Phosphate (Pi) starvation effect on the cytosolic Pi concentration and Pi exchanges across the tonoplast in plant cells: an in vivo 31P-nuclear magnetic resonance study using methylphosphonate as a Pi analog.

James Pratt,Anne-Marie Boisson,Richard Bligny,Elisabeth Gout,Roland Douce,Serge Aubert

doi:10.1104/pp.109.144626

Abstract

In vivo (31)P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60-80 mum instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.

Highlights

In vivo 31P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60–80 mM instead of .1 mM) and that it dropped very rapidly following the onset of Pi starvation
This signal disappeared after removing external Pi, whereas another Pi signal appeared symmetrically at 2.35 ppm (Fig. 2). These results strongly suggest that the signal at 2.20 ppm corresponds to cytsol-Pi, whereas the signal at 2.35 ppm corresponds to orgmp-Pi, permitting us to measure the size of these two Pi pools in sycamore and Arabidopsis cells (Table I)
Assuming that the cytoplasm-to-cell ratio is 0.15 in these cells and that the volume of mitochondria plus plastids estimated from electron micrographs represents approximately 12% of the volume of cytoplasm in sycamore cells (Bligny and Douce, 1976) and 20% in Arabidopsis cells, it was calculated that the cytsol-Pi concentration was 60 to 80 mM and 55 to 75 mM in sycamore and Arabidopsis cells, respectively

Summary

Introduction

In vivo 31P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60–80 mM instead of .1 mM) and that it dropped very rapidly following the onset of Pi starvation. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. Pi Starvation and Homeostasis in Plant Cells (Bligny et al, 1989), triggers a decrease of hexose phosphates These data suggested the existence of a cyt-Pi pool able to rapidly fluctuate according to the Pi supply from external and vacuolar stores and to the Pi demand for metabolism. As the Pi concentration of mitochondria and plastids is tightly regulated (Scarpa, 1979; Plaxton and Carswell, 1999), we hypothesized that the appearance of early Pi deprivation effects correlated with low Pi supply may originate from rapid Pi changes in cytosol. This is addressed in this study using in vivo 31P-NMR

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Discussion

Conclusion