Phosphorylation events controlling low oxygen-signaling in Arabidopsis thaliana

暴露在低氧条件下的植物，例如由淹水引起的，表现出广泛的新陈代谢重新编程。首先，有氧呼吸(使用分子氧)切换到无氧代谢，这仍然确保以ATP形式产生能量当量，但效率较低。低氧适应特异性基因的诱导受乙烯反应因子(ERF)家族转录因子的调控，其中第VII组ERF在这一过程中起着至关重要的作用。它们的功能受限于低氧，因为它们依赖于氧从质膜上定位的酰基COA结合蛋白(ACBPS)解离，然后转位到细胞核。我们已经证明，激活的脂肪酸酰基-COA触发ERFVII的转位，并表明细胞酰基-CoA池的某些变化引发低氧反应。然而，由于低氧诱导的酰基辅酶A池的变化需要长达三个小时的时间，并且与低氧下观察到的基因诱导相比，在空气中应用酰基辅酶A只能引起轻微的反应，因此独立于酰基辅酶A的第二个平行触发器对于诱导ERFVII的移位和调节ERFVII的活性是必不可少的。本研究的目的是揭示磷酸化在(1)诱导ERFVII移位到细胞核中和(2)以氧依赖的方式调节ERFVII的活性中的作用。通过探索磷酸化在依赖ERFVII的低氧信号中的影响，我们将极大地扩展我们对低氧下多种信号整合的理解，ACBP和ERFVII是中心界面。我们的初步工作使用体外激酶活性分析、体内相互作用研究、植物磷酸蛋白质组分析和反式激活分析表明，依赖ERFVII的信号级联的组成部分的磷酸化在这两个过程中都是重要的。此外，我们还发现，低氧促进了植物中ACBPS的磷酸化，并可以识别特定的低氧激活的丝裂原活化蛋白激酶(MPK)，这些蛋白激酶针对ERFVII信号级联的几个组成部分。我们打算研究这些信号元件的磷酸化状态究竟如何影响它们相互作用的能力、它们的活性以及它们在植物低氧耐受性中的作用。此外，我们还想研究在什么氧浓度下通过MPKs在植物中发生磷酸化。由于这些MPK本身被低氧下的活性氧物种(ROS)激活，我们将调查哪些细胞内ROS来源在ERFVII信号级联的诱导中起作用。为此，我们将进行分子生物学、生化和遗传策略。

Plants exposed to low oxygen conditions, e.g. caused by flooding, show extensive reprogramming of their metabolism. First of all, aerobic respiration (using molecular oxygen) switches to anaerobic metabolism, which still ensures the production of energy equivalents in the form of ATP but with less efficiency. The induction of genes specific to hypoxic adaptation is under the control of transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family, of which group VII ERFs play a crucial role in this process. Their function is restricted to hypoxia by their oxygen-dependent dissociation from plasma membrane-localized ACYL-COA-BINDING PROTEINS (ACBPs) and subsequent translocation to the nucleus.We already demonstrated that acyl-CoAs, activated fatty acids, trigger translocation of ERFVIIs and showed that certain changes in the cellular acyl-CoA pool induce hypoxic responses. However, because hypoxia-induced changes of the acyl-CoA pool take up to three hours to occur, and acyl-CoA application can induce only mild responses in air as compared to gene induction observed under hypoxia, a second, parallel trigger operating independently of acyl-CoAs is essential for inducing translocation of ERFVIIs and modulating ERFVII activity.The aim of this proposal is to unravel the role of phosphorylation in (1) inducing translocation of ERFVIIs into the nucleus and (2) modulating activity of ERFVIIs in an oxygen-dependent manner. By exploring the impact of phosphorylation in ERFVII-dependent hypoxia signaling we will greatly extend our understanding of the integration of multiple signals under hypoxia with ACBP and ERFVIIs being the central interface.Our preliminary work using in vitro kinase activity assays, in vivo interaction studies, in planta phosphoproteomic analyses and transactivation assays indicates that phosphorylation of components of the ERFVII-dependent signaling cascade is important for both processes. Furthermore, we showed that low oxygen promotes phosphorylation of ACBPs in planta and could identify specific low oxygen-activated MITOGEN-ACTIVATED PROTEIN KINASES (MPKs), which target several components of the ERFVII signaling cascade. We intend to investigate how exactly the phosphorylation status of the signaling components impacts on their ability to interact, their activity and their contribution to low oxygen tolerance in planta. In addition, we want to study under which oxygen concentrations phosphorylation via MPKs occurs in planta. As these MPKs themselves are activated by reactive oxygen species (ROS) under low oxygen, we will investigate to which extend intracellular ROS sources contribute to the induction of the ERFVII signaling cascade. To this aim, we will conduct molecular biological, biochemical as well as genetic strategies.