Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea.
Polyclonal rat antibodies were raised to a bovine serum albumin-sinigrin conjugate and used to immunolocalize sinigrin (2-propenylglucosinolate) in imbibed seeds and developing seedlings of Brassica juncea. (L.) Czern. Sinigrin was localized to protein bodies in aleurone-like cells but shown to be absent from myrosin cells. Double labelling techniques were used to co-localize both myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) and sinigrin. Myrosin grains were labelled only with the anti-myrosinase antibody, but aleurone cells were labelled with both anti-myrosinase and anti-sinigrin antibodies. High-performance liquid chromatographic analysis of conventionally fixed and dehydrated seed tissues (4 h post imbibition in water), indicated a high proportion of sinigrin was retained in fixed tissues. Over a time course of 100 h, protein bodies within aleurone-like cells degraded, fused to form the cell vacuole and lost all myrosinase labelling but retained residual sinigrin labelling. The degradation of protein bodies corresponded to a decrease in retention of sinigrin in the fixed tissues. The results describe for the first time the co-localization of a plant enzyme and its substrate, a secondary metabolite.
Department of Biological Sciences, Wye College, University of London, Ashford, Kent, UK.
P J
PJ
Kelly
A
A
Bones
J T
JT
Rossiter
D000906:Antibodies / Q000276:immunology; D001937:Brassica / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005961:Glucosinolates / Q000032:analysis* / Q000276:immunology; D015151:Immunoblotting; D012639:Seeds / Q000737:chemistry / Q000648:ultrastructure
D000906:Antibodies; D005961:Glucosinolates; C010330:sinigrin