15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

Abstract

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed (15)N2 and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to (15)N2, showed that highest initial (15)N2-labelling was into NH 4 (+) . After 12 min little further increase in the NH 4 (+) label occurred while that in the amide group of glutamine and in glutamate continued to increase. The (15)N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of (15)N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total (15)N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which (14)N2 was added to cephalodia previously exposed to (15)N2, showed that the NH 4 (+) pool rapidly became depleted of (15)N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The (15)N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the (15)N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.