Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria

Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: “What regulates flux through this pathway in vivo?” Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis : the NADP-TRX reductase a and b double mutant ( ntra ntrb ) and the mitochondrially located thioredoxin o1 ( trxo1 ) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when ¹³C-glucose, ¹³C-malate, or ¹³C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function. Significance The present work extends redox-based change in enzyme activity to the TCA cycle of plant mitochondria. Thioredoxin (TRX) was found to regulate the activity of enzymes of the mitochondrial cycle (succinate dehydrogenase and fumarase) and of an enzyme associated with it (ATP-citrate lyase) by modulating thiol redox status. A combination of experiments based on mutant and carbon isotope labeling analyses provides evidence that flux through this pathway is coordinately modulated by TRX at the enzyme level of both mitochondria and cytosol. The results provide in vivo confirmation of earlier in vitro results and further show that mitochondria resemble plastids in using