Characterization of two glutamate decarboxylase cDNA clones from Arabidopsis.
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 Ca2+/calmodulin but not by Ca2+ 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 NH4Cl, 5 mM NH4NO3, 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO3. 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.
United States Department of Agriculture, Agricultural Research Service, Climate Stress Laboratory, Beltsville, Maryland 20705, USA. fturano@asrr.arsusda.gov
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; D009584:Nitrogen; D002118:Calcium