Biology subjects

Bak, S.

Publications and source records attributed to Bak, S..

9 recordsLinked to original sources

bioRxiv2026

Glycosylation-dependent Turnover of Triterpenoid Saponins Controls Insect Deterrence

Background and AimsPlants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. MethodsLarvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC- qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key ResultsLarvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. ConclusionsInsect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Issue SectionOriginal article

biochemistry↗

bioRxiv2026

A genomic and functional framework for the rapid domestication of the wild plant Chenopodium album

Global reliance on a small number of genetically uniform crops makes our food system increasingly vulnerable to pests, diseases, and climate change, highlighting the need to develop resilient local species as crops. Chenopodium album, a stress-tolerant, protein-rich wild plant whose seeds were part of prehistoric Northern European diets and whose leaves are still foraged worldwide, remains undomesticated despite its agrifood potential. We established a Danish collection of 143 accessions and combined seed metabolomics, ploidy assessment and genomics to uncover the molecular basis of key nutritional and anti-nutritional traits. Seed profiling revealed substantial variation in protein content (14-22%), comparable to or higher than major crops, and 16 distinct triterpenoid saponins, which are widespread bitter and anti-nutritional compounds. Seed production of field-grown lines reached up to 1.5 t/ha in trials conducted in Denmark, demonstrating promising yield potential. A high-quality tetraploid genome of a low-saponin line was assembled and contrasted with resequencing of a diploid high-saponin line in order to uncover the genetic basis of saponin variation in C. album. Comparative genomic, phylogenetic, and transcriptomic analyses identified structural variants and candidate genes associated with saponin biosynthesis, and functional validation confirmed the coordinated activity of a {beta}-amyrin synthase, three CYP716 cytochromes P450, and a glucuronosyltransferase that reconstitute the core C. album saponin pathway. Together, these results define the genomic and biochemical foundation of C. album, establishing a platform for its rapid domestication as a locally adapted, high-protein seed crop and a model for translating wild plant diversity into future food security.

plant biology↗

bioRxiv2025

Degradation of biopesticidal triterpenoid saponins by the soil bacterium Arthrobacter sp. α-11c

BackgroundSaponins, a diverse group of glycosylated triterpenoid and steroid compounds produced by plants, exhibit potent insecticidal activity and are promising candidates for sustainable pest management. However, their potential persistence in soil raises concerns about ecological impacts, highlighting the need to understand microbial degradation rates and pathways. ResultsThis study reports the isolation of Arthrobacter sp. 11c, and its ability of fully degrading two hederagenin-based pentacyclic triterpenoid saponins: -hederin, hederacoside C, and the corresponding sapogenin hederagenin. All three compounds were fully metabolized within six hours when provided as carbon source. Fractionation experiments confirmed intracellular uptake and degradation of the saponins. Genome analysis of 11c revealed diverse and multiple glycosidase genes, yet basal glucosidase activity remained unchanged when cultured with these saponins. Comparative genomics with Arthrobacter sp. 12b, a closely related strain unable to degrade saponins, revealed two unique glycosidase gene clusters in 11c, suggesting a role in adaptation to saponin metabolism. ConclusionThese findings enhance our understanding of bacterial degradation of plant triterpenoid saponins and provide a foundation for evaluating their environmental fate. This knowledge supports the safe and sustainable use of saponin-based biopesticides in agriculture by identifying microbial partners that contribute to their breakdown in soil ecosystems.

microbiology↗

bioRxiv2025

Metabolic engineering of Barbarea vulgaris identifies β-amyrin synthase LUP5 as a determinant of saponin-mediated insect resistance

