CHROMATOGRAPHIC AND MICROSCOPIC DETECTION OF GLUCOSINOLATES IN RAPESEED USING N,2,6-TRICHLORO-p- BENZOQUINONEIMINE

Abstract

A new histochemical method for detecting glucosinolates in rapeseed (Brassica napus L. ’Midas’) methanolic extracts and rapeseed (Brassica campestris L. ’Candle’) hand-cut sections has been developed using N,2,6-trichloro-p-benzoquinoneimine (TCQ) as the staining reagent. The specificity of TCQ was established by the following experiments. (1) TCQ was used as a spray reagent for detecting thio sugars separated from (a) crude and (b) partially purified rapeseed extracts on silica gel coated chromatograms. By comparison, the reaction of TCQ was relatively more selective than that of alkaline silver nitrate as it detected fewer spots on the chromatogram. (2) Rapeseed extracts and glucosinolate (GS) standards were analyzed on the same plate by one- and two-dimensional chromatography. After spraying with TCQ two of the three spots originated from the extract had the same color (yellow) reaction and similar Rf values as those of 3-butenyl GS and 2-hydroxy-3-butenyl GS. (3) Gas chromatography was used to identify the TCQ-reactive compounds present in the rapeseed extract. 3-butenyl GS and 2-hydroxy-3-butenyl GS were present as major constituents while 4-pentenyl GS, 2-hydroxy-4-pentenyl GS and an hydroxyindolylmethyl GS formed the minor entity. The use of TCQ for detecting glucosinolates in situ was established by the following microscopic studies. Hand-cut sections were prepared, stained with TCQ and examined microscopically. Protein bodies found inside cells of the cotyledon were the only stained (yellow in color) structures. The number of yellow-colored protein bodies decreased if the sections were left untreated at room temperature for more than 10 min prior to staining. Treatment of the sections with distilled water, 70% methanol or myrosinase before staining diminished the stainability of the protein bodies.Key words: Rapeseed structure, glucosinolates, chromatographic analyses, N,2,6-trichloro-p-benzoquinoneimine

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

“…A localization of glucosinolates in vacuoles of horseradish (Armoracia lapathifolia = A. rusticana) and oilseed rape (B. napus) has been reported (Grob and Matile 1979;Matile 1980;Helmlinger et al 1983;Yiu et al 1984). Only one immunohistochemical localization of glucosinolates has been reported (Kelly et al 1998).…”

Correlation of Glucosinolate Content to Myrosinase Activity in Horseradish (Armoracia rusticana)

“…Studies have shown that in plant tissue, glucosinolates are present in an intact form and sequestered in the vacuole (22). Intact glucosinolates have no allelochemical property against plant pests or biological functions against human diseases (23,24).…”

Glucosinolate Uptake by Developing Rapeseed Embryos

“…At maturity, the embryo comprises from 80 to 90% of the seed dry weight and is the site where most of the glucosinolates contained in the seed are located (7,14). The subcellular location of the glucosinolates within the embryo is not known for certain, although they are thought to be localized in globoid bodies associated with aleurone grains (17), structures which form within and eventually fill the vacuoles. Inheritance studies (7,14) and grafting experiments (9) have indicated that seed glucosinolate content is maternally controlled.…”