Opinion TRENDS in Plant Science Vol.9 No.3 March 2004

GABA in plants: just a metabolite?

Nicolas Bouche´ 1 and Hillel Fromm2

1Commissariat a` l’Energie Atomique, Direction des Sciences du Vivant, Service de Bioe´ nerge´ tique, 91191 Gif-sur-Yvette, France 2Department of Plant Sciences, Tel Aviv University, 69978 Tel Aviv, Israel

For decades, GABA in plants has been treated merely as and the characterization of the encoded as a 2þ a metabolite, mostly in the context of the response to Ca -dependent (CaM)-binding protein [5]. stress. Recent evidence from the exploitation of Arabi- Subsequently, it was found that GAD activity in dopsis functional genomic tools points towards a new extracts from various plant species [6–14] is modulated possible role of GABA as a signal molecule and provides by Ca2þ –CaM. Detailed molecular analysis of the further insights into the role of the GABA metabolic CaM-binding domain of petunia GAD [10] and the pathway in response to stress and carbon:nitrogen characterization of the purified recombinant protein as . The challenge now is to uncouple the sig- aCa2þ –CaM-regulated enzyme [15] provided a work- naling and metabolic roles of GABA, and to identify the ing model to explain the rapid stimulation of GAD molecular components and their mode of action. activity in response to various stress situations that elicit changes in cytosolic Ca2þ concentrations. Evi- g-Aminobutyric acid (GABA; see Glossary) is a four-carbon dence to support this hypothesis was provided by non-protein amino acid conserved from bacteria to plants demonstrating that stimulation of GAD activity in response and vertebrates. It was discovered in plants more than half a to anoxia [11] and cold [12] involves Ca2þ–CaM. Taken century ago [1], but interest in GABA shifted to animals together, the regulation of GAD activity by Ca2þ–CaM has when it was revealed that GABA occurs at high levels in the been demonstrated in vitro [13–17] and in vivo [11,12]. brain, playing a major role in neurotransmission. There- Recently, the three-dimensional structure of CaM bound to after, research on GABA in vertebrates focused mainly on its the petunia GAD CaM-binding domain has been determined role as a signaling molecule, particularly in neurotrans- bynuclear magneticresonance[18], revealingan interesting mission. In plants and in animals, GABA is mainly complex of CaM with two peptides of the CaM-binding metabolizedvia a short pathway composedofthreeenzymes, domain. This suggests a role for CaM in regulating the called the GABA shunt because it bypasses two steps of the formation or stability of the GAD protein complex, as tricarboxylic acid (TCA)cycle(Figure 1). The pathway is previously suggested based on immunodetection of native composed of the cytosolic enzyme GAD complexes (,500 kDa) in transgenic plants expressing (GAD) and the mitochondrial GABA transaminase the full-length or truncated GAD lacking the CaM-binding (GABA-T) and succinic semialdehyde dehydrogenase domain [8]. However, recent evidence also suggests the (SSADH).Theregulation of thisconservedmetabolicpathway occurrence of a rice GAD isoform lacking a CaM-binding seems to have particular characteristics in plants. Indeed, domain [19]. Consequently, it should be interesting to interest in the GABA shunt in plants emerged mainly from investigate whether such an isoform is typical of monocots experimental observations that GABA is largely and rapidly or whether it is present in some dicots as well. The use of produced in response to biotic and abiotic stresses [2–4].The transgenic plants ectopically expressing either GAD or a GABA shunt has since been associated with various mutant GAD lacking the ability to bind CaM provided physiological responses, including the regulation of cytosolic further evidence for the importance of CaM binding to pH, carbon fluxes into the TCA cycle, nitrogen metabolism, GAD in vivo [8]. deterrence of insects, protection against oxidative stress, Since the completion of the sequencing of the osmoregulation and signaling (Box 1). In this article, we Arabidopsis genome [20], five GAD have been attempt to link these and other findings that have identified by sequence comparisons [2]. At least two of accumulated during the half-century since the discovery of the GAD isoforms (GAD1 and GAD2) differ in their organ GABA in plants with recent evidence, mainly from Arabi- distribution: GAD1 is root specific, whereas GAD2 is dopsis functional genomic approaches, pointing towards the possible role of GABA as a signal molecule in plants, as well Glossary as roles in plant responses to stress and in the carbon:nitro- CaM: calmodulin. gen (C:N) balance. C:N: carbon:nitrogen. GABA: g-aminobutyric acid. GAD: glutamate decarboxylases. Metabolism of GABA in plants GABA-T: GABA transaminase. Glutamate decarboxylase GHB: g-hydroxybutyric acid. Studies of the function of GAD and its regulation in plants AtGLRs: Arabidopsis glutamate receptors. ROIs: reactive oxygen intermediates. have been stimulated by the cloning of the petunia GAD SSADH: succinic semialdehyde dehydrogenase. TCA cycle: tricarboxylic acid cycle. Corresponding author: Hillel Fromm ([email protected]). www.sciencedirect.com 1360-1385/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2004.01.006 Opinion TRENDS in Plant Science Vol.9 No.3 March 2004 111

