Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?

1γ-Aminobutyric acid (GABA) is a four-carbon (C) non-proteinogenic amino acid (AA) that was first discovered in plants over half a century ago. GABA was later revealed in mammalian brain and soon main interest in GABA shifted to animals when it was shown to play a major role in neurotransmission. GABA has since been investigated in several organisms including bacteria, fungi, plants and animals. In both eukaryotes and bacteria GABA is a significant component of the free AA pool and the enzymes involved in GABA metabolism are conserved. Similar enzymes (e.g., glutamate decarboxylase) are found also in archaea, however their roles in GABA metabolism are questionable. Interest in plant GABA increased mainly following observations of rapid elevation of its levels under abiotic stresses. Nevertheless, the roles of GABA under these conditions is not clear. Recent combined genetics and physiological studies of the GABA shunt indicate that its function is required for proper growth in response to abiotic stresses such as low light and salt.

2γ-Aminobutyric acid is metabolized via a short pathway known as the GABA shunt (Figure 1), which bypasses two steps of the tricarboxylic acid cycle (TCAC). GABA is mainly produced by the irreversible reaction of the cytosolic enzyme glutamate decarboxylase (GAD; EC 4.1.l.15) that consumes a proton and releases CO2. However, GABA synthesis may also occur via polyamine (putrescine and spermidine) degradation and possibly by a non-enzymatic reaction from proline under oxidative stress. GABA catabolism occurs in the mitochondrial matrix of multicellular organisms by the action of GABA transaminase (GABA-T; EC 2.6.1.19) to produce succinic semi-aldehyde (SSA) with the possible participation of several amino acceptors such as α-ketoglutarate (AKG), pyruvate or glyoxylate. Subsequently, SSA is converted by another mitochondrial enzyme, SSA dehydrogenase (SSADH; EC 1.2.1.16) to succinate that acts both as an electron donor to the mitochondrial electron transport chain (ETC) and as a component of the TCAC. Alternatively, SSA can be converted to γ-hydroxybutyric acid (GHBA) through a GHB dehydrogenase (GHBDH) that was reported in animals and plants and more recently in E. coli. Because AKG may serve as a precursor of glutamate and subsequently of GABA, this metabolic pathway may be viewed as bypassing two enzymatic steps of the TCAC (Figure 1), AKG dehydrogenase (AKGDH) and Succinyl Co-A ligase (SCOAL), thus termed the GABA shunt.

3Direct functional association of the GABA shunt and the TCAC was demonstrated in transgenic tomato plants with reduced activity of SCOAL and in potato slices treated with a specific chemical inhibitor of AKGDH. Both enzymes are bypassed by the GABA shunt and in both cases elevated flux through the GABA shunt compensated for the lost activity of these TCAC enzymes. Reciprocally, mutants of the Arabidopsis GABP (mitochondrial GABA transporter) resulted in reduced uptake of GABA into mitochondria and increased TCAC activity. Much data accumulated regarding plant TCAC in illuminated leaves where it seems to “lose” its classical cyclic metabolic form. Evidence suggests that citrate, stored from the TCAC activity of the previous night, is mainly shuttled for N assimilation. Interestingly, the GABA shunt, as one of the bridges between N and C metabolism, is significantly active in illuminated Xanthium strumarium (cocklebur) leaves as demonstrated by the high metabolic flux through it. On the other hand, a metabolic flux from AKG to succinate through the TCAC was almost undetectable (; Figure 1). In other words, the GABA shunt is apparently the major source for succinate in leaves at day time, contrary to the night-type heterotrophic plant metabolism where TCAC functions in its classical cyclic form.

4An interesting transcriptional co-response was demonstrated with Arabidopsis genes that encode the GABA shunt pathway (GAD2 and SSADH) and other primary C metabolism associated genes, providing further support for the association of GABA with primary C metabolism in plants at both the metabolome and transcriptome levels. It should be noted that the genes encoding the mitochondrial GABA permease, AtGABP and the GABA catabolic enzyme SSADH are also highly co-expressed in response to stress. Changes in the cellular distribution and quantity of GABA were shown also to affect levels of several AAs in different plant species and even in seeds. As a molecule synthesized mainly from glutamate, yet intimately associated with the TCAC, GABA is continuously suggested to be an important component in the balance between C and nitrogen (C:N) pools of plant cells.

5A signaling role for GABA in plants is continuously suggested and discussed and was especially highlighted following the discovery of the interesting role of GABA in pollen tube growth and guidance and by identifying genes that encode putative receptors that possess domains, which are structurally homologous to mammalian GABAB receptors. Also, the role of GABA in regulating expression of several genes was reported: ACC-synthase, arginine-decarboxylase, 14-3-3, and genes encoding secreted and cell wall associated proteins. Consistent with a role in ACC-Synthase regulation, exogenously applied GABA was shown to induce ethylene emission in Stellaria longipes and sunflower. Nevertheless, in our opinion, the most compelling evidence suggesting that GABA is a signaling molecule in plants is the detection of Quantum dot (QD)-mediated GABA binding sites on the surface of plant pollen and somatic protoplasts, implying the existence of GABA receptors. Importantly, the binding of the QD-GABA probes caused transient elevation in intracellular Ca2+ levels (; Figure 1), which is known to induce signaling pathways in plants. Notably, it was recently shown that this increase in Ca2+ levels is the result of the modulation of Ca2+-permeable channels to enable proper pollen tube growth, further supporting the suggestion that the requirement for a GABA gradient along pollen tube is related to its signaling role rather than to a metabolic one.

