**2. Mitochondrial Organic Acid Transporters Are Important to Central Carbon Metabolism**

Metabolite partitioning between the cytosol and mitochondria, mediated by mitochondrial carriers, is a distinguishing feature of eukaryotic metabolism that necessitates flexibility in the IMM transport direction and kinetics. These transport processes are primarily driven by an electrochemical gradient or proton motive force (PMF), which is comprised of a proton gradient (ΔpH) and membrane potential (ΔΨ) generated across the IMM by proton pumps of the electron transport chain [13]. The location of mitochondrial carriers in three-dimensional space in the context of physical compartmentalization of plant metabolism makes them strategic points for metabolic regulation and control, e.g., in the TCA cycle. In vitro biochemical data regarding mitochondrial organic acid transporters showed that these mitochondrial carriers are most likely responsible for shuttling of TCA cycle intermediates, i.e., between the cytosol and the mitochondria. This strongly suggests that mitochondrial organic acid transporters play a role in central carbon metabolism. However, *in planta* interrogations of the physiological roles of these mitochondrial organic acid transporters are yet to be achieved. To date, there are no reported changes to plant metabolism when mitochondrial carrier loss-of-function plants have been characterized. Recent work on the Arabidopsis TCA cycle interactome shows that a putative phosphate transporter interacted with TCA cycle enzymes [31,32]. However, to date, the physiological significance of these protein–protein interactions remains unknown.

In plants, there are three mitochondrial carriers most likely to be relevant to TCA cycle operation under different flux modes. These are: (1) dicarboxylate carriers (DICs); (2) dicarboxylate/tricarboxylate carriers (DTC); and (3) succinate/fumarate carrier (SFC). To recapitulate the evolutionary relationships among these three transporters in the context of the entire mitochondrial carrier superfamily, we sampled sequences that showed similarity to an amino acid profile of MCF sequences. To this end, we aligned previously known MCF protein sequences and some close paralogs and using MUSCLE [33] built a protein profile using hmmbuild [34] after selecting conserved regions of the alignment via GBLOCKS. The HMM protein profile was queried against the complete proteome files of 69 species to detect protein sequences with similarity to the MCF profile. The resulting matches were aligned (hmmalign) against the protein profile. Homosites with more than 20% missing values, as well as the misaligned N- and C- terminus regions, were removed from the alignment. The phylogenetic relationships were inferred based on Maximum Likelihood using RAxML [35], and branch supports were calculated using BOOSTER [36]. Our analysis showed that DICs, DTC, and SFC are not monophyletic (Figure 1). Mitochondrial organic acid transporter formed two distinct clades. In the first clade, DICs and DTC grouped with 2-OG carriers (OGCs). The SFC formed the second organic acid clade with other non-plant organic acid transporters including oxodicarboxylate carriers (ODCs), citrate carriers (CiCs), and yeast suppressor of HM (histone-like proteins in yeast mitochondria) mutant 2 (YHM2). While these non-plant organic acid transporters likely play a vital role in these species, they will not be discussed in this review. Biochemical data would insinuate that DTC and CiC must be closely related as they both transport citrate; phylogenetic analysis revealed SFC and not DTC, is more similar to CiC (Figure 1). Based on available biochemical data, it appears that transport functions of CiC and DTC have evolved independently but perhaps convergently.

**Figure 1.** An unrooted phylogenetic tree of mitochondrial carrier families (MCFs) across Domain Eukaryota. The tree was inferred based on Maximum Likelihood using the RAxML software and visualized using iTOL (https://itol.embl.de/), see text for details. UCP: uncoupling proteins, SAMT: S-adenosyl methionine transporter, OATC-I/II: organic acid transporters clade I/II, DICs: dicarboxylate transporters, OGCs: 2-oxoglutarate carriers, DTCs: dicarboxylate/tricarboxylate carriers, ODC: oxodicarboxylate carriers, YHM2-like: yeast HM mutant 2-like transporters, SFCs: succinate/fumarate carriers, CiCs: citrate transporters.

