Hydroxynitrile Glucosides Research Articles

Magnetic separation hydroxynitrile glucoside of Orostachys malacophyllus

A double benefit is a kind of strategy for separate efficiency. By selecting the appropriate method, the small molecule in plant extracts can be effectively extracted. CCK-8 method was employed to bioassay-guide. Magnetic separation was adopted as extraction probe to capture and enrich potent cytotoxicity constituent. Borate functionalized magnetic solid (BFMS) was performed for two purposes to wrap hydrophobic and to leave hydrophilic component from the ethyl acetate extract of O. malacophyllus, fortunately, (R)-lotastralin, 1-O-alpha-D-glucopyranosyl-beta-D-fructopyranose and 3-O-alpha-D-glucopyranosyl-beta-D-fructo furanose was obtained. As a result, O. malacophyllus is much well-known that they also contain hydroxynitrile glucoside. This provides a route to enrich hydroxynitrile glucoside in plant analysis.

Hydroxynitrile Glucosides: Bioactive Constituent Recovery from the Oil Residue of Prinsepia utilis.

The seed oil of Prinsepia utilis is extensively used as an edible oil by the nationalities of Naxi, Tibetan, and Mosuo in China, which is particularly good for beauty care and has a health protection function. A large amount of industrial waste is thrown away during the production process of seed oil. Therefore, to recover bioactive compounds from the oil residue of P. utilis is environmentally friendly and economically important. For this purpose, the chemical constituents of the P. utilis oil residue were investigated in our research, and five new compounds, prinsepicyanosides F-I (1-4) and prinamoside A (5), together with 16 known compounds (6-21) were isolated. The structures of the new compounds (1-5) were unambiguously confirmed by extensive spectroscopic techniques. Preliminary in vitro pharmacological studies showed that the hydroxynitrile glucosides (3, 9, and 10) exhibited weak α-glucosidase inhibitory activity. To a certain extent, our research provides some evidence for the pharmacological function of γ-hydroxynitrile glucosides and proposes new ideas for recycling of the oil residue of P. utilis.

LC-MS Profile, Gastrointestinal and Gut Microbiota Stability and Antioxidant Activity of Rhodiola rosea Herb Metabolites: A Comparative Study with Subterranean Organs.

Golden root (Rhodiola rosea L., Crassulaceae) is a famous medical plant with a one-sided history of scientific interest in the roots and rhizomes as sources of bioactive compounds, unlike the herb, which has not been studied extensively. To address this deficiency, we used high-performance liquid chromatography with diode array and electrospray triple quadrupole mass detection for comparative qualitative and quantitative analysis of the metabolic profiles of Rhodiola rosea organs before and after gastrointestinal digestion in simulated conditions together with various biochemical assays to determine antioxidant properties of the extracts and selected compounds. R. rosea organs showed 146 compounds, including galloyl O-glucosides, catechins, procyanidins, simple phenolics, phenethyl alcohol derivatives, (hydroxy)cinnamates, hydroxynitrile glucosides, monoterpene O-glucosides, and flavonol O-glycosides, most of them for the first time in the species. The organ-specific distribution of compounds found for catechins, procyanidins, and cinnamyl alcohols and glucosides was typical for underground organs and flavonoids and galloylated glucoses concentrated in the herb. Extracts from rhizomes, leaves and flowers showed high phenolic content and were effective scavengers of free radicals (2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), O2•−, •OH) and protected β-carotene in a bleaching assay. Digestion in the gastric and intestine phase influenced the composition of R. rosea extracts negatively, affecting the content of catechins, procyanidins, and galloyl glucoses, and therefore, the antioxidativity level. After gut microbiota treatment, the antioxidant capacity of rhizome extract was lower than leaves and flowers due to the aglycone composition found in the colonic phase of digestion. Our study demonstrated that the herb of R. rosea is a rich source of metabolites with high antioxidant properties and could be a valuable plant for new bioactive products.

