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

Abstract.

The hybrid poplar (Populus tremula × P.␣alba) was transformed to express the Escherichia coli gene for γ-glutamylcysteine synthetase (EC 6.3.2.2: γ-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 γ-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS). Using the antiserum prepared against the purified His-tagged E.␣coliγ-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 γ-ECS activities of the γ-ECS transformants were in quantitative agreement with the protein levels. Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher γ-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold. The lines strongly overexpressing γ-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), γ-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold). Foliar thiol contents in ggs17 were no different to those of untransformed plants.