Mitochondrial Cytosol matrix + NH4

Inner mitochondrial GS/GOGAT + membrane NH4 (chloroplast and cytosol)

GDH α-Ketoglutarate Glutamate Glutamate

H+ α-KGDH Glutamate α-Ketoglutarate TCA cycle Succinyl-CoA GAD

GABA-TK Succinyl-CoA CO 2 Fumarate Succinic Succinate GABA GABA semialdehyde

SSADH GABA-TP NADH + H+ Ca2+ÐCaM

Alanine Pyruvate NAD+

Respiratory chain ATP

ATP synthase Outer mitochondrial membrane

Succinic SSR GHB semialdehyde

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 isthe principal nitrogen assimilation pathway into glutamate and amino acids in plants. The glutamate dehydrogenase (GDH) is thought to function primarily in glutamate cata- bolism but can also function in the opposite direction. The GABA shunt is composed of three enzymes (purple). Glutamate decarboxylase (GAD) is a cytosolic enzyme regu- lated (green) by the Ca2þ –calmodulin (CaM) complex, which catalyses the irreversible 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 neurotransmit- ter, whereas its role in plants is unknown. expressed in all organs [13,14]. Together, these findings GABA-TK enzyme of plants remains to be identified, imply that the different GAD isoforms are expressed in a whereas the pyruvate-dependent GABA-TP was partially tissue-dependent manner and might have specific purified from tobacco and a homologous Arabidopsis gene functions. was subsequently cloned [21]. The recombinant Arabi- dopsis GABA-TP characterized in vitro uses pyruvate but GABA transaminase not a-ketoglutarate, and shares little homology with non- GABA is converted to succinic semialdehyde (SSA) by plant GABA-Ts [21]. Arabidopsis knockouts disrupted in two sorts of GABA-Ts that use either a-ketoglutarate the corresponding gene have a GABA content elevated (GABA-TK) or pyruvate (GABA-TP) as amino acid 100-fold in flowers compared with the wild type, confirm- acceptors, producing glutamate or alanine (Figure 1). In ing that GABA-TP is a functional enzyme of the GABA mammals, only the GABA-TK seems to be present, shunt in vivo [22]. In this mutant, the increase of GABA whereas both enzyme activities can be detected in levels in other organs (e.g. leaves) is limited, implying that crude plant extracts [2]. The a-ketoglutarate-dependent the GABA-TP has a specialized function in flowers and www.sciencedirect.com 112 Opinion TRENDS in Plant Science Vol.9 No.3 March 2004

