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

在C3工厂A.在拟南芥中，光合作用过程中产生的苹果酸水平在白天增加，并在液泡中积累，直到感觉到代谢需求。在夜间，苹果酸在线粒体中被利用来提供TCA循环的一部分并补充TCA循环中间体的库。这一功能通过NAD依赖性苹果酸酶（NAD-ME）和苹果酸脱氢酶的协同作用来实现。NAD-ME除了参与苹果酸呼吸外，还参与了某些C4植物光合代谢过程中维管束鞘细胞内卡尔文循环的CO2提供。NAD-ME催化苹果酸的氧化脱羧，产生丙酮酸、CO2和NADH，并且仅定位于线粒体中。到目前为止，还没有关于存在仅参与C4代谢的特异性NAD-ME亚型的报道。这就提出了一个问题，如果NAD-ME在C4植物中执行双重功能，或者如果C4特异性同种型存在。基因复制将是C4代谢进化的先决条件，因为它允许植物保留原始基因，而复制版本可以获得有利的改变。有趣的是，C3 T。hassleriana和C4 G.一个早在1370万年前就发生了全基因组重复的gynandra Cleome物种。更确切地说，在两种Cleome物种中发现了一个NAD-ME β亚基的基因重复。因此，我们假设，NAD-ME b-亚基的复制是一个增强的进化事件，有助于重复的NAD-ME亚型C4光合作用在这个属的进化。在这个项目中，我们将分析NAD-ME从TCA循环相关酶到束鞘细胞中C4脱羧酶的进化过渡，使用作为模式生物的属醉蝶花。我们的主要目标是描述NAD-ME进入C4通路的募集机制。为了回答这些问题，我们提出了一个工作计划，结合蛋白质生物化学方法（蛋白质组学，酶学，结构分析）与体内功能丧失互补的方法和免疫组织化学，转录组学和系统发育分析。

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.