Effects of phosphinotricin treatment on glutamine synthetase isoforms in Scots pine seedlings

The occurrence of GS isoenzymes has been investigated in Scots pine (Pinus sylvestris) seedlings. A transient increase of glutamine synthetase (GS, EC 6.3.1.2) activity was observed in the cotyledon whorl of plants treated wi th the herbicide phosphinotricin (PPT). The increase in GS activity was acc ompanied by a parallel accumulation of GS1 protein, which remained at high levels throughout the PPT treatment. Two-dimensional SDS-PAGE western analy sis showed that pine extracts contained two GS1 polypeptides which differ i n their corresponding isoelectric points. Analysis of crude extracts by ion -exchange chromatography led to the separation of two GS isoforms. The firs t peak (GS1-a) eluted from the columns at a low ionic strength (0.15-0.18 M KCl), whereas the second one (GS1-b) was detected at 0.5 M KCl. A detailed molecular study of both GS holoenzymes confirmed that their subunits were similar in size (about 41 kDa) but different in charge. All these data clea rly demonstrate the presence of two GS1 forms in Scots pine cotyledons. Mor eover, a comparison of isolated GS isoproteins with the recombinantly expre ssed Scots pine cytosolic subunit suggests that GS1-a corresponds to the pr eviously characterized cDNA (pGSP114) whereas GS1-b is a minor GS isoenzyme with increased relative abundance in phosphinotricin treated plants. (C) E lsevier, Paris.