Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Effector-mediated relocalization of a maize lipoxygenase protein triggers susceptibility to Ustilago maydis.

As the gall-inducing smut fungus Ustilago maydis colonizes maize (Zea mays) plants, it secretes a complex effector blend that suppresses host defense responses, including production of reactive oxygen species (ROS) and redirects host metabolism to facilitate colonization. We show that the U. maydis effector ROS burst interfering protein 1 (Rip1), which is involved in pathogen-associated molecular pattern (PAMP)-triggered suppression of host immunity, is functionally conserved in several other monocot-infecting smut fungi. We also have identified a conserved C-terminal motif essential for Rip1-mediated PAMP-triggered suppression of the ROS burst. The maize susceptibility factor lipoxygenase 3 (Zmlox3) bound by Rip1 was relocalized to the nucleus, leading to partial suppression of the ROS burst. Relocalization was independent of its enzymatic activity, revealing a distinct function for ZmLox3. Most importantly, whereas Zmlox3 maize mutant plants showed increased resistance to U. maydis wild-type strains, rip1 deletion strains infecting the Zmlox3 mutant overcame this effect. This could indicate that Rip1-triggered host resistance depends on ZmLox3 to be suppressed and that lox3 mutation-based resistance of maize to U. maydis requires functional Rip1. Together, our results reveal that Rip1 acts in several cellular compartments to suppress immunity and that targeting of ZmLox3 by Rip1 is responsible for the suppression of Rip1-dependent reduced susceptibility of maize to U. maydis.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

The Bitter Truth: How Insects Cope with Toxic Plant Alkaloids.

Plants are unique organisms that have developed ingenious strategies to cope with environmental challenges, such as herbivorous insects. One of these strategies is the synthesis of a vast array of chemical compounds, known as specialized metabolites, that serve many ecological functions. Among the most fascinating and diverse groups of specialized metabolites are the alkaloids, which are characterized by the presence of a nitrogen atom within a heterocyclic ring. While some have medicinal and recreational applications, others are highly unpalatable and/or toxic. The effects of alkaloids on both humans and insects can be very diverse, affecting their physiology and behavior. Insects that feed on alkaloid-containing plants have evolved diverse mechanisms to cope with the consequences of these toxins. These include sequestration, where insects store alkaloids in specialized tissues or organs, enzymatic detoxification through enzymes such as cytochrome P450 monooxygenases and glutathione S-transferases, and behavioral adaptations such as selective feeding. In this review, we explore the relationships between plant alkaloids and the evolutionary adaptations that enable insects to exploit alkaloid-rich plants as food sources and ecological niches minimizing the harmful effects of these natural compounds. We aim to provide a comprehensive and updated overview of this fascinating and complex ecological interaction.

Unraveling metabolic patterns and molecular mechanisms underlying storability in sugar beet.

BACKGROUND: Sugar beet is an important crop for sugar production. Sugar beet roots are stored up to several weeks post-harvest waiting for processing in the sugar factories. During this time, sucrose loss and invert sugar accumulation decreases the final yield and processing quality. To improve storability, more information about post-harvest metabolism is required. We investigated primary and secondary metabolites of six sugar beet varieties during storage. Based on their variety-specific sucrose loss, three storage classes representing well, moderate, and bad storability were compared. Furthermore, metabolic data were visualized together with transcriptome data to identify potential mechanisms involved in the storage process. RESULTS: We found that sugar beet varieties that performed well during storage have higher pools of 15 free amino acids which were already observable at harvest. This storage class-specific feature is visible at harvest as well as after 13 weeks of storage. The profile of most of the detected organic acids and semi-polar metabolites changed during storage. Only pyroglutamic acid and two semi-polar metabolites, including ferulic acid, show higher levels in well storable varieties before and/or after 13 weeks of storage. The combinatorial OMICs approach revealed that well storable varieties had increased downregulation of genes involved in amino acid degradation before and after 13 weeks of storage. Furthermore, we found that most of the differentially genes involved in protein degradation were downregulated in well storable varieties at both timepoints, before and after 13 weeks of storage. CONCLUSIONS: Our results indicate that increased levels of 15 free amino acids, pyroglutamic acid and two semi-polar compounds, including ferulic acid, were associated with a better storability of sugar beet taproots. Predictive metabolic patterns were already apparent at harvest. With respect to elongated storage, we highlighted the role of free amino acids in the taproot. Using complementary transcriptomic data, we could identify potential underlying mechanisms of sugar beet storability. These include the downregulation of genes for amino acid degradation and metabolism as well as a suppressed proteolysis in the well storable varieties.

