Opinion

TRENDS in Plant Science

Vol.9 No.3 March 2004

GABA in plants: just a metabolite?

1 and Hillel Fromm2 Nicolas Bouche
1 2

Commissariat a ` lEnergie Atomique, Direction des Sciences du Vivant, Service de Bioe nerge tique, 91191 Gif-sur-Yvette, France Department of Plant Sciences, Tel Aviv University, 69978 Tel Aviv, Israel

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. g-Aminobutyric acid (GABA ; see Glossary) is a four-carbon non-protein amino acid conserved from bacteria to plants and vertebrates. It was discovered in plants more than half a century ago [1], but interest in GABA shifted to animals when it was revealed that GABA occurs at high levels in the brain, playing a major role in neurotransmission. Thereafter, research on GABA in vertebrates focused mainly on its role as a signaling molecule, particularly in neurotransmission. In plants and in animals, GABA is mainly metabolized via a short pathway composed of three enzymes, called the GABA shunt because it bypasses two steps of the tricarboxylic acid (TCA ) cycle (Figure 1). The pathway is composed of the cytosolic enzyme glutamate decarboxylase (GAD ) and the mitochondrial enzymes GABA transaminase (GABA-T ) and succinic semialdehyde dehydrogenase (SSADH ). The regulation of this conserved metabolic pathway seems to have particular characteristics in plants. Indeed, interest in the GABA shunt in plants emerged mainly from experimental observations that GABA is largely and rapidly produced in response to biotic and abiotic stresses [24]. The GABA shunt has since been associated with various physiological responses, including the regulation of cytosolic pH, carbon uxes into the TCA cycle, nitrogen metabolism, deterrence of insects, protection against oxidative stress, osmoregulation and signaling (Box 1). In this article, we attempt to link these and other ndings that have accumulated during the half-century since the discovery of GABA in plants with recent evidence, mainly from Arabidopsis functional genomic approaches, pointing towards the possible role of GABA as a signal molecule in plants, as well as roles in plant responses to stress and in the carbon:nitrogen (C:N ) balance. Metabolism of GABA in plants Glutamate decarboxylase Studies of the function of GAD and its regulation in plants have been stimulated by the cloning of the petunia GAD
Corresponding author: Hillel Fromm (hillelf@post.tau.ac.il).

gene and the characterization of the encoded enzyme as a Ca2-dependent calmodulin (C aM )-binding protein [5]. Subsequently, it was found that GAD activity in extracts from various plant species [6 14] is modulated by Ca2 CaM. Detailed molecular analysis of the CaM-binding domain of petunia GAD [10] and the characterization of the puried recombinant protein as a Ca2 CaM-regulated enzyme [15] provided a working model to explain the rapid stimulation of GAD activity in response to various stress situations that elicit changes in cytosolic Ca2 concentrations. Evidence to support this hypothesis was provided by demonstrating that stimulation of GAD activity in response to anoxia [11] and cold [12] involves Ca2 CaM. Taken together, the regulation of GAD activity by Ca2 CaM has been demonstrated in vitro [1317] and in vivo [11,12]. Recently, the three-dimensional structure of CaM bound to the petunia GAD CaM-binding domain has been determined by nuclear magnetic resonance [18], revealing an interesting complex of CaM with two peptides of the CaM-binding domain. This suggests a role for CaM in regulating the formation or stability of the GAD protein complex, as previously suggested based on immunodetection of native GAD complexes (, 500 kDa) in transgenic plants expressing the full-length or truncated GAD lacking the CaM-binding domain [8]. However, recent evidence also suggests the occurrence of a rice GAD isoform lacking a CaM-binding domain [19]. Consequently, it should be interesting to investigate whether such an isoform is typical of monocots or whether it is present in some dicots as well. The use of transgenic plants ectopically expressing either GAD or a mutant GAD lacking the ability to bind CaM provided further evidence for the importance of CaM binding to GAD in vivo [8]. Since the completion of the sequencing of the Arabidopsis genome [20], ve GAD genes have been identied by sequence comparisons [2]. At least two of the GAD isoforms (GAD1 and GAD2) differ in their organ distribution: GAD1 is root specic, whereas GAD2 is
Glossary
CaM: calmodulin. C:N: carbon:nitrogen. GABA: g-aminobutyric acid. GAD: glutamate decarboxylases. GABA-T: GABA transaminase. GHB: g-hydroxybutyric acid. AtGLRs: Arabidopsis glutamate receptors. ROIs: reactive oxygen intermediates. SSADH: succinic semialdehyde dehydrogenase. TCA cycle: tricarboxylic acid cycle.

