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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The female gametophyte is an absolutely essential structure for angiosperm reproduction. It produces the egg cell and central cell (which give rise to the embryo and endosperm, respectively) and mediates several reproductive processes including pollen tube guidance, fertilization, the induction of seed development, and perhaps also maternal control of embryo development. Although much has been learned about these processes at the cytological level, specific molecules mediating and controlling megagametogenesis and female gametophyte function have not been identified. A genetic approach to the identification of such molecules has been initiated in Arabidopsis and maize. Although genetic analyses are still in their infancy, mutations affecting female gametophyte function and specific steps of megagametogenesis have already been identified. Large-scale genetic screens aimed at identifying mutants affecting every step of megagametogenesis and female gametophyte function are in progress; the characterization of genes identified in these screens should go a long way toward defining the molecules that are required for female gametophyte development and function.

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle.

BACKGROUND: Age-related macular degeneration (AMD) is a common cause of blindness worldwide. Neovascular AMD (nAMD) is an advanced form of the disease, in which excess vascular endothelial growth factor (VEGF) induces growth of new blood vessels that leak fluid, accounting for 90% of vision loss in AMD. Dysfunction of the retinal pigment epithelium likely initiates AMD. Retinal pigment epithelial cells express a G protein-coupled receptor, GPR143, which downregulates VEGF in response to levodopa. Anti-VEGF therapy effectively treats nAMD, suggesting that excessive VEGF activity drives the pathology. METHODS:In an open-label pilot study, in patients with newly diagnosed nAMD and naı ¨ve to anti-VEGF injections (Cohort-1), the effects of carbidopa-levodopa on vision and anatomic outcomes were evaluated for 4 weeks. Then patients were followed 5 months further with ascending levodopa doses. Patients previously treated with anti-VEGF injection therapy (Cohort-2) were also treated with ascending levodopa doses and evaluated for 6 months. RESULTS: Levodopa was safe, well tolerated, and delayed anti-VEGF injection therapy while improving visual outcomes. In the first month, retinal fluid decreased by 29% (P = .02, n = 12) without anti-VEGF treatment. Through 6 months the decrease in retinal fluid was sustained, with a mean frequency of 0.38 injections/month. At month 6, mean visual acuity improved by 4.7 letters in Cohort-1 (P = .004, n = 15) and by 4.8 letters in Cohort-2 (P = .02, n = 11). Additionally, there was a 52% reduction in the need for anti-VEGF injections in Cohort-2 (P = .002). CONCLUSIONS: Our findings suggest efficacy and support the pharmacological targeting of GPR143 with levodopa for the treatment of nAMD in future studies.

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