Deletion of biosynthetic genes, specific SNP patterns and differences in transcript accumulation cause variation in hydroxynitrile glucoside content in barley cultivars

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides, potentially involved in alleviating pathogen and environmental stresses. These compounds include the cyanogenic glucoside epiheterodendrin. The biosynthetic genes are clustered. Total hydroxynitrile glucoside contents were previously shown to vary from zero to more than 10,000 nmoles g−1 in different barley lines. To elucidate the cause of this variation, the biosynthetic genes from the high-level producer cv. Mentor, the medium-level producer cv. Pallas, and the zero-level producer cv. Emir were investigated. In cv. Emir, a major deletion in the genome spanning most of the hydroxynitrile glucoside biosynthetic gene cluster was identified and explains the complete absence of hydroxynitrile glucosides in this cultivar. The transcript levels of the biosynthetic genes were significantly higher in the high-level producer cv. Mentor compared to the medium-level producer cv. Pallas, indicating transcriptional regulation as a contributor to the variation in hydroxynitrile glucoside levels. A correlation between distinct single nucleotide polymorphism (SNP) patterns in the biosynthetic gene cluster and the hydroxynitrile glucoside levels in 227 barley lines was identified. It is remarkable that in spite of the demonstrated presence of a multitude of SNPs and differences in transcript levels, the ratio between the five hydroxynitrile glucosides is maintained across all the analysed barley lines. This implies the involvement of a stably assembled multienzyme complex.

Mass Spectrometry Based Imaging of Labile Glucosides in Plants.

Mass spectrometry based imaging is a powerful tool to investigate the spatial distribution of a broad range of metabolites across a variety of sample types. The recent developments in instrumentation and computing capabilities have increased the mass range, sensitivity and resolution and rendered sample preparation the limiting step for further improvements. Sample preparation involves sectioning and mounting followed by selection and application of matrix. In plant tissues, labile small molecules and specialized metabolites are subject to degradation upon mechanical disruption of plant tissues. In this study, the benefits of cryo-sectioning, stabilization of fragile tissues and optimal application of the matrix to improve the results from MALDI mass spectrometry imaging (MSI) is investigated with hydroxynitrile glucosides as the main experimental system. Denatured albumin proved an excellent agent for stabilizing fragile tissues such as Lotus japonicus leaves. In stem cross sections of Manihot esculenta, maintaining the samples frozen throughout the sectioning process and preparation of the samples by freeze drying enhanced the obtained signal intensity by twofold to fourfold. Deposition of the matrix by sublimation improved the spatial information obtained compared to spray. The imaging demonstrated that the cyanogenic glucosides (CNglcs) were localized in the vascular tissues in old stems of M. esculenta and in the periderm and vascular tissues of tubers. In MALDI mass spectrometry, the imaged compounds are solely identified by their m/z ratio. L. japonicus MG20 and the mutant cyd1 that is devoid of hydroxynitrile glucosides were used as negative controls to verify the assignment of the observed masses to linamarin, lotaustralin, and linamarin acid.

Biosynthesis of the leucine derived α-, β- and γ-hydroxynitrile glucosides in barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non-cyanogenic HNGs are the β-HNG epidermin and the γ-HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP-glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co-expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi-enzyme complex.

Editorial: Physiology and Cellular Mechanisms of Isothiocyanates and Other Glucosinolate Degradation Products in Plants.

Editorial: Physiology and Cellular Mechanisms of Isothiocyanates and Other Glucosinolate Degradation Products in Plants.

Diversified glucosinolate metabolism: biosynthesis of hydrogen cyanide and of the hydroxynitrile glucoside alliarinoside in relation to sinigrin metabolism in Alliaria petiolata.

