Abstract
Low molecular weight compounds are typically used by insects and plants for defence against predators. They are often stored as inactive β-glucosides and kept separate from activating β-glucosidases. When the two components are mixed, the β-glucosides are hydrolysed releasing toxic aglucones. Cyanogenic plants contain cyanogenic glucosides and release hydrogen cyanide due to such a well-characterized two-component system. Some arthropods are also cyanogenic, but comparatively little is known about their system. Here, we identify a specific β-glucosidase (ZfBGD2) involved in cyanogenesis from larvae of Zygaena filipendulae (Lepidoptera, Zygaenidae), and analyse the spatial organization of cyanide release in this specialized insect. High levels of ZfBGD2 mRNA and protein were found in haemocytes by transcriptomic and proteomic profiling. Heterologous expression in insect cells showed that ZfBGD2 hydrolyses linamarin and lotaustralin, the two cyanogenic glucosides present in Z. filipendulae. Linamarin and lotaustralin as well as cyanide release were found exclusively in the haemoplasma. Phylogenetic analyses revealed that ZfBGD2 clusters with other insect β-glucosidases, and correspondingly, the ability to hydrolyse cyanogenic glucosides catalysed by a specific β-glucosidase evolved convergently in insects and plants. The spatial separation of the β-glucosidase ZfBGD2 and its cyanogenic substrates within the haemolymph provides the basis for cyanide release in Z. filipendulae. This spatial separation is similar to the compartmentalization of the two components found in cyanogenic plant species, and illustrates one similarity in cyanide-based defence in these two kingdoms of life.
Highlights
Introduction βGlucosidases are ubiquitous glycosidases found in all kingdoms of life, involved in various processes such as biomass conversion in microbes, glycoside metabolism, cell wall lignification, phytohormone activation and chemical defence in plants and insects [1,2,3]. β-Glucosidases are categorized into the glycoside hydrolase families GH1, GH2, GH3, GH5, GH9, GH30 and GH116 [4], with GH1 constituting the largest family, and encompassing most characterized plant β-glucosidases [1]
Chemical defence mediated by β-glucosidases is an important driver of the herbivore–plant arms race, and is excellently illustrated by the phenomenon of cyanogenesis [12,13,14,15,16], which is the release of hydrogen cyanide (HCN) from cyanogenic glucosides (CNglcs) catalysed by β-glucosidase activity [17]
Expressed enzymes were retained in Sf9 cells, harvested by mild centrifugation, and the viability of cells was confirmed by Fluorescein Diacetate (FDA) staining
Summary
Introduction βGlucosidases are ubiquitous glycosidases found in all kingdoms of life, involved in various processes such as biomass conversion in microbes, glycoside metabolism, cell wall lignification, phytohormone activation and chemical defence in plants and insects [1,2,3]. β-Glucosidases are categorized into the glycoside hydrolase families GH1, GH2, GH3, GH5, GH9, GH30 and GH116 [4], with GH1 constituting the largest family, and encompassing most characterized plant β-glucosidases [1]. Arthropods may rely on HCN for defence [17,19], and here cyanogenesis may either proceed in special tissues morphologically separated from the rest of the body, such as defence secretions [20,21,22], or in tissues largely connected to the rest of the body, such as gut and haemolymph In this case, a prerequisite for cyanogenesis is the immediate enzymatic detoxification of HCN by β-cyanoalanine synthase [23,24,25]. Significant amounts of HCN in Z. filipendulae are released from crude larval haemolymph, whereas other tissues such as cuticular cavities containing defence droplets do not release HCN per se [29] This scenario renders a haemolymph-based β-glucosidase with activity against CNglcs likely, and Zygaena trifolii larvae have been shown to harbour cyanogenic β-glucosidase activity in the haemolymph [5]. The localization of the β-glucosidase and its substrates would unravel whether compartmentalization of the two components occurs in Zygaena insects
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