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Larvae of Chrysomelina beetles use a double barrier system to protect themselves. The first barrier is based on the nine pairs of glandular reservoirs located under their dorsal cuticle. Once encountered predators, they secrete defensive secretions from the glandular reservoirs to fend off the predators. Along with the host plant shifts, the deterrents in the glandular secretions and the ways to synthesize these deterrents vary accordingly. The evolutionary analysis showed the larvae had evolved gradually from autogenous synthesis via sequestration to the mixed state. This stepwise evolution was based on the hypothesis that the de novo species have a set of genes, which made the evolvement from de novo synthesis to take up glucosides from the host plant possible. The second barrier system in chrysomlina beetles is the 3-nitropropionic acid (3-NPA) based defensive compounds (isoxazolin-5-one glucoside and its 3-NPA conjugated ester) that are stored in the hemolymph of the larvae. Once the ingested larvae reached to the gut of the predators, the toxic 3-NPA moiety from the ester will be liberated by the esterase that is presented in the gut of the predators, and the free 3-NPA moiety can bind covalently to the succinate dehydrogenase in the mitochondrial respiratory system, and thus block the energy production in the mammal predators. In the dissertation, the de novo species P. cochleariae was used as the model organism for the investigation of the important enzymes that are involved in the biosynthesis of these defensive compounds.
With the combination of transcriptomics, proteomics and RNAi, two cytochrome P450s, namely CYP6BH5 and CYP347W1, were identified in the fat body of P. cochleariae. CYP6BH5 was identified as the geraniol 8-hydroxylase in iridoid biosynthesis. In addition to geraniol, CYP6BH5 also catalyzes hydroxylation of other monoterpenols, such as nerol and citronellol to the corresponding α, ω-dihydroxy compounds. Homology modeling suggested that the -OH group of the substrate plays an important role in coordination the substrates with CYP6BH5’s catalytic center. The second cytochrome P450, CYP347W1, was proved involved in the production of the 3-NPA moiety in the 3-NPA based defensive line. Moreover, P. cochleariae from different life stages except eggs was showed to share the same enzymatic system for 3-NPA production. This β-alanine and P450-dependent 3-NPA synthetic tactic in P. cochleariae is different from the aspartic acid and flavin-dependent oxidoreductase based biosynthetic mechanism in prokaryotic organisms. This indicates that different organisms may choose their favorable amino acids and enzymes to produce the same products, which contributes to the ubiquitous occurrence of 3-NPA in eukaryotic and prokaryotic kingdoms.
last updated on 2019-10-28