www.sciencedirect.com 1360-1385/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2004.01.006

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Mitochondrial matrix

Cytosol NH4+

NH4+ -Ketoglutarate -KGDH Glutamate TCA cycle Succinyl-CoA Succinyl-CoA ligase Fumarate Succinate Succinic semialdehyde GDH

Inner mitochondrial membrane

GS/GOGAT (chloroplast and cytosol)

Glutamate

Glutamate H+

-Ketoglutarate GAD

GABA-TK

CO2 GABA GABA

SSADH NADH + H+

GABA-TP

Ca2+CaM

Alanine NAD+ Respiratory chain

Pyruvate

ATP

ATP synthase

Outer mitochondrial membrane Succinic semialdehyde SSR GHB

NAD(P)H + H+

NAD(P)+
TRENDS in Plant Science

Figure 1. The g-aminobutyric acid (GABA) shunt metabolic pathway and its regulation in plants. The glutamine-synthetase/glutamate-synthase (GS/GOGAT) cycle is the principal nitrogen assimilation pathway into glutamate and amino acids in plants. The glutamate dehydrogenase (GDH) is thought to function primarily in glutamate catabolism but can also function in the opposite direction. The GABA shunt is composed of three enzymes (purple). Glutamate decarboxylase (GAD) is a cytosolic enzyme regulated (green) by the Ca2 calmodulin (CaM) complex, which catalyses the irreversible decarboxylation of glutamate to produce GABA. 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 tricarboxylic acid (TCA) cycle. Both ATP and NADH can inhibit the activity of the SSADH enzyme (green). The succinyl-CoA ligase and the a-ketoglutarate dehydrogenase (a-KGDH) are two enzymes (pink) of the TCA cycle bypassed by the GABA shunt and sensitive to oxidative stress. Succinic semialdehyde can instead be reduced to g-hydroxybutyric acid (GHB) via a succinic semialdehyde reductase (SSR) localized in the cytosol in animal cells and possibly in plants as well. In mammals, GHB is thought to be a neurotransmitter, whereas its role in plants is unknown.

expressed in all organs [13,14]. Together, these ndings imply that the different GAD isoforms are expressed in a tissue-dependent manner and might have specic functions. GABA transaminase GABA is converted to succinic semialdehyde (SSA) by two sorts of GABA-Ts that use either a-ketoglutarate (GABA-TK) or pyruvate (GABA-TP) as amino acid acceptors, producing glutamate or alanine (Figure 1). In mammals, only the GABA-TK seems to be present, whereas both enzyme activities can be detected in crude plant extracts [2]. The a-ketoglutarate-dependent
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GABA-TK enzyme of plants remains to be identied, whereas the pyruvate-dependent GABA-TP was partially puried from tobacco and a homologous Arabidopsis gene was subsequently cloned [21]. The recombinant Arabidopsis GABA-TP characterized in vitro uses pyruvate but not a-ketoglutarate, and shares little homology with nonplant GABA-Ts [21]. Arabidopsis knockouts disrupted in the corresponding gene have a GABA content elevated 100-fold in owers compared with the wild type, conrming that GABA-TP is a functional enzyme of the GABA shunt in vivo [22]. In this mutant, the increase of GABA levels in other organs (e.g. leaves) is limited, implying that the GABA-TP has a specialized function in owers and

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Box 1. Possible roles of GABA and the GABA shunt in plants Contributing to the C:N balance
Levels of g-aminobutyric acid (GABA) are high in certain tissues. For instance, GABA can reach as much as 50% of the free amino acid in cherry tomato fruits [54] and the concentrations of GABA can increase drastically in response to different stresses [2,3]. Moreover, Arabidopsis grows efciently on medium containing GABA as a sole source of nitrogen [55]. GABA is thus involved in the general nitrogen metabolism and possibly in the storage and/or transport of nitrogen. The GABA shunt is also a way to assimilate carbons from glutamate and to generate C:N uxes that enter the tricarboxylic acid (TCA) cycle. stress conditions that inhibit certain enzymes of the TCA cycle (Figure 1). In yeast, mutants knocked out in GABA-shunt genes seem to be more sensitive to H2O2 [25].

