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Barley produces several specialized metabolites, including five α-, β-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Obesity is a major health risk still lacking effective pharmacological treatment. A potent anti-obesity agent, celastrol, has been identified in the roots of Tripterygium wilfordii. However, an efficient synthetic method is required to better explore its biological utility. Here we elucidate the 11 missing steps for the celastrol biosynthetic route to enable its de novo biosynthesis in yeast. First, we reveal the cytochrome P450 enzymes that catalyse the four oxidation steps that produce the key intermediate celastrogenic acid. Subsequently, we show that non-enzymatic decarboxylation-triggered activation of celastrogenic acid leads to a cascade of tandem catechol oxidation-driven double-bond extension events that generate the characteristic quinone methide moiety of celastrol. Using this acquired knowledge, we have developed a method for producing celastrol starting from table sugar. This work highlights the effectiveness of combining plant biochemistry with metabolic engineering and chemistry for the scalable synthesis of complex specialized metabolites., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Lupins are high-protein crops that are rapidly gaining interest as hardy alternatives to soybean; however, they accumulate antinutritional alkaloids of the quinolizidine type (QAs). Lupin domestication was enabled by the discovery of genetic loci conferring low QA levels (sweetness), but the precise identity of the underlying genes remains uncertain. We show that pauper , the most common sweet locus in white lupin, encodes an acetyltransferase (AT) unexpectedly involved in the early QA pathway. In pauper plants, a single-nucleotide polymorphism (SNP) strongly impairs AT activity, causing pathway blockage. We corroborate our hypothesis by replicating the pauper chemotype in narrow-leafed lupin via mutagenesis. Our work adds a new dimension to QA biosynthesis and establishes the identity of a lupin sweet gene for the first time, thus facilitating lupin breeding and enabling domestication of other QA-containing legumes.

Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Phytocannabinoids are a group of plant-derived metabolites that display a wide range of psychoactive as well as health-promoting effects. The production of pharmaceutically relevant cannabinoids relies on extraction and purification from cannabis ( Cannabis sativa ) plants yielding the major constituents, Δ 9 -tetrahydrocannabinol and cannabidiol. Heterologous biosynthesis of cannabinoids in Nicotiana benthamiana or Saccharomyces cerevisiae may provide cost-efficient and rapid future production platforms to acquire pure and high quantities of both the major and the rare cannabinoids as well as novel derivatives. Here, we used a meta-transcriptomic analysis of cannabis to identify genes for aromatic prenyltransferases of the UbiA superfamily and chalcone isomerase-like (CHIL) proteins. Among the aromatic prenyltransferases, Cs aPT4 showed CBGAS activity in both N. benthamiana and S. cerevisiae . Coexpression of selected Cs aPT pairs and of CHIL proteins encoding genes with CsaPT4 did not affect CBGAS catalytic efficiency. In a screen of different plant UDP-glycosyltransferases, Stevia rebaudiana Sr UGT71E1 and Oryza sativa Os UGT5 were found to glucosylate olivetolic acid, cannabigerolic acid, and Δ 9 -tetrahydrocannabinolic acid. Metabolic engineering of N. benthamiana for production of cannabinoids revealed intrinsic glucosylation of olivetolic acid and cannabigerolic acid. S. cerevisiae was engineered to produce olivetolic acid glucoside and cannabigerolic acid glucoside.

Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis-the conversion of an amino acid to the corresponding aldoxime-is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nibbering et al.)

Dhurrin is the most abundant cyanogenic glucoside found in sorghum ( Sorghum bicolor) where it plays a key role in chemical defense by releasing toxic hydrogen cyanide upon tissue disruption. Besides this well-established function, there is strong evidence that dhurrin plays additional roles, e.g. as a transport and storage form of nitrogen, released via endogenous recycling pathways. However, knowledge about how, when and why dhurrin is endogenously metabolized is limited. We combined targeted metabolite profiling with matrix-assisted laser desorption/ionization-mass spectrometry imaging to investigate accumulation of dhurrin, its recycling products and key general metabolites in four different sorghum lines during 72 h of grain imbibition, germination and early seedling development, as well as the spatial distribution of these metabolites in two of the lines. Little or no dhurrin or recycling products were present in the dry grain, but their de novo biosynthesis started immediately after water uptake. Dhurrin accumulation increased rapidly within the first 24 h in parallel with an increase in free amino acids, a key event in seed germination. The trajectories and final concentrations of dhurrin, the recycling products and free amino acids reached within the experimental period were dependent on genotype. Matrix-assisted laser desorption/ionization-mass spectrometry imaging demonstrated that dhurrin primarily accumulated in the germinating embryo, confirming its function in protecting the emerging tissue against herbivory. The dhurrin recycling products, however, were mainly located in the scutellum and/or pericarp/seed coat region, suggesting unknown key functions in germination., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2 nd generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.

