Role of the chloroplast ubiquitin E3 ligase SP1 in abiotic stress tolerance in plants

With the human population growing rapidly and set to reach 9 billion by 2050, and because of ever increasing pressure on natural resources, the drivers for increased crop yields and for resilience to climate change and sub-optimal growing conditions are stronger than ever. To meet these demands it will be essential to develop improved crop varieties. Through research on the model plant thale cress (Arabidopsis thaliana), we recently made a significant breakthrough that may have important implications for food security: We discovered a gene called SP1 that controls important aspects of plant growth, and found it to be important in plant responses to adverse environmental conditions such as water stress and high salinity (collectively, abiotic stresses). By modifying SP1 expression, thale cress plants can be made more tolerant of such stresses. In this project, we will study the SP1 gene to elucidate how it is involved in stress responses, and investigate its potential use for crop improvement by conducting studies in wheat.

The SP1 gene regulates the development of structures inside plant cells called chloroplasts. Chloroplasts are normal cellular constituents (i.e., they are organelles), and in many ways they define plants. They contain the green pigment chlorophyll and are responsible for photosynthesis - the vital process that captures sunlight energy and uses it to power the activities of the cell, for example by converting carbon dioxide from the air into sugars. 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. But photosynthesis also has the potential to generate toxic "reactive oxygen species" (ROS), particularly when conditions are challenging, and so chloroplasts have a critical role in stress responses too.

Although chloroplasts do contain DNA (a relic of their evolutionary origins as free-living photosynthetic bacteria) and so can make some of their own proteins, most of the thousands of different proteins needed to form a chloroplast are encoded by genes in the cell nucleus. These nucleus-encoded proteins are made outside of the chloroplast in the cellular matrix known as the cytosol. As chloroplasts are each surrounded by a double membrane envelope, they have evolved sophisticated protein import machinery that drives the uptake of proteins from the cytosol. This machinery comprises two molecular machines: one in the outer envelope membrane called TOC (an abbreviation of "Translocon at the outer membrane of chloroplasts") and another in the inner membrane called TIC. Each machine is composed of several different proteins that cooperate during import.

The SP1 gene encodes a type of regulatory factor called a "ubiquitin E3 ligase". Such regulators work by labelling-up unwanted proteins to target them for removal. The SP1 E3 ligase specifically acts on components of the TOC machinery, thereby controlling TOC composition and function so that only the desired proteins are imported. Such control is important when chloroplasts must undergo major functional changes, for example during adaptation to stress. We believe that SP1 acts during stress to limit the import of new components of the photosynthetic apparatus, in order to attenuate photosynthetic activity and so mitigate the negative effects of stress. By limiting photosynthesis during stress, SP1 reduces the potential for ROS overproduction such that plants are less likely to suffer serious or fatal stress-related damage.

Knowledge gained during this project will improve our understanding of plant responses to adverse environments, and may enable improved resilience of crops to such conditions. Drought and salinity are among the most significant factors affecting crop yields, with annual global crop losses due to drought alone estimated at $10bn. We believe that our work with SP1 may help to alleviate such losses.

SP1 is a ubiquitin E3 ligase in the plastid outer envelope. It regulates plastid protein import via ubiquitination and degradation of import machinery (TOC) components, and thus controls the proteome, biogenesis and functions of plastids. Unpublished data reveal a role for SP1 in abiotic stress tolerance, via a novel protein import control pathway that attenuates photosynthesis to avoid overproduction of reactive oxygen species (ROS). We will elucidate the mechanisms underlying SP1-mediated stress tolerance, using Arabidopsis (Objectives 1-4) and wheat (Objective 5) as models:

1. We will explore the regulatory mechanisms governing SP1 activation in stress, focusing on phosphorylation of its target TOC proteins. We will use phosphoproteomics to assess for changes in TOC modification under stress, and functionally assess the phosphorylation sites. In parallel LC-MS/MS work we will study TOC ubiquitination patterns and seek stress-dependent functional partners of SP1.

2. We will systematically study SP1's effect on the chloroplast proteome under stress using quantitative proteomics. We will assess whether SP1's role in stress is mediated by photosynthetic attenuation only, or additionally involves ROS detoxification.

3. We will explore the possibility that SP1, in addition to import regulation, has a complementary role in chloroplast protein autophagy in stress. We will use in vivo markers for autophagosomes and plastid-linked autophagic bodies to assess SP1's role in initiating chloroplast autophagy in stress.

4. We will assess for a role of SP1 homologue, SPL2, in stress. We will analyse plants with altered SPL2 expression in physiological and molecular studies, focusing on plant growth, ROS levels, TOC protein levels and protein import.

5. We will assess the role of SP1 in stress tolerance in wheat. We will analyse plants with altered SP1 expression for their stress responses during vegetative and reproductive growth, with a particular focus on yield.

Beneficiaries will include: (1) commercial stakeholders in agriculture; (2) the wider public and government; and (3) the academic community and research staff employed by the project. How these individuals will engage with and benefit from the research is summarized below.

1. Commercial stakeholders in agriculture. Abiotic stresses have major adverse effects on crop yields: annual global crop losses due to drought alone are estimated at $10bn. Manipulating SP1 expression improves stress tolerance and so could mitigate such losses, and may do so without compromising growth under normal conditions. Thus, SP1 has considerable potential as a technology and it is our goal to ensure that this is fully realized. The drivers for increased crop yields and resilience to climate change and sub-optimal growing conditions are stronger than ever, due to human population growth and pressure on natural resources. As well as offering more efficient food production in the UK and other developed agricultural economies, translation of our work into crops may also bring public good benefits to food production in developing countries by enhancing subsistence agriculture.
Current IP associated with SP1 is protected by a patent application and is licensed to PBL who are promoting the technology globally. New IP generated during the project will be similarly protected and, with the assistance of PBL and Isis Innovation (the University's technology transfer company), we will work to promote uptake of the SP1 technology by the agbiotech industry (several major companies have already expressed an interest). We will seek Follow-on Funding to facilitate development and commercialization of the technology at the end of year 2.

2. Wider public and government. Scientific information has enriching and educational quality of life benefits for society. Thus, we will work in partnership with the Oxford Botanic Garden and Harcourt Arboretum, Oxford Natural History Museum, and the Oxford Sparks online resource, which are all excellent avenues for science-related outreach, to develop and deliver a range of innovative, high-quality engagement activities and educational resources centred on the themes of the project. These activities will not only inform and educate the public, as mentioned, but will also benefit the aforementioned partner organizations by promoting their bilateral engagement with the academic community and public.
Through publications and associated press releases and media coverage, and via our attendance at the SET for Britain event attended by Members of both Houses of Parliament at Westminster, we will engage government. Opportunities for political dialogue that arise through the Oxford Martin Programme on the Future of Food will also be exploited. Our aim will be to highlight the importance of scientific research and plant biotechnology in relation to major societal challenges such as food security and climate change, and to influence policy in a positive way.

3. Academic community and research staff. Academic impact will be large due to the work's interdisciplinarity and fundamental significance, as detailed in the Academic Beneficiaries section. This will manifest itself in several ways: a) The work will provide new knowledge with relevance in several overlapping fields and disciplines. b) The project will contribute to the health of UK plant science by generating publicity, fostering interactions and enabling engagement activities designed to stimulate enthusiasm for plant biology among school students and teachers. c) The research staff will receive advanced training in bioscience research, further contributing to the health of UK plant science, reinforcing the UK's position as a leading country for academic research, and aiding transition to a Knowledge Based Bio-Economy. Training will also result from our supervision of graduate students with related projects, who will have daily interaction with the PI and research staff.