Effects of NH4+, NO3- and HCO3- on apoplast pH in the outer cortex of rootzones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

A fluorimetric ratio technique was elaborated to measure apoplastic pH in t he outer root cortex of maize (Zea mays L.) grown hydroponically. A newly s ynthesized fluorescent probe, fluorescein boronic acid (pK(a) = 5.48), whic h covalently binds to the cell wall of the outer cell layers, was used. Und er conditions of saturating ion concentrations the apoplastic pH was determ ined along the root axis ranging from I to 30 mm behind the root: tip. Apop lastic pH was recorded for root segment areas (1 mm(2)), and pH values of h igh statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. A t an external pH of 8.6, the difference between the external pH and the apo plastic pH was considerably more, with a pH of 5.2-5.3 in all root zones. A ddition of 1 mM NH4+ caused a small apoplastic pH decrease (0.05 of a pH un it) in all root zones. Apoplastic alkalization upon application of 6 mM NO3 - was highest (0.3 of a pH unit) in the zone where root hairs emerge; in th e division and early elongation zones, apoplastic pH increased only transie ntly. In the presence of 10 mM HCO3-, NO3- elicited a higher and persistent alkalization (0.06-0.25 of a pH unit) in all root zones. Application of fu sicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, wh ile inhibition of the Ht-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the r oot axis upon differential N supply and application of HCO3- provide eviden ce that this new pH technique is a useful tool with which to measure apopla stic pH, and in future may permit measurements at microsites at the cell le vel by use of microscope imaging.