Comprehensive Genome-Wide Identification and Transcript Profiling of GABA Pathway Gene Family in Apple (Malus domestica)

γ-Aminobutyric Acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. GABA is involved in pH regulation, maintaining C/N balance, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization via anion flux. Glutamate decarboxylase (GAD, EC 4.1.1.15) and GABA transaminase (GABA-T, EC 2.6.1.19) are two key enzymes involved in the synthesis and metabolism of GABA. Recently, GABA transporters (GATs), protein and aluminium-activated malate transporter (ALMT) proteins which function as GABA receptors, have been shown to be involved in GABA regulation. However, there is no report on the characterization of apple GABA pathway genes. In this study, we performed a genome-wide analysis and expression profiling of the GABA pathway gene family in the apple genome. A total of 24 genes were identified including five GAD genes (namely MdGAD 1–5), two GABA-T genes (namely MdGABA-T 1,2), 10 GAT genes (namely GAT 1–10) and seven ALMT genes (namely MdALMT1–7). These genes were randomly distributed on 12 chromosomes. Phylogenetic analyses grouped GABA shunt genes into three clusters—cluster I, cluster II, and cluster III—which had three, four, and five genes, respectively. The expression profile analysis revealed significant MdGAD4 expression levels in both fruit and flower organs, except pollen. However, there were no significant differences in the expression of other GABA shunt genes in different tissues. This work provides the first characterization of the GABA shunt gene family in apple and suggests their importance in apple response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of these gene families.

Phytosulfokine α (PSKα) delays senescence and reinforces SUMO1/SUMO E3 ligase SIZ1 signaling pathway in cut rose flowers (Rosa hybrida cv. Angelina)

Roses are widely used as cut flowers worldwide. Petal senescence confines the decorative quality of cut rose flowers, an impressively considerable economic loss. Herein, we investigated the SUMO1/SUMO E3 ligase SIZ1 signaling pathway during bud opening, and petal senescence of cut rose flowers. Our results exhibited that the higher expression of SUMO1 and SUMO E3 ligase SIZ1 during bud opening was accompanied by lower endogenous H2O2 accumulation arising from higher expression and activities of SOD, CAT, APX, and GR, promoting proline accumulation by increasing P5CS expression and activity and enhancing GABA accumulation by increasing GAD expression and activity. In harvested flowers, lower expressions of SUMO1 and SUMO E3 ligase SIZ1 during petal senescence were associated with higher endogenous H2O2 accumulation due to lower expression and activities of SOD, CAT, APX, and GR. Therefore, promoting the activity of the GABA shunt pathway as realized by higher expression and activities of GABA-T and SSADH accompanied by increasing OAT expression and activity for sufficiently supply proline in rose flowers during petal senescence might serve as an endogenous antisenescence mechanism for slowing down petals senescence by avoiding endogenous H2O2 accumulation. Following phytosulfokine α (PSKα) application, postponing petal senescence in cut rose flowers could be ascribed to higher expression of SUMO1 and SUMO E3 ligase SIZ1 accompanied by higher expression and activities of SOD, CAT, APX, and GR, higher activity of GABA shunt pathway as realized by higher expression and activities of GAD, GABA-T, and SSADH, higher expression and activities of P5CS and OAT for supplying proline and higher expression of HSP70 and HSP90. Therefore, our results highlight the potential of the PSKα as a promising antisenescence signaling peptide in the floriculture industry for postponing senescence and extending the vase life of cut rose flowers.

Not Just a Cycle: Three gab genes Enable the Non-cyclic Flux Toward Succinate Via GABA Shunt in ‘Candidatus Liberibacter asiaticus’-infected citrus

Although the mitochondria retain all required enzymes for an intact tricarboxylic acid (TCA) cycle, plants might shift the cyclic flux from the TCA cycle to an alternative non-cyclic pathway via γ-aminobutyric acid (GABA) shunt under specific physiological conditions. We hypothesize that several genes may ease this non-cyclic flux and contribute to the citrus response to the phytopathogenic bacterium ‘Candidatus Liberibacter asiaticus’, the causal agent of Huanglongbing in citrus. To test this hypothesis, we used multi-omics techniques (metabolomics, fluxomics, and transcriptomics) to investigate the potential role(s) of putative gab homologies from Valencia sweet orange (Citrus sinensis). Our findings showed that ‘Ca. L. asiaticus’ significantly increased the endogenous GABA and succinate content but decreased ketoglutarate in infected citrus plants. Citrus genome harbors three putative gab genes including amino-acid permease (aka GABA permease; CsgabP), GABA transaminase (CsgabT), and succinate-semialdehyde dehydrogenase (aka GABA dehydrogenase; CsgabD). The transcript levels of CsgabP, CsgabT, and CsgabD were upregulated in citrus leaves upon the infection with ‘Ca. L. asiaticus’ and after the exogenous application of GABA or deuterium-labeled GABA isotope (GABA-D6). Moreover, our finding showed that exogenously applied GABA is quickly converted to succinate and fed into the TCA cycle. Likewise, the fluxomics study showed that GABA-D6 is rapidly metabolized to succinate-D4. Our work proved that GABA shunt and three predicated gab genes from citrus, support the upstream non-cyclic flux toward succinate rather than an intact TCA cycle and contribute to citrus defense responses to ‘Ca. L. asiaticus’.

Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

Physiological and Biochemical Characterization of the GABA Shunt Pathway in Pea (Pisum sativum L.) Seedlings Under Drought Stress

The physiological and biochemical role of the γ-aminobutyric acid (GABA) shunt pathway in green pea seedlings (Pisum sativum L.) was studied in response to soil water holding capacity levels: 80%, 60%, 40%, 20%, and 10% grown under continuous light at 25 °C for 7 days and 14 days, separately. Characterization of seeds germination pattern, seedlings growth (plant height, fresh and dry weight, and chlorophyll contents), GABA shunt metabolite (GABA, glutamate, and alanine) levels, total protein and carbohydrate levels, and oxidative damage (MDA level) were examined. Data showed a significant effect of drought stress on seed germination, plant growth, GABA shunt metabolites level, total protein and carbohydrate contents, and MDA level. A significant decline in seed germination percentage was recorded at a 20% drought level, which indicated that 20% of soil water holding capacity is the threshold value of water availability for normal germination after 14 days. Seedling fresh weight, dry weight, and plant height were significantly reduced with a positive correlation as water availability was decreased. There was a significant decrease with a positive correlation in Chl a and Chl b contents in response to 7 days and 14 days of drought. GABA shunt metabolites were significantly increased with a negative correlation as water availability decreased. Pea seedlings showed a significant increase in protein content as drought stress was increased. Total carbohydrate levels increased significantly when the amount of water availability decreased. MDA content increased slightly but significantly after 7 days and sharply after 14 days under all water stress levels. The maximum increase in MDA content was observed at 20% and 10% water levels. Overall, the significant increases in GABA, protein and carbohydrate contents were to cope with the physiological impact of drought stress on Pisum sativum L. seedlings by maintaining cellular osmotic adjustment, protecting plants from oxidative stress, balancing carbon and nitrogen (C:N) metabolism, and maintaining cell metabolic homeostasis and cell turgor. The results presented in this study indicated that severe (less than 40% water content of the holding capacity) and long-term drought stress should be avoided during the germination stage to ensure proper seedling growth and metabolism in Pisum sativum L.

Enantiomers of 2-methylglutamate and 2-methylglutamine selectively impact mouse brain metabolism and behavior

Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of l-Glu or l-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous l-Glu. (R)-2MeGlu was transported less efficiently into brain and synaptosomes but was not released by membrane depolarization. Each enantiomer of 2MeGlu had limited activity across a panel of over 30 glutamate and GABA receptors. While neither enantiomer of 2MeGlu was metabolized along the GABA shunt, (S)-2MeGlu was selectively converted to (S)-2-methylglutamine, or (S)-2MeGln, which was subsequently slowly hydrolyzed back to (S)-2MeGlu in brain. rac-2MeGln was also transported into brain, with similar efficiency as (S)-2MeGlu. A battery of behavioral tests in young adult wild type mice showed safety with up to single 900 mg/kg dose of (R)-2MeGlu, (S)-2MeGlu, or rac-2MeGln, suppressed locomotor activity with single ≥ 100 mg/kg dose of (R)-2MeGlu or (S)-2MeGlu. No effect on anxiety or hippocampus-dependent learning was evident. Enantiomers of 2MeGlu and 2MeGln show promise as potential pharmacologic agents and imaging probes for cells that produce or transport l-Gln.

A non-canonical role for glutamate decarboxylase 1 in cancer cell amino acid homeostasis, independent of the GABA shunt

Glutamate decarboxylase 1 (GAD1) is best known for its role in producing the neurotransmitter γ-amino butyric acid (GABA) as part of the “GABA shunt” metabolic pathway, an alternative mechanism of glutamine anaplerosis for TCA cycle metabolism (Yogeeswari et al., 2005). However, understanding of the metabolic function of GAD1 in non-neuronal tissues has remained limited. Here, we show that GAD1 supports cancer cell proliferation independent of the GABA shunt. Despite its elevated expression in lung cancer tissue, GAD1 is not engaged in the GABA shunt in proliferating non-small cell lung cancer (NSCLC) cells, but rather is required for regulating amino acid homeostasis. Silencing GAD1 promotes a broad deficiency in amino acid uptake, leading to reduced glutamine-dependent TCA cycle metabolism and defects in serum- and amino acid-stimulated mTORC1 activation. Mechanistically, GAD1 regulates amino acid uptake through ATF4-dependent amino acid transporter expression including SLC7A5 (LAT1), an amino acid transporter required for branched chain amino acid (BCAA) uptake. Overexpression of LAT1 rescues the proliferative and mTORC1 signalling defects of GAD1-deficient tumor cells. Our results, therefore, define a non-canonical role for GAD1, independent of its characterised role in GABA metabolism, whereby GAD1 regulates amino acid homeostasis to maintain tumor cell proliferation.