MEASUREMENT OF INTRACELLULAR NITRATE CONCENTRATIONS IN CHARA USING NITRATE-SELECTIVE MICROELECTRODES

Nitrate-selective microelectrodes have been made using a quaternary ammonium sensor, methyltridodecylammonium nitrate, in a polyvinylchloride matrix. These electrodes showed a log-linear response from 0.1 to 100 mol . m-3 nitrate with a typical slope of 55.6 mV per decade change in nitrate concentration. The only physiologically significant interfering anion was chloride but the lower limit of nitrate detection was 0.5 mol . m-3 in the presence of 100 mol . m-3 chloride which means this interference will not be important in most physiological situations. These microelectrodes were used to measure nitrate concentrations in internodal cells of Chara corallina cultured under low nitrate and nitrate-replete conditions for 6 to 30 weeks. Cells maintained in low nitrate only showed measurements which were less than the detection limit of the electrodes, while cells grown under nitrate-replete conditions showed two populations of measurements having means of 1.6 and 6.2 mol . m-3. Chemical analysis of the high-nitrate cells indicated that they contained a mean nitrate concentration of 5.9 mol . m-3. As vacuolar nitrate concentration would dominate this whole-cell measurement, it is concluded that the higher concentration measured with the electrodes represents vacuolar nitrate concentration and the lower value represents the cytoplasmic concentration. This intracellular distribution of nitrate could only be achieved passively if the electrical potential difference across the tonoplast is between + 25 and + 35 mV.