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First published online November 7, 2002; 10.1104/pp.008243

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Plant Physiol, December 2002, Vol. 130, pp. 1927-1937

Control of Demand-Driven Biosynthesis of Glutathione in Green Arabidopsis Suspension Culture Cells1

Andreas J. Meyer2* and Mark D. Fricker

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom

We have investigated what limits demand-driven de novo glutathione (GSH) biosynthesis in green Arabidopsis suspension culture cells. GSH is the most abundant low-molecular weight thiol in most plants and can be quantified using monochlorobimane to fluorescently label GSH in live cells. Progress curves for labeling reached a plateau as all the cytoplasmic GSH was conjugated. In the presence of excess monochlorobimane, a second, almost linear phase of labeling was observed, after a lag of 2 to 3 h, that was then maintained for an extended period. The increase in fluorescence was shown to be because of de novo GSH biosynthesis by high-performance liquid chromatography analysis and was eliminated by DL-buthionine-[S,R]-sulfoximine, a specific inhibitor of GSH biosynthesis, or reduced by inhibitors of transcription and translation. The rate of GSH biosynthesis during the linear phase was 8.9 ± 1.4 nmol g fresh weight-1 min-1 and was not affected by addition of glutamate, glycine, or cysteine, the immediate precursors needed for GSH biosynthesis. Likewise, the synthesis rate was not affected by pretreatment with aminotriazole, menadione, jasmonic acid, or cadmium, all of which cause oxidative stress and up-regulate expression of GSH biosynthetic genes. The lag phase was markedly reduced by aminotriazole and menadione and marginally by jasmonic acid, suggesting the system was primed to react faster after mild stress. In contrast to the other feeding experiments, exclusion of SO42- from the medium abolished the second phase completely. This suggests demand-driven GSH biosynthesis is directly coupled to uptake of SO42- and that the linear increase in fluorescence reflects flux through the entire SO42- assimilation pathway.

1 This work was partially supported by Aventis Crop Science Ltd.

2 Present address: Institut für Forstbotanik und Baumphysiologie, Professur für Baumphysiologie, Universität Freiburg, Georges-Köhler-Allee 053, D-79085 Freiburg, Germany.

* Corresponding author; e-mail Andreas.Meyer{at}sonne.uni-freiburg.de; fax 49-761-2038302.

© 2002 American Society of Plant Biologists


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