In recent years, ionic liquids and deep eutectic solvents (DESs) have gained increasing attention due to their ability to extract and solubilize metabolites and biopolymers in quantities far beyond their solubility in oil and water. The hypothesis that naturally occurring metabolites are able to form a natural deep eutectic solvent (NADES), thereby constituting a third intracellular phase in addition to the aqueous and lipid phases, has prompted researchers to study the role of NADES in living systems. As an excellent solvent for specialized metabolites, formation of NADES in response to dehydration of plant cells could provide an appropriate environment for the functional storage of enzymes during drought. Using the enzymes catalyzing the biosynthesis of the defense compound dhurrin as an experimental model system, we demonstrate that enzymes involved in this pathway exhibit increased stability in NADES compared with aqueous buffer solutions, and that enzyme activity is restored upon rehydration. Inspired by nature, application of NADES provides a biotechnological approach for long-term storage of entire biosynthetic pathways including membrane-anchored enzymes., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Based on recent in vitro data, a relatively large number of the plant nitrate transporter 1/peptide transporter family (NPF) proteins have been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes, wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities toward organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis suggests distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed., (Copyright © 2019 Wulff, Ernst, Jørgensen, Lambertz, Maierhofer, Belew, Crocoll, Motawia, Geiger, Jørgensen, Mirza and Nour-Eldin.)

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products., (Copyright © 2019 International Metabolic Engineering Society. All rights reserved.)

Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.

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.

One application of synthetic biology is the redesign of existing biological systems to acquire new functions. In this context, expanding the chemical code underlying key biosynthetic pathways will lead to the synthesis of compounds with new structures and potentially new biological activities. Terpenoids are a large group of specialized metabolites with numerous applications. Yet, being synthesized from five-carbon units, they are restricted to distinct classes that differ by five carbon atoms (C10, C15, C20, etc.). To expand the diversity of terpenoid structures, we engineered yeast cells to synthesize a noncanonical building block with 11 carbons, and produced 40 C11 terpene scaffolds that can form the basis for an entire terpenoid class. By identifying a single-residue switch that converts C10 plant monoterpene synthases to C11-specific enzymes, we engineered dedicated synthases for C11 terpene production. This approach will enable the systematic expansion of the chemical space accessed by terpenoids.

Vitamin D 3 is a secosterol hormone critical for bone growth and calcium homeostasis, produced in vertebrate skin by photolytic conversion of the cholesterol biosynthetic intermediate provitamin D 3 . Insufficient levels of vitamin D 3 especially in the case of low solar UV-B irradiation is often compensated by an intake of a dietary source of vitamin D 3 of animal origin. Small amounts of vitamin D 3 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 D 5 in the model plant Arabidopsis thaliana. This plant secosterol is a UV-B mediated derivative of provitamin D 5 , the precursor of sitosterol. The present work will allow a further survey of vitamin D distribution in plant species.

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., (© 2018 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

Almond and sweet cherry are two economically important species of the Prunus genus. They both produce the cyanogenic glucosides prunasin and amygdalin. As part of a two-component defense system, prunasin and amygdalin release toxic hydrogen cyanide upon cell disruption. In this study, we investigated the potential role within prunasin and amygdalin and some of its derivatives in endodormancy release of these two Prunus species. The content of prunasin and of endogenous prunasin turnover products in the course of flower development was examined in five almond cultivars - differing from very early to extra-late in flowering time - and in one sweet early cherry cultivar. In all cultivars, prunasin began to accumulate in the flower buds shortly after dormancy release and the levels dropped again just before flowering time. In almond and sweet cherry, the turnover of prunasin coincided with increased levels of prunasin amide whereas prunasin anitrile pentoside and β-D-glucose-1-benzoate were abundant in almond and cherry flower buds at certain developmental stages. These findings indicate a role for the turnover of cyanogenic glucosides in controlling flower development in Prunus species.

