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.

Light-Dark Changes in Cytosolic Nitrate Pools Depend on Nitrate Reductase Activity in Arabidopsis Leaf Cells

“…Zhen et al, 1991;Miller and Smith, 1992;van der Leij et al, 1998). Values for the cytosol in leaf cells are similar to those in root cells and are in the low millimolar range (Table I).…”

Electrochemical Potential Gradients of H+, K+, Ca2+, and Cl- across the Tonoplast of the Green Alga Eremosphaera Viridis

“…Only when the technique of liquid-membrane ion-selective microelectrodes was applied to a variety of plant cells was it realized that the impalement of the vacuole is a rare event (Felle and Bertl, 1986a;Felle, 1993). Studies based on this technique generally reported tonoplast potentials in the range from -8 to -27 mV (Rona et al, 1982;Miller and Zhen, 1991;Zhen et al, 1991). It is interesting that a recent study reported a value as high as +50 mV (Okazaki et al, 1994).…”

“…Although not measured directly, there were some indications that the tonoplast potential was negligible (Trebacz et al, 1994). Measurements with NO,--selective microelectrodes were performed in Chara and in barley roots Zhen et al, 1991). Despite the high accumulation of NO,-inside the vacuole in both plants, the measured tonoplast potential was low.…”

Effects of anion channel blockers on xylem nitrate transport in barley seedlings

“…Interestingly, an anion channel that allows nitrate influx was identified in wheat root protoplasts (Skerett and Tyerman 1994). However, taking into account the mM level of cytosolic nitrate concentrations and negative membrane potentials of -110m V in epidermal and cortical cells of barley roots (Zhen et al 1991;Miller and Smith 1992), passive nitrate influx via channels may occur at higher external nitrate concentrations (> 300 mM). The possible involvement of anion channels in nitrate uptake at low concentrations in the medium remains to be elucidated.…”