Alliaria petiolata (garlic mustard, Brassicaceae) contains the glucosinolate sinigrin as well as alliarinoside, a γ-hydroxynitrile glucoside structurally related to cyanogenic glucosides. Sinigrin may defend this plant against a broad range of enemies, while alliarinoside confers resistance to specialized (glucosinolate-adapted) herbivores. Hydroxynitrile glucosides and glucosinolates are two classes of specialized metabolites, which generally do not occur in the same plant species. Administration of [UL-14C]-methionine to excised leaves of A. petiolata showed that both alliarinoside and sinigrin were biosynthesized from methionine. The biosynthesis of alliarinoside was shown not to bifurcate from sinigrin biosynthesis at the oxime level in contrast to the general scheme for hydroxynitrile glucoside biosynthesis. Instead, the aglucon of alliarinoside was formed from metabolism of sinigrin in experiments with crude extracts, suggesting a possible biosynthetic pathway in intact cells. Hence, the alliarinoside pathway may represent a route to hydroxynitrile glucoside biosynthesis resulting from convergent evolution. Metabolite profiling by LC-MS showed no evidence of the presence of cyanogenic glucosides in A. petiolata. However, we detected hydrogen cyanide (HCN) release from sinigrin and added thiocyanate ion and benzyl thiocyanate in A. petiolata indicating an enzymatic pathway from glucosinolates via allyl thiocyanate and indole glucosinolate derived thiocyanate ion to HCN. Alliarinoside biosynthesis and HCN release from glucosinolate-derived metabolites expand the range of glucosinolate-related defenses and can be viewed as a third line of defense, with glucosinolates and thiocyanate forming protein being the first and second lines, respectively.

How Does Garlic Mustard Lure and Kill the West Virginia White Butterfly?

As it pertains to insect herbivores, the preference-performance hypothesis posits that females will choose oviposition sites that maximize their offspring's fitness. However, both genetic and environmental cues contribute to oviposition preference, and occasionally "oviposition mistakes" occur, where insects oviposit on hosts unsuitable for larval development. Pieris virginiensis is a pierine butterfly native to North America that regularly oviposits on an invasive plant, Alliaria petiolata, but the caterpillars are unable to survive. Alliaria petiolata has high concentrations of the glucosinolate sinigrin in its tissues, as well as a hydroxynitrile glucoside, alliarinoside. We investigated sinigrin as a possible cause of mistake oviposition, and sinigrin and alliarinoside as possible causes of larval mortality. We found that sinigrin applied to leaves of Cardamine diphylla, a major host of P. virginiensis that does not produce sinigrin, had no effect on oviposition rates. We tested the effect of sinigrin on larval performance using two host plants, one lacking sinigrin (C. diphylla) and one with sinigrin naturally present (Brassica juncea). We found no effect of sinigrin application on survival of caterpillars fed C. diphylla, but sinigrin delayed pupation and decreased pupal weight. On B. juncea, sinigrin decreased survival, consumption, and caterpillar growth. We also tested the response of P. virginiensis caterpillars to alliarinoside, a compound unique to A. petiolata, which was applied to B. oleracea. We found a significant reduction in survival, leaf consumption, and caterpillar size when alliarinoside was consumed. The 'novel weapon' alliarinoside likely is largely responsible for larval failure on the novel host A. petiolata. Sinigrin most likely contributes to the larval mortality observed, however, we did not observe any effect of sinigrin on oviposition by P. virginiensis females. Further research needs to be done on non-glucosinolate contact cues, and volatile signals that may induce P. virginiensis oviposition.

Accumulation of secondary metabolites in healthy and diseased barley, grown under future climate levels of CO2, ozone and temperature

Plants produce secondary metabolites promoting adaptation to changes in the environment and challenges by pathogenic microorganisms. A future climate with increased temperature and CO2 and ozone levels will likely alter the chemical composition of plants and thereby plant–pathogen interactions. To investigate this, barley was grown at elevated CO2, temperature and ozone levels as single factors or in combination resembling future climatic conditions. Increased basal resistance to the powdery mildew fungus was observed when barley was grown under elevated CO2, temperature and ozone as single factors. However, this effect was neutralized in the combination treatments. Twenty-five secondary metabolites were putatively identified in healthy and diseased barley leaves, including phenylpropanoids, phenolamides and hydroxynitrile glucosides. Accumulation of the compounds was affected by the climatic growth conditions. Especially elevated temperature, but also ozone, showed a strong impact on accumulation of many compounds, suggesting that these metabolites play a role in adaptation to unfavorable growth conditions. Many compounds were found to increase in powdery mildew diseased leaves, in correlation with a strong and specific influence of the climatic growth conditions. The observed disease phenotypes could not be explained by accumulation of single compounds. However, decreased accumulation of the powdery mildew associated defense compound p-coumaroylhydroxyagmatine could be implicated in the increased disease susceptibility observed when barley was grown under combination of elevated CO2, temperature and ozone. The accumulation pattern of the compounds in both healthy and diseased leaves from barley grown in the combination treatments could not be deduced from the individual single factor treatments. This highlights the complex role and regulation of secondary metabolites in plants’ adaptation to unfavorable growth conditions.