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/β glycan linkage within oligosaccharides remains a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS 2 ). These findings allow for integration of MS 2 with ion mobility spectrometry (IM-MS 2 ) and lead to a strategy to distinguish α- and β-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii , in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13 R -manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13 R -manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana . The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales . Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.

The vast diversity and membrane-bound nature of plant P450s makes it challenging to study the structural characteristics of this class of enzymes especially with respect to accurate intermolecular enzyme-substrate interactions. To address this problem we here apply a modified hybrid structure strategy for homology modeling of plant P450s. This allows for structural elucidation based on conserved motifs in the protein sequence and secondary structure predictions. We modeled the well-studied Sorghum bicolor cytochrome P450 CYP79A1 catalyzing the first step in the biosynthesis of the cyanogenic glucoside dhurrin. Docking experiments identified key regions of the active site involved in binding of the substrate and facilitating catalysis. Arginine 152 and threonine 534 were identified as key residues interacting with the substrate. The model was validated experimentally using site-directed mutagenesis. The new CYP79A1 model provides detailed insights into the mechanism of the initial steps in cyanogenic glycoside biosynthesis. The approach could guide functional characterization of other membrane-bound P450s and provide structural guidelines for elucidation of key structure-function relationships of other plant P450s., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts., (Copyright © 2016, American Association for the Advancement of Science.)

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.

In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches.

Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.

An effective method for the chemical synthesis of cyanogenic glucosides has been developed as demonstrated by the synthesis of dhurrin, taxiphyllin, prunasin, sambunigrin, heterodendrin, and epiheterodendrin. O-Trimethylsilylated cyanohydrins were prepared and subjected directly to glucosylation using a fully acetylated glucopyranosyl fluoride donor with boron trifluoride-diethyl etherate as promoter to afford a chromatographically separable epimeric mixture of the corresponding acetylated cyanogenic glucosides. The isolated epimers were deprotected using a triflic acid/MeOH/ion-exchange resin system without any epimerization of the cyanohydrin function. The method is stereocontrolled and provides an efficient approach to chemical synthesis of other naturally occurring cyanogenic glucosides including those with a more complex aglycone structure.

Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

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-(14)C]-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.

Flowers and leaves of Lotus japonicus contain α-, β-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by β-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

Cyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic hydrogen cyanide (HCN) upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC-MS/MS and high resolution MS (HR-MS) complemented by ion-mobility MS was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di- and tri-glycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and 'anitriles' derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants., (© 2015 Authors; published by Portland Press Limited.)

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 allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in North America, was chemically synthesized using an efficient and practical synthetic strategy based on a simple reaction sequence. Commercially available 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose was converted into prop-2-enyl 2',3',4',6'-tetra-O-acetyl-β-D-glucopyranoside and subjected to epoxidation. In a one-pot reaction, ring-opening of the epoxide using TMSCN under solvent free conditions followed by treatment of the formed trimethylsilyloxy nitrile with pyridine and phosphoryl chloride, afforded the acetylated β-unsaturated nitriles (Z)-4-(2',3',4',6'-tetra-O-β-D-glucopyranosyloxy)but-2-enenitrile and its isomer (E)-4-(2',3',4',6'-tetra-O-β-D-glucopyranosyloxy)but-2-enenitrile. Deacetylation of Z- and/or E-isomers afforded the target molecules alliarinoside and its isomer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

