Analysis of singlet oxygen-mediated chloroplast-nucleus signaling in Arabidopsis
Final Report Abstract
Production of different ROS occurs continuously and simultaneously throughout the whole life cycle of plants. The major production sites are PS I and PS II in the chloroplasts capable of generating ROS levels that are lethal for the cell. Chloroplastic ROS, however, also exhibit a signaling function leading to a transcriptional reprogramming in the nucleus whereas the ROS themselves are unlikely to be the transmitted signal because of their short lifetime. To date, the nature of the signal(s) elicited in the chloroplast and altering transcription in the nucleus as well as the underlying mechanism are unknown. The present work aimed at the identification of the mechanisms underlying 1O2 signaling by using the conditional flu mutant in which EX1 is a crucial regulatory node of early 1O2 signaling events. Therefore exploring the function of EX1 has a high potential to unravel the molecular events upon 1O2 release. Initial approaches were seeking for interaction partners of EX1 and identified several proteins interacting with EX1. Those experiments were performed with the presumption that EX1 localizes to the stroma. In the course of the project it became apparent that EX1 is associated with the thylakoid membrane and therefore all previous finding had to be re‐evaluated. In addition, new experimental approaches had to be tested and optimized. After optimization of the IP conditions and solubilization of thylakoid membrane proteins the D1 and D2 proteins of the PS II were identified as interacting partners of EX1. Moreover, a mass spectrometry analysis of eluates derived from IPs is planned to identify possible other interaction partner of EX1. The D1 and D2 proteins are central components of the PS II core and serve as scavengers of 1O2. Once damaged by 1O2 the PS II core is translocated from the grana stacks to the grana lamella where it undergoes a repair cycle to replace the photo‐damaged D1 and D2 proteins. One hypothesis resulting from this observation is that EX1 could monitor the status of the D1 and D2 proteins. Once passing a certain damage threshold EX1 could trigger signaling events leading to an acclimation response. A possible mechanism could be the ratio of PS II in the grana stacks vs. grana lamella. Experiments to isolate PS II from grana stacks and lamella in order investigate a possible shift of EX1 upon 1O2 release are in preparation. EX1 possess two domains, an UVR B/C and a DUF3506 domain, that might facilitate protein‐protein interaction and/or might be tightly linked to the function of EX1. Preliminary experiments where domain deletion mutants of EX1 were transiently expressed in N. benth. did not show an abrogation of the interaction with D1 and D2. Also different pointmutations that have been introduced in the EX1 cDNA did not abolish the D1 and D2 interactions. However, stable transgenic lines in Arabidopsis flu mutants will be available soon and a set of experiments will be performed to investigate whether any of the pointmutations/domain deletions in EX1 have an impact on the flu phenotype and/or EX1 localization and protein interactions upon 1O2 release. These experiments should provide insight about how early 1O2 signaling is triggered and mediated from the chloroplasts to the nucleus.
