Decoding the dual function of NAD-malic enzyme: from a universal role in malate respiration to a specific function in C4 photosynthesis

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In the C3 plant A. thaliana, malate levels produced during photosynthesis increase during the day and accumulate in the vacuole until metabolic demands are sensed. During the night, malate is utilized in the mitochondria to furnish part of the TCA cycle and to replenish the pool of TCA cycle intermediates. This function is fulfilled through the concerted action of NAD-dependent malic enzyme (NAD-ME) and malate dehydrogenase. Apart from its role in malate respiration, NAD-ME was co-opted to provide CO2 for the Calvin cycle in the bundle sheath cells during the photosynthetic metabolism in some C4 plants. The NAD-ME catalyzes the oxidative decarboxylation of malate, producing pyruvate, CO2, and NADH and is localized exclusively in mitochondria. Until now, there are no reports on the existence of a specific NAD-ME isoform only involved in C4 metabolism. This poses the question if NAD-ME performs a dual function in C4 plants or if a C4 specific isoform exist. Gene duplication would be a pre-condition for the evolution of the C4 metabolism as it allows the plant to retain an original gene while a duplicate version can acquire advantageous alterations. Interestingly, the C3 T. hassleriana and C4 G. gynandra Cleome species underwent a whole genome duplication that occurred aPproximately 13.7 mya. More precisely, a gene duplication of one NAD-ME beta-subunit is found in both Cleome species. We thus hypothesize that the duplication of the NAD-ME b-subunit was a potentiating evolutionary event that aided the repeated evolution of the NAD-ME subtype C4 photosynthesis in this genus. During this project, we will analyse the evolutionary transition of NAD-ME from a TCA cycle-associated enzyme to a C4 decarboxylase in bundle sheath cells using as model organisms the genus Cleome. Our main objective is to describe the mechanisms behind the recruitment of NAD-ME into the C4 pathway. To answer these questions, we propose a work program that combines protein biochemistry methods (proteomics, enzymology, structural analyses) with an in vivo loss-of-function complementation approach and immunohistochemical, transcriptomics, and phylogenetic analyses.