Characterization of Two Glutamate Decarboxylase cDNA Clones from
Arabidopsis
Frank J. Turano* and
Tung K. Fang
United States Department of Agriculture, Agricultural Research
Service, Climate Stress Laboratory, Beltsville, Maryland 20705
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.
*
Corresponding author; e-mail fturano{at}asrr.arsusda.gov; fax
1-301-504-7521.
Plant Physiol. (1998) 117: 1411-1421
Copyright Clearance Center: 0032-0889/98/117//11
© 1998 American Society of Plant Physiologists