Glucosinolate-related glucosides in Alliaria petiolata: sources of variation in the plant and different metabolism in an adapted specialist herbivore, Pieris rapae.

Specialized metabolites in plants influence their interactions with other species, including herbivorous insects, which may adapt to tolerate defensive phytochemicals. The chemical arsenal of Alliaria petiolata (garlic mustard, Brassicaceae) includes the glucosinolate sinigrin and alliarinoside, a hydroxynitrile glucoside with defensive properties to glucosinolate-adapted specialists. To further our understanding of the chemical ecology of A. petiolata, which is spreading invasively in North America, we investigated the metabolite profile and here report a novel natural product, petiolatamide, which is structurally related to sinigrin. In an extensive study of North American populations of A. petiolata, we demonstrate that genetic population differences as well as developmental regulation contribute to variation in the leaf content of petiolatamide, alliarinoside, sinigrin, and a related glycoside. We furthermore demonstrate widely different metabolic fates of these metabolites after ingestion in the glucosinolate-adapted herbivore Pieris rapae, ranging from simple passage over metabolic conversion to sequestration. The differences in metabolic fate were influenced by plant β-glucosidases, insect-mediated degradation, and the specificity of the larval gut transport system mediating sequestration.

The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous β-glucosidases resulting from a crucial amino acid substitution.

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α-hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic γ- and β-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L.japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.

Visualizing metabolite distribution and enzymatic conversion in plant tissues by desorption electrospray ionization mass spectrometry imaging

In comparison with the technology platforms developed to localize transcripts and proteins, imaging tools for visualization of metabolite distributions in plant tissues are less well developed and lack versatility. This hampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon may be used to accurately image the distribution of even labile plant metabolites such as hydroxynitrile glucosides, which normally undergo enzymatic hydrolysis by specific β-glucosidases upon cell disruption. This fast and simple sample preparation resulted in no substantial differences in the distribution and ratios of all hydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a β-glucosidase mutant plant that lacks the ability to hydrolyze certain hydroxynitrile glucosides. In wild-type, the enzymatic conversion of hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restricted area of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme in hydroxynitrile glucoside biosynthesis in L.japonicus leaves, CYP79D3, was found to be highly expressed during the early stages of leaf development, and the hydroxynitrile glucoside distribution in mature leaves reflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated using cryo-sections of cassava (Manihot esculenta) tubers. The hydroxynitrile glucoside levels were highest in the outer cell layers, as verified by LC-MS analyses. The unexpected discovery of a hydroxynitrile-derived di-glycoside shows the potential of DESI-MSI to discover and guide investigations into new metabolic routes.

Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)

Glycosyl hydrolase family 1 β-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving β-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this β-glucosidase were found to be 45°C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K (m) and V (max) were found to be 38.59 μM and 0.8237μM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 μM and 0.1037μM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of -5.52kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

Occurrence of Sarmentosin and Other Hydroxynitrile Glucosides in Parnassius (Papilionidae) Butterflies and Their Food Plants

Sequestration of plant secondary metabolites is a widespread phenomenon among aposematic insects. Sarmentosin is an unsaturated γ-hydroxynitrile glucoside known from plants and some Lepidoptera. It is structurally and biosynthetically closely related to cyanogenic glucosides, which are commonly sequestered from food plants and/or de novo synthesized by lepidopteran species. Sarmentosin was found previously in Parnassius (Papilionidae) butterflies, but it was not known how the occurrence was related to food plants or whether Parnassius species could biosynthesize the compound. Here, we report on the occurrence of sarmentosin and related compounds in four different Parnassius species belonging to two different clades, as well as their known and suspected food plants. There were dramatic differences between the two clades, with P. apollo and P. smintheus from the Apollo group containing high amounts of sarmentosin, and P. clodius and P. mnemosyne from the Mnemosyne group containing low or no detectable amounts. This was reflected in the larval food plants; P. apollo and P. smintheus larvae feed on Sedum species (Crassulaceae), which all contained considerable amounts of sarmentosin, while the known food plants of the two other species, Dicentra and Corydalis (Fumariaceae), had no detectable levels of sarmentosin. All insects and plants containing sarmentosin also contained other biosynthetically related hydroxynitrile glucosides in patterns previously reported for plants, but not for insects. Not all findings could be explained by sequestration alone and we therefore hypothesize that Parnassius species are able to de novo synthesize sarmentosin.

