Plants have evolved to produce diverse bioactive specialized metabolites as part of their defense mechanisms against herbivorous insects and microbial pathogens. Frequently, these metabolites are produced as inactive glucosylated forms and require hydrolytic activation by specific β-glucosidases (BGLUs). Plants from Brassicaceae family produce thio-glucosides known as glucosinolates that have co-evolved with unique thio-glucosidases known as myrosinases. During immune response in Arabidopsis thaliana, PENETRATION 2 (PEN2/BGLU26) myrosinase hydrolyzes Trp-derived indol-3-ylmethyl glucosinolate (I3G) and its 4-methoxy derivative (4MI3G). This hydrolysis can be monitored in planta by the production of indol-3-ylmethyl amine (I3A), raphanusamic acid (RA), and 4-O-glucoside of indol-3-yl formamide (4GlcI3F). Closely related with PEN2 AtBGLU18-BGLU32 have been suggested to act as myrosinases based on the presence of conserved substrate recognition R/K residues.
To reveal if these putative myrosinases can replace PEN2 in pathogen-triggered IG metabolism, we expressed AtBGLU18 and AtBGLU27 fused in frame with the sequence encoding PEN2-specific C-terminal tail, essential to anchor this protein in mitochondrial membrane, under the control of PEN2 promoter in pen2 mutant. Our study also included BrBABG (brassinin-associated β-glucosidase) from Brassica rapa, which has been reported to hydrolyze I3G. AtBGLU27 and BrBABG exhibited activity towards I3G in planta, as confirmed by I3A and RA accumulation in transgenic A. thaliana lines. Comparing to BrBABG, AtBGLU27 showed higher activity towards 4MI3G as shown by elevated 4GlcI3F accumulation. AtBGLU18 was not detected in the investigated transgenic plants despite confirmed transgene expression in respective lines. This could be because of the presence of post-translational modifications (disulfide bonds and N-glycosiation sites), which are conserved in the vast majority of BGLUs, but not in PEN2, AtBGLU27 and BrBABG. Overall, our findings suggest that PEN2 and its closest homologs lost post-translational modifications to function in pathogen-triggered IG metabolism.