Plant-insect coevolution has been a major driver of specialized metabolite diversification, yet the genetic basis of natural variation in defensive chemistry remains poorly understood. The wild crucifer Barbarea vulgaris comprises two ecotypes, an insect-resistant G-type and a susceptible P-type, characterized by distinct triterpenoid saponin profiles. To investigate the causal relationship between saponin composition and insect resistance, we established a stable transformation system for B. vulgaris. Expression of the G-type {beta}-amyrin synthase gene LUP5 in the susceptible P-type conferred up to a 95% reduction in Plutella xylostella feeding, accompanied by increased accumulation of three hederagenin-derived monodesmosidic saponins. Comparison of LUP5 expression driven by its native promoter and by the constitutive 35S promoter revealed that the native promoter leads to increased hederagenin accumulation and is activated later in development, which may prevent early metabolic stress and allow coordinated expression of downstream pathway genes. In contrast, silencing of CYP72A552 by RNAi decreased total hederagenin levels by approximately 40% without affecting resistance, indicating threshold-dependent defense. Our results provide direct in planta evidence that LUP5 is a key determinant of natural variation in insect resistance in B. vulgaris, underscoring the pivotal role of the saponin backbone in herbivore deterrence. By linking promoter activity to metabolite structural diversity, this work provides mechanistic and conceptual insight into how plants coordinate specialized metabolism and defense.

plant biology↗

bioRxiv2025

Sterols govern membrane susceptibility to saponin-induced lysis

Saponins are natural detergents that interact with cellular membranes, causing deterioration leading to membrane disruption. The magnitude of these effects depends on both the saponin structure and target membrane composition, where sterols play a key modulating role in saponin-membrane interaction. We investigated the influence of different sterol classes on saponin-induced membrane lysis. The bioactive, cytotoxic saponin -hederin induced permeability in membranes containing zoosterol and mycosterol, whereas phytosterol-containing membranes were resistant to lysis in vitro. Similarly, in yeast, -hederin caused significant cell lysis, while in the ergosterol-deficient erg3{Delta} and pdr18{Delta} mutants, cell lysis was minimal. Supplementing phytosterols to yeast provided resistance to -hederin-induced lysis. Molecular dynamics simulations provide novel mechanistic insights, showing that the efficacy of the activity of -hederin is proportional to the sterol type in the membrane. Our findings reveal that while zoosterols and mycosterols render membranes vulnerable to bioactive saponins, phytosterols protect membranes from saponin-induced lysis in vitro, in vivo and in silico.

molecular biology↗

bioRxiv2025

Gene loss associated with plasticity-first evolution in Heliconius butterflies

Phenotypic plasticity occurs when a genotype can produce more than one phenotype under different environmental conditions. Genetic accommodation allows plastic phenotypes to be tuned to new environments and eventually to even be lost. The colourful and toxic Heliconiini butterflies have biochemical plasticity - they either sequester their cyanogenic glucosides (CG) from their larval hostplant or biosynthesize them when compounds for sequestration are not available. Here, we trace the evolution of CG biosynthesis in Heliconiini butterflies, a fundamental component of this biochemical plasticity. We first reconstructed the evolutionary history of biochemical plasticity in Heliconiini butterflies using chemical data from over 700 individuals, demonstrating that plasticity was ancestral in the tribe and further lost in a few clades, such as the Sapho clade that only sequester CGs. In lepidopterans, CG biosynthesis has been fully characterized in the moth Zygaena filipendulae, and is composed of CYP405A2, CYP332A3, and UGT33A1. Thus, we CRISPR-edited the gene CYP405 in Heliconius erato, and confirmed that CYP405-knockout caterpillars do not biosynthesize CGs. We identified the CYP405s and CYP332s in other lepidopterans and found that both genes were independently co-opted into CG biosynthesis in the Heliconiinae butterflies and Zygaena moths. Most lepidopterans have a CYP332, but CYP405 is mostly restricted to butterflies and has been duplicated in all Heliconius species. Although several CYP405 copies were found in the Sapho clade, they lack structurally important P450 domains, which explain they loss of biochemical plasticity via specialization in CG sequestration. Our findings represent one of the few examples of plasticity-first evolution in which the genetic mechanisms associated with its accommodation/assimilation are known.