Considering the staggering diversity of bioactive natural products present in plants, insects are only able to sequester a small number of phytochemicals from their food plants. The mechanisms of how only some phytochemicals are sequestered and how the sequestration process takes place remains largely unknown. In this study the model system of Zygaena filipendulae (Lepidoptera) and their food plant Lotus corniculatus is used to advance the knowledge of insect sequestration. Z. filipendulae larvae are dependent on sequestration of the cyanogenic glucosides linamarin and lotaustralin from their food plant, and have a much lower fitness if reared on plants without these compounds. This study investigates the fate of the cyanogenic glucosides during ingestion, sequestration in the larvae, and in the course of insect ontogeny. To this purpose, double-labeled linamarin and lotaustralin were chemically synthesized carrying two stable isotopes, a (2)H labeled aglucone and a (13)C labeled glucose moiety. In addition, a small amount of (14)C was incorporated into the glucose residue. The isotope-labeled compounds were applied onto cyanogenic L. corniculatus leaves that were subsequently presented to the Z. filipendulae larvae. Following ingestion by the larvae, the destiny of the isotope labeled cyanogenic glucosides was monitored in different tissues of larvae and adults at selected time points, using radio-TLC and LC-MS analyses. It was shown that sequestered compounds are taken up intact, contrary to earlier hypotheses where it was suggested that the compounds would have to be hydrolyzed before transport across the gut. The uptake from the larval gut was highly stereo selective as the β-glucosides were retained while the α-glucosides were excreted and recovered in the frass. Sequestered compounds were rapidly distributed into all analyzed tissues of the larval body, partly retained throughout metamorphosis and transferred into the adult insect where they were distributed to all tissues. During subsequent mating, isotope labeled cyanogenic glucosides were transferred from the male to the female in the nuptial gift., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Reproducible and quantitative gas chromatography-mass spectrometry (GC-MS)-based metabolomics analysis of complex biological mixtures requires robust and broad-spectrum derivatization. We have evaluated derivatization of complex metabolite mixtures using trimethylsilyl cyanide (TMSCN) and the most commonly used silylation reagent N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). For the comparative analysis, two metabolite mixtures, a standard complex mixture of 35 metabolites covering a range of amino acids, carbohydrates, small organic acids, phenolic acids, flavonoids and triterpenoids, and a phenolic extract of blueberry fruits were used. Four different derivatization methods, (1) direct silylation using TMSCN, (2) methoximation followed by TMSCN (M-TMSCN), (3) direct silylation using MSTFA, and (4) methoximation followed by MSTFA (M-MSTFA) were compared in terms of method sensitivity, repeatability, and derivatization reaction time. The derivatization methods were observed at 13 different derivatization times, 5 min to 60 h, for both metabolite mixtures. Fully automated sample derivatization and injection enabled excellent repeatability and precise method comparisons. At the optimal silylation times, peak intensities of 34 out of 35 metabolites of the standard mixture were up to five times higher using M-TMSCN compared with M-MSTFA. For direct silylation of the complex standard mixture, the TMSCN method was up to 54 times more sensitive than MSTFA. Similarly, all the metabolites detected from the blueberry extract showed up to 8.8 times higher intensities when derivatized using TMSCN than with MSTFA. Moreover, TMSCN-based silylation showed fewer artifact peaks, robust profiles, and higher reaction speed as compared with MSTFA. A method repeatability test revealed the following robustness of the four methods: TMSCN > M-TMSCN > M-MSTFA > MSTFA.

