Strategies and tools for studying the metabolism and ...

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Strategies and tools for studying the metabolism and function of γ-aminobutyrate in plants. II. Integrated analysis Barry J. Shelp, Gale G. Bozzo, Adel Zarei, Jeffrey P. Simpson, Christopher P. Trobacher, and Wendy L. Allan

Abstract: g-Aminobutyrate (GABA) is a ubiquitous nonprotein amino acid that accumulates in plants in response to abiotic and biotic stresses. In a companion paper, we discussed the origin of GABA from glutamate and subsequent catabolism to succinic semialdehyde and either succinate or g-hydroxybutyrate (GHB), and the characteristics of genes and proteins responsible for GABA permease, glutamate decarboxylase, GABA transaminase, succinic semialdehyde dehydrogenase, and succinic semialdehyde reductase activities. In this paper, we explore gene expression and transcript–metabolite relationships during the response to abiotic stress, and describe phenotypes of genetic mutants and relationships of GABA metabolism to other plant functions. Evidence indicates that both gene-dependent and -independent processes are involved in the response of the GABA pathway to abiotic stresses. Study of stress-specific responses and their interplay with the C/N network and various signalling pathways would be more informative if circadian rhythms and light–dark transitions upon imposition of the stress were always taken into account, and relevant genes and metabolites simultaneously profiled in wild-type plants or genetic mutants. Key words: abiotic stress, metabolomics, mutants, signaling, transcriptomics, translatomics. Résumé : Le g-Aminobutyrate (GABA) est un acide aminé non protéinique ubiquiste s’accumulant dans les plantes en réaction à des stress abiotiques et biotiques. Dans une publication antécédente apparentée, les auteurs ont discuté de l’origine du GABA à partir du glutamate et son catabolisme subséquent en semi-aldéhyde succinique et en succinate ou g-hydroxybutyrate (GHB), ainsi que des caractéristiques gènes et protéines responsables des activités GABA perméase, glutamate décarboxylase, GABA transaminase, déshydrogénase semi-aldéhyde succinique et réductase semi-aldéhyde succinique. Ici, les auteurs explorent l’expression des gènes et les relations transcript-métabolite au cours de la réaction à un stress abiotique et ils décrivent les phénotypes des mutants génétiques ainsi que les relations du métabolisme du GABA avec d’autres fonctions de la plante. La preuve indique que des processus dépendants aussi bien qu’indépendants des gènes sont impliqués dans la réaction du sentier GABA aux stress abiotiques. Les études de réactions spécifiques au stress et leurs interactions avec le réseau C/N ainsi que divers sentiers de signalisation fourniraient de meilleures informations si les rythmes circadiens et les transitions lumière-noirceur au moment d’appliquer le stress étaient prises en compte, et que les gènes et les métabolites impliqués étaient simultanément profilés chez les plantes de type sauvage et les mutant génétiques. Mots‐clés : stress abiotique, métabolomique, mutants, signalisation, transcriptomique, translatomique. [Traduit par la Rédaction]

Introduction Numerous studies have demonstrated that g-aminobutyrate (GABA) accumulates in plants upon exposure to abiotic and biotic stresses (Shelp et al. 1999, 2009; Kinnersley and Turano 2000) so that GABA pathway genes are now often included in expression studies. For example, transcriptional

induction of glutamate decarboxylase (GAD) or GABA transaminase (GABA-T) in Arabidopsis roots (Klok et al. 2002; Deeken et al. 2006), grape (Vitis vinifera L.) shoot tips (Cramer et al. 2007), orange (Citrus sinesis L.) flavedo tissues (Pasentsis et al. 2007), and ginseng (Panax ginseng C.A. Meyer) stems (Lee et al. 2010) has been demonstrated in response to low O2, water deficit, salinity, or Agrobacte-

Received 13 January 2012. Accepted 10 April 2012. Published at www.nrcresearchpress.com/cjb on 17 August 2012. B.J. Shelp, G.G. Bozzo, A. Zarei, J.P. Simpson, C.P. Trobacher, and W.L. Allan. Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada. Corresponding author: Barry J. Shelp (e-mail: [email protected]). Botany 90: 781–793 (2012)

doi:10.1139/B2012-041

Published by NRC Research Press

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rium infection. Over the past decade, there has been increasing emphasis on integrated approaches to provide a better overall picture of plant processes. In this article, we explore these studies to improve our understanding of the role of GABA metabolism during abiotic stress. Emphasis is on gene expression, transcript–metabolite relationships, mutant phenotypes, and relationships with other functions.