Box 1. Possible roles of GABA and the GABA shunt in plants

Contributing to the C:N balance stress conditions that inhibit certain enzymes of the TCA cycle (Figure 1). Levels of g-aminobutyric acid (GABA) are high in certain tissues. In yeast, mutants knocked out in GABA-shunt genes seem to be more For instance, GABA can reach as much as 50% of the free amino acid sensitive to H2O2 [25]. in cherry tomato fruits [54] and the concentrations of GABA can increase drastically in response to different stresses [2,3]. Moreover, Defense against insects Arabidopsis grows efficiently on medium containing GABA as a sole Because GABA is a in vertebrates and invertebrates, it source of nitrogen [55]. GABA is thus involved in the general was speculated that GABA could be produced by the plant to deter nitrogen metabolism and possibly in the storage and/or transport of insect feeding. GABA levels are elevated by mechanical stimulation or nitrogen. The GABA shunt is also a way to assimilate carbons from damage [59], and even by insects crawling on leaves [60], and it is glutamate and to generate C:N fluxes that enter the tricarboxylic acid possible that the ingested GABA interferes with the normal develop- (TCA) cycle. ment of insects [2,3]. For instance, transgenic tobacco plants containing elevated GABA levels were suggested to be resistant to the root-knot Regulation of cytosolic pH nematode [61] and tobacco budworm larvae [62]. Moreover, the In bacteria, there seems to be a role for the GABA shunt in acid pathogen-induced oxidative burst is associated with an increase in resistance [56,57]. When is exposed to such stress, GABA content in isolated Asparagus cells [63]. 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 GABA as an osmoregulator plants, GAD is activated by acidic pH [6,15] and GABA accumulates in Proline transporters from both Arabidopsis (i.e. AtProT2 [55]) and response to cytosolic acidification [2], so it is possible that GAD activity tomato (i.e. LeProT1 [64]) also transport GABA. AtProT2 is strongly could participate in regulating the cytosolic pH of plants. induced during water or salt stress [65]. Proline–GABA transporters can transport organic osmolytes, also called compatible solutes, which Protection against oxidative stress serve several protective roles. In Arabidopsis, mutants disrupted in succinic semialdehyde dehydro- genase are more sensitive to environmental stress because they are GABA as a signaling molecule

