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All plants flower late in their life cycle. For example, in Arabidopsis, the shoot undergoes a transition and produces reproductive flowers after the adult phase of vegetative growth. Much is known about genetic and environmental processes that control flowering time in mature plants. However, little is understood about the mechanisms that prevent plants from flowering much earlier during embryo and seedling development. Arabidopsis embryonic flower (emf1 and emf2) mutants flower soon after germination, suggesting that a floral repression mechanism is established in wild-type plants that prevents flowering until maturity. Here, we show that polycomb group proteins play a central role in repressing flowering early in the plant life cycle. We found that mutations in the Fertilization Independent Endosperm (FIE) polycomb gene caused the seedling shoot to produce flower-like structures and organs. Flower-like structures were also generated from the hypocotyl and root, organs not associated with reproduction. Expression of floral induction and homeotic genes was derepressed in mutant embryos and seedlings. These results suggest that FIE-mediated polycomb complexes are an essential component of a floral repression mechanism established early during plant development.

The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA (MEA) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals.

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis, f644, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA (MEA), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446-450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

A fundamental problem in biology is to understand how fertilization initiates reproductive development. Higher plant reproduction is unique because two fertilization events are required for sexual reproduction. First, a sperm must fuse with the egg to form an embryo. A second sperm must then fuse with the adjacent central cell nucleus that replicates to form an endosperm, which is the support tissue required for embryo and/or seedling development. Here, we report cloning of the Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) gene. The FIE protein is a homolog of the WD motif-containing Polycomb proteins from Drosophila and mammals. These proteins function as repressors of homeotic genes. A female gametophyte with a loss-of-function allele of fie undergoes replication of the central cell nucleus and initiates endosperm development without fertilization. These results suggest that the FIE Polycomb protein functions to suppress a critical aspect of early plant reproduction, namely, endosperm development, until fertilization occurs.

The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.

We identified a tobacco stigma-specific gene, designated STIG1. The STIG1 gene is developmentally regulated and expressed specifically in the stigmatic secretory zone. We used a chimeric STIG1-GUS gene to show that the stigma-specific STIG1 gene expression pattern is controlled primarily at the transcriptional level. We constructed a stigma-specific cytotoxic gene by fusing the STIG1 gene 5' regulatory region with the coding sequence of the Bacillus amyloliquefaciens barnase gene, to assess the role of the stigmatic secretory zone in the pollination process. Pistils of transgenic STIG1-barnase tobacco plants undergo normal development, but lack the stigmatic secretory zone and are female sterile. Pollen grains germinate on the ablated 'stigmatic' surface, but are unable to penetrate the transmitting tissue of the style. Application of stigmatic exudate from wild-type pistils to the ablated surface increases the efficiency of pollen tube germination and growth and restores the capacity of pollen tubes to penetrate the style. Our data demonstrate the importance of the stigmatic secretory zone in the pollination process and provide an approach to identify compounds produced by the stigma that are critical for successful pollination and fertilization to occur.

We introduced a 17.1-kilobase soybean DNA fragment containing the lectin gene and at least four nonseed protein genes into the tobacco genome. As in soybean plants, lectin mRNA is present in tobacco seeds, accumulates and decays during tobacco seed development, and is translated into a protein that accumulates prior to dormancy. Each soybean nonseed protein mRNA is present in tobacco leaves, roots, stems, and seeds at levels similar to that found in soybean plants. We conclude that a differentially expressed soybean gene cluster is correctly regulated in transformed tobacco plants and that sequences controlling their expression are recognized by regulatory factors present in tobacco cells.