Expression of genes encoding proteins of GABA metabolism Schmid et al. (2005) conducted a comprehensive study of the developmental control of expression of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) genes, including those associated with the GABA pathway. Microarray analysis revealed that GAD genes are differentially expressed as follows: GAD1 is abundant in roots only, although much lower levels are present in other tissues such as hypocotyl, young rosette leaves, and sepals; GAD2 is abundant in the root, shoot, flower parts, and immature siliques; GAD3/4 is present in young leaves, sepals, carpels, and immature siliques, but at much lower levels than those for GAD2; and GAD5 transcript is present at moderate levels in stamens and mature pollen only (Supplementary Fig. S11). GABA-T, glyoxylate reductase/succinic semialdehyde reductase 1 (GLYR1), glyoxylate reductase/succinic semialdehyde reductase 2 (GLYR2), succinic semialdehyde dehydrogenase (SSADH), and GABA permease (GABP) transcripts are distributed throughout the plant, including the root and imbibed seed, in decreasing order of abundance. Interestingly, GLYR2 is more highly associated than GLYR1 with rosette leaves, which are known to highly express photorespiratory genes (Suppl. Fig. S11; see also Foyer et al. 2009). Miyashita and Good (2008) confirmed the differential expression of the five GAD genes and ubiquitous expression of the GABA-T in various organs of Arabidopsis using quantitative polymerase chain reaction (qPCR). The expression response of GABA pathway genes to low O2 stress has been extensively studied using Arabidopsis. Supplementary Fig. S21 compares microarray results from three separate studies of young seedlings (Loreti et al. 2005; Branco-Price et al. 2005, 2008). Interestingly, the transcript abundance of none of these genes is upregulated in leaves within 2 h of removing O2 and CO2, although control genes known to respond to anoxia such as alcohol dehydrogenase 1 (ADH1) and alanine aminotransferase 1 are markedly upregulated. It is evident, however, that the transcription of some of the genes is in some cases, upregulated slightly over the longer term or during recovery from low O2 stress (Suppl. Fig. S21). Branco-Price et al. (2008) demonstrated that low O2 stress promotes adjustments in the levels of polysomes in Arabidopsis and allows translation of only a subset of the cellular mRNAs, thereby promoting the conservation of ATP. Recently, capture of epitope-tagged ribosomes was used to assess the remodeling of the translatome following 2 h exposure to an argon environment (Mustroph et al. 2009). This study confirmed that the expression of GABA metabolism genes in tissues (including both shoot and roots, and re1Supplementary

productive parts) of Arabidopsis, with the possible exception of GAD3/4 in the leaf vascular system, is rarely induced during the early response to low O2 stress (Suppl. Fig. S31). Miyashita and Good (2008) used qPCR analysis to investigate the expression response of roots from Arabidopsis plants treated with hypoxia for 24 h under dark conditions to eliminate photosynthetic O2 evolution. Their experimental design does not allow correction for changes in transcript levels that occur during the light–dark transition, although this is perhaps unimportant when dealing with root processes. Like the studies mentioned above for anoxia, hypoxia rapidly induces ADH1 expression, but has no effect on GABA-T expression. However, differential responses are observed for the five GADs; GAD1 expression is unchanged, whereas GAD2 expression declines progressively after 2 h and GAD4 expression increases steadily so that its level is the highest after 8 and 24 h (Suppl. Fig. S41). Another study revealed that the expression of GLYR1 and GLYR2 in rosette leaves is upregulated by 1- to 5-fold after submergence of 3- to 4-week-old Arabidopsis plants for 4 h, but the response is transient (Allan et al. 2012). Kilian et al. (2007) conducted a microarray study of the temporal response of gene expression in young seedlings of Arabidopsis to various stresses (see description of stress conditions in legend for Fig. 1). Experimental and control plants were handled similarly so that circadian rhythms can be taken into account. For the shoot, the various GAD genes respond differentially to stress; GAD1 and GAD5 are slightly to moderately upregulated in response to most of the stresses tested, whereas GAD2 is only slightly upregulated, if at all. Also, GAD3/4 is markedly upregulated by salt, UV-B, osmotic, drought, and wounding, and slightly upregulated by cold. Over the 24 h studied, only heat stress does not affect GAD expression, and other stresses such as drought, UV-B, and wounding often elicit a transient or biphasic response, perhaps due, at least in part, to the cessation of the stress after a brief exposure. The expression of GABA-T is slightly upregulated by osmotic and salt stresses, and SSADH and GABP are slightly upregulated by cold, whereas GLYR1 and GLYR2 are unaffected or slightly downregulated by all stresses tested. For the root, there is no significant effect of any stress on the expression of GAD1 or GAD2, whereas GAD3/4 is moderately upregulated by salt and slightly by cold, osmotic, and UV-B stresses, and GAD5 is slightly upregulated by salt and heat stresses. The other genes exhibit only slight upregulation, if at all, in response to the stresses. From these studies, it is evident that time course studies with appropriate controls for circadian rhythms or light–dark transitions are essential for identifying stress-specific expression responses of GABA pathway genes and to some degree, the response is dependent on the tissue and developmental stage of the plants. Nevertheless, some generalizations are possible: (i) in the short-term (2 h or less), these genes respond only slightly or not at all, whereas in the longer term (up to 24 h), changes in expression can become evident; (ii) expression of the shoot genes seems to be more responsive than corresponding root genes; and (iii) genes associated with the anabolic phase of the GABA pathway are more responsive than those associated with the catabolic phase. In

data are available with the article through the journal Web site (http://nrcresearchpress.com/doi/suppl/10.1139/b2012-041) Published by NRC Research Press

