The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate–citrate exchange

Abstract

Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate-citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilisation in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.

Physiological functions of malate shuttles in plants and algae

“…Arabidopsis protein sequences were used first as baits. Note that the function assigned to mitochondrial malate translocators in arabidopsis is only based on in vitro experiments except for DIC2 which has recently been characterized in vivo [87]. Other players in the malate shuttle can be found in the Box 2 and 3.…”

“…These DICs were proposed to be involved in mitochondrial malate-OAA shuttle [86]. Very recently, the physiological function of DIC2 has been demonstrated in arabidopsis as being a high affinity malate-citrate antiporter in the mitochondria, through evidence from the reverse genetic approach in combination with comprehensive in vitro, in organello, and in vivo analyses [87]. On the other hand, in addition to dicarboxylates (malate, OAA, 2-OG and succinate), DTC was shown to transport also tricarboxylates (such as citrate or isocitrate) [85].…”

Metabolic evidence for distinct pyruvate pools inside plant mitochondria

“…Using selective reaction monitoring-mass spectrometry (SRM-MS) assays to trace the fate of two substrates simultaneously (2,22), we assessed the relative contribution of MPC1 and NAD-ME to metabolites derived from the pyruvate pool in mitochondria from Col-0. Isolated mitochondria were subjected to a series of 13 C3-pyruvate concentrations ranging from 0 to 500 µM with a fixed concentration of 500 µM malate at pH 6.4 to determine if a high NAD-ME activity competes with MPC for supplying pyruvate to the TCA cycle in vitro.…”

Comprehensive In Silico Analysis and Transcriptional Profiles Highlight the Importance of Mitochondrial Dicarboxylate Carriers (DICs) on Hypoxia Response in Both Arabidopsis thaliana and Eucalyptus grandis

“…The transcriptional up-regulation elicited by touch was proposed to address a physiological change since a significant reduction in DIC substrates such as succinate and citrate was observed in treated plants [9]. In this context, AtDIC2 was recently shown to promote the export of cytosolic malate in exchange for mitochondrial citrate [10], an activity that contributes for the maintenance of metabolic homeostasis, especially under stressful conditions. Interestingly, mutant plants lacking AtDIC2 were impaired in growth [10], a phenotype that further supports the importance of this carrier to normal metabolic function.…”

“…In this context, AtDIC2 was recently shown to promote the export of cytosolic malate in exchange for mitochondrial citrate [10], an activity that contributes for the maintenance of metabolic homeostasis, especially under stressful conditions. Interestingly, mutant plants lacking AtDIC2 were impaired in growth [10], a phenotype that further supports the importance of this carrier to normal metabolic function. Collectively, these findings suggest that modulation of DIC activity and expression might be part of the strategy of plant metabolic reprogramming required to overcome adverse growth conditions.…”