"Regulation of gamma-aminobutyric acid synthesis by nitrogen availability on tobacco transgenic plants."

Transgenic tobacco plants were transformed with the human glutamic acid decarboxylase (GAD) gene (isoform 65KDa) to obtain edible organs for oral somministration of the major IDDM (human insulin-dependent diabetes) autoantigen. This insertion generated a line with altered phenotype, showing a reduced growth and delayed flowering. A similar phenotype was already reported in other tobacco plants transformed with petunia GAD, deleted of its CaM-binding domain (Baum et al. 1996 EMBO J. 15,2988-2996). Since hGAD65 lacks CaM-binding domain it is reasonable that the observed transgenic line could be due to this insertion. We hypothesized that nitrogen availability can trigger the dwarfism, affecting plant growth by modification of the C/N ratio, and altering the distribution of metabolites in various organs. To verify our hypothesis, control and transgenic plants (normal and altered phenotype) have been grown in hydroponics culture with different nutritional solution, i.e. different nitrogen concentrations. Plant organs have been tested to define the distribution of gamma-aminobutyric acid, glutamic acid, total aminoacids content and soluble sugars. GAD enzymes have been detected enzymatically and by immunodetection of hGAD with western blot and immunoblot analysis. The results indicate a different pattern of distribution of the metabolites between altered transgenic plants and both the control and transgenic normal lines. The dwarf plants do not show particular changes of metabolites amounts during treatments, while aminoacids and soluble sugars change in control and non-dwarf transgenic lines. The higher GAD activity observed in source organs of transgenic plants indicates a fast translocation of GABA in the sink organs or GABA utilization in the source (via GABA-Shunt).