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First published online February 22, 2005; 10.1104/pp.104.058453 Plant Physiology 137:901-910 (2005) © 2005 American Society of Plant Biologists Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1Technische Universität Kaiserslautern, Pflanzenphysiologie, D–67653 Kaiserslautern, Germany (M.A.H., T.K., T.G., H.E.N.); Universität Zürich, Institut für Pflanzenbiologie, CH–8008 Zurich, Switzerland (S.J.S., E.M.); and Instituto de Biofisica, Consiglio Nazionale delle Ricerche, I–16149 Genova, Italy (M.B., F.G.)
Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH– to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.
1 This work was supported by the Deutsche Forschungsgemeinschaft, Graduate Research School 845 (to the laboratory of H.E.N.); by the European Union project Novel Ion Channels in Plants (NICIP; EU HPRN–CT–00245; BBW 01.0598 to F.G. and E.M.); and by the Italy-Switzerland Consiglio Nazionale delle Ricerche/Swiss National Foundation bilateral cooperation program. E.M. was financially supported by the Swiss National Foundation. 2 These authors contributed equally to the paper. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058453. * Corresponding author; e-mail neuhaus{at}rhrk.uni-kl.de; fax 0631–205–2600. Received December 16, 2004; returned for revision January 14, 2005; accepted January 17, 2005. This article has been cited by other articles:
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