Possible evolution of alliarinoside biosynthesis from the glucosinolate pathway in Alliaria petiolata

Nitrile formation in plants involves the activity of cytochrome P450s. Hydroxynitrile glucosides are widespread among plants but generally do not occur in glucosinolate producing species. Alliaria petiolata (garlic mustard, Brassicaceae) is the only species known to produce glucosinolates as well as a γ-hydroxynitrile glucoside. Furthermore, A. petiolata has been described to release diffusible cyanide, which indicates the presence of unidentified cyanogenic glucoside(s). Our research on A. petiolata addresses the molecular evolution of P450s. By integrating current knowledge about glucosinolate and hydroxynitrile glucoside biosynthesis in other species and new visions on recurrent evolution of hydroxynitrile glucoside biosynthesis, we propose a pathway for biosynthesis of the γ-hydroxynitrile glucoside, alliarinoside. Homomethionine and the corresponding oxime are suggested as shared intermediates in the biosynthesis of alliarinoside and 2-propenyl glucosinolate. The first committed step in the alliarinoside pathway is envisioned to be catalysed by a P450, which has been recruited to metabolize the oxime. Furthermore, alliarinoside biosynthesis is suggested to involve enzyme activities common to secondary modification of glucosinolates. Thus, we argue that biosynthesis of alliarinoside may be the first known case of a hydroxynitrile glucoside pathway having evolved from the glucosinolate pathway. An intriguing question is whether the proposed hydroxynitrile intermediate may also be converted to novel homomethionine-derived cyanogenic glucoside(s), which could release cyanide. Elucidation of the pathway for biosynthesis of alliarinoside and other putative hydroxynitrile glucosides in A. petiolata is envisioned to offer significant new knowledge on the emerging picture of P450 functional dynamics as a basis for recurrent evolution of pathways for bioactive natural product biosynthesis.

Characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry

Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints.

Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism.

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (alpha-hydroxynitrile glucosides) by specific beta-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the beta-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related beta-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.

Supinaionosides A and B: Megastigmane Glucosides and Supinanitrilosides A-F: Hydroxynitrile Glucosides from the Whole Plants of Euphorbia supina RAFINESQUE

From whole plants of Euphorbia supina, two new megastigmane glucosides, named supinaionosides A and B (1, 2), six new hydroxynitrile glucosides, named supinanitrilosides A-F (3-8), and six known compounds were isolated. The structures of the new compounds were elucidated on the basis of spectroscopic and chemical evidence.

Hydroxynitrile glucosides

β- and γ-Hydroxynitrile glucosides are structurally related to cyanogenic glucosides (α-hydroxynitrile glucosides) but do not give rise to hydrogen cyanide release upon hydrolysis. Structural similarities and frequent co-occurrence suggest that the biosynthetic pathways for these compounds share common features. Based on available literature data we propose that oximes produced by CYP79 orthologs are common intermediates and that their conversion into β- and γ-hydroxynitrile glucosides is mediated by evolutionary diversified multifunctional orthologs to CYP71E1. We designate these as CYP71 βγ and CYP71 αβγ; in combination with the classical CYP71 α (CYP71E1 and orthologs) these are able to hydroxylate any of the carbon atoms present in the amino acid and oxime derived nitriles. Subsequent dehydration reactions and hydroxylations and a final glycosylation step afford the unsaturated β- and γ-hydroxynitrile glucosides. This scheme would explain the distribution patterns of α-, β- and γ-hydroxynitrile glucosides found in plants. The possible biological functions of these hydroxynitriles are discussed.