Defense against insects

Because GABA is a neurotransmitter in vertebrates and invertebrates, it was speculated that GABA could be produced by the plant to deter insect feeding. GABA levels are elevated by mechanical stimulation or damage [59], and even by insects crawling on leaves [60], and it is possible that the ingested GABA interferes with the normal development of insects [2,3]. For instance, transgenic tobacco plants containing elevated GABA levels were suggested to be resistant to the root-knot nematode [61] and tobacco budworm larvae [62]. Moreover, the pathogen-induced oxidative burst is associated with an increase in GABA content in isolated Asparagus cells [63].

Regulation of cytosolic pH
In bacteria, there seems to be a role for the GABA shunt in acid resistance [56,57]. When Escherichia coli is exposed to such stress, glutamate decarboxylase (GAD) activity is induced, thus virtually removing protons by catalysing the decarboxylation of glutamate (Figure 1). The GABA produced is then exported from the cells [58]. In plants, GAD is activated by acidic pH [6,15] and GABA accumulates in response to cytosolic acidication [2], so it is possible that GAD activity could participate in regulating the cytosolic pH of plants.

GABA as an osmoregulator
Proline transporters from both Arabidopsis (i.e. AtProT2 [55]) and tomato (i.e. LeProT1 [64]) also transport GABA. AtProT2 is strongly induced during water or salt stress [65]. Proline GABA transporters can transport organic osmolytes, also called compatible solutes, which serve several protective roles.

Protection against oxidative stress

In Arabidopsis, mutants disrupted in succinic semialdehyde dehydrogenase are more sensitive to environmental stress because they are unable to scavenge H2O2 [26]. The last step of the GABA shunt can provide both succinate and NADH to the respiratory chain (Figure 1). It was therefore hypothesized that the degradation of GABA could limit the accumulation of reactive oxygen intermediates under oxidative

GABA as a signaling molecule

GABA is a neurotransmitter in animals with a clearly dened role in signaling. Similarly, GABA could be a signaling molecule in plants [4,45]. There are indications that GABA receptors exist in plants or that plant glutamate receptors use GABA as a modulatory ligand.

that other GABA-Ts might degrade GABA in the rest of the plant. Therefore, in plants, GABA transamination occurs via different types of GABA-T, which probably have specialized functions. Succinic semialdehyde dehydrogenase The rst cloned SSADH gene from plants has been biochemically analysed [23]. Antibodies raised against puried recombinant Arabidopsis SSADH localized SSADH to the mitochondria of potato tubers and Arabidopsis cell cultures, consistent with previous biochemical studies of SSADH localization in soybean cells [24]. Subcellular localization of SSADH to the mitochondria is common in various organisms, although exceptions have been described; in yeast, for example, the enzyme is in the cytosol [25]. In vitro assays revealed that SSADH is specic for SSA and exclusively uses NAD to produce NADH. Importantly, both ATP and NADH negatively regulate the activity of the enzyme. Both products of the reaction catalysed by SSADH (i.e. succinate and NADH) are substrates of the mitochondrial respiratory chain, which produces ATP as a nal product. Therefore, regulation of SSADH by ATP suggests a tight feedback control of the rate of substrates provided by the GABA shunt to the respiratory chain. Consequently, the feedback regulation might also play a role in controlling the steadystate levels of GABA and hence possible functions of GABA via pathways other than the TCA cycle, such as signaling pathways. In Arabidopsis, disruption of the unique SSADH gene results in plants undergoing necrotic cell death when exposed to environmental stresses, owing to the abnormal
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accumulation of reactive oxygen intermediates (ROIS ). For example, ssadh mutants exposed to white light (100 mmol m2 s21) appear to be dwarfed with necrotic lesions (see Figures 2,3 in Ref. [26]). Indeed, ssadh mutants are sensitive to at least two types of environmental stresses: both ultraviolet irradiation (particularly ultraviolet B) and heat cause a rapid increase in the levels of H2O2 in ssadh mutants, and this is associated with enhanced cell death and necrosis of leaves [26]. The phenotype of the ssadh mutants could be caused by a lack of certain metabolites (e.g. loss of NADH or succinate to the respiratory chain), an excess of a metabolite derived from the GABA shunt with a possible toxic effects [e.g. SSA or g-hydroxybutyric acid (GHB ), see below] or an imbalance in signaling molecules derived from the GABA shunt (including GABA). In plant mitochondria, several enzymes of the TCA cycle, including those that are bypassed by the GABA shunt (Figure 1) (the succinylCoA ligase and the a-ketoglutarate dehydrogenase), are degraded under oxidative stress conditions [27]. In the brain, inhibition of a-ketoglutarate dehydrogenase during H2O2-induced oxidative stress crucially limits the amount of NADH [28], and a role has also been suggested for the GABA shunt in protecting yeast against oxidative stress [25]. Recent studies revealed a relationship between Ca2 signaling and ROIs in plants [29 32]. The GABA shunt, whose activity is regulated by Ca2 via GAD, is thus activated under stress conditions that cause enhanced production of ROIs. These studies of the ssadh mutants and the SSADH enzyme suggest the possible involvement of the GABA shunt in tolerance to oxidative stress.

