The Arabidopsis vacuolar malate channel is a member of the ALMT family

Summary In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter ( Arabidopsis thaliana tonoplast dicarboxylate transporter, At tDT) was identified that did not correspond to the well‐characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum‐activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that At ALMT9 ( A. thaliana ALMT9) is targeted to the vacuole. Promoter‐GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell‐type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch‐clamp analysis of an Atalmt9 T‐DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize At ALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of At ALMT9‐GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of At ALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that At ALMT9 is a vacuolar malate channel. Deletion mutants for At ALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that At tDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.