Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses

doi:10.1073/pnas.0913689107

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses

^aInstitute of Biotechnology, University of Cambridge, Cambridge CB2 1QT, United Kingdom;
^bDepartment of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia;
^cHeidelberger Institut für Pflanzenwissenschaften, Universität Heidelberg, 69120 Heidelberg, Germany;
^d Centre for Crop Genetic Improvement, Rothamsted Research, Harpenden Herts AL5 2JQ, United Kingdom;
^e Liverpool School of Tropical Medicine, Molecular and Biochemical Parasitology, Pembroke Place, Liverpool L3 5QA, United Kingdom; and
^fCentre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom

Communicated by Bob B. Buchanan, University of California, Berkeley, CA, December 2, 2009 (received for review July 8, 2008)

Abstract

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

Footnotes

¹To whom correspondence should be addressed. E-mail: smaughan{at}venrock.com.
Author contributions: S.C.M., M.P., N.C. and A.J. Meyer designed research; S.C.M., M.P., N.C., G.K., T.B., R.J., F.H., J.N., B.L., C.M., E.S.-S., C.K., M.O., and A.J. Miller performed research; S.C.M., M.P., E.S.-S., R.H., A.J. Miller, P.B., C.H.F., J.A.H.M., A.J. Meyer, and C.S.C. contributed new reagents/analytic tools; S.C.M., M.P., N.C., G.K., T.B., R.J., F.H., J.N., B.L., C.M., E.S.-S., C.K., M.O., J.A.H.M., A.J. Meyer, and C.S.C. analyzed data; and S.C.M., J.A.H.M., and C.S.C. wrote the paper.
↵ ²Present address: Venrock, 3340 Hillview Avenue, Palo Alto, California, 94304.
↵ ³M.P. and N.C. contributed equally to this work.
↵ ⁴Present address: Cardiff School of Biosciences, Life Sciences Building, Museum Avenue, Cardiff CF10 3AX, United Kingdom.
↵ ⁵Present address: Amélioration des Plantes et Biotechnologies Végétales, UMR118, INRA-AgroCampus, Rennes, BP 35327 35653 Le Rheu Cedex, France.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0913689107/DCSupplemental.