Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling

Abstract

Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m(-3) nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m(-3) (n=19) and 41.8 ± 2.6 mol · m(-3) (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m(-3) (n = 4) and 72.8 ± 8.4 mol · m(-3) (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m(-3) (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m(-3) (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m(-3), while the vacuole accumulated nitrate to 30-50 mol · m(-3). The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.