Epsilon Archive for Student Projects

Abstract

Glucosinolates are sulphur and nitrogen containing secondary metabolites which are present
in the order Brassicales. These compounds are hydrolysed by enzymes called myrosinases
upon tissue damage and serve to protect plants against insects and pathogens. Protein
extracts from leaves of fully developed Arabidopsis thaliana Col-0 plants and the ascorbic acid
deficient mutants vtc1-1 and vtc4-1 were utilised to characterize effects on basal myrosinase
expression. The catalytic activity of myrosinases against the glucosinolate sinigrin was lower
in vtc4-1 compared to Col-0 and vtc1-1. The content of myrosinase and myrosinase related
proteins in Col-0, vtc1-1 and vtc4-1 was analysed by Western blotting and
immunoprecipitation using monoclonal and polyclonal antibodies. Differences were found
regarding myrosinase and myrosinase related proteins in the evaluated genotypes. However,
further analysis is required to clearly identify all the proteins present upon Western blot and
IP analysis. His-tagged recombinant Arabidopsis myrosinases TGG1 and TGG4 were overexpressed
in Pichia pastoris, extracted and purified through immobilised metal affinity
chromatography. The effect of the sinigrin product allyl isothiocyanate (AITC) for enzyme
inactivation (“suicide inactivation”) of TGG1 and TGG4 was measured. Decrease of myrosinase
activity was observed both for TGG1 and TGG4 in the presence of 1 mM AITC. The catalytic
activity of TGG1 was completely inhibited by 5 mM and 10 mM AITC. On the other hand, 5 mM
and 10 mM AITC caused a reduction of activity in TGG4 but more than 60 % activity was still
remaining. Accordingly it seems that glucosinolate products can affect myrosinase activity in
plant tissues if not finding other nucleophiles to react with which may represent an
endogenous regulatory mechanism.