Benefits of isochoric freezing for carrot juice preservation.

Isochoric freezing (IF) at -5°C/77 and -10°C/100 MPa was used to preserve carrot juice for 12 weeks. The juice qualities were compared to those using heat treatment (HT) at 95°C for 15 s followed by cold storage at 4°C. The native population of total aerobic bacteria, yeasts, and molds in isochoric frozen juice remained below the detection limit for 12 weeks. In comparison, microbes started to grow in heat-treated juices after 3 weeks of refrigeration. The color of isochoric frozen juice appeared more deep orange than the fresh juice due to an increase in carotenoid extractability. IF was not effective in reducing the activities of peroxidase, polyphenol oxidase, and pectin methyl esterase compared with HT. However, the isochoric samples showed higher carotenoid content, polyphenol content, and antioxidant capacity compared to the fresh and heat-treated juices. PRACTICAL APPLICATION: Isochoric freezing was used to produce carrot juice with extended shelf life. Isochoric freezing could be a beneficial alternative to conventional heat treatment for carrot juice processing as the applied pressures reached total inactivation levels of spoilage microorganisms. Moreover, the low processing temperatures better retained desirable compounds and quality attributes of fresh juice throughout its shelf life.

Recruitment of distinct UDP-glycosyltransferase families demonstrates dynamic evolution of chemical defense within Eucalyptus L'Hér.

The economic and ecologically important genus Eucalyptus is rich in structurally diverse specialized metabolites. While some specialized metabolite classes are highly prevalent across the genus, the cyanogenic glucoside prunasin is only produced by c. 3% of species. To investigate the evolutionary mechanisms behind prunasin biosynthesis in Eucalyptus, we compared de novo assembled transcriptomes, together with online resources between cyanogenic and acyanogenic species. Identified genes were characterized in vivo and in vitro. Pathway characterization of cyanogenic Eucalyptus camphora and Eucalyptus yarraensis showed for the first time that the final glucosylation step from mandelonitrile to prunasin is catalyzed by a novel UDP-glucosyltransferase UGT87. This step is typically catalyzed by a member of the UGT85 family, including in Eucalyptus cladocalyx. The upstream conversion of phenylalanine to mandelonitrile is catalyzed by three cytochrome P450 (CYP) enzymes from the CYP79, CYP706, and CYP71 families, as previously shown. Analysis of acyanogenic Eucalyptus species revealed the loss of different ortholog prunasin biosynthetic genes. The recruitment of UGTs from different families for prunasin biosynthesis in Eucalyptus demonstrates important pathway heterogeneities and unprecedented dynamic pathway evolution of chemical defense within a single genus. Overall, this study provides relevant insights into the tremendous adaptability of these long-lived trees.

An Independent Evolutionary Origin for Insect Deterrent Cucurbitacins in Iberis amara.

Pieris rapae and Phyllotreta nemorum are Brassicaceae specialists, but do not feed on Iberis amara spp. that contain cucurbitacins. The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Using de novo assembled transcriptomics from I. amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase (IaCPQ). In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyze the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the draft genome assembly of I. amara showed that the IaCPQ was localized to the same scaffold together with CYP708A15 but spanning over 100 kb, this contrasts with the highly organized cucurbitacin gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This study thus provides new insight into the mechanism of recurrent evolution and diversification of a plant defensive chemical.

Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates.

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale.

Texture of Hot-Air-Dried Persimmon (Diospyros kaki) Chips: Instrumental, Sensory, and Consumer Input for Product Development.

Persimmon (Diospyros kaki) is an underutilized tree fruit. Previous studies have shown the feasibility of making a hot-air-dried, chip-style product from persimmon. However, the texture of this type of product has not been explored or connected to consumer preference. Thus, for dried samples representing 37 cultivars, this study aimed to (1) predict trained sensory panel texture attributes from instrumental measurements, (2) predict consumer liking from instrumental measurements and sensory texture attributes, and (3) elucidate whether astringency type affects dried product texture. Partial least-squares regression models of fair-to-good quality predicted all measured sensory texture attributes (except Tooth Packing) from instrumental measurements. Modeling also identified that consumer preference is for a moist, smooth texture. Lastly, while astringency type has significant (p < 0.05) effects on several individual texture attributes, astringency type should not be used a priori to screen-in or -out persimmon cultivars for processing into a hot-air-dried product.