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Fig. 1. Relative expression of genes associated with g-aminobutyrate metabolism in Arabidopsis seedlings exposed to various stresses. Absolute expression levels in treated tissue are corrected for levels in control tissues subjected to similar conditions, and the difference expressed as fold-increase (not a ratio) compared to the zero time control. The heat map is based on log2-transformed ratios, which can be thought of as two to the exponent of the given value, as indicated in the colour key. Expression data were accessed from the abiotic stress series microarray data set organized on the Bio-Array Resource for Arabidopsis Functional Genomics (http://bar.utoronto.ca/) (Toufighi et al. 2005). Plants were grown in tissue culture for 10 days under a typical light–dark regime and the stresses applied as follows: cold, continuously at 4 °C in the presence of control light; drought, 15 min dry air stream and 10% decline in fresh mass followed by recovery; heat, 3 h at 38 °C, followed by recovery; osmotic, 300 mmol·L–1 mannitol in medium; salt, 150 mmol·L–1 NaCl in medium; UV-B, 15 min exposure followed by recovery; wounding, punctured with pins followed by recovery (Kilian et al. 2007). The choice of probes did not permit the separation of GAD3 and GAD4. Abbreviations: GAD, glutamate decarboxylase; GABA-T, g-aminobutyrate transaminase; GLYR, glyoxylate/succinic semialdehyde reductase; SSADH, succinic semialdehyde dehydrogenase; GABP, GABA permease. The TAIR accession numbers are given to the right of the gene names.

particular, the GAD genes are the most responsive of the GABA pathway genes to the suite of abiotic stress conditions tested so far. Interestingly, GAD2 is the most abundant constitutively expressed isoform throughout the plant; however, it is not induced in shoots and roots in response to abiotic stress. By contrast, GAD1 is constitutively expressed at a high level in roots and hypocotyls only, but can be highly induced in shoots by abiotic stress. Constitutive expression of GAD3/4 is negligible throughout the plant, but it can be highly induced in shoots and roots by abiotic stress; with low O2 stress, this induction can be attributed specifically to GAD4. With the exception of pollen, constitutive expression of GAD5 is negligible throughout the plant; however, it is moderately induced in shoots and roots by abiotic stress.

Also, there appears to be some cases of coordinated stressinduced upregulation of GAD and other genes in the GABA pathway (e.g., cold induction of GAD4, SSADH, and GABP in shoots). Closer examination of these data may reveal motifs and elements that are common among genes of the GABA pathway, and between genes of the GABA pathway and other plant processes (Howell et al. 2009). Further study of gene expression under additional conditions such as long-term drought and oxidative stresses, and in vegetative versus reproductive plants and organs, using appropriate controls to account for nonstress effects, and monitoring the distinct GAD isoforms, would be helpful in identifying specific stress-induced responses of genes in the GABA pathway. Published by NRC Research Press

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Fig. 2. Response of GABA pathway metabolites and enzyme transcripts in rice seed germinated under anoxia. Data are plotted as a ratio of the level in plants treated with nitrogen in the dark at 30 °C versus plants supplied with air under the same conditions so that a value of one represents no change. Data were accessed from the supplementary tables in Narsai et al. (2009) and the Metabolome Express Web site (https://www.metabolome-express.org/); putative GLYR isoforms are reported in Shelp et al. (2012). Abbreviations for genes accessed are given in Fig. 1; the genes are denoted by their respective TIGR gene loci. Abbreviations for metabolites: GABA, g-aminobutyrate; GHB, ghydroxybutyrate; NAD(P)+, oxidized nicotinamide adenine dinucleotide (phosphate); NAD(P)H, reduced nicotinamide adenine dinucleotide (phosphate); SSA, succinic semialdehyde; SSAR, succinic semialdehyde reductase.

Transcript–metabolite relationships in the GABA pathway Miyashita and Good (2008) demonstrated that hypoxia rapidly enhances the expression of GAD4 in roots of wildtype Arabidopsis plants (shoots were not analyzed) and this increases steadily over a 24-h period; there is no effect on the expression of GAD1, the predominant form in the absence of hypoxia. Surprisingly, there is no evidence for the accumulation of GABA, as measured by an enzyme-linked assay, in response to hypoxia in either roots or shoots. Because GABA levels are known to be influenced by light intensity (Fait et al. 2005) and circadian rhythm (Espinoza et al. 2010), this result may be attributed to the use of a dark period for imposing hypoxia. Also, it may be associated with differences in adaptation of metabolism to hypoxia versus anoxia (Geigenberger 2003). Bouché et al. (2004) demonstrated

that disruption of GAD1 reduces GABA levels in Arabidopsis roots and prevents the typical accumulation of GABA that is observed after a 2-h period at 39 °C. Narsai et al. (2009) monitored metabolite and transcript levels by gas chromatography – mass spectrometry (GC– MS) and microarray, respectively, to compare rice germination under normoxia and anoxia. Their experimental design accounts for circadian variation. The GABA concentration increases steadily over the first 12 h of anoxia, followed by a transient decline over the next 36 h (Fig. 2). Two of five putative GAD transcripts exhibit a transient increase in abundance over the first 24 h, with peak abundance occurring at 12 h. The abundance of both GABA-T and SSADH transcripts exhibits a slight increase at 3 h, and then markedly declines over the next 21 h. The peak GHB concentration occurs rapidly within 4 h and this is correlated with the abundance of two putative GLYR transcripts, identified on the basis of their Published by NRC Research Press

