Development of an In Vitro Assay for Chloroplast Protein Targeting in the Moss, Physcomitrella patens

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Approximately 90% of the proteins in chloroplasts are encoded in the nucleus, translated on cytoplasmic ribosomes and imported post-translationally across the envelope membranes. A large subset of these proteins are further targeted to one of the numerous internal plastidic compartments. Most of our knowledge of this process comes through in vitro experiments performed with isolated chloroplasts and thylakoids. More recently, a relatively small number of mutants have been described which are defective in plastid protein import, allowing genetic analysis of the import process. However, it has been difficult to combine both genetic and biochemical analyses in the same plant, as those that are convenient for genetic use are poor biochemical subjects, and vice versa. The moss Physcomitrella patens is unique among land plants in its ability to incorporate introduced DNA by homologous recombination. This opens the possibility to examine plant functions by site-directed mutation and allele replacement. The utility of this model plant system would be significantly enhanced if it were to prove to be a good organism for biochemical as well as genetic analysis. The objectives of this project are to collect the tools required to perform in vitro analysis of chloroplast protein targeting in the moss, and then to optimize the procedures for isolation of protein import-competent chloroplasts and thylakoids. It is hoped that this work will lead to the development of the first plant model in which biochemical and genetic tools can be combined without difficulty in the same organism.