Effects of NH 4 + , NO 3 − and HCO 3 − on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

Abstract

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK(a) = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm(2)), and pH values of high 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. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2-5.3 in all root zones. Addition of 1 mM NH(4)(+) caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO(3)(-) was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO(3)(-), NO(3)(-) elicited a higher and persistent alkalization (0.06-0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H(+)-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO(3)(-) provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging.

Rapid Ammonia Gas Transport Accounts for Futile Transmembrane Cycling under NH3/NH4 + Toxicity in Plant Roots

“…A relatively minor channel/carrier-mediated flux of NH 4 + may also occur across both membrane systems. NH 4 + concentrations are a function of NH 3 equilibration and compartment pH (Roberts et al, 1982;Walker et al, 1996;Kosegarten et al, 1997;Kosegarten et al, 1999). PIP, Plasmalemmaintrinsic protein; TIP, tonoplast-intrinsic protein; AMT, ammonium transporter; Kir, K + inward rectifier; NSCC, nonselective cation channel.…”

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

“…Hence, although the fraction of NH 3 in solution at pH 6-7 is very small (approx. 0.07-0.6%), the transient alkalinisation of the cytosol reported after NH 3 uptake can be attributed to rapid diffusion of NH 3 across the plasma membrane and its subsequent protonation within the cytosol [49,50]. The increased NH 3 concentration will therefore consume the established Δ μ H+ , thereby contributing to a higher energetic cost to balance it.…”

Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1[W]

“…YFP has a pK a value above 7.1, with fluorescence emission abrogated below pH 6.0 (Llopis et al, 1998), meaning that the fluorescing N-YFP-AUX1 fusion protein is unlikely to be exposed to the pH conditions (4.8 to 5.3) measured in the apoplastic space of root cells (Kosegarten et al, 1999). Indeed, transgenic root cells continued to emit YFP fluorescence even when the external media was buffered to pH 5.0 ( Figures 2G to 2I).…”