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Fig. 3. Response of GABA pathway metabolites and enzyme transcripts in the aerial portions of cold-acclimated Arabidopsis plants. Data are plotted as a function of the level in the zero time control so that a value of one indicates no change; no correction was made for circadian rhythms. Some data were accessed from Kaplan et al. (2007) and the remainder was provided by Charles Guy, personal communication. Plants were grown for 21 days and then subjected to cold treatment under the same light–dark conditions. Abbreviations and TAIR accession numbers are given in Fig. 1.

structural similarity to known Arabidopsis GLYRs (Shelp et al. 2012). Thus, the oscillations in GABA and GHB may be associated with the abundance of particular GAD and GLYR transcripts. However, the accumulation of GHB precedes that for GABA, a surprising result considering that GHB is derived from GABA. This may be interpreted as support for inhibition of SSADH activity and stimulation of GLYR activity by altered redox balance under anoxia (Allan et al. 2008, 2012). If so, the activity of the Krebs cycle also declines, as indicated by decreases in isocitrate, citrate, and 2-oxoglutarate (Narsai et al. 2009), and the steady accumulation of succinate, glutamate, alanine, and glycine over the time course can be attributed to one or more of the following biochemical mechanisms: operation of a reductive Krebs cycle (Vanlerberghe et al. 1989); inhibition of alanine aminotransferase

degradative activity by limiting 2-oxoglutarate supply (Miyashita et al. 2007); and inhibition of protein synthesis by a restricted energy supply (Geigenberger 2003). It is noteworthy that metabolite pool sizes by themselves are not very informative in addressing mechanisms. Allan et al. (2012) monitored metabolites by high performance liquid chromatography (HPLC) with or without MS, transcripts by qPCR, and pyridine nucleotides by an enzymecycling assay. Six-hour time course experiments revealed that GHB accumulates in wild-type Arabidopsis in response to submergence and this is accompanied by elevated levels of GABA and alanine, ratios of NADH/NAD+ and NADPH/NADP+, and abundance of GLYR1 and GLYR2 transcripts (Allan et al. 2008, 2012). The experimental design accounts for changes that may occur in response to Published by NRC Research Press


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the light–dark transition imposed during submergence. The use of glyr1 and glyr2 knockout mutants demonstrated that GHB production during submergence is mediated via both GLYR isoforms and the loss of either GLYR1 or GLYR2 activity influences redox status (Suppl. Fig. S51). However, the levels of the wild-type GLYR transcript are not highly correlated with GHB levels and the levels of GABA are decreased in the mutants, providing support for feedback control of GABA-T by SSA accumulation. These results were interpreted as evidence for biochemical control of SSADH and GLYR activities by increasing NADH and NADPH levels, resulting in GHB production. Other studies investigated the impact of cold stress on GABA pathway transcripts and metabolites. For example, the GABA concentration in frost-resistant barley (Hordeum vulgare L.) seedlings increases approximately 15-fold in response to cold or freezing stress (i.e., –3 °C for 16 h or –8 °C for 21 h in dark), without any accompanying changes in the expression of GAD, GABA-T, and SSADH (Mazzucotelli et al. 2006). By contrast, the expression of GAD, GABA-T, and SSADH is induced during cold acclimation (3 °C for 21 days), resulting in further GABA accumulation during subsequent exposure to the freezing stress. A frost-sensitive genotype has elevated GABA, as well as glutamate decarboxylation during cold stress, but the genes are not induced and glutamate is nearly depleted. Kaplan et al. (2004, 2007) used GC–MS and microarray analysis to monitor metabolite and transcript levels in the aerial portions of 21-day-old Arabidopsis during acclimation at 4 °C. They found that the abundance of GAD4 transcript in cold-acclimated plants increases within 12 h of treatment and precedes the peak GABA concentration at 24 h (Fig. 3). By contrast, the abundance of SSADH transcript peaks after 24 h, concomitantly with declining GABA concentration, and is maintained during the peak succinate concentration. GHB concentration increases markedly by 48 h, and then declines, without any increases in the abundance of both GLYR1 and GLYR2 transcripts. Thus, the transient increase in GABA concentration at 24 h is apparently linked to the coordinated increase of GAD4 and SSADH transcripts, but the transient increase in GHB is independent of GLYR abundance. This suggests that diversion of SSA from succinate to GHB is controlled at the post-transcriptional level, probably by an alteration in redox balance (Allan et al. 2008), and that the succinate level is maintained by a reductive Krebs cycle (Vanlerberghe et al. 1989). Interestingly, over the time course there is a steady increase in concentrations of glutamate, the substrate for GAD, as well as glycine and alanine, potential products of GABA-T activity, even though the abundance of GABA-T transcript is essentially unchanged. Surprisingly, these findings are similar to those for low O2 stress, suggesting that common biochemical mechanisms are involved. Inhibition of glycine decarboxylation by NADH accumulation also may contribute to glycine accumulation (Bourguignon et al. 1988). Espinoza et al. (2010) demonstrated that GABA levels in the Arabidopsis rosette exhibit a diurnal fluctuation at 20 °C, with a sharp peak at (24 h) or closely following an 8-h dark period (52 h); this is not accompanied by changing GAD2 transcript abundance. At 4 °C, GABA levels also oscillate but with less amplitude, and the peak accumulation


