RT-PCR analysis and stress response capacity of transgenic gshI-poplar clones (Populus x canescens) in response to paraquat exposure

Abstract

Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) ofuntransf ormed hybrid poplar, Populus x canescens (P. tremula x P. alba), and two transgenic lines overexpressing γ-ECS (γ-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 x 10Ð9 to 4.0 x 10Ð6 m) for 21 days. Significant differences at sublethal (4.0 x 10Ð7 m) and bleaching (4.0 x 10Ð6 m) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at Fmax), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR ofthe cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression ofthe constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities ofgene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression ofthe paraquat treated (4.0 x 10Ð7 m) samples ofthe transgenic poplar line (cyt-ECS).