Manipulation of glutathione and amino acid biosynthesis in the chloroplast

Poplars (Populus tremula x Populus alba) were transformed to overexpress Escherichia coli gamma-glutamylcysteine synthetase (gamma-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic gamma-ECS activity markedly increased gamma-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic gamma-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing gamma-ECS in the chloroplast. High chloroplastic, but not cytosolic, gamma-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased gamma-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.