Dissecting the regulation of plant mitochondrial metabolism and its impact on cyto-nuclear NAD signaling

线粒体在大多数复杂生物体中扮演着中心能量转换器的角色，并深深地嵌入到细胞代谢网络中。线粒体代谢在植物中特别灵活，可以在不同的程序之间快速切换，以适应不断变化的环境条件。在这个项目的前一阶段，我们研究了在暗-光转换过程中重新协调线粒体新陈代谢的生理动力学和调节机制。我们发现线粒体苹果酸代谢是灵活“远程”控制细胞-核NAD状态的热点。这不仅对亚细胞新陈代谢有影响，而且对细胞信号也有影响。在这个合作项目的第二阶段，我们将解决细胞-核NAD状态将线粒体代谢灵活性与核基因表达的重新编程联系起来的假设。结合来自三个实验室的独特专业知识，我们将解决以下问题：(1)线粒体新陈代谢如何调节细胞核NAD状态，以及(2)线粒体衍生的NAD动态对核基因表达有何影响。我们将在拟南芥中通过反向遗传来调节线粒体苹果酸脱氢酶(MMDH)和NAD苹果酸酶(NAD-ME)的能力，以改变亚细胞代谢的路线。为了了解线粒体控制的胞核NAD状态调节的生理机制，我们将探索翻译后修饰和相互作用对mMDH和NAD-ME的调节。改变代谢流量对NAD状态的影响将使用体内和动态荧光蛋白生物传感在暗光循环和激发子暴露期间进行评估，我们最近发现这对NAD状态有深远影响。同时监测NADH/NAD+和NAD+浓度将以高精度定义体内NAD信号，并为NAD信号转录学研究提供信息，重点是核NAD依赖的脱乙酰酶sirtuin 1作为NAD动力学的潜在表观遗传整合因子。因此，我们将解开植物线粒体新陈代谢的灵活性和设计驯化的信号程序之间的接口。

Mitochondria act as central energy converters in most complex organisms and are deeply embedded into the cellular metabolic network. Mitochondrial metabolism is particularly flexible in plants and can rapidly switch between different programmes to integrate changing environmental conditions. In the previous phase of this project, we investigated physiological dynamics and regulatory mechanisms that operate to re-orchestrate mitochondrial metabolism during dark-light transitions. We identified mitochondrial malate metabolism as a hotspot of flexible ‘remote’ control of cyto-nuclear NAD status. This has consequences not only for subcellular metabolism, but also for cell signalling. In the second phase of this collaborative project we will address the hypothesis that cyto-nuclear NAD status links mitochondrial metabolic flexibility to reprogramming of nuclear gene expression. Bringing together unique expertise from three laboratories we will address the questions of (1) how mitochondrial metabolism modulates cyto-nuclear NAD status, and (2) what impact mitochondria-derived NAD dynamics have on nuclear gene expression. We will modulate mitochondrial malate dehydrogenase (mMDH) and NAD malic enzyme (NAD-ME) capacities by reverse genetics in Arabidopsis to re-route subcellular metabolism. To understand the physiological mechanisms behind mitochondria-controlled modulation of cyto-nuclear NAD status, we will explore the regulation of mMDH and NAD-ME by post-translational modifications and interactors. The impact of the re-routed metabolic flux on cyto-nuclear NAD status will be assessed using in vivo and dynamically fluorescent protein biosensing during dark-light cycles and elicitor exposure, for which we have recently found profound impact on NAD status. Monitoring both, NADH/NAD+ and NAD+ concentration will define NAD in vivo signatures at high precision and inform transcriptomic investigation of NAD signalling, with a focus on the nuclear NAD-dependent deacetylase sirtuin 1 as a potential epigenetic integrator of NAD dynamics. As a result, we will unravel the interface between the flexibility of plant mitochondrial metabolism and signalling programs to choreograph acclimation.