Abstract
Cyanide is generated in larvae of the glucosinolate-specialist Pieris rapae (Lepidoptera:Pieridae) upon ingestion of plant material containing phenylalanine-derived glucosinolates as chemical defenses. As these glucosinolates were widespread within ancient Brassicales, the ability to detoxify cyanide may therefore have been essential for the host plant shift of Pierid species from Fabales to Brassicales species giving rise to the Pierinae subfamily. Previous research identified β-cyanoalanine and thiocyanate as products of cyanide detoxification in P. rapae larvae as well as three cDNAs encoding the β-cyanoalanine synthases PrBSAS1-PrBSAS3. Here, we analyzed a total of eight species of four lepidopteran families to test if their cyanide detoxification capacity correlates with their feeding specialization. We detected β-cyanoalanine synthase activity in gut protein extracts of all six species tested, which included Pierid species with glucosinolate-containing host plants, Pierids with other hosts, and other Lepidoptera with varying food specialization. Rhodanese activity was only scarcely detectable with the highest levels appearing in the two glucosinolate-feeding Pierids. We then amplified by polymerase chain reaction (PCR) 14 cDNAs encoding β-cyanoalanine synthases from seven species. Enzyme characterization and phylogenetic analysis indicated that lepidopterans are generally equipped with one PrBSAS2 homolog with high affinity for cyanide. A second β-cyanoalanine synthase which grouped with PrBSAS3 was restricted to Pierid species, while a third variant (i.e., homologs of PrBSAS1), was only present in members of the Pierinae subfamily. These results are in agreement with the hypothesis that the host shift to Brassicales was associated with the requirement for a specialized cyanide detoxification machinery.
Highlights
The glucosinolate-myrosinase system serves as an activated chemical defense in plants of the order Brassicales
In case of Z. filipendulae, gene-specific primers were designed based on transcriptome data [26] and used to amplify cDNA fragments with similarity to β-cyanoalanine synthase cDNAs. cDNA ends were completed by 30 and 50 RACE and ORFs confirmed by independent amplification of the entire sequence in one polymerase chain reaction (PCR)
Among the lepidopteran larvae analyzed, only the glucosinolate-feeding specialists possessed considerable rhodanese activity, while β-cyanoalanine synthase activity was present in gut extracts of all Pierid and non-Pierid species analyzed
Summary
The glucosinolate-myrosinase system serves as an activated chemical defense in plants of the order Brassicales. The ability to colonize glucosinolate-containing plants may of Pierid and non-Pierid species with different feeding preferences and evolutionary histories We applied a polymerase chain reaction and non-Pierid species with different feeding preferences and evolutionary histories (Figure 2) for (PCR)-based strategy to clone cDNAs encoding β-cyanoalanine synthases from the seven species that β-cyanoalanine synthase and rhodanese activities. Applied a polymerase chain reaction had not been studied previously This allowed us toWe compare the kinetic properties of the purified (PCR)-based strategy to clone cDNAs encoding β-cyanoalanine synthases from the seven species that recombinant enzymes in relation to their phylogenetic relationships.
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