GABA and GHB Metabolism

In plants, GABA is derived primarily via the H+-consuming a-decarboxylation of glutamate in an irreversible reaction catalyzed by cytosolic-localized glutamate decarboxylase (GAD) that proceeds optimally at acidic pH (Fig.1; Shelp et al. 1999). While increasing cytosolic H+ concentration can result in GABA accumulation, there is abundant evidence for a mechanism involving Ca++-dependent binding of calmodulin to GAD proteins at neutral pH, thereby relieving the enzyme from autoinhibition and stimulating enzymatic activity (reviewed by Shelp et al. 1999). Thus, calmodulin links GABA accumulation with increasing cytosolic Ca2+, which typically accompanies stress. Research has identified multiple GAD genes from Petunia, tomato, tobacco, Arabidopsis and rice, and differential organ localization of two isoforms in both Arabidopsis and tobacco (Shelp et al. 1999; Yevtushenko et al. 2003; Akama and Takaiwa 2007; Miyashita and Good 2008) + implying that they may have specific functions. For example, GAD1 is predominantly expressed in roots, while GAD2 expression is evident in all organs; expression of the other three GAD genes is weak (Miyashita and Good 2007). Phenotypic analysis of loss-of-function gadl mutants revealed that GABA levels in roots are dramatically lower than in wild-type roots, and that heat-induced GABA accumulation is prevented in gadl mutants (Bouché et al. 2004). Moreover, antisense suppression of GAD results in the accumulation of glutamate in transgenic tomato fruit (Kisaka et al. 2006) . Transcriptional induction of one or more GAD forms is often observed in response to low oxygen, water deficit, salinity or Agmbacterium infection (Klok et al. 2002; Deeken et al. 2006; Cramer et al. 2007; Miyashita and Good 2007; Pasentsis et al. 2007).

GABA is then transaminated to succinic semialdehyde (SSA) via a mitochondrial-localized GABA transaminase (GABA-T) that is probably reversible (Fig . 1 ; Van Cauwenberghe and Shelp 1999; Van Cauwenberghe et al. 2002). Both pyruvate- and 2-oxoglutarate-dependent activities are found in crude tobacco plant extracts; however, only the gene for pyruvate-dependent activity (GABA-T1) in Arabidopsis has been identified to date (Van Cauwenberghe et al. 2002). Research has identified highly homologous proteins in pepper, tomato and rice (Ansari et al. 2005; Wu et al. 2006), although protein function has not been examined. The expression of GABA-T1 is detected in all Arabidopsis organs and the vegetative phenotype appears normal, but a gaba-t1 mutant lacks a GABA gradient from the stigma to the embryo sac and pollen tube growth is misdirected, thereby causing a reduced-seed phenotype, while GABA-T activity is decreased to negligible levels in both shoots and roots and GABA accumulates in roots (Palanivelu et al. 2003; Miyashita and Good 2007). Significant transcriptional change typically occurs in GABA-T1 under low oxygen, water deficit and salinity (Klok et al. 2002; Cramer et al. 2007), although not always (Miyashita and Good 2007).

SSA dehydrogenase (SSADH) catalyzes the irreversible, NAD-dependent oxidation of SSA to succinate in the mitochondrion (Fig. 1). The enzyme is competitively inhibited by NADH and AMP, noncompetitively inhibited by ATP, and inhibited by ADP via both competitive and noncompetitive means (Busch and Fromm 1999). SSADH occurs as a single-copy gene in Arabidopsis; and ssadh mutants contain elevated levels of reactive oxygen species, are hypersensitive to heat and light stress, and have a stunted and necrotic phenotype (Bouché et al. 2003a).

Other research suggests an additional coping mechanism for the detoxification of SSA, which involves its reduction to GHB (Fig; 1} . A number of strategies, including complementation of a SSADH-deficient yeast mutant with an Arabidopsis cDNA library, recombinant expression in Escherichia coli, and transient expression in tobacco BY-2 cells, were used to identify two highly homologous proteins (designated as Arabidopsis glyoxylate reductases 1 and 2, or AtGR1 and AtGR2) that catalyze the conversion of both SSA to GHB and glyoxylate to glycolate via an essentially irreversible, NADPH-based ordered mechanism, although they are located in different cellular compartments (cytosol, plastid) (Breitkreuz et al. 2003; Hoover et al. 2007a; b; Simpson et al. 2008) ; NADP+ is an effective competitive inhibitor with respect to NADPH, suggesting that the ratio of NADPH/NADP; regulates the activities of both isoforms in planta. Time course experiments revealed that GHB accumulates in leaves of both Arabidopsis and tobacco plants subjected to stress, and that this accumulation is associated with higher GABA levels, higher NADPH/NADP+ ratios, and lower glutamate levels (Fig; 2 ; see also Allan et al. 2008). Expression analysis of Arabidopsis leaves revealed that the relative abundance of the AtGRl and AtGR2transcripts is enhanced by stress (Allan et al. 2008). Thus, it was proposed that the AtGR isoforms are involved in redox homeostasis, and that they represent alternative means for the detoxification of SSA as well as glyoxylate, but further in planta work is required to substantiate this hypothesis.

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