In‐situ localization of cyanogenic β‐glucosidase (linamarase) gene expression in leaves of cassava (Manihot esculenta Cranz) using non‐isotopic riboprobes

Abstract

The release of hydrogen cyanide (cyanogenesis) from damaged plant tissue depends upon the sequential action of a β‐glucosidase and an α‐hydroxynitrilase on cyanoglucosides. The non‐isotopic digoxigenin labelling system was used to visualize the presence of cyanogenic β‐glucosidase (linamarase) mRNA in cells of young leaves of Manihot esculenta Cranz (cassava). Strong hybridization to antisense riboprobes produced from the cDNA clone pCAS5, indicates localization of linamarase gene expression in laticifers (latex vessels). This is supported by the demonstration of linamarase mRNA in exuded latex. In contrast, in‐situ localization of the control gene pGLF4, showed expression in all leaf mesophyll cells. High levels of linamarase activity were demonstrated in the latex of leaf petioles and this activity was shown to be dependent on the presence of attached leaflets. Assays of α‐hydroxynitrilase activity in exuded latex and whole leaves shows that, unlike linamarase, this enzyme is present at very low levels in latex and must be located elsewhere in the leaf.