Defining the scope and components of ubiquitin-dependent chloroplast-associated protein degradation

The human population is growing rapidly and set to exceed 9bn by 2050. This presents significant challenges to food security, and places ever increasing pressure on natural resources. Thus, the drivers for increased crop yields with resilience to sub-optimal growing conditions are stronger than ever. To meet these demands it will be essential to develop improved crops. Through research on the model plant thale cress, we recently made some significant breakthroughs: We discovered a new regulatory process, named "CHLORAD", that controls vital aspects of plant growth, including plant responses to environmental stresses like drought and salinity. Significantly, modifying CHLORAD activity makes plants more tolerant of such stresses. In this project, we will define the molecular targets and mechanisms of CHLORAD, and in so doing develop a better understanding of how it can be used to deliver novel crop improvement strategies.

CHLORAD (which stands for "chloroplast-associated protein degradation") regulates the development and operation of structures inside plant cells called chloroplasts, which are normal cellular constituents (i.e., organelles). They define plants, contain the green pigment chlorophyll, and are responsible for photosynthesis, harnessing 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. Chloroplasts also have critical roles in plant responses to stress, and so are ideal targets for engineering resilient crops.

Chloroplasts are composed of thousands of different proteins, most of which are encoded by genes in the cell nucleus and so are made outside of the organelle in the cellular matrix known as the cytosol. As chloroplasts are surrounded by a double-membrane "envelope", sophisticated machinery is needed to enable the import of 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.

Currently, CHLORAD is known to act on the TOC machinery, breaking up its constituent proteins in order to control which other proteins are imported into the organelle (this in turn influences organelle development and operation). Known components of the CHLORAD machinery are proteins called SP1, SP2 and CDC48. The first, SP1, is a "ubiquitin E3 ligase" that labels-up unwanted proteins to target them for removal. The SP2 protein forms a channel in the chloroplast outer membrane, providing the exit route for removal of proteins labelled by SP1. Lastly, CDC48 is a molecular motor that drives extraction of the unwanted to the cytosol, where they are then broken down.

Our unpublished results have revealed that additional proteins (or "cofactors") work together with CDC48 in CHLORAD. We believe that these cofactors are required for the docking of CDC48 onto the unwanted TOC proteins, and for the subsequent release of those proteins from CDC48. Here, we will study these cofactors in detail, to better understand how unwanted chloroplast proteins are removed in CHLORAD. Furthermore, we have additional new results showing that CHLORAD acts on a much larger number of target proteins than previously envisaged (i.e., not just TOC proteins), including proteins of the chloroplast interior. We will systematically identify these novel targets, and seek to understand how they are processed by CHLORAD even when deep inside the organelle. Informed by information on the identity of the targets, we will also explore the broader physiological significance of CHLORAD, for plant growth and development.

Overall, the knowledge gained will enhance our understanding of CHLORAD, and will be important for the development of crops with improved chloroplast performance.

We discovered a novel pathway designated chloroplast-associated protein degradation (CHLORAD) (Science, 2019). In CHLORAD, substrates pass through several steps: ubiquitination by SP1 (Science, 2012); and retrotranslocation by SP2 and CDC48 (Science, 2019). Unpublished data show that CHLORAD acts on a greater diversity of targets than previously envisaged, including proteins of the organelle interior; and that it involves several uncharacterized CDC48 cofactors. Thus, we will define the full target range of CHLORAD (1, 2), and study these cofactors in detail (3, 4), in Arabidopsis. We will:

1. Characterize the chloroplast ubiquitinome. PTMScan Technology and LC-MS/MS will be used to define the chloroplast ubiquitinome, in two physiological contexts (steady-state; abiotic stress). And, we will use tagged, chloroplast-localized ubiquitin to test whether ubiquitination may occur inside chloroplasts.

2. Identify and analyse new targets of CHLORAD. Proteomic analysis of CHLORAD-defective mutants will be used to identify new targets, building on our unpublished data. Putative targets that are also identified in 1 above will be studied in detail to confirm that they are bona fide CHLORAD substrates. Informed by the data, we will explore the broader physiological significance of CHLORAD, for chloroplast biogenesis and metabolism.

3. Functionally define CDC48 adapter complexes involved in CHLORAD. The chloroplast-association and interactions (with each other, and CDC48) of the adapters will be defined. And, their functions in CHLORAD will be studied by assessing their biochemical properties, and their importance for chloroplast biogenesis and target retrotranslocation in vivo.

4. Identify and functionally define deubiquitinases (DUBs) involved in CHLORAD. Candidates will be identified pharmacologically, and their participation in CHLORAD will be studied by conducting interaction tests (with known CHLORAD components and targets), and genetic and functional assays.