The promoters of MEA (FIS1), FIS2, and FIE (FIS3), genes that repress seed development in the absence of pollination, were fused to ␤-glucuronidase (GUS) to study their activity pattern. The FIS2::GUS product is found in the embryo sac, in each of the polar cell nuclei, and in the central cell nucleus. After pollination, the maternally derived FIS2::GUS protein occurs in the nuclei of the cenocytic endosperm. Before cellularization of the endosperm, activity is terminated in the micropylar and central nuclei of the endosperm and subsequently in the nuclei of the chalazal cyst. MEA::GUS has a pattern of activity similar to that of FIS2::GUS, but FIE::GUS protein is found in many tissues, including the prepollination embryo sac, and in embryo and endosperm postpollination. The similarity in mutant phenotypes; the activity of FIE, MEA, and FIS2 in the same cells in the embryo sac; and the fact that MEA and FIE proteins interact in a yeast two-hybrid system suggest that these proteins operate in the same system of control of seed development. Maternal and not paternal FIS2::GUS, MEA::GUS, and FIE::GUS show activity in early endosperm, so these genes may be imprinted. When fis2, mea, and fie mutants are pollinated, seed development is arrested at the heart embryo stage. The seed arrest of mea and fis2 is avoided when they are fertilized by a low methylation parent. The wild-type alleles of MEA or FIS2 are not required. The parent-of-origin-determined differential activity of MEA, FIS2, and FIE is not dependent on DNA methylation, but methylation does control some gene(s) that have key roles in seed development.

We have identified mutant alleles of two sporophytically acting genes, HAIKU2 (IKU2) and MINISEED3 (MINI3). Homozygotes of these alleles produce a small seed phenotype associated with reduced growth and early cellularization of the endosperm. This phenotype is similar to that described for another seed size gene, IKU1. MINI3 encodes WRKY10, a WRKY class transcription factor. MINI3 promoter::GUS fusions show the gene is expressed in pollen and in the developing endosperm from the two nuclei stage at Ϸ12 hr postfertilization to endosperm cellularization at Ϸ96 hr. MINI3 is also expressed in the globular embryo but not in the late heart stage of embryo development. The early endosperm expression of MINI3 is independent of its parent of origin. IKU2 encodes a leucine-rich repeat (LRR) KINASE (At3g19700). IKU2::GUS has a similar expression pattern to that of MINI3. The patterns of expression of the two genes and their similar phenotypes indicate they may operate in the same genetic pathway. Additionally, we found that both MINI3 and IKU2 showed decreased expression in the iku1-1 mutant. IKU2 expression was reduced in a mini3-1 background, whereas MINI3 expression was unaltered in the iku2-3 mutant. These data suggest the successive action of the three genes IKU1, IKU2, and MINI3 in the same pathway of seed development.autoregulation ͉ endosperm development S eed development involves a complex of processes, including the expansion and growth of the maternal integuments of the ovule and the development of the diploid zygote after the union of the maternal egg cell with one of the two sperm cells delivered to the embryo sac by the pollen tube. It also involves the development of a triploid endosperm after the union of the two nuclei of the homodiploid central cell of the embryo sac with the second sperm cell (1). In eudicots such as Arabidopsis, endosperm development progresses through phases of syncytial growth, cellularization, and cell death. The syncytial phase is characterized by successive divisions of the triploid nuclei without cytokinesis (2). The endosperm cytoplasm is initially compartmentalized into nuclear cytoplasmic domains (3), and subsequently cellularization occurs after the eighth round of syncytial mitoses, initially in the region surrounding the embryo, and proceeding toward the chalazal region (4). Viable seed formation results from the integrated growth and development of the genetically diverse integument, embryo, and endosperm tissues.Major seed controls are provided by genes that define the development of the maternal integument and the new-generation embryo and endosperm. A number of mutations have been described that impair integument development (5, 6), and genes disrupting embryo pattern formation have also been described (7-9). Endosperm development controls are represented by the FIS loci, MEA, FIS2, and FIE, as well as MSI1 (10-12). These genes code for proteins that are components of a chromatin-associated polycomb complex that prevents endosperm development before double fertilization....