Embryogenesis in <i>Arabidopsis thaliana</i>: Mutant library construction and embryo mutant identification and characterization
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.
0307: Molecular biology