2 ISOFORMS OF GLUTAMATE-DECARBOXYLASE IN ARABIDOPSIS ARE REGULATED BYCALCIUM CALMODULIN AND DIFFER IN ORGAN DISTRIBUTION/

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kD a) and sharing 82% identical amino acid sequences, were determined. Th e recombinant proteins bound [S-35] calmodulin (CaM) in the presence o f calcium, and a region of 30-32 amino acids from the C-terminal of ea ch isoform was sufficient for CaM binding when fused to glutathione S- transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect ce lls infected with recombinant baculovirus vectors. Recombinant protein s were partially purified by CaM affinity chromatography and were foun d to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD g ene-specific probes suggest that Arabidopsis possesses one gene relate d to GAD1 and one to GAD2. Northern hybridization and western blot ana lysis revealed that GAD1 was expressed only in roots and GAD2 in roots , leaves, inflorescence stems and flowers. Our study provides the firs t evidence for the occurrence of multiple functional Ca2+/CaM-regulate d GAD gene products in a single plant, suggesting that regulation of A rabidopsis GAD activity involves modulation of isoform-specific gene e xpression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.