Using 31P nuclear magnetic resonance spectroscopy, we followed cytoplasmic and vacuolar pH in pea (Pisum sativum cv Alaska) internode segments during treatment with indoleacetic acid (IAA) or fusicoccin (FC) in continuously perfused, oxygenated buffer. Although IAA and FC (21,22,34,37) and allows FC to counteract responses to permeant acids (34, 42). It seems possible that such an increase in pHcyt also stimulates polysaccharide synthase activity and cell wall synthesis (3,5,25) and activates gene expression (39). A contrasting proposal is that the primary action of FC (1, 15) and auxin (4,8,10,11) is to cause cytoplasmic acidification, which stimulates a pH-sensitive H+ efflux pump and thereby induces H+ extrusion.The direct evidence for the cytoplasmic acidification hypothesis is from measurements on absorbed dyes and with micro-pH electrodes, yielding data For high-resolution measurements of proton extrusion (Fig. 3), the third internode's cuticle was abraded by rubbing gently with an aqueous suspension of No. 305 emery powder (Edmund Scientific, Barrington, NJ) before cutting the segment.For NMR measurements, about 150 segments were loaded into a 12 mm diameter NMR tube, open at both ends, in 5 layers of about 30 segments each. During the measurement, the segments were perfused, in a recirculating arrangement, with a continuously oxygenated 1 mm MES buffer, initial pH 6.5, containing 1 mM KCI and 15 mM sucrose, at a flow rate of about 20 ml/ min. After recording initial NMR spectra, IAA, FC, or NA was added from a stock solution to give the indicated concentration.NMR spectra were initially measured with a modified Bruker HXS 360 spectrometer operating at 145 MHz in the Fourier transform mode. Chemical shifts were measured with respect to 0.5 M MDP located in a co-axial capillary tube. Segments were equilibrated in the spectrometer and an initial spectrum was accumulated for 15 min, after which growth substances were added to treated samples, and spectra were recorded for an additional 1 to 3 h. The pH of the buffer solution was checked before and after each experiment. This check showed that FC and IAA did induce H+ extrusion from pea segments in the NMR spectrometer.In later experiments, high-resolution spectra were obtained using a General Electric GN 500 NMR system operating at 202 MHz. The -y-phosphate group of tissue ATP (peak 4, Fig. 1), whose chemical shift (-21.45 ppm relative to MDP) is insensitive to pH changes above pH 7.0, was used as an internal reference peak for these spectra. Six 5 min spectra were accumulated, after which either IAA or NA was added, and spectra were accumulated for an additional 30 min.NMR measurements were made in an air-conditioned room at 22 to 24°C, continuously perfusing the tissue sample from a reservoir containing 10 L or more of perfusion fluid which further buffered the sample against temperature changes. Typical spectrometer parameters were pulse width 500, pulse interval 0.9 sec, spectral width + 3000 Hz, 8K data points, digital resolution about ...