Harmonizing biosynthesis with post-ingestive modifications to understand the ecological functions of plant natural products

Abstract

Covering: up to 2022The recent dramatic advances in our understanding of the biosynthetic pathways that produce diverse bouquets of plant-derived natural products have far surpassed our understanding of the function of these compounds for plants: how they influence a plant's Darwinian fitness in nature. Our understanding of their mechanisms, the life-processes targeted by these compounds, is similarly poorly resolved. Many plant specialized metabolites (PSMs) are further modified after ingestion by herbivores, and these post-ingestive modifications are frequently essential for PSM function. Here we summarize the biosynthesis and functional mechanisms of 17-hydroxygeranyllinalool diterpene glycosides in the ecological model plant Nicotiana attenuata, and summarize the post-ingestive modifications known from other two-component PSMs. We propose that parallel comparisons of plant natural product biosynthetic pathways and insect post-ingestive metabolism of the same plant tissues ("frassomics") will facilitate the often-elusive identification of the molecular targets of these effective chemical defenses, contribute to elucidations of post-ingestive metabolite interactions in insect guts, and predicate the rapid evolutions of resistance against insecticides inspired by PSMs. We highlight the value of conducting these parallel investigations at the level of the entire metabolome so as to include the multiple interacting pathways in both natural product biosynthesis as well as their post-ingestive processing. We introduce the concept of frass metabolite QTL (fmQTL) analysis that integrates powerful forward genetic approaches with frassomics, and suggest that insect-guided high-throughput forward- and reverse-genetics approaches in natural habitats will advance our understanding of PSM biosynthesis and function.

Fig. 1. Frassomic analyses elucidate post-ingestive modifications of HGL-DTGs and their diverse functional consequences. When Manduca sexta larvae consume leaves of their native host plant, Nicotiana attenuata, they produce frass, which can be subjected metabolomics analyses and compared with similar analyses of the ingested leaves. HGL-DTGs metabolites from leaves (green) and frass (gray) are clustered into different groups based on their neutral loss (NL) and normalized dot product (NDP, representing mass fragment) similarities. Line thickness reflects the degree of similarity among different compounds. Deglycosylation of ingested HGL-DTGs reflects a detoxification process for the larvae but has the downside of increasing larval vulnerability to spider predators. Hydroxylations of ingested HGL-DTGs results in active toxins responsible for the compounds defensive function against herbivores as well as their toxicity to the producing plants, when the normal patterns of hydroxylation or glucosylations during their biosynthesis are abrogated. Hydroxycinnamoylations thwart the toxic effects of both ingested HGL-DTGs and chlorogenic acid, and functions as a detoxification process by mechanisms that require further study. Demalonylation occurs in the alkaline environment of the larval midgut without noticeable functional consequences for the herbivore, but when malonylation steps in the biosynthesis of HGL-DTGs in the plant are thwarted, normal stylar development is abrogated, revealing a developmental role for pathway intermediates.

Fig. 2. Post-ingestive modifications of defensive PSMs. (A) When plant cells are damaged, myrosinases cleave the glucose from GLS, generating the unstable GLS aglycone. Using nitrile-specifier proteins (NSP), insect herbivores can transform the aglycone to nitriles, which prevents their spontaneous transformations into more toxic isothiocyanates. Some other insects can directly modify GLS by adding extra glucoses via sucrase (SUC), or by de-sulphatation via glucosinolate sulphatase (GSS). (B) 2R-DIMBOA-Glc is converted into toxic aglycone, 2R-DIMBOA, by plant glucosidase. The 2S-DIMBOA, epimer of 2R-DIMBOA, can be re-glucosylated by insect glycosyltransferases to produce the less toxic epimer, 2S-DIMBOA-Glc that is inert to plant glucosidase. (C) Linamarin, a representative cyanogenic glucoside, is hydrolyzed by plant glucosidases and hydroxynitrile lyase to liberate HCN. Insects can transform HCN into β-cyanoalanine by conjugating cysteine via β-cyanoalanine synthase. Whiteflies can modify linamarin with extra glucoses and/or phosphates to prevent the formation of hydrogen cyanide. (D and E) Illustrate PSMs with specialized targets: (D) nicotine and (E) 20 hydroxyecdysone. Text in green and orange indicates enzymes from plants and insects, respectively. Red highlights moieties that are subjected to transformation. Inhibition symbols indicate the chemicals are inert to the corresponding enzymes.

Fig. 3. An insect-guided forward-genetics framework to study the function of PSMs. As described in Fig. 1, comparative leaf and frass metabolomics can elucidate the post-ingestive modification of PSMs, and contribute to our understanding of PSM functions in both producers and consumers of PSMs. In addition to the reverse-genetics approaches used in a frassomics context (for examples, see references in Fig. 1 caption), forward-genetics tools, such as the Recombinant Inbred Lines (RILs) that can capture a majority of the natural variation in a species in a structured manner (e.g., Multiparent Advanced Generation Intercross (MAGIC) RIL populations) can be used in a frassomic context to conduct frass metabolite QTL (fmQTL) imputations that can bridge the gap between frass metabolites and plant genetics, and provide a powerful tool with which to identify plant enzymes that control post-ingestive modification of PSMs. When released into natural habitats, these forward genetic tools can be used to study the frassomics of multitrophic interactions mediated by PSMs.