The female gametophyte is an essential structure for angiosperm reproduction that mediates a host of reproductive functions and, following fertilization, gives rise to most of the seed. Here, we describe a rapid method to analyze Arabidopsis female gametophyte structure using confocal laser scanning microscopy (CLSM). We present a comprehensive description of megagametogenesis in wild-type Arabidopsis. Based on our observations, we divided Arabidopsis megagametogenesis into eight morphologically distinct stages. We show that synergid cell degeneration is triggered by pollination, that dramatic nuclear migrations take place during the fournucleate stage, and that megagametogenesis within a pistil is fairly synchronous. Finally, we present a phenotypic analysis of the previously reported Gf mutant (Redei 1965) and show that it affects an early step of megagametogenesis.

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2 , with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.

The female gametophyte (embryo sac or megagametophyte) plays a critical role in sexual reproduction of angiosperms. It is the structure that produces the egg cell and central cell which, following fertilization, give rise to the seed's embryo and endosperm, respectively. In addition, the female gametophyte mediates a host of reproductive processes including pollen tube guidance, fertilization, and the induction of seed development. Several major events occur during megagametogenesis, including syncitial nuclear divisions, cellularization, nuclear migration and fusion, and cell death. While these events have been described morphologically, the molecules regulating them in the female gametophyte are largely unknown. We discuss a genetic screen based on reduced seed set and segregation distortion to identify mutations affecting megagametogenesis and female gametophyte function. We report on the isolation of four mutants (fem1, fem2, fem3, and fem4) and show that the four mutations map to different locations within the genome. Additionally, we show that the fem1 and fem2 mutations affect only the female gametophyte, while the fem3 and fem4 mutations affect both the female and male gametophyte. We analyzed female gametophyte development in these four mutants as well as in the gfa2, gfa3, gfa4, gfa5, and gfa7 mutants. We found that the fem2, fem3, gfa4, and gfa5 mutants abort development at the one-nucleate stage, while the fem1, fem4, gfa2, gfa3, and gfa7 mutants are affected in processes later in development such as polar nuclei fusion and cellularization. The establishment of a genetic screen to identify mutants and the development of a rapid procedure for analyzing mutant phenotypes represent a first step in the isolation of molecules that regulate female gametophyte development and function.

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