Erickson 9316915 Photosynthetic organisms capable of converting light energy into chemical energy are the primary source of renewable biomass on earth. In plants and eukaryotic algae, this conversion takes place in the chloroplast. The photosynthetic reaction centers, pigment-protein complexes found in the chloroplast thylakoid membrane, harness light energy to drive the photosynthetic electron transfer chain. The goal of this proposal is to study how the structure of the core D1 reaction center polypeptide of the photosystem ll (PSII) reaction center affects chloroplast PSII assembly/stability, and PSII function including the photooxidation of water to molecular oxygen. PSII, and the oxygen-evolving complex in particular, is labile under conditions of environmental stress, contributing to loss of productivity in the field. An increased understanding of how chloroplast PSII function is regulated in response to environmental cues may provide a basis for improving crop yields. This proposal describes a new approach to studying structure-function relationships of the chloroplast PSII of a eukaryotic organism. Random mutagenesis targeted in vitro to specific sub-regions of the D1 chloroplast gene and subsequent co-transformation of the chloroplast genome of the green alga Chlamydomonas reinhardtii with the mutagenized gene and a selectable drug-resistance marker gene will be followed by a classical genetic screen of drug-resistant transformants for those exhibiting an aberrant fluorescence phenotype. The genetic screen relies on measurements of chlorophyll fluorescence which provide a rapid and sensitive assay of PSII function. Hence, this method will allow for the ready identification of all D1 amino acid residues in each targeted region which, when altered, affect PSII function as monitored by chlorophyll fluorescence. The regions targeted will include sequences coding for amino acid residues found in a) the fifth transmembrane helix through to the COOH terminus, b) the lumenal loop between the first and second transmembrane helices, and c) the lumenal loop between the third and fourth transmembrane helices. The use of random mutagenesis in select regions of the D1 gene followed by a phenotypic screen of resulting transformant algae should provide a major advance in this field. By letting the transformed algae identify (through expression of an observable altered phenotype) which amino acid residues are important for function, the laborintensive work involved in a classic site-directed mutagenesis approach is avoided, as is the prejudicial selection of specific residues as site-directed targets. Detailed molecular, biochemical and biophysical analysis of resulting transformants of interest will provide a correlation between D1 structure and PSII stability and function. These studies should yield significant insight into a biochemical process crucial to our ecosystem. Results of these investigations may ultimately allow for the educated design and manipulation of macromolecules of biological relevance to agricultural productivity. Finally, the light-driven photosynthetic electron transfer reactions studied here are absolutely essential for the production of renewable biomass on earth which is used as food, fiber and fuel. %%% Life on earth as we know it is sustained by the important chemical process known as photosynthesis. Using this process, plants, algae and some bacteria are able to live on water, minerals and air, receiving the energy they need from sunlight. The capture of light energy by plants and algae takes place within the cell in a structure called the chloroplast. Inside the chloroplast, specific proteins assemble with each other and with cofactor molecules to form a large membrane-associated complex called Photosystem ll (PSII) which carries out the first steps of photosynthesis. In these first steps, light energy is stored up and then used to break apart water molecules, producing oxygen. When the water molecules are split, electrons are removed from water and are then transferred, in an orderly fashion, through a series of other molecules which are in the correct position to receive and pass on the electrons. It is ultimately this carefully coordinated electron transfer chain that produces the chemical energy that the plant cell needs to survive. However, in order for the electron transfer chain to work properly, the electron acceptor molecules must be lined up in the proper order and at the correct distance from each other. We want to find out where these acceptor molecules are located within the protein complex, and how they assemble with the different protein subunits to form the functional PSII complex. These questions are of particular importance because under certain conditions of environmental stress, including strong light, PSII becomes damaged and plants are unable to photosynthesize. This can result in reduced crop yield and in some cases plant death. One particular subunit of the PSII protein, called the D1 subunit, appears to be the part of PSII that is specifically damaged and subsequently degraded. Without Dl, PSllis unstable. Without PSII, plants can not survive. Because of its central role in PSII assembly, function and stability, the Dl subunit will be the focus of our studies. Our approach to studying how D1 functions in PSII will involve the molecular manipulation of the DNA molecules which carry the information dictating the structure of the Dl protein subunit. We are using a unicellular green alga as a model experimental system for our studies because it contains a single large chloroplast, and we are able to introduce DNA into the chloroplast of living algal cells. Thus, we will mutate the DNA sequences which code for D1, put the mutant DNA back into the chloroplast of cells to produce mutant algae, and then see what the mutants do. A video imaging system that records the fluorescence light emitted by algal cells will be used to look at thousands of m utant algae in a very short period of time. Because the fluorescence signal emitted by the algae comes from PSII, this system will allow us to identify mutants with defects in PSII function. Once these mutants are identified, we will characterize them in detail to find out what changes are present in the mutant D1 subunit, and exactly how PSII function is different. We particularly want to find out whether PSII can assemble, whether the electron acceptor molecules are correctly positioned, and how quickly PSII is damaged in the mutant algae. In this way, we will identify regions of the D1 subunit that are critical for PSII function and that affect PSII stability. The long-term goal of our studies is to understand in detail how PSII works so that it might be possible to produce plants which are more resistant to PSII damage under environmental conditions, more active in photosynthesis, and hence more productive. ***
