Award details

Defining the role of SUMO in regulating chloroplast biogenesis and functions

Reference	BB/W015021/1
Principal Investigator / Supervisor	Professor Paul Jarvis
Co-Investigators / Co-Supervisors	Dr Yi Sun
Institution	University of Oxford
Department	Biology
Funding type	Research
Value (£)	641,662
Status	Current
Type	Research Grant
Start date	06/02/2023
End date	05/02/2026
Duration	36 months

Abstract

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.

Summary

The human population is set to exceed 9bn by 2050, presenting significant challenges to food security and placing ever increasing pressure on natural resources. Thus, the need for increased crop yields with resilience to sub-optimal growing conditions is stronger than ever. To meet these demands it will be essential to develop improved varieties of our staple crops. Through research on the model plant thale cress, it is well established that the extent of protein modification by "SUMO" (which stands for "small ubiquitin-like modifier") increases in response to different abiotic stresses, including high salinity, high temperature, freezing, drought, and oxidative and heavy metal stresses; and that such "SUMOylation" is a vital aspect of plant stress responses. Previous studies identified over a thousand SUMO targets in thale cress, most of which are located in a central cellular structure called the nucleus. However, our latest results show that SUMO also acts on different parts of the plant cell called chloroplasts. In this project, we will define how and why SUMO acts on chloroplasts, and in so doing understand how it may be used to deliver more resilient crops. Chloroplasts are the cellular constituents (or organelles) that define plants. They contain the green pigment chlorophyll, and are the site of photosynthesis - the process which harnesses sunlight energy to power the activities of the cell and the growth of the plant. As photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of huge importance, not just to plants but to all life on Earth. Moreover, chloroplasts have critical roles in plant responses to stress, and so they are ideal targets for crop improvement. Chloroplasts are composed of thousands of different proteins, most of which are encoded by genes in the cell nucleus and, therefore, are made outside of the organelle in the cellular matrix known as the cytosol. As chloroplasts are surrounded by adouble-membrane "envelope", sophisticated machinery is needed to import these proteins into the organelle; this comprises molecular machines in both membranes, called TOC (for "Translocon at the Outer membrane of Chloroplasts") and TIC. Each machine is composed of several proteins that work cooperatively to drive the import process. We recently made some significant breakthroughs in this area: We discovered that the constituent proteins of the TOC machinery are broken-down by a novel regulatory process named "CHLORAD" (which stands for "chloroplast-associated protein degradation"). In CHLORAD, unwanted TOC proteins are tagged with a protein modifier named ubiquitin, which targets them for removal and break-down. Thus, CHLORAD regulates the import of other proteins into the organelle, which in turn influences the development and operation of the organelle. Significantly, modifying CHLORAD activity makes plants more tolerant of stress. Now, we have new results revealing that the SUMO system also acts on chloroplasts. We believe that such SUMOylation destabilizes the TOC machinery to regulate protein import. In this project, we will study the mechanisms of SUMO-dependent chloroplast regulation in detail. We will define the SUMO pathway that acts on TOC proteins, and elucidate its physiological significance. Furthermore, inspired by our latest data suggesting that SUMO actually acts on a large number of chloroplast proteins, we will systematically identify the full range of SUMOylated chloroplast proteins, and study the effects of such SUMOylation. Lastly, we will investigate whether there is crosstalk between the SUMO and CHLORAD systems in TOC regulation. Together, our experiments will shed unprecedented new light on the mechanisms and significance of SUMO-dependent control of chloroplast functions and, in turn, plant development. This knowledge will be invaluable for the development of crops with improved chloroplast performance and stress resilience.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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