RGD-mediated membrane-matrix adhesion triggers agarose-induced embryoid formation in sunflower protoplasts

Agarose embedding of sunflower (Helianthus annuus L.) hypocotyl protoplasts induces an asymmetric division pattern and subsequent polarized developmen t leading to embryoid formation. We cultured protoplasts in media with diff erent mannitol concentrations. Induction of plasmolysis of agarose-embedded protoplasts by increasing the mannitol concentration lowered the proportio n of embryoids formed. This indicates that adhesion sites between the plasm a membrane and the agarose matrix are involved in embryoid formation. The i nvolvement of such adhesion sites was tested by incubating embedded protopl asts with RGD peptide. 1 mu M RGD heptapeptide reduced embryoid formation b y 50% as compared to the control DGR peptide. We also showed that RGD hepta peptide acts on the cytoskeleton by disrupting cortical microtubules. The r esults are discussed in terms of a model in which the anchorage of the prot oplast membrane to the agarose matrix is mediated by RGD-binding proteins c onnected with microtubules, determining asymmetric division of the cell and polarized development.