Defining the role of SUMO in regulating chloroplast biogenesis and functions

Our latest results (eLife, 2021) reveal that the SUMO system plays an important role in chloroplast biogenesis, by acting directly on TOC proteins to negatively regulate their abundance, and potentially other chloroplast-resident proteins too. Here, we will unveil the molecular mechanisms and functional significance of such regulation, and define the chloroplast SUMOylome in detail, in Arabidopsis. Specifically, we will: 1. Determine which E3 SUMO ligase(s) act on TOC proteins. This will be an important step in defining the TOC-SUMOylation pathway, and will facilitate downstream experiments. 2. Establish the functional significance of TOC SUMOylation. Building on our genetic data that already demonstrated that TOC SUMOylation is functionally important, we will more deeply define the role of TOC SUMOylation, focusing in two critical areas: chloroplast protein import and abiotic stress tolerance, and the relationship between them. 3. Systematically characterize the chloroplast SUMOylome. We will use two complementary strategies ("three-step purification"; PTMScan Technology) to enrich SUMO conjugates from chloroplast protein samples; then analyse the samples by LC-MS/MS to define the chloroplast SUMOylome. We will do this under steady-state and abiotic stress conditions; and SUMO modification sites will be verified. 4. Determine the effects of SUMOylation on chloroplast proteostasis. We will assess the effects of SUMO on the steady-state levels and turnover of selected targets from Obj. 3. Then, UPS involvement in such protein turnover will be assessed in vivo; we will determine whether SUMOylation promotes the ubiquitination and UPS-mediated degradation of the selected targets. 5. Explore the interplay between SUMOylation and CHLORAD. The functional relationship between the SUMO and CHLORAD systems will be investigated using genetic and biochemical experiments. And, the possible involvement of a new type of ubiquitin ligase will be investigated.