Homosporous ferns such as Ceratopteris richardii produce only one type of spore; irrespective of the genotype, spores can develop into either ameristic males, or meristic, hermaphroditic gametophytes. Determination of sexual identity occurs after spore germination by the action of the hormone antheridiogen, which promotes male development and is secreted by the hermaphrodite. Meristem development antagonizes the antheridiogen response, whereas activation of maleness by antheridiogen involves the specific repression of female-associated traits, such as the meristem and the archegonia. To elucidate the possible relationship between the meristem and the antheridiogen sex determination pathway, I cloned a Ceratopteris gene (CKNOX-A or CK) homologous to the homeobox-containing, meristem-specific KNOTTED1 or KN1 gene. CK encodes a putative protein that contains an 88-aa region 76% identical to the KN1 homeodomain. Expression studies performed by RT-PCR and in situ hybridization showed that CK is most strongly expressed in tissues that contain a meristem, such as hermaphroditic gametophytes, and sporophyte apices and leaves. However, CK RNA was also detected in antheridial cells in males. Surprisingly, the cellular localization of the CK hybridization signal differed depending on the regions of the CK gene used as a probe. Using the CK 3$\sp\prime$ UTR probe, the hybridization signal localized to the nuclei, in contrast to the cytoplasmic localization with a homeobox-containing probe. Functional homology of the CK gene to KN1 was assessed by overexpressing CK in Arabidopsis plants (ecotype Columbia). Transformant phenotypes ranged between two classes: in one, leaf size was severely reduced and plants formed petite rosettes. In the other, plants displayed a loss of apical dominance, by overproducing rosette leaves that developed without a visible phyllotactic pattern, or by forming several inflorescences simultaneously. Contrary to the KN overexpressing phenotypes in Arabidopsis, leaf shape was not altered by lobe formation in CK transformants. In conclusion, CK can be recognized by the molecules that control the initiation and maintenance of the Arabidopsis meristem, which suggests that Ceratopteris CK may play a similar role to the KNOTTED gene of maize.

Embryogenesis plays a central role in the plant life cycle. It starts after fertilization. A single zygote divides asymmetrically, giving rise a small apical cell and a larger basal cell. The small apical cell undergoes precise cell divisions, passes through 2, 4 and 8 cell stages, followed by the protoderm, globular, heart, torpedo and cotyledon stage, in the process of forming a mature embryo. In embryo development a large number of genes are estimated to be involved and interact with each other during embryogenesis. We demonstrated that the RSH gene was required for normal embryo development of Arabidopsis. Its essential role was determined by disrupting expression of the gene by the Ac/ DsE two-element transposon system, which caused the embryo mutation. The abnormal phenotype was traced to the first asymmetrical division of the zygote and the embryo development lost its precise programmed cell division pattern. The rsh mutant showed both apical-basal and radial pattern defects. The RSH gene mapped to chromosome I. The gene was cloned and sequenced. It encoded a HRGP-type protein of predicted size, 49 k Dalton. The pre-protein contained a signal peptide and 13 almost identical repeats. Each repeat had 28 amino acids. The rescue of homozygous rsh mutants by complementation with the wild-type RSH gene demonstrated that the rsh mutation is the consequence of the DsE insertion. The gene was found to be expressed through out the developing embryo. It was expressed in a tissue specific manner after embryo germination. The RSH gene expression pattern was first profiled using the GUS reporter gene assay. The Northern and RT-PCR confirmed the results. To localize the RSH protein at the cellular level, an EGFP gene was linked to the C-terminus of the RSH gene and expressed in wild-type Arabidopsis . Confocal microscopy showed that the fusion protein was localized to the cell wall. Wild type transformants expressing RSH-EGFP showed mutant phenotypes. All these results support the conclusion that the RSH protein plays an important role in cell division during embryogenesis.