activities of proteins involved in C and N transport and metabolism, allowing plants to optimize the use of energy resources. Recent studies, which have involved molecular-genetic, genomic, and cell biological approaches, have begun to uncover the signals and components of C:N sensing and signaling mechanisms in plants. For sugar sensing, analysis of Arabidopsis mutants has revealed intersections with hormone and nitrogen signaling. For nitrogen sensing/signaling, recent progress has identified transcriptional and posttranslational mechanisms of regulation. In all, a complex picture is emerging in which C:N signaling systems are subject to a 'matrix effect' in which downstream responses are dependent upon cell-type, developmental, metabolic, and/or environmental conditions.
We have isolated full-length cDNAs for two distinct isoforms of glutamate decarboxylase (GAD), designated OsGAD1 and OsGAD2 from a rice shoot cDNA library. Open reading frames found in OsGAD1 and OsGAD2 cDNAs encode putative proteins of 501 (56.7 kDa) and 500 amino acids (55.6 kDa), respectively. They show 69% identity to each other and 67-78% identity to dicotyledonous counterpart sequences determined so far. Comparative analysis of relevant genomic clones obtained from the rice genomic library with these cDNAs as probes demonstrated that the number and sizes of introns deduced for these two genes differ considerably. Interestingly, in the regions in the putative gene products corresponding to the C-terminal 30-amino-acid peptide known as the calmodulin-binding domain of plant GADs, OsGAD1 possesses a typical motif, while OsGAD2 contains several substitutions of amino acids that contribute strongly to the binding of calmodulin (CaM). An in vitro CaM-binding assay of these proteins over-expressed in Escherichia coli revealed that OsGAD1 can in fact bind specifically to bovine CaM but OsGAD2 cannot. RNA analysis showed that transcripts of OsGAD1 and OsGAD2 were present in all tissues examined, but their expression was differentially regulated, at least in roots and maturing seeds.
Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.