unable to scavenge H2O2 [26]. The last step of the GABA shunt can GABA is a neurotransmitter in animals with a clearly defined role in provide both succinate and NADH to the respiratory chain (Figure 1). It signaling. Similarly, GABA could be a signaling molecule in plants was therefore hypothesized that the degradation of GABA could limit [4,45]. There are indications that GABA receptors exist in plants or that the accumulation of reactive oxygen intermediates under oxidative plant glutamate receptors use GABA as a modulatory . that other GABA-Ts might degrade GABA in the rest of the accumulation of reactive oxygen intermediates (ROIS). For plant. Therefore, in plants, GABA transamination occurs example, ssadh mutants exposed to white light via different types of GABA-T, which probably have (100 mmol m2 s21) appear to be dwarfed with necrotic specialized functions. lesions (see Figures 2,3 in Ref. [26]). Indeed, ssadh mutants are sensitive to at least two types of environmen- Succinic semialdehyde dehydrogenase tal stresses: both ultraviolet irradiation (particularly The first cloned SSADH gene from plants has been ultraviolet B) and heat cause a rapid increase in the levels biochemically analysed [23]. Antibodies raised against of H2O2 in ssadh mutants, and this is associated with purified recombinant Arabidopsis SSADH localized enhanced cell death and necrosis of leaves [26]. The SSADH to the mitochondria of potato tubers and Arabi- phenotype of the ssadh mutants could be caused by a lack dopsis cell cultures, consistent with previous biochemical of certain metabolites (e.g. loss of NADH or succinate to studies of SSADH localization in soybean cells [24]. the respiratory chain), an excess of a metabolite derived Subcellular localization of SSADH to the mitochondria is from the GABA shunt with a possible toxic effects [e.g. SSA common in various organisms, although exceptions have or g-hydroxybutyric acid (GHB), see below] or an imbalance been described; in yeast, for example, the enzyme is in the in signaling molecules derived from the GABA shunt cytosol [25]. In vitro assays revealed that SSADH is (including GABA). In plant mitochondria, several specific for SSA and exclusively uses NADþ to produce enzymes of the TCA cycle, including those that are NADH. Importantly, both ATP and NADH negatively bypassed by the GABA shunt (Figure 1) (the succinyl- regulate the activity of the enzyme. Both products of the CoA ligase and the a-ketoglutarate dehydrogenase), reaction catalysed by SSADH (i.e. succinate and NADH) are degraded under oxidative stress conditions [27].In are substrates of the mitochondrial respiratory chain, the brain, inhibition of a-ketoglutarate dehydrogenase which produces ATP as a final product. Therefore, during H2O2-induced oxidative stress crucially limits regulation of SSADH by ATP suggests a tight feedback the amount of NADH [28], and a role has also been control of the rate of substrates provided by the GABA suggested for the GABA shunt in protecting yeast shunt to the respiratory chain. Consequently, the feedback against oxidative stress [25]. Recent studies revealed a regulation might also play a role in controlling the steady- relationship between Ca2þ signaling and ROIs in plants state levels of GABA and hence possible functions of GABA [29–32]. The GABA shunt, whose activity is regulated by via pathways other than the TCA cycle, such as signaling Ca2þ via GAD, is thus activated under stress conditions pathways. that cause enhanced production of ROIs. These studies of In Arabidopsis, disruption of the unique SSADH gene the ssadh mutants and the SSADH enzyme suggest the results in plants undergoing necrotic cell death when possible involvement of the GABA shunt in tolerance to exposed to environmental stresses, owing to the abnormal oxidative stress. www.sciencedirect.com Opinion TRENDS in Plant Science Vol.9 No.3 March 2004 113 g-Hydroxybutyric acid glutamate were the only metabolites for which the diurnal In an alternative reaction to the GABA shunt, SSA can be changes are shifted by 6 h between source and sink. This reduced to GHB by an SSA reductase (SSR; Figure 1). In finding suggests that the production of GABA is tightly the brain, GHB can be converted to SSA via another linked to the glutamate content, and the authors hypoth- enzyme, the GHB dehydrogenase (GHBDH). GHB is esized that GABA has a dominant role in buffering the normally found in small quantities in the mammalian production of glutamate [40]. This might confirm previous brain, representing ,0.2% of the levels of its parent observations about the role of GABA as a transient compound, GABA [33]. GHB is released by neuronal nitrogen storage metabolite [2,3]. Following this idea, it depolarization in a Ca2þ-dependent manner [33] and high is also tempting to speculate that GAD activity is regulated affinity GHB binding sites are present in specific brain by the level of glutamate [2]. regions (e.g. thalamus). Furthermore, the pharmacologi- To control their C:N balance, plants can sense their cal action of GHB is to reduce the release of , reduced nitrogen status and regulate the uptake and probably via GHB receptors [34,35] and possibly through reduction of nitrate adequately [42]. Still, the identity of the GABAB receptors, of which GHB is a weak agonist. In the metabolite(s) that plants use as sensor(s) in that humans, GHB and GABA levels are noticeably increased process is unclear. The role played by glutamine is still a in patients deficient for SSADH activity, a rare genetic matter of debate and other metabolites, including amino