PI: Matthew Evans (Carnegie Institution of Washington) CoPIs: Donald Auger (South Dakota State University; subawardee), John Fowler and Scott Givan (Oregon State University; subawardees), Erik Vollbrecht (Iowa State University; subawardee)
Collaborator: Kelly Beck, Gabriel Garcia (Stanford University; subawardees)
An understanding of the genetic basis of gametophyte function will have important implications for agriculture, as gametophytes are central to plant reproduction. The female and male gametophytes make up the haploid phase of the angiosperm life cycle, which immediately succeeds meiosis and precedes formation of the seed (embryo and endosperm). Although gametophytes are small and undergo few cell divisions, they are crucial for reproduction, as they produce gametes, control the fertilization process, and influence development of the seed. However, because mutations that are deleterious to these haploid tissues are difficult to recover and maintain, relatively little is known about the genetic and cellular mechanisms underlying gametophyte function and development, especially in crop plants. This project seeks to overcome this limitation using genetic tools unique to the model crop Zea mays (maize) to accomplish a genomic-scale investigation of gametophytically-required genes. Trisomic stocks that transmit duplicate chromosomal regions through the gametophyte will be used to screen for Activator transposon-tagged gametophyte-lethal mutants. The phenotype of these mutants will be characterized in male and female gametophytes, and the corresponding DNA sequences of the mutated genes identified. Verification of the identity of a select group of candidate genes will be accomplished using multiple alleles and RNA expression analyses. Complementary aims in expression profiling of gametophytes and in bioinformatics - to identify orthologous genes in other plant models and integrate the data generated by the project - will allow an assessment of the genetic basis of gametophyte function. Finally, the ability to predict gametophytic functions across species boundaries will be tested using RNA interference to target select genes in the dicot model Arabidopsis thaliana.
Broader Impacts:
This project is relevant to many agricultural objectives seeking to influence plant reproduction - for example, controlling pollen fertility for hybrid seed production, limiting pollen-mediated transgene flow, and inducing apomixis. Because the tools and stocks created, and sequences generated, will be freely available to the scientific community, the project will enable other researchers to better investigate gametophytes through creation of a gametophyte-specific sequence-indexed mutant collection. All stocks created by the project will be deposited in the Maize Coop Stock Center (maizecoop.cropsci.uiuc.edu). All Ac transposon flanking sequences will be searchable by BLAST, both at PlantGDB (www.plantgdb.org), and at the project''s database: ZGamDB (maizegametophyte.org). Phenotypic and expression data will also be accessible at ZGamDb. For long-term storage and broad community access, phenotypic and genetic data will be incorporated into MaizeGDB (www.maizegdb.org), and expression data will be deposited at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) and the Plant Expression Database (www.plexdb.org).
The project will help train 10 undergraduates to serve as science mentors for K-12 students, through exposing them to plant science research. In partnership with Stanford University''s Haas Center for Public Service, these undergraduate mentors will be placed in communities with large populations from under-represented groups, and help develop science experiences for K-12 students. In addition, the project will train two postdoctoral researchers and a graduate student in genomic-scale approaches to plant biology, and will introduce undergraduates and high school students to genomic science.
Mechanisms of sexual reproduction in plants differ from those in animals. While in animals the haploid cells produced by meiosis differentiate directly into the gametes, in plants post-meiotic haploid plant cells divide mitotically to produce the haploid gametophytes (e.g. the male pollen grain and the female embryo sac of flowering plants), in which a subset of cells differentiates into the gametes. The cellular growth and differentiation events after meiosis require extensive gene activity that is required for successful fertilization. Since the global food chain depends upon plant fertilization and the production of fruits and seeds, it is critical to have a better understanding of the gametophyte phase of the life cycle, including: what the makeup is of the gametophyte transcriptomes (i.e. what genes have RNAs present in gametophyte cells), how the plant genome is reprogrammed during sexual reproduction, and which genes are required for gametophyte functions as revealed by genetic analysis. In this project, experiments to answer these questions were performed in the important crop, corn. Comparison of the transcriptome of the male and female gametophytes to tissues of the diploid phase of the life cycle revealed several themes. The male gametophyte has a highly specialized transcriptome distinct from other tissues, and the female gametophyte transcriptome is enriched for several classes of small, signaling proteins and a few gene families that regulate transcription of other genes. The gametophytes, particularly the female, also produce, at a higher level than other tissues, RNA of repetitive elements including transposable elements that constitute the majority of the maize genome especially the space between what have traditionally been considered the functional genes in the genome. This gametophyte expression of these elements has implications, which are still being analyzed, about how plants control these transposable elements to maintain genome structure and how this control influences the expression of other genes. Analysis of different types of mutants affecting gametophyte function has also revealed a few broad themes. Despite the dramatic differences between the male and female gametophyte transcriptomes, many mutations affect both the male and female. This may reflect the large number of genes that are active in all tissues with the earliest detectable defects in these mutants occurring in the gametophytes. A subset of mutants demonstrates that some genes are required before fertilization to control developmental events after fertilization including endosperm patterning and embryo development. Because these so-called maternal effect mutants do not all affect seed development in the same way, multiple aspects of seed development are likely influenced by maternal gene activity. Analysis of mutants and gene expression studies also revealed that the plant hormone auxin, which was previously known to affect many other aspects of plant growth and development, also plays a role in the development of a subset of cells in the female gametophyte.