occurs earlier (20 and 36 h, respectively), is greater, and is maintained over a longer interval; the initial peak is preceded by an increase in GAD2 transcript abundance, which is maintained even though GABA declines during the intervening light period. Expression of SSADH exhibits diurnal and circadian oscillations at 20 °C, but this is inversely related to the oscillations in GABA accumulation. The diurnal oscillations of SSADH are stopped in the cold. Thus, at 20 °C the oscillating GABA levels are correlated with SSADH abundance, whereas at 4 °C the levels are correlated with GAD2 abundance. Renault et al. (2010) investigated the response of tissueculture-grown Arabidopsis plants to salt stress using qPCR and enzymatic activity to monitor expression of GAD1-5, GABA-T, and SSADH, and GC–MS and high performance liquid chromatography to profile metabolites. They found that exposure to 150 mmol·L–1 NaCl increases GABA levels in whole plantlets by approximately 50% and 280% within 2 and 4 days, respectively, and this increase is greater in shoots than roots after 4 days (13-fold vs. 0.5-fold). Within the first 2 days in vitro GABA-T activity increases, whereas GAD activity does not, even though a dose-dependent increase in GAD4 expression is evident after 24 h treatment with 0– 150 mmol·L–1 NaCl. These data suggest that gene-independent mechanisms such as Ca2+/ CaM are responsible for GABA accumulation, at least during the early response to stress. Interestingly, the expression of GAD4 is also induced by drought (Urano et al. 2009), as well as O2 deficiency (Miyashita and Good 2008) and cold (Kaplan et al. 2007). Also, the expression of GAD2, GABA-T, and SSADH is upregulated in a co-ordinated fashion at 100 and 150 mmol·L–1 NaCl, perhaps to prevent the accumulation of SSA (Renault et al. 2010). Our survey of the literature revealed only a few reports that provide a reasonably complete picture of transcript–metabolite relationships in the GABA pathway. These reports suggest that both gene-dependent and -independent processes are involved in the response to low O2, low temperature or salinity. To more clearly identify stress–specific responses, interpretation of experimental results should not be complicated by the influence of diurnal rhythms and light–dark transitions that may be imposed with the stress treatment (e.g., Mazzucotelli et al. 2006; Kaplan et al. 2007; Miyashita and Good 2008). Moreover, it is important to monitor the multiple forms of GAD, GABA-T, SSADH, and GLYR that may be present in the plant species under consideration, as well as the relevant metabolites.

Phenotypes of GABA pathway mutants Kinnersley and Turano (2000) attempted to identify stressspecific trends via a survey of the time-course kinetics of peak GABA accumulation in several experimental plant systems: (i) stresses causing metabolic and (or) mechanical disruptions (e.g., O2 deficiency), which result in cytosolic acidification and cause an acidic pH-dependent stimulation of GAD and GABA synthesis; and (ii) “other stresses including cold, heat salt, and mild or transient environmental factors, such as touch, wind, rain”, which increase cytosolic Ca2+ and activate calmodulin (CaM)-dependent GAD activity and GABA synthesis (see Shelp et al. 2012). This comparaPublished by NRC Research Press


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tive survey was complicated by differing tissues and type and duration of stress. More recently, Arabidopsis was subjected to salinity, heat, cold, or submergence, and the rosette GABA levels were found to increase significantly within 2– 10 h (Suppl. Fig. S61, Allan et al. 2008). By contrast, GABA levels increased under drought conditions after 3– 6 days, probably due to time required for the pot medium to dry sufficiently to elicit a plant response. Interestingly, with all five stresses investigated, GHB accumulation was evident within 2–4 h, even preceding the accumulation of GABA under four of the five stresses. This suggests that GHB accumulation is not simply a consequence of GABA accumulation. One approach for identifying the relative importance of acid- and Ca2+/CaM-stimulated GABA production after exposure to various stress conditions is the use of GAD mutants. Transgenic tobacco plants constitutively overexpressing petunia or tobacco GAD possess more GABA and less glutamate than the wild-type (Baum et al. 1996; McLean et al. 2003). In the first of these studies, plants with a mutant GAD lacking the C-terminal CaM-binding domain have the highest GABA concentrations, but are sterile and exhibit severe morphological abnormalities (Baum et al. 1996), whereas in the second study similar transgenic plants are fertile, phenotypically normal, and moderately resistant to the northern root-knot nematode, tobacco budworm, and Agrobacterium tumefaciens (McLean et al. 2003; MacGregor et al. 2003; Chevrot et al. 2006). These findings have been attributed to varying transgene expression due to the use of different promoters or the presence of multiple transgene copies (McLean et al. 2003). By contrast, transgenic rice plants overexpressing OsGAD2 lacking the C-terminal extension dramatically accumulate GABA even though the full-length recombinant protein does not bind CaM (Akama et al. 2001; Akama and Takaiwa 2007). Curiously, antisense suppression of GAD in tomato fruit typically results in glutamate accumulation, but does not decrease the GABA concentration (Kisaka et al. 2006). T-DNA knockouts of the root-specific GAD1 in Arabidopsis are phenotypically unaffected, but GABA levels are dramatically decreased and GABA accumulation in response to heat stress is prevented (Bouché et al. 2004). Other mutants may also be useful for understanding the regulation of the GABA pathway. Overexpression of the mitochondrial GABA-T in Arabidopsis has no effect on the phenotype under normal growth conditions, but decreases the short-term, low temperature-induced increase in leaf GABA concentrations (Simpson et al. 2010). By contrast, gaba-t mutants accumulate GABA under normal growth conditions, exhibit a reduced seed production phenotype, and altered response to C-6 volatiles, are less resistant to infection by Pseudomonas syringae, and are oversensitive to ionic stress (Palanivelu et al. 2003; Mirabella et al. 2008; Miyashita and Good 2008; Clark et al. 2009; Park et al. 2010; Renault et al. 2010). Arabidopsis ssadh mutants accumulate GABA, GHB, and reactive oxygen intermediates when exposed to stress and appear dwarfed with necrotic lesions when exposed to white light (Bouché et al. 2003; Fait et al. 2005). Inhibition of GABA-T by treatment of the plant with vigabatrin, a GABA analog, or by disrupting the GABA-T gene prevents accumulation of GHB and reactive oxygen species, prevents

