43. Cloning of two cDNAs encoding the HAP3 subunit protein from developing rice seeds
K.MIYOSHI and N. KURATA
National Institute of Genetics, Mishima, 411-8540 Japan

     During seed development, several discrete phenomena, such as embryogenesis, accumulation of storage reserves and acquisition of desiccation tolerance and dormancy, progress in a coordinated manner. Recent genetic studies using several mutants affecting seed development identified genes that control many distinct aspects of morphogenesis and maturation of the seed, however little is known at the molecular level about how discrete processes that occur during seed development are coordinated.

     The LEC1 gene is thought to be a central regulator of seed development in Arabidopsis thaliana. LEAFY COTYLEDON1 encodes the HAP3 subunit protein, a component of the transcription factor CBF (CAAT-binding factor), which is ubiquitously present in many eukaryotes (Lotan et a!. 1998). The recessive mutation that occurred at LEC1 locus causes pleiotropic defects in both morphogenesis and maturation of the seed (West eta!. 1994). In addition, overexpression of LEC1 has been shown to cause ectopic embryogenesis on leaves in transgenic Arabidopsis plants. These results suggest that LEC1 regulates expression of a set of genes required for embryo formation including morphogenesis and maturation process in a precisely coordinated manner. Analysis of plant HAP3 gene(s) would provide useful information for understanding zygotic and somatic embryogenesis in monocot

plants. Here, we report a preliminary study of rice cDNAs encoding the HAP3 subunit - protein.

    A rice cDNA library constructed from developing seeds at 3 days alter pollination (DAP) was screened using a maize HAP3 cDNA fragment as the probe. Two independent cDNA clones (#3, #35) which show homology to the HAP3 gene were isolated (Fig.l). Sequence analysis revealed that the two clones encoded amino acid sequences homologous to the B domain of the maize HAP3 protein, which contained motifs for DNA binding and protein interaction. The amino acid sequences of B domain encoded by the two cDNAs showed 86.7% identity to each other. In contrast to high conservation in the N-terminal portion, the deduced amino acid sequences of the C-terminal portion were variable between the two cDNA clones. Fig. 2 shows a comparison of the predicted amino acid sequences of the B domain among four HAP3 homologs from three plant species. Significant homology was observed between rice cDNAs and those of other species. Among the four plant B domain sequences, 50 of 90 amino acids are identical. Database search identified another rice EST clone similar to cDNA clone #3 and #35. This EST clone was isolated from a cDNA library of panicles at ripening stage. These results suggest that the rice HAP3 constitutes a multigene family, and at least three members of the family are expressed at early stages of seed development. Molecular characterization of these cDNAs is in progress.