BIPHASIC EXPRESSION OF A VICIA-FABA LEGUMIN-B-GENE IN DEVELOPING SEEDS OF TRANSGENIC TOBACCO

We analysed the spatial and temporal expression of the Vicia faba legu min gene LeB4 in developing seeds of transgenic tobacco plants by mean s of in situ hybridization and immunohistostaining. The results indica te that the expression of the LeB4 gene is regulated in embryo and end osperm in a biphasic manner. Legumin accumulation starts during early seed development at low amounts in single cells of the integument. Acc umulation proceeds successively in the proembryo, suspensor and endosp erm reaching maximum levels in all cells of these organs around 10 to 12 DPP With the beginning of the heart stage (around 13 DAP) legumin r eserves disappear completely from all parts of the seed. The described early phase of legumin accumulation occurs in embryo and endosperm st ill before the onset of the cell expansion phase in a period of contin uous mitotic activity A second expression phase starts in embryo and e ndosperm around 18 DAP as the embryo enters late torpedo stage. Within the embryo the accumulation is restricted to the parenchyma cells of cotyledons and hypocotyl, while the embryonic root and provascular tis sue do not accumulate LeB4 protein. In addition, a low percentage (<10 %) of cells from protoderm and calyptra was found to be involved in l egumin accumulation. A comparison of LeB4 mRNA and protein distributio n in embryo and endosperm revealed corresponding patterns for both exp ression phases. The biphasic expression of the LeB4 gene in transgenic tobacco seeds is identical to that found in V. faba for the legumin a nd vicilin genes during early embryogenesis (Panitz et al., 1995), but differs from that of tobacco 12S globulin genes, which are expressed in the embryo not before the late heart stage.