TARGETING OF GLUTAMINE-SYNTHETASE TO THE MITOCHONDRIA OF TRANSGENIC TOBACCO

Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) gamma-polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia-beta-F1 ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS-gamma-polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the beta-F1 ATPase has successfully targeted the GS-gamma-polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the gamma-polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the gamma-polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.