Signaling pathways involved in the phosphorylation of membrane proteins in response to N and C

氮是植物生长所必需的大量营养元素。此外，氮还通过触发中枢代谢和根发育中的特定适应而具有信号功能。虽然硝酸盐通过质膜转运系统的基本摄取机制是众所周知的，并且过去已经研究了氮有效性变化的全球转录反应，但硝酸盐摄取调控的详细机制以及转录上游的信号转导事件仍然很少被揭示。我们的工作将致力于通过研究硝酸盐转运蛋白NRT2.1和硝酸盐反应蛋白激酶的调控来缩小这一差距。磷酸化是研究最多的具有调控功能的翻译后修饰。它可以诱导蛋白质活性的变化，为蛋白质相互作用提供对接位点或诱导亚细胞位置的变化。硝酸盐转运体在过去被证明是受蛋白质磷酸化影响的，特别是NRT1.1，蛋白质磷酸化在转运亲和开关中的作用已经被很好地描述。另外，对于铵转运体，近年来阐明了磷酸化依赖的激活/失活机制。因此，研究蛋白磷酸化在硝酸盐主要转运体NRT2.1调控中的作用是本项目的重点。在过去的几年里，总的情况已经出现，NO3-和NH4+转运蛋白都是通过翻译后修饰在蛋白质水平上控制的，以确保在质膜上直接对高度波动的环境条件做出快速反应。因此，该合作项目的科学目标是(i)表征响应C和N可用性的与N代谢相关的靶蛋白的条件依赖性磷酸化。它将涉及鉴定新的C/N调节蛋白以及已知NRT2.1磷酸化位点的作用的具体特征。该项目旨在（ii）以NRT2.1和选定的氮代谢相关蛋白为靶点，寻找候选蛋白激酶和/或磷酸酶；（iii）在体内表征感兴趣的蛋白激酶和/或磷酸酶对NRT2.1和选定的氮代谢相关蛋白磷酸化的影响。我们还将开始研究与选定的蛋白激酶和/或磷酸酶相互作用的蛋白作为诱饵。我们相信，通过双方合作团队的专业知识互补，并明确关注NRT2.1在蛋白质水平上的调节，我们将在植物的关键功能，即氮吸收的调节方面获得新的有价值的见解。

Nitrogen is an essential macronutrient for plant growth. In addition, nitrogen also has signaling functions by triggering specific adaptations in central metabolism and root development. While basic uptake mechanisms for nitrate through plasma membrane located transport systems are well known and global transcriptional responses to changes in nitrogen availability have been studied in the past, the detailed mechanisms in regulation of nitrate uptake, as well as signal transduction events upstream of transcription are still barely revealed. Our work will aim at closing this gap by particularly focusing on studying the regulation of nitrate transporter NRT2.1 and nitrate responsive protein kinases. Phosphorylation is the most well studied post-translational modification with regulatory function. It can induce changes in protein activity, provide docking sites for protein-protein interaction or induce changes in subcellular location. Nitrate transporters were in the past shown to be subject to protein phosphorylation, and especially for NRT1.1 the role of protein phosphorylation in switching of transport affinity has been well described. Also for ammonium transporters, a phosphorylation-dependent activation/inactivation mechanism was elucidated in recent years. Therefore, a particular focus of this project is to study the role of protein phosphorylation for the regulation of the major nitrate transporter NRT2.1.Within the last couple of years, the general picture has emerged that both NO3- and NH4+ transporters are controlled at the protein level by posttranslational modification to ensure fast responses directly at the plasma membrane to highly fluctuating environmental conditions. Therefore, the scientific aim of this collaborative project is (i) to characterize the condition-dependent phosphorylation of target proteins related to N metabolism in response to C and N availability. It will involve both the identification novel C/N regulated proteins as well as the specific characterisation of the role of the known NRT2.1 phosphorylation sites. The project aim at (ii) finding candidate protein kinases and/or phosphatases using both NRT2.1 and selected nitrogen-metabolism related proteins as targets and (iii) characterising in vivo the impact of the interesting protein kinases and/or phosphatases on the phosphorylation of NRT2.1 and selected nitrogen metabolism related proteins. We will also start to investigate the proteins interacting with the selected protein kinases and/or phosphatases as baits. We are convinced that with the complementary expertise of both partner groups and with a clear focus on NRT2.1 regulation on protein level, we will gain novel valuable insights into a crucial plant function, namely the regulation of nitrogen uptake.