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cell death and improves growth in ssadh mutants (Fait et al. 2005; Ludewig et al. 2008). Arabidopsis ssadh and gaba-t mutants exhibit abnormalities in the polarity of the adaxial– abaxial axis in leaf primordia (Toyokura et al. 2011). Arabidopsis glyr1 and glyr2 mutants have no visual phenotype in the absence of stress, but possess lower GHB levels under short-term submergence than the wild-type control (Allan et al. 2012). Currently, efforts in the authors’ laboratories are using single and double mutants of Arabidopsis (GABA-T overexpression, GLYR1 overexpression, gaba-t knockout, glyr1 knockout, glyr2 knockout) to further investigate the function of GABA and GHB metabolism under stress and to determine whether GLYR1 and GLYR2 activities are the sole sources of GHB in plants. Together, these studies indicate that it is not easy to distinguish stress conditions under which acid- and (or) Ca2+/CaM-dependent mechanisms would be elicited in the plant so that the rate of GABA production is increased. While GAD mutants offer a useful approach, the occurrence of multiple GAD isoforms, some of which may not bind CaM can complicate their use (also see Shelp et al. 2012). Interpretation of such studies would be facilitated by complementary estimates of H+ and calcium levels in the cytosol (e.g., Cholewa et al. 1997; Schulte et al. 2006; Couldwell et al. 2009; Bose et al. 2011). As various other GABA pathway mutants become available interesting insights are being uncovered from their phenotypes under ambient conditions and their responses to short- or long-term stress conditions such as O2 deficiency and low temperature. They have also served as important tools in uncovering a role for GABA in communication between plants and other plants and organisms (Bown et al. 2006; Shelp et al. 2007).

Relationship of GABA pathway with other functions The use of microarray and qPCR analysis of gene expression and GC–MS determination of metabolites during late development and ripening of citrus fruit flesh revealed that glutamate is utilized for both glutamine production and catabolism via the GABA shunt, and static glutamate and smaller GABA pools are associated with reductions in both citrate concentration and cytosolic acidity (Cercós et al. 2006). Similar analysis of developing tomato fruit suggested that at the start of ripening, GABA is converted via SSA to malate, which enters a shunt involving pyruvate and citrate; in turn the stored citrate serves as a substrate for respiration during fruit ripening (Yin et al. 2010). Another study demonstrated that chemical inhibition of aconitase activity in juice vesicle callus induces the levels of extractable succinic semialdehyde reductase and SSADH activities, as well as alanine and aspartate transaminases (Degu et al. 2011), suggesting that excess citrate is converted to amino acids with the induction of GABA metabolism. Recent studies using mutants or antisense plants of Arabidopsis indicated that compromising TCA cycle enzymes involved in the synthesis of succinate, such as succinyl-CoA ligase and 2-oxoglutarate dehydrogenase, enhances GABA shunt activity, whereas compromising enzymes involved in its degradation, such as NAD+-dependent isocitrate dehydroPublished by NRC Research Press

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Fig. 4. Clustering in operonic arrangements of various GABA catabolic genes in bacteria known to communicate with plants. Genes of completed bacterial genomes in the SEED database are colour coded according to known GabT (GABA-T, red arrow) and GabD (SSADH, blue arrow) genes of Rhizobium leguminosarum. MerR (brown arrow) is proposed to be a transcriptional regulator of GabTD gene expression (Prell et al. 2002). GabR (yellow arrows) genes are those with homology to the transcriptional activator of the GABTD operon in Bacillus subtilus in the presence of exogenous GABA (Belitsky and Sonenshein 2002). Nonconserved genes are coloured grey. Arrows indicate transcriptional direction; overlapping arrows indicate translational coupling.

