Characterization of Two Glutamate Decarboxylase cDNA Clones from Arabidopsis

Abstract

Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58-and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35-and 13-fold, respectively, by Ca 2؉ /calmodulin but not by Ca 2؉ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mM NH 4 Cl, 5 mM NH 4 NO 3 , 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO 3 . The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

“…However, our results clearly indicate that the predicted inhibition of the GABA shunt activity does not affect the isotope between GABA and succinate. In contrast, the data support the hypothesis that the consumption of protons by Glu decarboxylase for the production of GABA can help to stabilize the cytosolic pH during exposure to different stress conditions, including hypoxia (Turano and Fang, 1998;Shelp et al, 2012).…”

Salinity Stress Affects Assimilate Metabolism at the Gene-expression Level during Fruit Development and Improves Fruit Quality in Tomato (Solanum lycopersicum L.)

“…5). GABA is formed from glutamate by glutamate decarboxylase (GAD), which is activated by Ca 2+ /CaM (Aurisano et al, 1995;Snedden et al, 1995Snedden et al, , 1996Turano and Fang, 1998) and acidic pH (Johnson et al, 1997;Snedden et al, 1996). Although salinity stress increased the acidity of the fruit, as described above, we could not determine whether it directly enhanced GABA accumulation because it has been reported that the cellular Ca 2+ level generally increases with salinity stress in plant cells (Sanders et al, 1999).…”

Screening for γ-aminobutyric Acid (GABA)-rich Tomato Varieties

“…There have also been reports of a crucial role for Ca 2+ and calmodulin in the regulation of GAD activity in many plants (Baum et al, 1996;Snedden et al, 1995;Turano and Fang, 1998). It is possible that an increase in intracellular Ca 2+ caused by salinity stress stimulates GAD activity, resulting in increased fruit GABA levels.…”