The Significance of Organellar Membrane Contact Sites in Photosynthetic Metabolism of Plant Cells

植物细胞代谢可以根据环境条件在自养和异养生长模式之间切换。在光呼吸过程中，几个细胞器之间的代谢协同作用是植物生存所必需的。在这里，有害的乙醇酸产生的加氧酶活性的核酮糖-1，5-二磷酸羧化酶/加氧酶（RuBisCO）是通过八个酶之间的过氧化物酶体，线粒体和chloroplasts.The建立的光呼吸的生化水平的知识面临着缺乏知识的空间细胞器组织的功能和特定的膜接触点（MCS）的形成。而叶绿体，线粒体和过氧化物酶体之间的密切联系已经监测显微镜下几十年来，它们的分子结构和植物代谢和性能的相关性仍然未知，在这里，我们调查的假设，即在植物中的MCSs的基础代谢重塑和重组细胞有效地应对不断变化的环境条件下提供的基础。为了解决光呼吸细胞器之间的特定和动态MCS形成的问题，我们建立了一套遗传编码的体内邻近生物传感器，分别基于双分子荧光互补（BiFC）和Förster共振能量转移/荧光寿命成像（FRET/FLIM）。此外，为了解决MCS驻留蛋白的分子身份和功能，我们创建了一组候选蛋白，其将通过使用混杂生物素连接酶构建体（BioID技术）标记MCS驻留蛋白来扩增。通过设计细胞器对的合成拴系结构，以及通过研究候选MCS驻留蛋白的突变植物系，我们将干扰MCS的形成。为了量化特定MCSs的生物学相关性，我们使用植物生长表型、光呼吸能力和代谢产物以及光合参数作为读数，我们的研究结果将解决长期存在的MCSs的形成和生理相关性的问题，这些MCSs与光呼吸和植物在光/暗转换期间的表现有关。我们将定义细胞器的接近是基于特定的关联，而不是空间限制。我们期待我们的工作，以建立细胞生物学之间的联系，细胞器之间的途径分区，和植物代谢可塑性。此外，由于光呼吸是理解和优化光合植物性能的主要目标，我们希望我们的工作与未来的生物技术方法相关。

Plant cell metabolism can switch between autotrophic and heterotrophic growth modes, depending on environmental conditions. In the light, metabolic cooperativity between several organelles is necessary for plant survival due to the process of photorespiration. Here, noxious glycolate produced by the oxygenase activity of Ribulose-1,5-bisphosphat carboxylase/oxygenase (RuBisCO) is recycled via eight enzymes partitioned between peroxisomes, mitochondria and chloroplasts.The established knowledge on the biochemical level of photorespiration faces a lack of knowledge regarding the function of spatial organelle organisation and the formation of specific membrane contacts sites (MCS). Whereas close associations between chloroplasts, mitochondria and peroxisomes have been monitored microscopically since decades, their molecular structure and their relevance for plant metabolism and performance remains unknown.Here we investigate the hypothesis that MCSs in plants underpin metabolic remodelling and provide the basis for reorganising the cell to respond effectively to changing environmental conditions. To address the question of specific and dynamic MCS formations between the photorespiratory organelles, we establish a set of genetically-encoded in vivo proximity biosensors, based on Bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer/ fluorescence lifetime imaging (FRET/FLIM), respectively. In addition, to address the molecular identity and function of MCS-resident proteins, we created a set of candidate proteins that will be expanded by labelling MCS-resident proteins using a promiscuous biotin ligase construct (BioID technique). By designing synthetic tethering constructs for organelle pairs, as well as by investigating mutant plant lines of candidate MCS-resident proteins, we will perturb MCS formation. In order to quantify the biological relevance of specific MCSs, we use plant growth phenotypes, photorespiratory capacity and metabolites, as well as photosynthetic parameters as a readout.Our results will solve the long-standing question of formation and physiological relevance of MCSs regarding photorespiration and plant performance during light/dark transitions. We will define were organelle proximity is based on specific association rather than spatial constraints. We anticipate our work to create links between cell biology, pathway partitioning between organelles, and plant metabolic plasticity. Furthermore, as photorespiration is a major target to understand and optimize photosynthetic plant performance, we expect our work to be relevant for future biotechnological approaches.