Eugene, Oregon
University of Oregon
NSF plant genome project: Functional genomics of chloroplast biogenesis --Barkan, A, Stern, D1, Hanson, M2

1Boyce Thompson Institute
2Cornell University

Through funding by the NSF Plant Genome Research Program, we are developing a genetic resource in maize that is tailored for the study of chloroplast biogenesis and function ( The goals of the project are to assemble a saturated collection of Mu-induced non-photosynthetic mutants, and to develop the collection for use in two ways: (i) as an efficient reverse genetic resource to identify loss-of-function alleles of nuclear genes suspected of influencing chloroplast functions; and (ii) as a forward genetic resource to identify and clone "new" genes involved in chloroplast biogenesis.

The Mutant Collection. Mutants are identified by screening the F2 progeny of high copy Mu lines for the segregation of seedlings with chlorophyll-deficient (pale green, yellow, albino, virescent, albescent, striate, zebra-stripe), or high chlorophyll fluorescent leaves. Prior studies with maize and other organisms indicate that these phenotypes report defects in most aspects of chloroplast biogenesis and function, including defects in the expression of chloroplast or nuclear genes encoding components of the photosynthetic apparatus; assembly of photosynthetic enzymes; protein targeting to and within the chloroplast; plastid pigment/ lipid/ prosthetic group synthesis; light signaling; and bundle sheath cell differentiation.

To ensure that mutants in the collection are independently-arising, Mu-active F0 founder plants are progeny-tested to determine whether they carry mutations that cause any of our selected phenotypes. If the progeny of a self-pollination segregate any such phenotype, the corresponding F1 outcross is not used for the generation of F2 screening material. Many different founder plants have been used, to maximize coverage of the genome. Complete lineage information for each mutant is stored in a database that is available on-line.

There are currently ~2000 mutants in the collection. We believe that we are nearing saturation because those reverse genetic screens that are successful generally yield multiple alleles. We intend to add ~500 additional mutants in the coming year and anticipate that the collection will be complete at that time.

The Reverse Genetic Screening Service. The reverse-genetic screening service involves PCR screens of pooled mutant DNAs for Mu insertions in genes of known sequence. The fact that these screens are limited to mutants with phenotypes offers the advantage that mutant alleles recovered generally have phenotypes, obviating the need to sort through numerous "silent" insertions in introns and UTRs. Of course, if disruption of the targeted gene does not cause one of the collected phenotypes, alleles are unlikely to be recovered. Therefore, we are limiting screens to genes predicted to encode chloroplast-localized proteins, or for which there is genetic or biochemical evidence that the mutant phenotype will be within the spectrum of those in our collection. A more detailed discussion of the advantages and disadvantages of this resource in comparison with various related resources can be found on our web site.

In pilot screens we have recovered numerous useful alleles, including (a) mutant alleles of genes identified in 2-hybrid screens involving genetically-defined chloroplast gene expression factors; (b) new mutant alleles of chloroplast biogenesis genes previously cloned by transposon-tagging; (c) mutant alleles of genes whose sequences are related to known chloroplast biogenesis genes; (d) mutant alleles of genes with bacterial homologs that function in conserved processes in common to bacteria and plastids.

Users design and test primers according to the protocol on our web site. Screens and heritability tests are then done at the University of Oregon and seed from positive families are provided to the user for propagation and analysis. There will be no charge for this service through the end of 2002. Users should anticipate a turn around time of 2-3 weeks.

The Phenotype Database. The phenotype database contains information on leaf pigmentation, and chloroplast protein and RNA deficiencies. For mutants that are not completely lacking in chlorophyll, a snapshot of chloroplast protein deficiencies is obtained by probing immunoblots with an antibody cocktail that simultaneously detects one core subunit of each of the major photosynthetic enzyme complexes. These characterizations are nearing completion. To detect defects in chloroplast RNA metabolism, plastid RNAs encoding missing proteins are being analyzed by Northern hybridization; these data will be added to the database as they become available.

Users can search the database for pigment or protein defects of interest and request mutant seed for further study and/or cloning.

The Web Site. Our web site (http://chloroplast.uoregon. edu) provides an overview of the mutant collection and its potential applications, instructions for using the reverse genetic screening service, and access to the phenotype and lineage databases.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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