A phosphorus-31 nuclear magnetic resonance study of phosphate uptake and storage in cultured Catharanthus roseus and Daucus carota plant cells.

P Brodelius,H J Vogel

doi:10.1016/s0021-9258(19)83658-x

Abstract

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus and Daucus carota cells grown in suspension cultures were obtained using a solenoidal perfusion probe. The spectra showed resonances for various phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose, and sugar phosphates. The relative levels of the phosphorylated metabolites remained constant throughout the growth curve. No resonances for storage compounds such as polyphosphates, pyrophosphate, or phytates were observed. Two resolved resonances for Pi indicated an intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles, respectively. The time course of Pi uptake and storage during growth in fresh culture medium was followed by studying the level of vacuolar Pi with 31P NMR (145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed with radioactive 32P. C. roseus quickly takes up all the Pi from the culture medium (maximum rate 1.7 mumol min-1 g-1 (dry weight of cells]. The Pi is first stored in the vacuoles; subsequently, one part of this pool is used to keep a constant cytoplasmic Pi level while another part is apparently accumulated as an NMR invisible Pi store, probably in another cell organelle. In contrast, D. carota does not accumulate Pi in the vacuoles and consequently it takes up Pi from the medium at a much slower rate (0.05 mumol min-1 g-1 (dry weight of cells].

Highlights

High resolution “P NMR spectra (103.2 MHz) of these reasons it is of interest to compare Pi uptake, storage, oxygenated Catharanthus roseu8 and Daucus carota metabolism, and compartmentalization in various cultured cells grown in suspension cultures were obtained using a solenoidal perfusion probe
Rate 1.7 pmol min” g”).The Pi is Since NMR is a relatively insensitive technique it is often first stored in the vacuoles; subsequently, one part of necessary to accumulate individual spectra over extended time this pool is used to keep a constant cytoplasmic Pi level periods
Note that the same resonances are observed in a ratiovery similar to thatof C. roseus (Fig. 1).This ratio for the major phosphorylated metabolites resembles the composition for a model plant cell tissue as reported by Bieleski (6)

Summary

THEJOURNAOLF BIOLOGICACHLEMISTRY

0 1985 by The American Society of Biological Chemists, Inc. Vol 260,No., Issue of March 25.pp. 355&3560 1985 Printed in L.I.s.A. Rate 1.7 pmol min” g” (dry weight of cells)).The Pi is Since NMR is a relatively insensitive technique it is often first stored in the vacuoles; subsequently, one part of necessary to accumulate individual spectra over extended time this pool is used to keep a constant cytoplasmic Pi level periods. This creates the practical problem of keeping the while another part is apparently accumulated as an dense cell suspension oxygenated and supplied with nutrients.

MATERIALS ANDMETHODS

SUGAR PHOSPHATES

PP M

RESULTS

31PNMR Studies of Plant Cell Suspensions

CONCLUSIONS