Modification of thiol contents in poplars (Populus tremula x P-alba) overexpressing enzymes involved in glutathione synthesis

The hybrid poplar (Populus tremula x P. alba) was transformed to express the Escherichia coli gene for gamma-glutamylcysteine synthetase (EC 6.3.2.2: gamma-ECS) in the cytosol. Four transformed lines of poplar were obtained. These were phenotypically indistinguishable from untransformed poplars. Three lines, ggs28 (Noctor et al. 1996, Plant Physiol 112: 1071-1078), ggs11 and ggs5 possessed high levels of bacterial gene transcripts. Line ggs17 had lower transcript levels. Antisera were prepared against bacterial gamma-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS). Using the antiserum prepared against the purified His-tagged E. coli gamma-ECS, lines ggs28, ggs11 and ggs5 were shown to possess abundant quantities of the bacterial protein, whereas ggs17 contained lower amounts. The antiserum prepared against the purified His-tagged E. coli GS was also effective in screening poplars transformed with the E. coli gene coding for this enzyme. Immunoblots of leaf extracts from poplars overexpressing GS using this antibody revealed two bands. The extractable foliar gamma-ECS activities of the gamma-ECS transformants were in quantitative agreement with the protein levels. Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher y-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold. The lines strongly overexpressing gamma-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), gamma-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold). Foliar thiol contents in ggs17 were no different to those of untransformed plants.