The chloroplast is an essential organelle whose biogenesis is a complex, orchestrated process central to plant growth and development. Many hundreds of nuclear genes are involved in the import of nucleus-encoded proteins, the intra-organellar sorting of nucleus- and chloroplast-encoded proteins, the expression of chloroplast genes, the assembly of chloroplast enzymes, and the regulation of these processes. However, only a small proportion of such genes have been characterized in any detail. The goal of this project is to develop, use, and disseminate a set of complementary and powerful resources for the genetic and biochemical dissection of this complex process.
Maize offers an ideal set of attributes for this project. In addition to its superb genetic tools and the capacity to generate mutations at high frequency with Mu transposons, this project exploits the fact that non-photosynthetic mutant tissue can readily be obtained for biochemical analysis. The core resource that will be developed is a saturated collection of transposon-tagged chloroplast-defective maize mutants. Mutants will be selected from Mu-active maize lines based upon their chlorophyll-deficient leaves and/or increased chlorophyll fluorescence, an indication of a block in photosynthetic electron transport. Previous studies support the notion that one of these easily identified phenotypes will result from a disruption of most aspects of chloroplast biogenesis (import of proteins into the organelle, lipid, pigment, and prosthetic group synthesis, chloroplast gene expression, intra-chloroplast protein sorting, assembly of the photosynthetic apparatus).
The mutant collection will be used in two ways: (i) To determine the role of genes of known sequence but unknown function; and (ii) To discover new genes that play critical roles in chloroplast biogenesis and function.
(i) Genome sequencing projects have unmasked thousands of predicted chloroplast-localized proteins, the majority of which have no known function. To determine the roles of such proteins, the mutant collection will be used to develop a reverse genetics resource, called Photosynthesis Mutant Search (PMS). PMS, already functioning on a small scale, consists of DNA pools from plants with chloroplast defects caused by Mu insertions. 1200 independently-arising mutants are currently in the collection; this number will be increased to ~2000, at which point it is predicted that the collection will be saturated. The small number of DNA pools can be screened in a cost-effective manner to find mutant alleles of genes of known sequence with suspected roles in chloroplast biogenesis. Mutants will be identified and provided as a service. Users will analyze the mutant phenotypes to elucidate the function of the disrupted gene.
(ii) To discover new genes that play critical roles in chloroplast biogenesis and function, the same mutant lines will undergo "snapshot" characterization of visual phenotype and chloroplast protein and RNA defects. The description of each mutant will be incorporated into a web site and the mutants will be made available to other researchers. From these snapshots, users will order specific lines for further study and cloning of the disrupted gene.
In addition, the team of collaborators will form a synergistic partnership to use these tools to focus on one aspect of chloroplast biogenesis: chloroplast gene expression and its control. The study of many other aspects of chloroplast biology by the community-at-large will be facilitated by the unique genetic resources that will be produced. In addition to the agronomic relevance of genes that function in chloroplast processes, it is anticipated that this project will impact fields ranging from evolutionary biology to basic cell and molecular biology.