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g-Hydroxybutyric acid In an alternative reaction to the GABA shunt, SSA can be reduced to GHB by an SSA reductase (SSR; Figure 1). In the brain, GHB can be converted to SSA via another enzyme, the GHB dehydrogenase (GHBDH). GHB is normally found in small quantities in the mammalian brain, representing , 0.2% of the levels of its parent compound, GABA [33]. GHB is released by neuronal depolarization in a Ca2-dependent manner [33] and high afnity GHB binding sites are present in specic brain regions (e.g. thalamus). Furthermore, the pharmacological action of GHB is to reduce the release of dopamine, probably via GHB receptors [34,35] and possibly through the GABAB receptors, of which GHB is a weak agonist. In humans, GHB and GABA levels are noticeably increased in patients decient for SSADH activity, a rare genetic disease called 4-hydroxybutyric aciduria [36,37] that leads to neurological abnormalities. In plants, a gene encoding a protein with SSR activity was recently cloned by complementing a SSADH-decient yeast strain with an Arabidopsis cDNA library [38]. The SSR of Arabidopsis shares little homology with its animal counterpart. The role of GHB in plants remains obscure but GHB seems to be produced in response to ooding and oxygen deciency [38]. Understanding the role of SSR and GHB in response to environmental stresses will require further investigations such as isolating SSR knockouts in Arabidopsis and measuring GHB levels in plants altered in the GABA shunt, like ssadh [26] or gaba-t [22] mutants.
GABA shunt and the metabolism of nitrogen Plants sense their nutrient environment and respond to it by modifying their uptake and metabolism using an intricate system of sensors, receptors, transporters, signal transduction components and gene expression regulators that collectively lead to changes in growth rates and development [39]. More specically, the complex mechanisms coordinating the C:N balance involve sensing and regulating sugar levels, nitrate uptake and internal contents of reduced nitrogen (e.g. ammonium and amino acids). The GABA shunt appears to be part of the metabolic pathways involved in the C:N balance and the metabolism of nitrogen, the extent of which is not fully understood. Impact of the GABA shunt on nitrogen metabolism Most inorganic nitrogen is assimilated through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway, producing glutamate, which is then the starting point for the synthesis of most amino acids, including GABA (Figure 1). Glutamate levels can be drastically affected when the function of GAD is modied. For instance, transgenic plants overexpressing a constitutively active GAD have signicantly reduced levels of glutamate [8]. Still, the importance of GAD in controlling glutamate levels in wild-type plants remains to be claried. This question was partially addressed in tobacco, in which the levels of several metabolites were followed during two day night cycles and compared between upper young leaves (i.e. sink) and lower old leaves (i.e. source) [40]. Diurnal rhythms were detected in both types of leaves for different amino acids [41]. Interestingly, GABA and
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glutamate were the only metabolites for which the diurnal changes are shifted by 6 h between source and sink. This nding suggests that the production of GABA is tightly linked to the glutamate content, and the authors hypothesized that GABA has a dominant role in buffering the production of glutamate [40]. This might conrm previous observations about the role of GABA as a transient nitrogen storage metabolite [2,3]. Following this idea, it is also tempting to speculate that GAD activity is regulated by the level of glutamate [2]. To control their C:N balance, plants can sense their reduced nitrogen status and regulate the uptake and reduction of nitrate adequately [42]. Still, the identity of the metabolite(s) that plants use as sensor(s) in that process is unclear. The role played by glutamine is still a matter of debate and other metabolites, including amino acids or sugars, are probably involved [41]. GABA could be one of these metabolites. Regulation of the GABA shunt by nitrogen availability Shifting plants from an unreduced nitrogen source (KNO3) to reduced forms of nitrogen (NH4Cl, NH4NO3, glutamate or glutamine) for a prolonged treatment of 4 days resulted in increased levels of GAD2 transcript and protein, and an increase in the total GAD specic activity in Arabidopsis leaf extracts [14]; GAD1 was not detected because it is a root-specic enzyme [13,14]. By contrast, in roots, no changes were observed in the levels of GAD1 and GAD2 transcripts, protein or total GAD activity in response to similar treatments [14]. Microarray analysis of the transcriptome of plants shifted from ammonium to 250 mM nitrate for a short period of 20 min did not reveal any changes in the transcription of the GAD genes or any of the other GABA-shunt genes, in roots or shoots [43]. Therefore, the links between GABA and nitrate or ammonium uptake and assimilation are still not clear and require further investigation, given that the GABA shunt is regulated at different levels, from transcription to metabolic control of enzyme activity. GABA as a signaling molecule in plants GABA and pollen tube growth In Arabidopsis, the pollen pistil-interaction2 ( pop2) mutant is affected in the guidance and growth of pollen tubes in pistils [44]. GABA was found to be of particular importance in this process, because the gene mutated in pop2 encodes a pyruvate-dependent GABA-T [22], which participates in GABA catabolism (GABA-TP; Figure 1). In the wild type, GABA concentrations increase along the path through which pollen tubes travel to female tissues, thus creating a gradient of GABA in the pistil. The gradient is disturbed in pop2 plants because they accumulate GABA in owers. Interestingly, both guidance and growth of the pollen tube can be restored in crosses between pop2 and wild-type plants. Thus, the pollen tube is accurately targeted to the ovule if either the pistil or the pollen tube itself can degrade GABA with an active POP2 enzyme (i.e. GABA-TP). Although the need to sustain proper levels of GABA in pollen tube development is clear, the mechanism involved is still vague and can be mediated by GABA receptors (i.e. signaling role) or by the

