MYROSINASE AND MYROSIN CELL DEVELOPMENT DUR EMBRYOGENESIS AND SEED MATURATION

Abstract

The occurrence and distribution of myrosin cells and myrosinases have been followed using immunochemical techniques on samples taken from pistils before and after pollination, and seeds harvested at three different stages of maturity. Two Brassica napus cultivars (Jet Neuf and Bolko) with a high and a low glucosinolate seed content, respectively, were used. Two methods recently developed in our laboratory were applied for detection of the enzyme using light microscopy (LM) and transmission electron microscopy (TEM). In the first case, paraffin-embedded sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase- and fluorescein-isothiocyanate-conjugated secondary antibodies. For the TEM localization a polyclonal antibody raised in rabbit against a highly purified myrosinase from white mustard was used. Myrosinase activity was detected in the early stage during embryogenesis in both cultivars but was higher in the high glucosinolate cultivar. In the early stages of development, typical embryonic myrosinase cells could not be detected. Later they appear in cotyledons, hypocotyls, and radicles. A typical, specific, and positive LM localization of myrosinase in myrosin cells could be detected both in radicles and the cotyledons. In the radicle the positively identified cells are located in the second outermost cell layer in the peripheral cortex; in the cotyledon positively identified myrosin cells were found scattered around in the tissue. The positive TEM localization demonstrated gold particles uniformly distributed over the matrix in the myrosin grains.