evolutionary biology↗

bioRxiv2023

Pollen-feeding delays reproductive senescence and maintains toxicity of Heliconius butterflies

Dietary shifts may act to ease energetic constraints and allow organisms to optimise life-history traits. Heliconius butterflies differ from other nectar-feeders due to their unique ability to digest pollen, which provides a reliable source of amino acids to adults. Pollen-feeding has been associated with prolonged adult lifespan and increased fertility, yet there is a lack of empirical data demonstrating how pollen consumption influences key fitness traits, including chemical defences and adult body weight, as well as fertility over their elongated lifespan. Here, we investigated the effect of pollen-feeding on fertility, weight and chemical defences, as well as offspring defences, controlling for butterfly age and sex. Recently emerged Heliconius erato butterflies of similar size were fed for 14 or 45 days on one of three diets: sugar solution only, or sugar solution replenished with either amino acid supplement or pollen. At the end of the experiment, oviposition assays were performed to evaluate fertility, and afterwards all butterflies and eggs were weighed and used for quantification of cyanogenic glucosides (CG). We found that there is an age-specific and sex-specific effect of pollen-feeding on butterfly weight, with both the sugar-only and amino-acid supplement diets reducing the weight of old females (45d), but not young females (14d) or males of any age. Females fed only sugar significantly reduced their egg-laying through adulthood, whereas females that had access to pollen maintained their fertility. Diet had a significant effect on the maintenance of the chemical defence of females, but not males. Curiously, even though females that have access to pollen were heavier, more toxic and laid more eggs, this did not translate into improvements in offspring defences, as eggs from butterflies of all ages and diet treatments had similar CG content. Our results emphasise the importance of controlling for age-specific and sex-specific effects in studies of life-history evolution and demonstrate that dietary novelty can relax energetic constraints.

evolutionary biology↗

bioRxiv2020

Phenotypic plasticity in chemical defence allows butterflies to diversify host use strategies

Hostplant specialization is a major force driving ecological niche partitioning and diversification in insect herbivores. The cyanogenic defences of Passiflora plants keeps most herbivores at bay, but not larvae of Heliconius butterflies, which can both sequester and biosynthesize cyanogenic compounds. Here, we demonstrate that both Heliconius cydno chioneus, a host plant generalist, and H. melpomene rosina, a specialist, have remarkable plasticity in their chemical defence. When feeding on Passiflora species with cyanogenic compounds they can readily sequester, both species downregulate the biosynthesis of these compounds. In contrast, when fed on Passiflora plants that do not contain cyanogenic glucosides that can be sequestered, both species increase biosynthesis. This biochemical plasticity comes at a significant fitness cost for specialist like H. m. rosina, as growth rates for this species negatively correlate with biosynthesis levels, but not for a generalist like H. c. chioneus. In exchange, H. m rosina has increased performance when sequestration is possible as on its specialised hostplant. In summary, phenotypic plasticity in biochemical responses to different host plants offers these butterflies the ability to widen their range of potential host within the Passiflora genus, while maintaining their chemical defences.

evolutionary biology↗

bioRxiv2019

Sequestration and functional diversification of cyanogenic glucosides in the life cycle of Heliconius melpomene

Heliconius butterflies are highly specialized in Passiflora, laying eggs and feeding as larvae only on these plants. Interestingly, Heliconius butterflies and Passiflora plants both contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been fully biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 are hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different development stages of Heliconius butterflies. We also established which kind of CNglcs H. melpomene larvae can sequestered from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we observed that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They were also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea was not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin was catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship and calling. Mature adults had a higher concentration of CNglcs than any other developmental stages due to intense de novo biosynthesis of linamarin and lotaustralin. All CYP405As were expressed in adults, whereas larvae mostly expressed CYP405A4. Our results shed light on the importance of CNglcs in Heliconius biology and for their coevolution with Passiflora.

biochemistry↗