Biosynthesis of cyanogenic glucosides in Phaseolus lunatus and the evolution of oxime-based defenses.

Lima bean, Phaseolus lunatus, is a crop legume that produces the cyanogenic glucosides linamarin and lotaustralin. In the legumes Lotus japonicus and Trifolium repens, the biosynthesis of these two α-hydroxynitrile glucosides involves cytochrome P450 enzymes of the CYP79 and CYP736 families and a UDP-glucosyltransferase. Here, we identify CYP79D71 as the first enzyme of the pathway in P. lunatus, producing oximes from valine and isoleucine. A second CYP79 family member, CYP79D72, was shown to catalyze the formation of leucine-derived oximes, which act as volatile defense compounds in Phaseolus spp. The organization of the biosynthetic genes for cyanogenic glucosides in a gene cluster aided their identification in L. japonicus. In the available genome sequence of P. vulgaris, the gene orthologous to CYP79D71 is adjacent to a member of the CYP83 family. Although P. vulgaris is not cyanogenic, it does produce oximes as volatile defense compounds. We cloned the genes encoding two CYP83s (CYP83E46 and CYP83E47) and a UDP-glucosyltransferase (UGT85K31) from P. lunatus, and these genes combined form a complete biosynthetic pathway for linamarin and lotaustralin in Lima bean. Within the genus Phaseolus, the occurrence of linamarin and lotaustralin as functional chemical defense compounds appears restricted to species belonging to the closely related Polystachios and Lunatus groups. A preexisting ability to produce volatile oximes and nitriles likely facilitated evolution of cyanogenesis within the Phaseolus genus.

Glucosinolate structural diversity, identification, chemical synthesis and metabolism in plants.

The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-ß-d-glucopyrano unit anomerically connected to an O-sulfated (Z)-thiohydroximate function. After enzymatic hydrolysis, the sulfated aglucone can undergo rearrangement to an isothiocyanate, or form a nitrile or other products. The number of GSLs known from plants, satisfactorily characterized by modern spectroscopic methods (NMR and MS) by mid-2018, is 88. In addition, a group of partially characterized structures with highly variable evidence counts for approximately a further 49. This means that the total number of characterized GSLs from plants is somewhere between 88 and 137. The diversity of GSLs in plants is critically reviewed here, resulting in significant discrepancies with previous reviews. In general, the well-characterized GSLs show resemblance to C-skeletons of the amino acids Ala, Val, Leu, Trp, Ile, Phe/Tyr and Met, or to homologs of Ile, Phe/Tyr or Met. Insufficiently characterized, still hypothetic GSLs include straight-chain alkyl GSLs and chain-elongated GSLs derived from Leu. Additional reports (since 2011) of insufficiently characterized GSLs are reviewed. Usually the crucial missing information is correctly interpreted NMR, which is the most effective tool for GSL identification. Hence, modern use of NMR for GSL identification is also reviewed and exemplified. Apart from isolation, GSLs may be obtained by organic synthesis, allowing isotopically labeled GSLs and any kind of side chain. Enzymatic turnover of GSLs in plants depends on a considerable number of enzymes and other protein factors and furthermore depends on GSL structure. Identification of GSLs must be presented transparently and live up to standard requirements in natural product chemistry. Unfortunately, many recent reports fail in these respects, including reports based on chromatography hyphenated to MS. In particular, the possibility of isomers and isobaric structures is frequently ignored. Recent reports are re-evaluated and interpreted as evidence of the existence of "isoGSLs", i.e. non-GSL isomers of GSLs in plants. For GSL analysis, also with MS-detection, we stress the importance of using authentic standards.

The dynamics of cyanide defences in the life cycle of an aposematic butterfly: Biosynthesis versus sequestration.

Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants 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 biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been 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 developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are 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 is 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 is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.

The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

No evidence of quantitative signal honesty across species of aposematic burnet moths (Lepidoptera: Zygaenidae).

