Different induction patterns of glutamate metabolizing enzymes in ripening fruits of the tomato mutant green flesh

The stay-green mutant of tomato green flesh (gf) is deficient in the chloroplast degradation machinery and displays an altered ripening profile. The ripening process of tomato (Lycopersicon esculentum Mill.) fruit is characterized by a substantial induction in glutamate dehydrogenase (GDH, EC 1.4.1.3) and a decline in glutamine synthetase (GS, EC 6.3.1.2) levels, associated with an increase in the relative glutamate content. In this study the level of free amino acids and enzymes associated with glutamate metabolism were followed in mature gf fruits at green and ripe stages. From these fruits, total RNA, protein extracts and intact plastids were isolated. Specific activities of GDH, GS and NADH- or ferredoxin-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14; Fd-GOGAT, EC 1.4.7.1) were measured and immunoblot analyses were performed. The total free amino acid contents of gf mature fruits decreased during ripening, contrasting with that observed in several cultivated varieties of tomatoes. The relative glutamate content, however, increased markedly in ripe gf fruits, as in all the tomato cultivars tested so far. Although GDH activity and polypeptides were found in green gf fruits, GDH transcripts were not detected in these fruits. In ripe gf fruits GDH was clearly present. NADH- and Fd-GOGAT activities, on the other hand, were barely measurable in gf fruits whereas Fd-GOGAT protein was detected in green gf fruits and, to a lower extent in ripe gf fruits. Levels of GS protein and transcripts were correlated in all fruits tested. Collectively, the expression of the enzymes involved in the primary glutamate metabolism seems to be differently regulated during the ripening process.