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Downregulation of the δ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis
(The Plant Cell, 2012-07) Nunes-Nesi, Adriano; Geisler, Daniela A.; Päpke, Carola; Obata, Toshihiro; Matthes, Annemarie; Schneitz, Kay; Maximova, Eugenia; Araújo, Wagner L.; Fernie, Alisdair R.; Persson, Staffan
The mitochondrial ATP synthase (F1Fo complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded Fo part to the matrix-facing central stalk of F1. We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.
Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria
(Proceedings of the National Academy of Sciences of the United States of America, 2015-02-02) Daloso, Danilo M.; Müller, Karolin; Obata, Toshihiro; Florian, Alexandra; Tohge, Takayuki; Bottcher, Alexandra; Riondet, Christophe; Bariat, Laetitia; Carrari, Fernando; Nunes-Nesi, Adriano; Buchanan, Bob B.; Reichheld, Jean-Philippe; Araújo, Wagner L.; Fernie, Alisdair R.
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 enzymeencoding 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 13 C- glucose, 13 C-malate, or 13 C-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.
Arabidopsis uses two gluconeogenic gateways for organic acids to fuel seedling establishment
(Nature Communications, 2015-04-10) Eastmond, Peter J.; Astley, Holly M.; Aubry, Sylvain; Williams, Ben P.; Menard, Guillaume N.; Craddock, Christian P.; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Hibberd, Julian M.; Parsley, Kate
Gluconeogenesis is a fundamental metabolic process that allows organisms to make sugars from non-carbohydrate stores such as lipids and protein. In eukaryotes only one gluconeogenic route has been described from organic acid intermediates and this relies on the enzyme phosphoenolpyruvate carboxykinase (PCK). Here we show that two routes exist in Arabidopsis, and that the second uses pyruvate, orthophosphate dikinase (PPDK). Gluconeogenesis is critical to fuel the transition from seed to seedling. Arabidopsis pck1 and ppdk mutants are compromised in seed-storage reserve mobilization and seedling establishment. Radiolabelling studies show that PCK predominantly allows sugars to be made from dicarboxylic acids, which are products of lipid breakdown. However, PPDK also allows sugars to be made from pyruvate, which is a major product of protein breakdown. We propose that both routes have been evolutionarily conserved in plants because, while PCK expends less energy, PPDK is twice as efficient at recovering carbon from pyruvate.

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