ISOLATION AND CHARACTERIZATION OF AN ARABIDOPSIS CDNA INVOLVED IN EARLY EMBRYOGENESIS

Abstract

Embryo development in the dicotyledonous plant typically passes through the different stages of octant, globular, heart and torpedo shape. Much effort has been devoted to the characterization of the cellular and biochemical changes that occur within the seed. In recent years, improved molecular techniques have provided us with a tool to look at this process at a more fundamental level, that is, at the level of gene control. Towards this goal, we have isolated from the Arabidopsis flower and seed cDNA libraries, a gene that is expressed predominantly at the early stages of embryogenesis. The gene isolated, designated GE9, is a single copy gene with a transcript size of 2.3 kb. At the proximal promoter region, there are multiple putative binding sites for enhancers/repressors. The transcript of G E9 encodes a polypeptide of 596 amino acids with a predicted size of 68.7 kDa. Sequence comparison showed that it is a novel gene with unknown function. The protein, however, contains a distinct feature at the carboxy terminus. Beginning at amino acid position 277, the polypeptide can be aligned into 8 repeat motifs of 36 amino acids. This feature is also shared by four other proteins, namely, YGL023, a yeast open reading frame; another yeast protein which is a putative suppressor; a human open reading frame and Pumilio from Drosophila. Of these, only Pumilio has been shown to have distinct mutant phenotype. Pumilio is believed to act in conjunction with another posterior determinant Nanos, in suppressing the translation of the maternally inherited transcript hunchback. This raises the possibility that GE9 may function in a similar manner as Pumilio, perhaps by binding to a nucleic acid. The polypeptide at the amino terminus, on the other hand, contains segments that are homologous to a number of different proteins. The most significant of these is a stretch of 153 amino acids that shows homology to ?-spectrin from Drosophila. Spectrin binds to actin in the formation of the membrane skeleton. Hence, it is possible that while the carboxy terminus of GE9 may be responsible for associating with a nucleic acid, the amino terminus binds to actin. Arabidopsis thaliana plants transformed with the chimeric GE9 promoter-GUS gene showed GUS activity in the pollen grains and ovules. RNA gel blot and Western blot analysis, however, have shown that the transcript and protein of GE9 is found predominantly in seeds containing young embryos. These results indicate that GE9 may be involved in more than one way in the formation and development of the Arabidopsis embryo. During the isolation of the genomic clones, the partial clone of a chaperonin ?-subunit gene was found approximately 3 kbp upstream of the GE9 gene. Sequence analysis revealed that this clone represents a new member of the chaperonin ?-subunit multigene family.