Aliphatic and benzenic glucosinolates in phylogenetically distinct Tropaeolum peregrinum, detection of ethylglucosinolate but lack of chain-elongated glucosinolates, and effects of a methyl group for detoxification by Pieris caterpillars

Understanding glucosinolate (GSL) structural evolution needs high-certainty determination of GSL profiles of conclusively identified plant species. Literature review found hydroxylated aliphatic GSLs to be uncertain in the family Tropaeolaceae from the basal part of order Brassicales phylogeny. To establish the GSL profile of Tropaeolum peregrinum , major seed GSLs were isolated using an optimized procedure and identified as 2-hydroxy-2-methylpropylGSL, 1-methylethylGSL, 4-hydroxybenzylGSL and 4-methoxybenzylGSL by 1 H and 13 C NMR and MS/MS analysis. Six accessions were investigated, two including leaves/roots. The four NMR-verified GSLs were detected in all accessions and plant parts. Presence or absence of numerous other GSLs was tested by UHPLC-MS/MS comparison to authentic references. Known GSLs from chain-elongated amino acids were consistently not detected. EthylGSL was detected and attributed to biosynthesis from a general metabolite, alpha-ketobutanoic acid, independent of chain elongation. In addition, 1-methylpropylGSL was prominent, with lower levels of 2-methylpropylGSL, benzylGSL, disubstituted GSLs such as 3,4-dimethoxybenzylGSL, and minute peaks possibly representing isomers and side-products of 2-hydroxy-2-methylpropylGSL. Plants were DNA-barcode characterized and identified, and a Tropaeolaceae phylogeny was prepared, with T. peregrinum in a distinct branch. Metabolism of 4-hydroxybenzylGSL in caterpillars of the butterfly Pieris rapae was known to produce 4-hydroxybenzylcyanide sulfate (HBC sulfate), while metabolism of 4-methoxybenzylGSL was not known. When P. rapae caterpillars were offered those two GSLs on brassicaceous leaves, both GSLs resulted in HBC sulfate in frass. However, molar levels of HBC sulfate were high from 4-hydroxybenzylGSL but 20-fold lower from the methyl derivative 4-methoxybenzylGSL, suggesting incomplete demethylation of intermediate 4-methoxybenzylcyanide in caterpillars.