FIGURE 4.

Distribution of 4MSOB glucosinolate in the body of adult P. armoraciae beetles shortly after ingestion (A) and influence of beetle gut content extracts on plant myrosinase activity in in vitro enzyme assays (B). (A) Beetles were allowed to feed for 1 min on Arabidopsis leaves with (wild type) or without (tgg) myrosinase activity and were dissected into gut and rest of body 5 min later (n = 3 per plant genotype). Beetles were extracted with 80% methanol and 4MSOB glucosinolate was quantified by LC-MS/MS using an external standard curve. The distribution of 4MSOB glucosinolate in gut and rest of body is expressed relative to the total amount detected in both samples (set to 100%). (B) Enzyme assays were performed with partially purified Sinapis alba myrosinase that was incubated with 4MSOB glucosinolate as a substrate in the presence of untreated gut content extract of P. armoraciae, boiled gut content extract, or buffer (n = 4 per treatment). Myrosinase activity was determined by quantifying the 4MSOB glucosinolate substrate in each assay after conversion to desulfo-glucosinolate and analysis by HPLC-DAD. Assays without myrosinase served as background controls and activities were subtracted from the corresponding samples. Myrosinase activity was compared by One-way ANOVA (p < 0.001). Different letters indicate significant differences between groups. The result of the statistical analysis is shown in Supplementary Table 1.