Cloning and characterization of a tobacco cDNA encoding calcium/calmodulin-dependent glutamate decarboxylase

In plants, gamma-aminobutyric acid (GABA), a major transmitter in the central nervous system in animals, is synthesized by glutamate decarboxylase (GAD), the activity of which is tightly modulated by Ca2+/calmodulin. To study the molecular mechanism of GAD regulation and examine the physiological role of GABA in plants, we isolated and characterized a 1771 bp tobacco cDNA clone, pNtGAD2. The 496 amino acid sequence deduced from pNtGAD2 showed 97, 92, and 81% identity to NtGAD1, petunia, and tomato GAD, respectively. The 26 amino acid sequence within the putative calmodulin binding domain at the carboxy terminus showed a typical alpha-helical structure with hydrophobic and charged amino acid clusters. The pNtGAD2-encoded 56 kDa protein interacted strongly with a monoclonal antibody against the petunia GAD and its GAD activity was stimulated markedly by the addition of exogenous calcium and calmodulin. The molecular sequence of pNtGAD2 and biochemical characteristics of the pNtGAD2-encoded protein confirmed that pNtGAD2 is a clone encoding a functional calmodulin-binding and Ca2+/calmodulin-dependent tobacco glutamate decarboxylase. This result indicates that tobacco plants also have Ca2+/calmodulin-dependent GADs.