Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast

Abstract

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 2 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.

CLC-mediated anion transport in plant cells

“…In both mutant alleles, reversal potentials were different from wildtype, in agreement with the absence of a transport system in mutant vacuoles that would impose its equilibrium potential. The lack of AtClCa did not affect vacuolar malate currents (Hafke et al 2003;Hurth et al 2005) that were similar in wild-type and clca-2 mutant vacuoles ( et al 2006) that is in agreement with the 2Cl K /1H C ratio reported for CLCec-1 (Accardi & Miller 2004). Thus, AtClCa is the first anion/proton antiporter belonging to the CLC family, identified and functionally characterized in plant cells.…”

“…In many of these processes, malate is accumulated into the vacuole (Martinoia & Ratajcsak 1997). The transport of malate to the vacuolar lumen has been shown to occur via specific transporters such as AttDT (Arabidopsis thaliana tonoplast dicarboxylate transporter; Hurth et al 2005) and via specific channels as well (Hafke et al 2003). The molecular identity of malate channels has been revealed only recently with the identification of the AtALMT9 protein (A. thaliana aluminium-activated malate transporter; Kovermann et al 2007).…”

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis

“…Along with these changes, an increase in total citrate concentration was observed in leaves from tdt knock-out mutants (14,19). However, the increase in citrate levels in these mutants could so far not be explained on the basis of the previously deciphered transport properties of tDT (14,19).…”

“…Surprisingly, compared to wild type plants, tDT loss-of-function mutants from Arabidopsis did not exhibit morphological alterations, nor developmental retardation or impaired stomata movement when exposed to different growth conditions (19). However, the mutant showed metabolic alterations clearly pointing to an in vivo malate transport activity.…”

“…These differences include decreased leaf malate levels, increased respiration, an altered respiratory coefficient and changes in mitochondrial metabolites. Furthermore, mutant plants were impaired in their ability to cope with acid stress (14,(19)(20)(21). These results suggest that vacuolar ALMTs can partially compensate for the loss of tDT and that corresponding mutants can adapt their metabolism to a situation where malate is less efficiently transferred to the vacuole mainly by increasing consumption of malate.…”