The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism

Abstract

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate (OAA) and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases (AlaAT) when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

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

“…Given the importance of mitochondria-supplied citrate to metabolism in green tissues ( Gauthier et al, 2010 ; Cheung et al, 2014 ; Igamberdiev, 2020 ), it is expected that a complete block in mitochondrial citrate synthesis and export would result in severe reduction in post-germination vegetative growth. Indeed, a simultaneous loss of NAD-ME, mitochondrial pyruvate carrier and alanine aminotransferase activities severely disrupts seedling development, most likely due to limited supply of pyruvate for mitochondrial citrate synthesis ( Le et al, 2021a ). Mutants lacking the activity in one of the subunits of mitochondrial pyruvate dehydrogenase complex (mPDC) or both mMDH isoforms, which catalyze acetyl-CoA and OAA formation, respectively, required for citrate synthesis, are significantly slower in vegetative growth ( Tomaz et al, 2010 ; Yu et al, 2012 ; Ohbayashi et al, 2019 ).…”

“…At night, NAD-dependent malic enzyme (NAD-ME) and dephosphorylated pyruvate dehydrogenase are activated, allowing optimal supply of acetyl-CoA and OAA for efficient citrate synthesis. While the identity of plant mitochondrial pyruvate carrier (MPC) has recently been confirmed ( Le et al, 2021a ), the exact molecular identity and in vivo function for malate, OAA, citrate and alanine transporters are not clear. AlaAT, alanine aminotransferase; CS, citrate synthase; MDH, malate dehydrogenase; MPC, mitochondrial pyruvate carrier; PDC, pyruvate dehydrogenase complex.…”

Legume Alternative Oxidase Isoforms Show Differential Sensitivity to Pyruvate Activation

“…While it is clear that different AOX isoforms have tissue-specific expression (eg Finnegan et al, 1997 ), different kinetics of pyruvate activation (this report), and also respond differently to other organic acid activators ( Selinski et al, 2018 ), we still lack precise knowledge of the concentrations of activators in planta and how they vary with tissue type and growth conditions. Organic acid concentrations have been estimated in whole tissue extracts from numerous plant species, but for AOX activation it is the intramitochondrial concentrations that are important, and these are very difficult to measure, especially pyruvate given that its import and generation within mitochondria can occur via multiple pathways ( Le et al, 2021a ). The advent of a pyruvate sensor for in vivo estimation of pyruvate levels ( Arce-Molina et al, 2020 ) may be of help in this context, although very recent data suggest that even within mitochondria distinct pools of pyruvate exist, and the fate of the different pyruvate pools may be predetermined by source, e.g., import vs in situ biosynthesis ( Le et al, 2021b ).…”

Validation of carbon isotopologue distribution measurements by GC-MS and application to13C-metabolic flux analysis of the tricarboxylic acid cycle inBrassica napusleaves

“…Finally, mitochondrial transaminases such as Alanine:2-oxoglutarate and Aspartate:2-oxoglutarate could also participle in this process. Indeed, Alanine:2-oxoglutarate aminotransferase is one of the three pathway supplying pyruvate to mitochondria and was notably solicited in the mpc1 mutants to compensate for the loss of mitochondrial pyruvate import (Le et al, 2021). Regarding Glutamate, its production from 13 C-2-Oxoglutarate and 12 C-Glutamine molecules in our metabolic network will necessarily introduce a 50% isotopic dilution between 12 C and 13 C molecules.…”