Zygaena filipendulae accumulates the cyanogenic glucosides linamarin and lotaustralin by larval sequestration from the food plant or de novo biosynthesis. We have previously demonstrated that the Z. filipendulae male transfers linamarin and lotaustralin to the female in the course of mating. In this study we report the additional transfer of 5-hydroxytryptophan glucoside (5-(β-d-glucopyranosyloxy)-L-Tryptophan) from the Z. filipendulae male internal genitalia to the female spermatophore around 5 h into the mating process. 5-Hydroxytryptophan glucoside is present in the virgin male internal genitalia, and production continues during the early phase of mating. Following initiation of 5-hydroxytryptophan glucoside transfer to the female, the amount in male internal genitalia is drastically reduced until after mating where it is slowly replenished. For unambiguous structural identification, 5-hydroxytryptophan glucoside was chemically synthesized and used as an authentic standard. The biological function of 5-hydroxytryptophan glucoside remains to be established, although we have indications that it may be involved in inducing the female to stay in copula and delay egg-laying to prevent re-mating of the female. To our knowledge 5-hydroxytryptophan glucoside has not previously been reported present in animal tissues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family--as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non cyanogenic hydroxynitriles, rhodiocyanoside A and D, with rhodiocyanoside A as the major rhodiocyanoside. Rhodiocyanosides are structurally related to cyanogenic glucosides but are not cyanogenic. In vitro administration of intermediates of the lotaustralin pathway to microsomes prepared from selected L. japonicus accessions identified 2-methyl-2-butenenitrile as an intermediate in the rhodiocyanoside biosynthetic pathway. In vitro inhibitory studies with carbon monoxide and tetcyclacis indicate that the conversion of (Z)-2-methylbutanal oxime to 2-methyl-2-butenenitrile is catalyzed by cytochrome P450(s). Carbon monoxide inhibited cyanogenic glucosides as well as rhodiocyanosides synthesis, but inhibition of the latter pathway was much stronger. These results demonstrate that the cyanogenic glucoside and rhodiocyanosides pathways share CYP79Ds to obtain (Z)-2-methylbutanaloxime from l-isoleucine, whereas the subsequent conversions are catalyzed by different P450s. The aglycon of rhodiocyanoside A forms the cyclic product 3-methyl-2(5H)-furanone. Furanones are known to possess antimicrobial properties indicating that rhodiocyanoside A may have evolved to serve as a phytoanticipin that following β-glucosidase activation and cyclization of the aglycone formed, give rise to a potent defense compound., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The cyanogenic glucoside profile of Eucalyptus camphora was investigated in the course of plant ontogeny. In addition to amygdalin, three phenylalanine-derived cyanogenic diglucosides characterized by unique linkage positions between the two glucose moieties were identified in E. camphora tissues. This is the first time that multiple cyanogenic diglucosides have been shown to co-occur in any plant species. Two of these cyanogenic glucosides have not previously been reported and are named eucalyptosin B and eucalyptosin C. Quantitative and qualitative differences in total cyanogenic glucoside content were observed across different stages of whole plant and tissue ontogeny, as well as within different tissue types. Seedlings of E. camphora produce only the cyanogenic monoglucoside prunasin, and genetically based variation was observed in the age at which seedlings initiate prunasin biosynthesis. Once initiated, total cyanogenic glucoside concentration increased throughout plant ontogeny with cyanogenic diglucoside production initiated in saplings and reaching a maximum in flower buds of adult trees. The role of multiple cyanogenic glucosides in E. camphora is unknown, but may include enhanced plant defense and/or a primary role in nitrogen storage and transport., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l-valine and l-isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside-specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2-hydroxy-2-methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co-occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co-expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine-tuning nitrogen assimilation in cassava., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Starch phosphate is important in starch metabolism and in order to deduce its location and structural effects in clusters and building blocks of amylopectin, these were isolated from a normal potato (WT) and two starches with antisense suppressed glucan water dikinase (asGWD) activity and starch branching enzyme (asSBE) activity possessing suppressed and increased phosphate contents, respectively. Neutral N-chains and phosphorylated P-chains of the amylopectin macromolecules were similar in WT and asGWD, whereas asSBE possessed considerably longer P-chains. Cluster β-limit dextrins were isolated by α-amylase treatment and successive β-amylolysis. Cluster sizes were generally smaller in asSBE. The building block composition of neutral N-clusters were very similar in WT and asGWD, while asSBE was different, containing less blocks with degree of polymerization (DP)>14. Phosphate content of the P-clusters of WT and asGWD was rather similar, while asSBE contained highly phosphorylated P-clusters with proportionally more P-chains and a low degree of branching. The average chain lengths of the P-clusters were, however, similar in all samples. Our data demonstrate only minor effect on the cluster structure in relation to phosphate deposition suggesting conserved reaction patterns of starch phosphorylation. Models are suggested to account for the principle structural and functional effects of starch phosphate esters., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine- and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A K(S) of approximately 0.9 μm was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min(-1) for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell layer. In fully unfolded leaves, the expression is pronounced in the cortex cell layer just beside the epidermis and in specific cells in the vascular tissue cortex cells. Thus, the transcripts of the CYP71E7 paralogs colocalize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in cassava.

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.