Role of protein phosphorylation in the maintenance of photosystem two in plants

The photosystem two (PSII) protein complex is widely considered to be one of the most remarkable molecular machines on Earth. PSII is found in plants, algae and cyanobacteria and performs the complex task of using sunlight to extract electrons from highly stable water molecules to allow oxygenic photosynthetic organisms to grow. At the same time PSII also produces the oxygen that we breathe. Unfortunately PSII is not a perfect machine; it sometimes breaks down, especially when the sunlight is very bright, and has to be repaired. Without this special repair mechanism PSII would be quickly inactivated in the light and plant growth and oxygen evolution would be inhibited. Despite the physiological importance of PSII repair, the details of the repair process are still unknown. A detailed understanding of PSII repair at the molecular level will provide us with important knowledge to help in the global effort to enhance photosynthesis in crop plants so that we can increase growth to help satisfy the ever increasing demand for more food and more biomass. In the case of land plants, we know that active PSII is located within the thylakoid membrane system of green chloroplasts found in leaves, but is segregated within the characteristic stacked regions of the membrane known as grana. For repair, the damaged PSII complex migrates outwards to the unstacked regions of the thylakoid system where the repair machinery is located. Here damaged PSII subunits can be degraded, newly synthesised subunits inserted and the PSII complex reactivated. How damaged PSII is specifically shuttled to the repair apparatus and how damaged subunits are specifically recognised for replacement is currently unknown. Over the years evidence has accumulated to suggest that the presence of negatively charged phosphate groups on four PSII core subunits might play a role in the repair process and in reshaping the membrane system in bright light to enhance repair. These previous studies have relied on the analysis of mutants that lack the kinase enzymes that phosphorylate PSII. However, it is now clear that these kinase mutants have effects outside PSII so it is still uncertain whether the specific lack of PSII phosphorylation is responsible for all the effects seen in the kinase mutants. In addition the kinase mutants block all PSII phosphorylation which has prevented analysis of the specific role of single subunit phosphorylation. In this application we propose to use a new approach to examine the role of protein phosphorylation in maintaining PSII activity in land plants. Rather than study kinase mutants, we will use chloroplast transformation technology to make tobacco plants that lack the amino-acid residue in each subunit that is normally phosphorylated. The kinase enzyme will still be active in these plants and so the effect of removing just one specific phosphate group from PSII can now be studied in isolation. In background work we have shown that this is a feasible strategy as we have already made tobacco plants unable to phosphorylate the D1 protein. Effects on the damage to PSII, its disassembly, its migration within the thylakoid membrane, the proteolytic degradation of damaged proteins, the reassembly of PSII and the impact on the structure of grana and plant growth under various illumination conditions will be performed use state-of-the-art approaches by a team of researchers with proven expertise in this area. We will study the maintenance of PSII in mutant plants lacking each of the four phosphorylation sites and also in engineered plants in which we remove increasing numbers of the phosphorylation sites to test for overlap of function. Overall our research will provide important new information on how the oxygen-evolving complex of photosynthesis is maintained in land plants and how the structure of the thylakoid membrane system is regulated.

The role of photosystem II core protein phosphorylation in the PSII repair cycle and in the folding of the thylakoid membranes in higher plant chloroplasts is still unclear. In this application we will study single and multiple phosphorylation site mutants of tobacco generated through chloroplast transformation. In background work we have constructed D1 mutants in which the phosphorylated threonine residue at position 2 has been replaced by either alanine, serine or aspartate. Objective 1 will use well established biochemical and spectroscopy assays to investigate the impact of these mutations on photosynthetic electron flow and PSII activity, the assembly status of PSII and different aspects of PSII repair including the rate of photodamage, rates of assembly and disassembly of PSII supercomplexes and rates of D1 synthesis and degradation. Various light and temperature regimes will be used to test for growth defects. Objective 2 will assess the impact of the D1-T2 mutations on thylakoid membrane structure using transmission electron microscopy combined with freeze-fracture and cryo-scanning electron microscopy. In this way a possible link to granal stacking will be tested. Fluoresence recovery after photobleaching will be used to determine changes in the migration of chlorophyll proteins in the thylakoid. Objective 3 will generate chloroplast mutants in which the single phosphorylation sites present in D2 and CP43 and the two found in PsbH are mutated. The assays used in Objectives 1 and 2 will be used to rigorously characterise the phenotypes of the mutants and in particular the possible role of PsbH phosphorylation in regulating electron transfer in PSII and CP43 in remodelling PSII supercomplexes. To test for overlap of function, Objective 5 will use Cre/loxP marker excision technology to ultimately generate a mutant lacking all 5 PSII phosphorylation sites plus mutants containing various combinations of phosphorylation sites.

Understanding the details of the repair of the oxygen-evolving complex of photosynthesis might in the long-term lead to the development of microalgae and plants with more robust and more efficient photosynthesis. In the agricultural sector, beneficiaries could include: companies involved in modifying or selecting plants to maintain and or improve crop yields under stress conditions either imposed by the changing environment or to exploit less fertile land; farmers who wish to develop new practices for similar reasons; governments and policy-makers interested in developing novel strategies to achieve food security; and the public who will benefit from food security. In the bioenergy sector, beneficiaries include: companies wishing to develop alternatives to fossil fuels; governments and policy makers who are interested in new routes to energy security and for new energy sources for developing countries; the military who are looking for alternative fuels for specific and niche uses; environmentalists who need to focus on rational long-term alternatives to fossil fuels. In the environmental and ecological sector, beneficiaries include those wishing to understand and mitigate loss of photosynthetic species or productivity in a changing environment. These include governments and policy makers, the tourism sector (coral reef bleaching) and fisheries (the loss of photosynthetic microbes at the start of the food chain).

Our research will also investigate at a fundamental level how proteins are assembled and disassembled in the thylakoid membrane and the factors controlling the stacking of membranes. Ultimately this knowledge will benefit those in the biotechnology sector who wish to develop microalage and other related photosynthetic organisms as solar biorefineries for the sustainable production of green chemicals and high-value products.

In the education sector, in museums and in the media, there will be benefits from publicising new advances in one of the most fundamental biological processes and one that has been taught at secondary school and so readily familiar to the general public.

Staff hired for the project will obtain training in cutting edge research in world-leading research centres. Peter Nixon is a member of the Photosynthesis Research Lab at Imperial College which includes several world leading experts in Photosynthesis including Bill Rutherford, Jim Barber, James Murray and Jasper van Thor, with expertise ranging from femtosecond spectroscopy to biofuel production through metabolic engineering of photosynthetic microbes.

The link with QMUL will further enhance the multidisciplinary training environment provided by the project, with expertise available in confocal and electron microscopy (Mullineaux) and computational modelling (Duffy). Here the project will provide cutting-edge training in techniques that are very widely applicable in the life sciences.
The project will benefit the professional career development of the technician working in Lawson's lab at Essex through training in a wide range of plant physiology skills, which has been recognized as skills shortage area by BBSRC. Many of the skills, such as data handling and analysis, independent planning and communication of results are transferable to other employment sectors. They will have experience in working in research intensive university environment with links to the commercial sector.

They will have the benefit of the excellent intellectual environment of a leading university with a tradition of close ties with engineers and industry.