Nitrate fluxes in soybean seedling roots and their response to amino acids: an approach using 15N

This study was undertaken to determine which of the two NO 3 - fluxes (influx or efflux) across plasma membranes of root cells is the target of those amino acids which have been shown to inhibit net NO 3 - uptake (Muller & Touraine 1992, Journal of Experimental Botany 43, 617-623). Parallel experiments were performed to measure either the time course of 15 NO 3 - release from roots of soybean seedlings previously labelled with this isotope into non-labelled solution, or the time course of 15 N accumulation from labelled 15 NO 3 - solution in non-labelled seedlings. Focusing on the fate of 15 NO 3 - in the cytoplasmic compartment, a model is developed to describe the time courses of the accumulation and release of tracer across the plasma membranes of root cells. Both time courses can be described by the sum of an exponential plus a linear term. In our material, the linear part of the accumulation time course is obscured by the NO 3 - fluxes exiting the cytoplasm, and the curve thus appears to be quasi-linear over several minutes. However, we show that the use of the net tracer accumulation rate during this time period as an estimate of NO 3 - influx does not provide accurate estimates of influx and efflux. By contrast, 15 NO 3 - efflux analysis permits calculation of the unidirectional fluxes across plasma membranes of root cells and the kinetic parameters of the cytoplasmic NO 3 - pool. Under our experimental conditions, efflux accounted for 30 to 50% of influx, and the cytoplasmic NO 3 - content was found to be in the 70-400 nmol g -1 fw range. Using this methodology, the effect of amino acid accumulation on unidirectional fluxes of nitrate was then examined. Pretreatments of the seedlings with an amino acid which has been shown to inhibit net NO 3 - uptake led to concomitant decreases in net accumulation rates of 15 NO 3 - and of reduced 15 N in roots and total 15 N in cotyledons. NO 3 - influx was markedly inhibited by these treatments, while NO 3 - efflux remained essentially unaffected, or even decreased. It is concluded that the target of the regulation of NO 3 - uptake by phloem-translocated amino acids is the influx system.