Many plant
phytochemicals constitute binary
enzyme-
glucoside systems and function in plant defence. In brassicas, the
enzyme myrosinase is confined to specific myrosin cells that separate the
enzyme from its substrate; the
glucosinolates. The
myrosinase-catalysed release of toxic and bioactive compounds such as
isothiocyanates, upon activation or tissue damage, has been termed 'the mustard oil bomb' and characterized as a 'toxic mine' in plant defence. The removal of myrosin cells and the
enzyme that triggers the release of
phytochemicals have been investigated by genetically modifying Brassica napus plants to remove
myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a
ribonuclease,
barnase. Transgenic plants ectopically expressing
barnase were embryo lethal. Co-expressing
barnase under the control of the Myr1.Bn1 promoter with the
barnase inhibitor,
barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of
myrosinase and negligible
myrosinase activity, and
autolysis experiments showing negligible production of
glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named 'MINELESS plants'. The epithiospecifier
protein profile and
glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35 kDa epithiospecifier
protein in myrosin cells and a reduced turnover of
glucosinolates in MINELESS plants.