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. 1989 Jan;89(1):19–26. doi: 10.1104/pp.89.1.19

Regulation of Vacuolar pH of Plant Cells

I. Isolation and Properties of Vacuoles Suitable for 31P NMR Studies

Yves Mathieu 1,2, Jean Guern 1,2, Armen Kurkdjian 1,2, Pierre Manigault 1,2, Jeanne Manigault 1,2, Teresa Zielinska 1,2, Brigitte Gillet 1,2, Jean-Claude Beloeil 1,2, Jean-Yves Lallemand 1,2
PMCID: PMC1055792  PMID: 16666513

Abstract

For the first time, the 31P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 106 vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H+ exchanges at the tonoplast. The 31P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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