genase, has no effect on GABA metabolism (Lemaitre et al. 2007; Nunes-Nesi et al. 2007; Studart-Guimarāes et al. 2007; Araújo et al. 2008; Fait et al. 2008). Isotope labelling experiments suggested that the GABA shunt can contribute significantly to the mitochondrial succinate requirement (Lemaitre et al. 2007; Araújo et al. 2008; Fait et al. 2008), and the use of publicly available databases of transcript and metabolite profiles suggested relations between GABA metabolism and other pathways (Fait et al. 2008). For example, co-response analysis of transcript levels revealed a strong correlation between GAD2 and SSADH expression, although neither is correlated to GABA-T. Furthermore, the expression of GAD2 and SSADH is strongly correlated with genes of C/N metabolism, in particular those associated with the Krebs cycle. Based on this work, Fait et al. (2008) suggested that the metabolism of glutamate to succinate can be regarded as part of the Krebs cycle per se. A combination of transcript and metabolite profiling of pea embryos overexpressing an amino acid permease (VfAAP1) indicated that GABA pathway activity is part of a complex C/N network that responds to aspects of abscisic acid metabolism (Weigelt et al. 2008). Biochemical characterization of recombinant GABA-Ts from Arabidopsis and tomato revealed that they can utilize glyoxylate, as well as pyruvate, as amino donors (Clark et al. 2009). Furthermore, recombinant Arabidopsis GLYRs can utilize glyoxylate more effectively than SSA (Hoover et al. 2007; Simpson et al. 2008). As glyoxylate is an intermediate in the photorespiratory pathway in green leaves, and the rate of photorespiration, like GABA accumulation, increases under conditions such as salinity and drought due to stomatal closure, it has been suggested that photorespiration might interact with GABA metabolism (Allan et al. 2009; Clark et al. 2009). By contrast, under O2 deficiency the rate of photores-

piration would be much lower and not expected to supply glyoxylate (Narsai et al. 2009). This situation would be further complicated by the control of GABA catabolism by NADH/NAD+ and NADPH/NADP+ ratios and pH (Allan et al. 2008, 2012; Simpson et al. 2010; Shelp et al. 2012). Time course studies would help to elucidate potential interactions between photorespiratory-derived glyoxylate and GABA-Ts or GLYRs in the absence or presence of various stress conditions. Metabolite profiling of wild-type Lotus japonicus [Regel] R.K. Larsen and an RNAi-induced mutant of nodular leghemoglobin (causing nitrogen deficiency) subjected to waterlogging suggested that alanine is derived from both GABAT- and alanine transaminase-mediated reactions, but succinate accumulation occurs due to the inhibition of succinate dehydrogenase activity in the Krebs cycle (Rocha et al. 2010). Also, transcriptome profiling of waterlogged maize roots suggested that the late stage of waterlogging stress is associated with the generation of glutamate and alanine for the regulation of cytoplasmic pH and the breakdown of carbon skeletons for the generation of intermediates for energy production (Zou et al. 2010). Other research indicates that exogenous GABA influences developmental processes and associated gene expression. For example, incubation of excised sunflower (Helianthus annuus L.) tissues with exogenous GABA enhances the expression of ACC synthase (Kathiresan et al. 1997), and exogenous GABA that is supplied to the root system enhances the expression of nitrate transporter 2 in canola (Brassica napus L.) (Beuvé et al. 2004) and nitrate reductase protein in Arabidopsis (Barbosa et al. 2010). Also, GABA supplied in the medium of tissue culture-grown Arabidopsis mutants represses the transcription of several 14-3-3 genes in a Ca2+-, Published by NRC Research Press


Shelp et al.

