Differential pH restoration after ammonia-elicited vacuolar alkalisation in rice and maize root hairs as measured by fluorescence ratio

Changes of vacuolar pH in hair cells of young rice (Oryza sativa L.) and maize (Zea mays L.) roots were measured after ammonia application at various levels of external pH. After loading the pH-sensitive, fluorescent dye Oregon green 488 carboxylic acid 6-isomer into the vacuoles of root hairs, ratiometric pH data of high statistical significance were obtained from root hair populations comprising hundreds of cells. The pH of the vacuole at external pH 5.0 was 5.32 +/- 0.08 (+/-SD, n = 15) and 5.41 +/- 0.13 (+/-SD n 15) in rice and maize, respectively. A moderate external ammonia concentration of 2 mM led to vacuolar alkalisation at both, low (pH 5.0) and high (pH 7.0-9.0) external pH, presumably due to NH3 permeation into the vacuole. With increasing external pH, ammonia application did not cumulatively increase vacuolar pH. In rice, the increase in vacuolar pH ranged from 0.1-0.8 pH units; in maize a more constant increase of 0.5 pH units was observed. The vacuolar pH increase was efficiently depressed in rice (especially at high external pH), but not in maize. Inhibition of the tonoplast H+-ATPase by concanamycin A raised vacuolar pH and increased the ammonia-elicited vacuolar alkalisation in both species, proving that vacuolar H+-ATPase activity counters the ammonia-elicited alkalisation effect. However, even under conditions of vacuolar H+-ATPase inhibition, rice was still able to restore an ammonia-elicited pH increase. High vacuolar pH levels as found in maize under conditions of high NH3 influx may derive from inefficient cytosolic ammonia assimilation and tonoplast proton pumping. Thus, in maize, prolonged reduction of the proton gradient between the cytosol and the vacuole may play an important role in NH3 toxicity.