disease called 4-hydroxybutyric aciduria [36,37] that leads acids or sugars, are probably involved [41]. GABA could be to neurological abnormalities. one of these metabolites. In plants, a gene encoding a protein with SSR activity was recently cloned by complementing a SSADH-deficient Regulation of the GABA shunt by nitrogen availability yeast strain with an Arabidopsis cDNA library [38]. The Shifting plants from an unreduced nitrogen source (KNO3) SSR of Arabidopsis shares little homology with its animal to reduced forms of nitrogen (NH4Cl, NH4NO3, glutamate counterpart. The role of GHB in plants remains obscure or glutamine) for a prolonged treatment of 4 days resulted but GHB seems to be produced in response to flooding and in increased levels of GAD2 transcript and protein, and an oxygen deficiency [38]. Understanding the role of SSR and increase in the total GAD specific activity in Arabidopsis GHB in response to environmental stresses will require leaf extracts [14]; GAD1 was not detected because it is a further investigations such as isolating SSR knockouts in root-specific enzyme [13,14]. By contrast, in roots, no Arabidopsis and measuring GHB levels in plants altered changes were observed in the levels of GAD1 and GAD2 in the GABA shunt, like ssadh [26] or gaba-t [22] mutants. transcripts, protein or total GAD activity in response to similar treatments [14]. Microarray analysis of the GABA shunt and the metabolism of nitrogen transcriptome of plants shifted from ammonium to Plants sense their nutrient environment and respond to it 250 mM nitrate for a short period of 20 min did not reveal by modifying their uptake and metabolism using an any changes in the transcription of the GAD genes or any intricate system of sensors, receptors, transporters, signal of the other GABA-shunt genes, in roots or shoots [43]. transduction components and gene expression regulators Therefore, the links between GABA and nitrate or that collectively lead to changes in growth rates and ammonium uptake and assimilation are still not clear development [39]. More specifically, the complex mechan- and require further investigation, given that the GABA isms coordinating the C:N balance involve sensing and shunt is regulated at different levels, from transcription to regulating sugar levels, nitrate uptake and internal metabolic control of enzyme activity. contents of reduced nitrogen (e.g. ammonium and amino acids). The GABA shunt appears to be part of the metabolic GABA as a signaling molecule in plants pathways involved in the C:N balance and the metabolism GABA and pollen tube growth of nitrogen, the extent of which is not fully understood. In Arabidopsis, the pollen–pistil-interaction2 ( pop2) mutant is affected in the guidance and growth of pollen Impact of the GABA shunt on nitrogen metabolism tubes in pistils [44]. GABA was found to be of particular Most inorganic nitrogen is assimilated through the importance in this process, because the gene mutated in glutamine synthetase/glutamate synthase (GS/GOGAT) pop2 encodes a pyruvate-dependent GABA-T [22], which pathway, producing glutamate, which is then the starting participates in GABA catabolism (GABA-TP; Figure 1). In point for the synthesis of most amino acids, including the wild type, GABA concentrations increase along the GABA (Figure 1). Glutamate levels can be drastically path through which pollen tubes travel to female tissues, affected when the function of GAD is modified. For thus creating a gradient of GABA in the pistil. The instance, transgenic plants overexpressing a constitu- gradient is disturbed in pop2 plants because they tively active GAD have significantly reduced levels of accumulate GABA in flowers. Interestingly, both guidance glutamate [8]. Still, the importance of GAD in controlling and growth of the pollen tube can be restored in crosses glutamate levels in wild-type plants remains to be between pop2 and wild-type plants. Thus, the pollen tube clarified. This question was partially addressed in tobacco, is accurately targeted to the ovule if either the pistil or the in which the levels of several metabolites were followed pollen tube itself can degrade GABA with an active POP2 during two day–night cycles and compared between upper enzyme (i.e. GABA-TP). Although the need to sustain young leaves (i.e. sink) and lower old leaves (i.e. source) proper levels of GABA in pollen tube development is [40]. Diurnal rhythms were detected in both types of leaves clear, the mechanism involved is still vague and can be for different amino acids [41]. Interestingly, GABA and mediated by GABA receptors (i.e. signaling role) or by the www.sciencedirect.com 114 Opinion TRENDS in Plant Science Vol.9 No.3 March 2004 modification of GABA homeostasis (i.e. metabolic imbal- 3 Snedden, W.A. and Fromm, H. (1999) Regulation of the g-aminobu- ance and toxicity). tyrate-synthesizing enzyme, glutamate decarboxylase, by calcium– calmodulin: a mechanism for rapid activation in response to stress. In Plant Responses to Environmental Stresses: From Phytohormones to GABA receptors Genome Reorganization (Lerner, H.R., ed.), pp. 549–574, Marcel In the mammalian central nervous system, GABA is the Dekker principal neurotransmitter mediating inhibitory synaptic 4 Kinnersley, A.M. and Turano, F.J. (2000) Gamma aminobutyric acid currents by binding to receptors localized in the pre- or (GABA) and plant responses to stress. Crit. Rev. Plant Sci. 19, 479–509 5 Baum, G. et al. (1993) A plant glutamate decarboxylase containing a postsynaptic membranes. 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