ethylene- and abscisic acid-dependent manner (Lancien and Roberts 2006). Recent evidence suggests that root-supplied GABA activates multiple mechanisms involved in signaling, protein degradation, hormone biosynthesis, and production of reactive oxygen species during the response of Caragana intermedia to salt stress (Shi et al. 2010). Although time course measurements were conducted, the experimental design and statistical treatment of the data are unclear, making the significance of the work difficult to assess. Other work using wild-type Arabidopsis and gaba-t mutants demonstrates that exogenous GABA inhibits growth of the primary root and dark-grown hypocotyl in tissue culture-grown material, and decreases the expression of genes that encode secreted and cell wall-related proteins (Renault et al. 2011). Plant and microbial mutants altered in their respective abilities to metabolize and respond to exogenous GABA provide evidence that this metabolite serves as a communication signal for both symbiotic (i.e., root nodulation) and pathogenic relationships with bacteria (Shelp et al. 2007, 2009; Prell et al. 2009; Sulieman 2011). For example, phloem-delivered GABA accumulates in root nodules of various legumes (0.36–5.72 mmol·g–1 FM, Sulieman and Schulze 2010; and references therein) and in Agrobacterium tumefaciens induced crown galls (6.5 mmol·g–1 FM) of Arabidopsis, whereas it is negligible in bacteria pre-infection (Deeken et al. 2006). Also, 15N2 supplied to intact nodulated pea plants is recovered as labelled GABA in both cytosol and bacteroid fractions following Percoll density centrifugation (Prell et al. 2009). A drawback of this approach is the harvest of nodules from roots, followed by extraction, without precautionary instant cryofreezing so a proportion of nodular amino acids may be a consequence of mechanical disruption (Shelp et al. 2012). However, unlike acid-tolerant E. coli and Listeria monogycetes that have a GAD gene (GabB; Castanie-Cornet et al. 1999; Cotter et al. 2001), and hence the capacity to produce endogenous GABA from glutamate, a BLAST query of published bacterial genomes revealed that GAD is absent in mutualistic bacteria such as Rhizobium leguminosarum and in infectious bacteria such as Agrobacterium tumefaciens and Pseudomonas syringae, which are known to interact with plants. Therefore, GABA accumulating within these interacting bacteria originates from plants. Active uptake of GABA by a branched chain amino acid transporter is the first step in plant–bacteria relations, as rhizobia containing a mutation in a GABA transporter gene (Bra) are deficient in either their capacity to grow on minimal medium containing GABA or to transport [3H]GABA, as determined via a rapid filtration technique (Hosie et al. 2002). Pea root nodules formed with R. leguminosarum bra mutants contain bacteroids that are N2-starved and exhibit a 70% reduction in N2-fixing capacity (Lodwig et al. 2003). Also, the overexpression of GAD in tobacco enhances plant GABA levels, which in turn, disrupt quorum-mediated signalling and Agrobacterium virulence (Chevrot et al. 2006). Pseudomonas syringae pv. tomato DC3000, a pathogen known to accumulate in Arabidopsis and tomato apoplastic fluids, uses GABA as a sole C/N source (Rico and Preston 2008), and P. syringae gabT triple mutants (deletion mutants of three GABA-Ts) are unable to grow on minimal medium containing GABA (Park et al. 2010). Inoculation of wildtype and gaba-t mutants of Arabidopsis with the P. syringae

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gabT triple mutants increases leaf GABA concentration by 2to 4-fold, and this is correlated with a reduction in elicitation of the hypersensitive response (Park et al. 2010).The ability of bacteria to metabolize plant GABA is dependent upon the capacity of this signal to induce the co-ordinated expression of GABA-T and SSADH genes (encoded by GabD; Prell et al. 2002, 2009). Sulieman (2011) postulated that phloemdelivered GABA serves as a feedback mechanism for symbiotic N2-fixation, whereby GABA cycled through a nodular GABA shunt serves as a signal of the legume host’s N status, while simultaneously supplying succinate for energy production via the Krebs cycle. Thus, a functional bacterial GABA catabolism pathway for use of plant-derived GABA appears to be a prerequisite for GAD-deficient bacteria. Rhizobium leguminosarum genes for GABA catabolism are clustered into an operon (Prell et al. 2002). The organization of neighbouring genes is highly conserved amongst prokaryotes (Hanson et al. 2010). A query of completed bacterial genomes available in the SEED database (http:// www.theseed.org/wiki/index.php/Main_Page) with R. leguminosarum GabT revealed Gab operons in other symbiotic and pathogenic bacteria, some of which are known to respond to GABA (Fig. 4). Gab gene clustering was identified in Pseudomonas aeruginosa PAO1 (Fig. 4), and although the infection of Arabidopsis by a related strain leads to soft rot symptoms in leaves (Rahme et al. 2000), and propagation through the vessel parenchyma confers a systemic disease response, a role for plant-derived GABA as a communication signal in this pathogenic interaction has not yet been studied. Together, these studies indicate that accumulated GABA is part of a stress-responsive C/N network and various signalling pathways associated with developmental processes and communication between plants and other organisms (also see Shelp et al. 2007). In general, however, they do not exclude the possibility that GABA has an important role even when accumulation is not evident, nor firmly establish the hierarchical nature of the relationships.

Concluding remarks Both gene-dependent and -independent processes are involved in the response of the GABA pathway to abiotic stresses. GABA accumulates after short-term exposure to stress even in the absence of elevated GAD expression, probably as a result of acidic pH- or Ca2+/CaM-dependent stimulation of GAD activity. Likewise, GHB can accumulate without elevated GLYR expression due to altered redox balance. However, GABA accumulation over the long term is typically associated with elevated levels of GAD4 expression and perhaps GAD1 or GAD2 expression depending on the tissue and stress. Elevated GABA-T and SSADH expression may be associated with a loss in GABA levels, but it is not a requirement, suggesting that biochemical mechanisms are also involved. Further insights into stress-specific responses of the GABA pathway and their interplay with the C/N network and various signalling pathways would be facilitated by accounting for circadian rhythms and light–dark transitions upon imposition of the stress in time course experiments, and simultaneously monitoring the suite of relevant genes and metabolites in wild-type plants or genetic mutants. Published by NRC Research Press

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Acknowledgements We thank Charles Guy, Joachim Killian, and Reena Narsai for sharing data that are not publicly available, and clarifying experimental design and calculations in their published papers. We gratefully acknowledge funding from the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs to research the metabolism and function of GABA in plants.


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