| Efficient assimilation of inorganic nitrogen into amino acids is fundamental to plant growth and development. Therefore, biosynthesis of amino acids in plants has been the subject of intensive research. Nevertheless, the complex nature of plant amino acid metabolism is not fully understood. The current study analyzed the enzymes involved in the metabolism of alanine, GABA and glutamate since uncertainty remains as to the physiological roles of these enzymes and metabolites. To characterize the in vivo functions of the corresponding enzymes, a reverse genetics approach was taken using Arabidopsis thaliana..;Glutamate dehydrogenase (GDH), a key player for the metabolism of glutamate, was also studied. Prior to this research, the in vivo role of this enzyme has been ambiguous for several decades. The physiological role of GDH was characterized here by analyzing a double mutant defective in the two known GDH genes. The increased susceptibility of the double mutant to prolonged darkness demonstrated the involvement of GDH in funneling glutamate carbon to the respiratory pathway. Furthermore, monitoring changes in the amino acid contents during dark treatments suggested the central role of GDH in the catabolism of many amino acids.;Regarding alanine and GABA, their increased syntheses under low oxygen stress were examined since this adaptive response undergoes a dramatic change in the metabolism of these amino acids. Firstly, alanine aminotransferase (AlaAT) was studied since this enzyme had been thought to be responsible for the accumulation of alanine under hypoxia. However, this was not supported in the experiment as the mutants defective in AlaAT accumulated alanine under hypoxia. Instead, the slowed decline in alanine during the post-hypoxic periods was observed in the alaat mutants, suggesting that the physiological role of AlaAT lies in the breakdown of alanine. Secondly, the involvement of the GABA-shunt in the hypoxic alanine accumulation was investigated since this pathway produces alanine along with the conversion of GABA to its downstream metabolites. The analyses of the mutants for the enzymes catalyzing the first and second steps of the GABA-shunt, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T), respectively, demonstrated the contribution of this pathway to the hypoxic alanine accumulation in roots. |