It has been established that the plant TCA cycle can also operate distinctly from the textbook cyclic mode [13,37] (Figure 2). The well-established cyclic mode of TCA flux most often associated with non-photosynthetic organisms is most likely to operate in leaves in the dark when there is a high demand for ATP through cellular respiration (Figure 2A) [13]. Import of pyruvate may be exclusively attributed to the mitochondrial pyruvate carriers (MPCs) [38]. However, malate/2-OG exchange could be undertaken by either DICs and/or DTC. On the other hand, fumarate efflux is probably catalyzed by SFC using 2-OG as counter-substrate as there is no net flux of succinate reported. The observation that these different non-cyclic modes are dictated by cellular metabolic demands suggests some level of control. However, whether regulation of the activity and/or expression of these transporters exist is

still an open question. Based on the available biochemical data on these relevant transporters (see below), we can begin to put forward some theories regarding their potential roles in central carbon metabolism. Several metabolites associated with the TCA cycle have been proposed to exchange across the IMM and thereby link the operation of several enzymes in mitochondrial to those in other cellular compartments. For example, citrate is suggested to be exported to the cytosol and then converted to 2-OG for redistribution to either chloroplasts or mitochondria [39]. Similarly, the malate-OAA shuttle can possibly mediate photorespiration through the mitochondrial malate dehydrogenase (mMDH) catalyzed reversible reaction after OAA import into the mitochondria [39,40].

**Figure 2.** The plant central metabolism related to the mitochondrial Organic Acid Transporters (**A**) In the dark, the TCA cycle most likely operate in the familiar cyclic mode. (**B**) Non-cyclic TCA flux mode has been modeled based on enzymatic kinetic analysis during the day [37,41]; flux in red is based on non-modeling literature. (**C**) A non-cyclic flux mode of the TCA cycle based on isotope labeling studies [42], an alternative metabolic route of stored citrate is shown as broken black arrows. Fluxes shown in green are either inactive or significantly reduced.

During the day, ATP demand from cellular respiration is low and enzyme kinetic analysis suggests that this results in the operation of a non-cyclic flux mode [37] (Figure 2). In this model, pyruvate entry into the TCA is reduced since the pyruvate dehydrogenase complex is inactivated in the light by phosphorylation. The model, furthermore, predicts that there is a net influx of malate accompanied by a net efflux of citrate, a metabolite exchange that is in congruence with the in vitro transport activity of DTC. Net import of OAA by the mitochondria during the day has been proposed on the basis of experiments described in the literature [13]. Such a net influx of OAA could also be mediated by DTC in exchange with citrate. We propose that DTC is sufficient to support this flux, partly explaining the very high DTC protein abundance in an average Arabidopsis mitochondrion [43] (Figure 2B). A non-cyclic flux mode of the TCA cycle has been proposed based on evidence from isotope labeling studies in illuminated leaves of *Xanthium strumarium* [42]. Here, there is a net influx of OAA as well as citrate. However, this influx has also proposed to be accompanied by net efflux of malate, fumarate, and 2-OG. In this model, the metabolite fluxes can only be supported by the concerted activities of DICs, DTC, and SFC. Citrate influx is mediated by DTC in exchange with OAA, malate, or 2-OG, while fumarate is transported by SFC using OAA or 2-OG as counter-substrate. DICs may also play a role in the transport of malate, 2-OG, and OAA. Alternatively, stored citrate can be converted to 2-OG via isocitrate in the cytosol [37]. In this case, the remaining metabolic fluxes can be attributed to the activities of DICs and SFC. The overlap of substrates allowed for transport by these transporters makes it challenging to pin down the responsible transporter for a specific metabolite exchange. While absolute quantification of the subcellular levels of organic acids remains a major analytical challenge, there is an acute need to define the *in planta* transport substrates and the directionality of the transport carried out by these MCs in order to complete our understanding of mitochondrial metabolism as they potentially dictate the metabolic fates of the TCA intermediates.
