Genetic suppressors of Arabidopsis chloroplast protein import mutations

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.

To identify new loci involved in plastid protein import, or its regulation, we conducted screens for second-site suppressors of two fundamentally different protein import mutations, ppi1 and tic40. We identified five recessive suppressor of ppi1 (sp) mutations, at two loci, and ten suppressor of tic40 (stic) mutations (five recessive, five semi-dominant). These screens were done using previous BBSRC funding. We seek new support to take these projects forward: the proposed work is a logical continuation of the previous work. The sp1 locus maps to the bottom of chr 1. We will identify sp1 by map-based cloning, and then characterize the gene using different approaches; the approaches taken will depend on the nature of the gene. We will map the sp2 locus with a view to its cloning in a future project. Characterization of the sp mutants was started previously (project 91/P12928). We will complete a more detailed study of two mutants, sp1-3 and sp2. Specifically, we will: (i) compare expression levels of key translocon genes in sp1, sp2 and ppi1; (ii) measure protein import efficiency in sp2 (sp1 is already known to import more efficiently than ppi1); (iii) quantify chloroplast ultrastructural recovery in the suppressors; (iv) investigate the specificity of suppression mediated by sp1 and sp2, by crossing them to tic40. We will map the stic loci at low resolution, and test for allelism by crossing the mutants in all combinations. We will identify one stic locus by map-based cloning. Selection of the locus for cloning will be influenced by the mapping, allelism and characterization experiments. The cloned gene will be studied using a range of approaches, which will be determined by the nature of the gene. Characterization of the stic mutants was started previously (project 91/C18638). We will complete a more detailed study of two stic mutants (one recessive, one semi-dominant). The experiments will be very similar to those described for the sp mutants.