Genetic suppressors of Arabidopsis chloroplast protein import mutations

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Chloroplasts are normal components of plant cells (such sub-cellular components are called organelles) that in many ways resemble free-living bacteria. They contain the green pigment, chlorophyll, and are exclusively responsible for the reactions of photosynthesis (the process that enables the capture of sunlight energy and its use to make organic products). Since photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of inestimable importance, not just to plants but to all life on Earth. Chloroplasts are also important in many other ways, since they play important roles in the biosynthesis of lipids, amino acids and starch. Although chloroplasts do contain DNA (a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and are therefore able to encode some of their own proteins, >90% of the 3000 or so proteins required to build a fully functional chloroplast are encoded on DNA within the cell nucleus. The majority of chloroplast proteins are therefore synthesized outside of the chloroplast, in the cellular matrix known as the cytosol. Since chloroplasts are each surrounded by a double membrane, or envelope, that is impervious to the passive movement of proteins, this presents a significant problem. In order to overcome the problem, chloroplasts have evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol, across the envelope, and into the chloroplast interior. This protein import apparatus comprises two molecular machines: one in the outer envelope membrane called Toc (an abbreviation of 'Translocon at the outer envelope membrane of chloroplasts'), and one in the inner envelope membrane called Tic. Over the last decade, a great deal of progress has been made in our understanding of how this protein import apparatus works. In particular, it seems likely that most of the main components of the machinery have now been identified. Nevertheless, substantial gaps remain in our knowledge. For example, while it is known that the import process is regulated throughout plant development, very little is known about the mechanisms that underlie this regulation. To fill in these gaps in our knowledge, completely new experimental approaches will need to be employed. The experiments described in this proposal are an entirely new way of studying chloroplast protein import. In previous work, we identified plants carrying genetic mutations that affect the efficiency of protein import; what we propose to do here is to identify the genes affected by these mutations. Because chloroplasts carry out essential functions, and because protein import is essential for chloroplast development, it should come as no surprise to learn that plants with defective chloroplast protein import machinery are unable to survive beyond the embryo stage. Thus, chloroplast protein import is an essential process for plants. Similarly, since we are all ultimately dependent upon plant products for survival, it follows that chloroplast protein import is essential on a global scale. What is more, since chloroplasts play an instrumental role in the synthesis of many economically important products (such as lipids and starch), a more complete understanding of how these organelles develop may enable us to enhance the productivity of crop plants, or otherwise manipulate their products.