Synthesis of glutathione in leaves of transgenic poplar overexpressing gamma-glutamylcysteine synthetase

Internode stem fragments of the poplar hybrid Populus tremula x Populus alba were transformed with a bacterial gene (gshl) for gamma-glutamylcysteine synthetase (gamma-ECS) targeted to the cytosol. Lines overexpressing gamma-ECS were identified by northern analysis, and the transformant with the highest enzyme activity was used to investigate the control of glutathione synthesis. Whereas foliar gamma-ECS activity was below the limit of detection in untransformed plants, activities of up to 8.7 nmol mg(-1) protein min(-1) were found in the transformant, in which the foliar contents of gamma-glutamylcysteine (gamma-EC) and glutathione were increased approximately 10- and 3-fold, respectively, without affecting either the reduction state of the glutathione pool or the foliar cysteine content. A supply of exogenous cysteine to leaf discs increased the glutathione content from both transformed and untransformed poplars, and caused the gamma-EC content of the transformant discs to increase still further. The following conclusions are drawn: (a) the native gamma-ECS of untransformed poplars exists in quantities that are limiting for foliar glutathione synthesis; (b) foliar glutathione synthesis in untransformed poplars is limited by cysteine availability; (c) in the transformant interactions between glutathione synthesis and cysteine synthesis operate to sustain the increased formation of gamma-EC and glutathione; and (d) the foliar glutathione content of the transformant is restricted by cysteine availability and by the activity of glutathione synthetase.