Journal of Experimental Botany, Vol. 42, No. 245, pp. 1541-1549, December 1991 Fate of Myrosin Cells: Characterization of Monoclonal Antibodies Against Myrosinase A.M. BONES', O. P. THANGSTAD 1 - 4 , O. A. HAUGEN 2 and T. ESPEVIK 3 1 UNIGEN—Centre lor Molecular Biology, Department of Botany, University of Trondheim, Medisinsk Teknisk Senter, N-7005 Trondheim, Norway 2 Department of Pathology, University of Trondheim, Regionsykehuset, N-70O0 Trondheim, Norway 3 Institute for Cancer Research, University of Trondheim, Medisinsk Teknisk Senter, N- 7005 Trondheim, Norway Received 21 March 1991; Accepted 27 June 1991 ABSTRACT Immunofluorescence labelling of myrosinase in paraffin sections was used to study the fate, appearance, and distribution of myrosin cells in Brassica napus after seeding. Labelling was achieved using monoclonal antibodies (MAbs) produced against myrosinase. Myrosinase was extracted and purified from seeds of rape {Brassica napus L.) to homogeneity. Mice were immunized with highly purified myrosinase and after fusion and cloning of the hybridoma cells, specificity was tested with highly purified myrosinase and the antibodies were characterized. One monoclonal antibody inhibited myrosinase activity. Precipitation of myrosinase activity was achieved with antibodies coupled to monosized magnetic polymer particles (immunomagnetic precipitation). Labelled cells were found in all organs and also in vascular tissue. A large proportion of the labelled cells were found in the marginal zone, both in radicles and hypocotyl, supporting the theory that myrosinase and its substrates, the glucosinolates, take part in the defence system of the plants. A description of the morphological development of myrosin cells during seedling growth is presented. Key words: |3-thioglucosidase, Brassica, monoclonal antibodies, myrosinase, myrosin cell, thioglucoside glucohydrolase. INTRODUCTION Myrosinase (/3-thioglucosidase, thioglucoside glucohydro- lase, E.C. 3.2.3.1) catalyses the hydrolysis of glucosinol- ates, a group of sulphur containing glycosides present in all Brassicaceae species examined. The glucosinolates are not deleterious themselves, but the cleavage products isothiocyanates, nitriles or thiocyanates (depending upon substrate and pH of the hydrolysis) can have undesirable effects in animal feedstuff's owing to their pungency and goitrogenic activity (Wilkinson, Rhodes, and Fenwick, 1984). Although a model for the compartmentation of glucosi- nolates and myrosinases was reported by Luthy and Matile (1984), little definitive evidence was available concerning how the glucosinolate-myrosinase system was held latent until the tissue was disrupted. Myrosinase has long been thought to be localized in myrosin cells (Bones and Iversen, 1985), and this has recently been shown to be the case (Thangstad, Iversen, Slupphaug, and Bones, 1990; Thangstad, Evjen, and Bones, 1991). This study deals with the morphological changes and the appearance of immunofluorescence-labelled myrosin cells during seed- ling growth. The purification and characterization of myrosinase from seeds of Brassica napus and Sinapis alba are reported in two recent reports (Bones and Slupphaug, 1989; Bones and Thangstad, 1991). Brassica napus has been reported to contain at least four different isoenzymes of myrosinase (Lonnerdal and Janson, 1973). The glyco- protein myrosinase C from rape consists of three forms Ca, Cb, and Cc with varying carbohydrate content. The molecular mass of the enzyme is approximately 154 kD with two subunits and isoelectric points of 4-94, 4-96, and 500 for Ca, Cb, and Cc, respectively (Bones and Slupphaug, 1989). We describe here the generation, identification, and * To whom correspondence should be addressed. Abbreviations: ELISA = Enzyme-linked immunosorbent assay; FITC = Fluorescein isothiocyanate; MAb = monoclonal antibody; PAGE-poly- acrylamide gel electrophoresis; SDS = sodium dodecyl sulphate. © Oxford University Press 1991