Light-dependent modulation of foliar glutathione synthesis and associated amino acid metabolism in poplar overexpressing γ-glutamylcysteine synthetase

Abstract.

Glutathione (GSH), γ-glutamylcysteine (γ-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula × P. alba) and poplars expressing Escherichia coli genes for γ-glutamylcysteine synthetase (γ-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing γ-ECS, foliar γ-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of γ-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and γ-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate γ-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, γ-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, γ-EC was negligible in the light but increased in the dark. Cysteine content was little affected by light in any of the poplar types. No light-dependent changes in the extractable activities of γ-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing γ-ECS at elevated CO₂ or low O₂ largely abolished the inverse light-dependent changes in γ-EC and GSH. Low O₂ did not affect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for γ-EC synthesis is augmented.