SUBCELLULAR IMMUNOLOCALIZATION OF THE GLUCOSINOLATE SINIGRIN IN SEEDLINGS OF BRASSICA-JUNCEA

Polyclonal rat antibodies were raised to a bovine serum albumin-sinigr in conjugate and used to immunolocalize sinigrin (2-propenylglucosinol ate) in imbibed seeds and developing seedlings of Brassica juncea. (L. ) Czern. Sinigrin was localized to protein bodies in aleurone-like cel ls but shown to be absent from myrosin cells. Double labelling techniq ues were used to co-localize both myrosinase (beta-thioglucoside gluco hydrolase, EC 3.2.3.1) and sinigrin. Myrosin grains were labelled only with the anti-myrosinase antibody, but aleurone cells were labelled w ith both anti-myrosinase and anti-sinigrin antibodies. High-performanc e liquid chromatographic analysis of conventionally fixed and dehydrat ed seed tissues (4 h post imbibition in water), indicated a high propo rtion of sinigrin was retained in fixed tissues. Over a time course of 100 h, protein bodies within aleurone-like cells degraded, fused to f orm the cell vacuole and lost all myrosinase labelling but retained re sidual sinigrin labelling. The degradation of protein bodies correspon ded to a decrease in retention of sinigrin in the fixed tissues. The r esults describe for the first time the co-localization of a plant enzy me and its substrate, a secondary metabolite.