6Furthermore, a role for GABA as a signal between plants and bacteria is already well established. GABA that accumulates in wounded plant tissues restricts quorum sensing of Agrobacterium tumefaciens by regulating expression of the bacterial attKLM operon. It was shown that plant GAD is responsible for the accumulation of the GABA signal that is transported into the bacteria via the bacterial Bra ABC transporter. Interestingly, plants with an ectopically functional GAD, which lacks the Ca2+/Calmodulin (Ca2+/CaM) binding domain, exhibited increased resistance to Agrobacterium infection. Increased GABA shunt activity was more recently also associated with tomato resistance to Botrytis cinerea. In pepper (Capsicum annuum), the Xanthomonas campestris pv. vesicatoria effector, AvrBsT, induces hypersensitive cell death. Investigation of this effector revealed that it interacts with a pepper arginine decarboxylase (CaADC1) and their joint expression is accompanied by polyamine, nitric oxide and hydrogen peroxide bursts. Treatment of pepper with GABA significantly reduced growth of avirulent Xanthomonas campestris, suggesting a role for GABA in cell death promotion. This is consistent with an older report exhibiting GABA as a modulator of soybean (Glycine max) arginine decarboxylase, ultimately affecting polyamine biosynthesis. The association of GABA and cell death was also demonstrated in Arabidopsis seeds expressing a constitutively active GAD where up-regulation of cell-death-associated genes was detected. Among the over-represented categories of genes expressed in the hyperaccumulating GABA seeds were those involved in Ca2+-mediated signaling, redox and cysteine proteases. The over representation of Ca2+-signaling associated genes is intriguing in light of the transient change in Ca2+ levels caused by applying GABA to protoplasts. Furthermore, mutation in GABA-T (which results in GABA accumulation) was shown also to suppress plant response to E-2-Hexenal, a herbivore and pathogen associated C6-volatile. This volatile was shown to increase GABA levels.

7γ-Aminobutyric acid accumulation also results in the down regulation of genes associated with cell-wall modifications. Cell wall modification are correlated with developmental changes which are exerted on plants by environmental constraints. Indeed, the association of increased GABA levels with altered plant development was demonstrated extensively. Ectopically active GAD (lacking its Ca2+/CaM regulated autoinhibitory domain) in transgenic tobacco results in developmental abnormalities that include shortened and more branched plants. Young developing leaves exhibited a delay in greening and were narrower than normal and the plants lacked pollen. Histological analysis revealed shorter cells in the stem cortex parenchyma of these lines, which could in part explain the short plant phenotype. These observations are consistent with more recent evidence from GABA-T mutants displaying cell elongation defects and dwarfism in Arabidopsis and tomato, especially under salt stress. Immuno-localization of GABA in pine seedlings suggests a role for GABA in vascular development. Moreover, Arabidopsis ssadh mutants are significantly dwarfed, a phenotype that can be rescued by a second mutation in GABA-T, which encodes the enzyme that functions upstream of SSADH in the mitochondrial GABA catabolism process. Interestingly, SSADH was implicated in Arabidopsis patterning along the abaxial/adaxial axis and these patterning defects were recovered in an ssadh/gaba-t double mutant. These authors suggested that it is the SSA intermediate between GABA and succinate that is mediating this patterning process.

8In conclusion, GABA levels can readily be affected by activating plant GAD (which is tightly regulated by Ca2+/CaM), by suppressing its catabolism (through suppression of GABA-T), by exogenous application of GABA or by interrupting the cellular C:N balance (through suppressing of primary metabolism related enzymes such as TCAC enzymes). Such changes in GABA levels result in plant responses at the metabolic, transcriptional and developmental levels, which are difficult to explain solely within a metabolic context. Although there is yet no direct evidence for a signaling role of GABA in plants, it’s impossible to ignore the numerous evidences that imply the existence of such signaling pathways.

9In order to obtain further insight into natural modulation of GABA levels, we discuss the current knowledge regarding GABA metabolism under plant starvation in comparison to metabolism in starved animals. We suggest that the metabolic and signaling functions of GABA evolved to be functionally entwined.