Many defended species use conspicuous visual warning signals to deter potential predators from attacking. Traditional theory holds that these signals should converge on similar forms, yet variation in visual traits and the levels of defensive chemicals is common, both within and between species. It is currently unclear how the strength of signals and potency of defences might be related: conflicting theories suggest that aposematic signals should be quantitatively honest, or, in contrast, that investment in one component should be prioritized over the other, while empirical tests have yielded contrasting results. Here, we advance this debate by examining the relationship between defensive chemicals and signal properties in a family of aposematic Lepidoptera, accounting for phylogenetic relationships and quantifying coloration from the perspective of relevant predators. We test for correlations between toxin levels and measures of wing colour across 14 species of day-flying burnet and forester moths (Lepidoptera: Zygaenidae), protected by highly aversive cyanogenic glucosides, and find no clear evidence of quantitative signal honesty. Significant relationships between toxin levels and coloration vary between sexes and sampling years, and several trends run contrary to expectations for signal honesty. Although toxin concentration is positively correlated with increasing luminance contrast in forewing pattern in 1 year, higher toxin levels are also associated with paler and less chromatically salient markings, at least in females, in another year. Our study also serves to highlight important factors, including sex-specific trends and seasonal variation, that should be accounted for in future work on signal honesty in aposematic species.

Vitamin D5 in Arabidopsis thaliana.

Vitamin D3 is a secosterol hormone critical for bone growth and calcium homeostasis, produced in vertebrate skin by photolytic conversion of the cholesterol biosynthetic intermediate provitamin D3. Insufficient levels of vitamin D3 especially in the case of low solar UV-B irradiation is often compensated by an intake of a dietary source of vitamin D3 of animal origin. Small amounts of vitamin D3 were described in a few plant species and considered as a peculiar feature of their phytochemical diversity. In this report we show the presence of vitamin D5 in the model plant Arabidopsis thaliana. This plant secosterol is a UV-B mediated derivative of provitamin D5, the precursor of sitosterol. The present work will allow a further survey of vitamin D distribution in plant species.

Development of an e-learning programme to improve knowledge of interprofessional education.

Evidence has highlighted the importance of interprofessional education in relation to the promotion of collaborative team working and the ultimate reduction in communication errors between health professionals. The establishment of interprofessional education in undergraduate healthcare programmes prepares students for interprofessional collaboration, which is vital to the delivery of safe patient care. Using a logic model approach an interprofessional education e-learning programme was developed and made available to healthcare students to undertake during their clinical practice experiences. This article describes how a logic model process was used to develop and implement this interprofessional education e-learning programme for use by undergraduate healthcare students.

Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55.

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.

Sex differences but no evidence of quantitative honesty in the warning signals of six-spot burnet moths (Zygaena filipendulae L.).

The distinctive black and red wing pattern of six-spot burnet moths (Zygaena filipendulae, L.) is a classic example of aposematism, advertising their potent cyanide-based defences. While such warning signals provide a qualitatively honest signal of unprofitability, the evidence for quantitative honesty, whereby variation in visual traits could provide accurate estimates of individual toxicity, is more equivocal. Combining measures of cyanogenic glucoside content and wing color from the perspective of avian predators, we investigate the relationship between coloration and defences in Z. filipendulae, to test signal honesty both within and across populations. There were no significant relationships between mean cyanogenic glucoside concentration and metrics of wing coloration across populations in males, yet in females higher cyanogenic glucoside levels were associated with smaller and lighter red forewing markings. Trends within populations were similarly inconsistent with quantitative honesty, and persistent differences between the sexes were apparent: larger females, carrying a greater total cyanogenic glucoside load, displayed larger but less conspicuous markings than smaller males, according to several color metrics. The overall high aversiveness of cyanogenic glucosides and fluctuations in color and toxin levels during an individual's lifetime may contribute to these results, highlighting generally important reasons why signal honesty should not always be expected in aposematic species.

Glutathione transferases catalyze recycling of auto-toxic cyanogenic glucosides in sorghum.

Cyanogenic glucosides are nitrogen-containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin-derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant-specific lambda class (GSTLs) to produce p-hydroxyphenyl acetonitrile. This is further metabolized to p-hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p-glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co-localized with expression of the genes encoding the p-hydroxyphenyl acetonitrile-metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.

Honeybees Tolerate Cyanogenic Glucosides from Clover Nectar and Flowers.