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modication of GABA homeostasis (i.e. metabolic imbalance and toxicity). GABA receptors In the mammalian central nervous system, GABA is the principal neurotransmitter mediating inhibitory synaptic currents by binding to receptors localized in the pre- or postsynaptic membranes. Reviews [4,45] have discussed the possible presence of GABA receptors in plants, which could mediate the action of GABA as a signaling molecule. Briey, genes highly homologous to the animal GABA receptors are not present in the Arabidopsis genome. Nevertheless, there are reasons to suspect that GABA could interact with a family of proteins (designated ATGLRS [46] for Arabidopsis glutamate receptors) sharing sequence and structural homology with the mammalian ionotropic glutamate receptors (iGluRs). AtGLRs can mediate Ca2 entry into plant cells when stimulated by glutamate [47,48] or glycine [49]. Nevertheless, in addition to the glutamate/glycine-binding domain, AtGLRs possess a domain known to bind modulatory ligand molecules, which share structural homology with domains of several receptors including the GABAB receptors [50 52]. Therefore, it is tempting to speculate that GABA could bind to this domain and modulate the activity of AtGLRs, in addition to their main ligands (i.e. glutamate or glycine). In addition, it should be noted that known agonists and antagonists of animal GABA receptors have contrasting effects on growth of duckweed (Lemna minor), again suggesting the possible presence of GABA-responsive receptors in plants [53]. Their exact nature and function need to be determined. In a recent article, Kang and Turano [47] have described the phenotype of plants in which the function of one of the putative glutamate receptors was suppressed by antisense technology. In these transgenic lines, several key enzymes involved in the control of the C:N balance were not regulated properly. Therefore, GABA could be involved in the control of C:N balance either via direct interaction with glutamate receptors (signaling role) or by controlling glutamate availability (metabolic role). Concluding remarks and perspectives The GABA shunt is a conserved pathway in eukaryotes and prokaryotes but, although the role of GABA as a neurotransmitter in mammals is clearly established, its role in plants is still vague, albeit with increasing evidence for its importance in various aspects of plant development, metabolism and responses to stress. GABA probably plays a dual role as both a signaling molecule and a metabolite. This dual role of GABA could be similar to those played by other metabolites, such as glutamate or sugars. Uncoupling and deciphering the signaling and metabolic functions of GABA will require intensive and exciting research work during the next few years.
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