10Metabolic profiling of Arabidopsis mutants impaired in enzymes that are essential for respiration under extended dark-induced C starvation revealed an increase in the content of several AAs associated with starvation-induced protein degradation. On the other hand, the levels of GABA and of the TCAC intermediate, succinate, increased significantly in these mutants although both metabolites are not the product of protein degradation. The authors suggested that an increase in GABA shunt activity produces mitochondrial succinate to maintain respiration in these mutants. This was also apparent in a different study that employed metabolic and transcriptional analyses to decipher mechanisms mediating the Arabidopsis response to dark-induced starvation. GABA was once again highlighted as the major non-proteinogenic AA with a tight correlation to succinate levels. Notably, at the transcriptional level, starving plants exhibited significant reduced expression of TCAC-associated SCOAS genes in parallel to a significant increase in SSADH expression, implying an induction of the GABA shunt bypass under these circumstances. Though succinate accumulation in starved plants may be explained by its use as an electron donor in the respiratory chain, the accumulation of GABA, if not merely a metabolic side effect, awaits explanation. One possibility is that the accumulation of GABA is a prerequisite for its signaling function. Let’s look at an example for such a metabo–signaling coupling of GABA under low energy status in an organism with a well-established signaling role of GABA.

11In mice, the brain is responsible for inducing a hunger sensation under low (or no) food availability that will result in a behavioral response of seeking and consuming food. An interesting report demonstrates how specific neurons (AgRP neurons of the arcuate nucleus) modulate feeding behavior in mice by providing GABAergic input into mice brainstem (the parabrachial nucleus; PBN) and how inactivation of GABA biosynthesis (by inactivating murine Gad1) in the arcuate nucleus, or block of GABA(A) receptors in the PBN, promotes mice anorexia. This elegantly shows how GABA neurotransmission participates in the maintenance of energy homeostasis of the whole organism (feeding behavior) and how it is directly affected by GABA biosynthesis through the action of Gad1.

12As autotrophic organisms, the two main aspects of plant “feeding” are photosynthesis and nutrient uptake by the root. GABA is one of few metabolites whose metabolic path is divided between the cytosol and the mitochondrion matrix. The possibility to target mitochondrial metabolism as a means to enhance photosynthesis was already discussed and shown. Notably, in silico analysis of optimal photosynthesis in cyanobacteria highlighted the importance of the GABA shunt, combined with an incomplete TCAC during autotrophic metabolism. Remarkably, some GABA-T isoforms of rice and tomato were shown to localize in plastids. This suggests a role for GABA or its derivatives in plastid-associated functions. Indeed exogenously applied GABA resulted in increased photosynthesis parameters is muskmelon seedlings, mainly under hypoxia stress.

13Nevertheless, in order to more accurately compare animal feeding with plant “feeding” it is more prudent to refer also to nutrient uptake by plant roots. Two reports suggest a signaling role for GABA in nitrate uptake in Brassica napus roots and more recently in Arabidopsis thaliana. An earlier report described how GABA enhances significantly the growth of Lemna minor plants by increasing mineral consumption. In contrast, isomers of GABA such as 3-aminobutyric acid and 2-aminobutyric acid inhibited plant growth. Remarkably, GABA mediated promotion of Lemna growth was inhibited by bicuculline and picrotoxin, which are competitive and non-competitive antagonists of GABA receptors in the mammalian central nerve system, respectively. Consistent with these findings, Baclofen, a known GABA agonist in animals, significantly increased GABA mediated promotion of Lemna growth suggesting the existence of GABA receptors in plants that participate in nutrient uptake and eventually affect plant growth. Moreover, a role for GABA in the up-regulation of nodule activity was also suggested followed by evidences for a role of GABA in increasing the efficiency of symbiotic N2 fixation in legumes. Thus, it seems that GABA levels increase during plant starvation and energetically demanding stresses. Importantly, GABA seems to induce plant responses that may aid in replenishing the energetic supply, very similar to the action of GABA in mammals where it modulates feeding behavior. The already mentioned effect of GABA on exerting cell-elongation arrest may be important in parallel in order to save cellular energy. Thus, the combined GABA associated effects of modulating photosynthesis and nutrient uptake, in parallel to growth arrest, may be pivotal to ensure plant survival under energetically demanding stresses.

14γ-Aminobutyric acid is a major metabolic component in the interface between C and N metabolism. As such, its levels are “sensitive” to the availability of both essential elements, which makes it an excellent sensor for the energetic state of the cell. Thus, GABA seems ideal to serve as a signal that participates in a pathway that “instructs” the organism whether to try and gain more energy, or whether to initiate processes to cope with excess energy. Also, it seems that GABA is a major alternative pathway to which C skeleton is directed depending on the environment, growth stage, and tissue specificity. These reports summarized here suggest that from an evolutionary perspective, GABA metabolism predated signaling, and that mechanistically, GABA metabolism underlies its signaling functions. This is consistent with a work that integrated metabolomics with transcript and enzyme activity profiling of plants undergoing diurnal cycles. The authors concluded that “…correlation between metabolites and transcripts are due to regulation of gene expression by metabolites, rather than metabolites being changed as a consequence of a change in gene expression”. Future research should shed light on the manner by which physiological increase in GABA levels, either by GAD induction, repression of catabolism or increased flux in the direction of the GABA shunt, affect plant resilience and development. Identification of the cell-surface localized GABA receptors, as well as identifying components that participate in GABA-mediated gene control is essential to this end.

15The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.