Aspects of brassica juncea meal toxicity : allyl isothiocyanate release and bioassay
Saini, Akal Rachna Kaur
Oilseed and oilseed meal extracted from members of Brassicaceae release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of Brassica juncea, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (Brassica juncea), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal. Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective. It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice. An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in Caenorhabditis elegans as a marker of toxicity. C.elegans strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-HSP70 antibody showed a marked increase in the expression of HSP70 protein in a dose-dependent manner. Assays of the expression of HSP70A mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of HSP70A mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of HSP70A mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected HSP70 transcript production, C. elegans was exposed to AITC or Brassica juncea cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (
DegreeMaster of Science (M.Sc.)
DepartmentApplied Microbiology and Food Science
ProgramApplied Microbiology and Food Science
SupervisorTyler, Robert T. (Bob); Reaney, Martin J. T.
CommitteeSmith, Peta; Shand, Phyllis J.; Wickstrom, Mark