Using confocal laser scanning microscopy to measure apoplastic pH change in roots of Lupinus angustifolius L. in response to high pH

A confocal laser scanning microscopy method to measure apoplastic pH in silts of the tap roots of Lupinus angustifolius L. is described. Pseudo-ratiometric imaging of cell walls of lupin tap roots was achieved by loading living roots with a pH-sensitive dye, C1-NERF-dextran, and a pH-insensitive dye, Texas Red-dextran, as a reference. In 5-d-old tap roots which had previously been grown at pH 5.4, the apoplastic pH in the elongating zone was 5.2 +/- 0.06. The apoplastic pH in roots grown at pH 7.5 increased to 5.5. The method was validated by supplying different forms of nitrogen in the nutrient solution. With NH4+ nutrition the apoplastic pH was lowered by 0.3 units while when NO3- was supplied the apoplastic pH increased by 0.2 units. Causes of the poor root growth of L. angustifolius at high pH are discussed. (C) 2000 Annals of Botany Company.