Honeybees (Apis mellifera) pollinate flowers and collect nectar from many important crops. White clover (Trifolium repens) is widely grown as a temperate forage crop, and requires honeybee pollination for seed set. In this study, using a quantitative LC-MS (Liquid Chromatography-Mass Spectrometry) assay, we show that the cyanogenic glucosides linamarin and lotaustralin are present in the leaves, sepals, petals, anthers, and nectar of T. repens. Cyanogenic glucosides are generally thought to be defense compounds, releasing toxic hydrogen cyanide upon degradation. However, increasing evidence indicates that plant secondary metabolites found in nectar may protect pollinators from disease or predators. In a laboratory survival study with chronic feeding of secondary metabolites, we show that honeybees can ingest the cyanogenic glucosides linamarin and amygdalin at naturally occurring concentrations with no ill effects, even though they have enzyme activity towards degradation of cyanogenic glucosides. This suggests that honeybees can ingest and tolerate cyanogenic glucosides from flower nectar. Honeybees retain only a portion of ingested cyanogenic glucosides. Whether they detoxify the rest using rhodanese or deposit them in the hive should be the focus of further research.

Synthesis of descriptive sensory attributes and hedonic rankings of dried persimmon (Diospyros kaki sp.).

This work aimed to characterize the sensory attributes of hot air-dried persimmon (Diospyros kaki) chips, correlate these attributes with consumer hedonic information, and, by doing so, present recommendations for cultivars that are most suitable for hot-air drying. A trained sensory panel evaluated dried persimmon samples (representing 40 cultivars) for flavor, taste/aftertaste, and texture. In addition, in each of two tests conducted in different years, more than 100 consumers provided hedonic evaluations of 21 unique samples in a ranking task with a balanced incomplete block design. A partial least squares regression model correlating the mean hedonic ranking to the trained panel data was developed using the data from the first consumer panel. The predictions from the model were correlated with the second panel to verify the model. It was found that including taste, aftertaste, and texture data (but not specific flavor attribute data) produced a predictive model (Spearman's ρ=0.83). This indicates that flavor is likely secondary to taste and texture in dried persimmon chips. Using the validated predictive model, 6 of the 40 persimmon cultivars tested are recommended for a dried chip product; these cultivars are 'Fuyu', 'Lycopersicon', 'Maekawa Jiro', 'Nishimura Wase', 'Tishihtzu', and 'Yotsumizo'.

Diurnal regulation of cyanogenic glucoside biosynthesis and endogenous turnover in cassava.

Cyanogenic glucosides are present in many plants, including eudicots, monocots, and ferns and function as defence compounds based on their ability to release hydrogen cyanide. In this study, the diurnal rhythm of cyanogenic glucoside content and of transcripts and enzymes involved in their biosynthesis was monitored in cassava plants grown in a glasshouse under natural light conditions. Transcripts of CYP79D1, CYP79D2, CYP71E7/11, and UGT85K5 were at minimal levels around 9 p.m., increased during the night and decreased following onset of early morning light. Transcripts of UGT85K4 and HNL10 showed more subtle variations with a maximum reached in the afternoon. Western blots showed that the protein levels of CYP71E7/11 and UGT85K4/5 decreased during the light period to a near absence around 4 p.m. and then recovered during the dark period. Transcript and protein levels of linamarase were stable throughout the 24-hr cycle. The linamarin content increased during the dark period. In the light period, spikes in the incoming solar radiation were found to result in concomitantly reduced linamarin levels. In silico studies of the promoter regions of the biosynthetic genes revealed a high frequency of light, abiotic stress, and development-related transcription factor binding motifs. The synthesis and endogenous turnover of linamarin are controlled both at the transcript and protein levels. The observed endogenous turnover of linamarin in the light period may offer a source of reduced nitrogen to balance photosynthetic carbon fixation. The rapid decrease in linamarin content following light spikes suggests an additional function of linamarin as a ROS scavenger.

Spatial separation of the cyanogenic ß-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of Zygaena larvae facilitates cyanide release.

Low molecular weight compounds are typically used by insects and plants for defence against predators. They are often stored as inactive ß-glucosides and kept separate from activating ß-glucosidases. When the two components are mixed, the ß-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 ß-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 ß-glucosidases, and correspondingly, the ability to hydrolyse cyanogenic glucosides catalysed by a specific ß-glucosidase evolved convergently in insects and plants. The spatial separation of the ß-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.