Erythroxylum in Focus: An Interdisciplinary Review of an Overlooked Genus.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.

Improved Synthesis of N-Methylcadaverine.

Alkaloids compose a large class of natural products, and mono-methylated polyamines are a common intermediate in their biosynthesis. In order to evaluate the role of selectively methylated natural products, synthetic strategies are needed to prepare them. Here, N-methylcadaverine is prepared in 37.3% yield in three steps. The alternative literature two-step strategy resulted in reductive deamination to give N-methylpiperidine as determined by the single crystal structure. A straightforward strategy to obtain the mono-alkylated aliphatic diamine, cadaverine, which avoids potential side-reactions, is demonstrated.

The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase.

The esterification of methylecgonine (2-carbomethoxy-3ß-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis.

The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders, while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring to their core ring structures. Throughout much of human history, humans have cultivated tropane- and granatane-producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis. In addition, modern approaches to producing some pharmaceutically important tropanes via metabolic engineering endeavors are discussed.

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the ß-oxidative or nonoxidative pathways. The first step in the ß-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the ß-oxidative pathway.

Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae.

The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3ß-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

The BAHD and the bold: the mitochondria's role in alkaloid artistry.

In a recent study, Zeng et al. uncovered 3ß-tigloyloxytropane synthase (TS) in Atropa belladonna, characterizing its mitochondrial localization and substrate specificity. The discovery of this enzyme opens up new bioengineering possibilities for tropane alkaloids (TAs), enhancing the potential for sustainable agriculture and expanding our knowledge of TA biosynthesis.

The evolution of tropane alkaloids: Coca does it differently.

It is undeniable that tropane alkaloids (TAs) have been both beneficial and detrimental to human health in the modern era. Understanding their biosynthesis is vital for using synthetic biology to engineer organisms for pharmaceutical production. The most parsimonious approaches to pathway elucidation are traditionally homology-based methods. However, this approach has largely failed for TA biosynthesis in angiosperms. In the recent decade, significant progress has been made in elucidating the TA synthesis pathway in Erythroxylum coca, highlighting the parallel development of TAs in both the Solanaceae and Erythroxylaceae families. This separate evolutionary path has uncovered substantial divergence in the TAs formed by E. coca and distinct enzymatic reactions that differ from the traditional TA biosynthetic pathway found in TA-producing nightshade plants.

Biosynthesis of the allelopathic alkaloid gramine in barley by a cryptic oxidative rearrangement.

The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

Plant synthetic biology: from inspiration to augmentation.

Although it is still in its infancy, synthetic biology has the capacity to face scientific and societal problems related to modern agriculture. Innovations in cloning toolkits and genetic parts allow increased precision over gene expression in planta. We review the vast spectrum of available technologies providing a practical list of toolkits that take advantage of combinatorial power to introduce/alter metabolic pathways. We highlight that rational design is inspired by deep knowledge of natural and biochemical mechanisms. Finally, we provide several examples in which modern technologies have been applied to address these critical topics. Future applications in plants include not only pathway modifications but also prospects of augmenting plant anatomical features and developmental processes.

Metabolic Profiling Identifies Changes in the Winter Wheat Grains Following Fusarium Treatment at Two Locations in Croatia.

Fusarium head blight (FHB) is one of the most dangerous diseases of winter wheat, resulting in reduced grain yield and quality, and production of mycotoxins by the Fusarium fungi. In the present study, changes in the grain metabolomics of winter wheat samples infected with Fusarium spp. and corresponding non-infected samples from two locations in Croatia were investigated by GC-MS. A Mann-Whitney test revealed that 24 metabolites detected were significantly separated between Fusarium-inoculated and non-infected samples during the variety by treatment interactions. The results confirmed that in grains of six FHB-resistant varieties, ten metabolites were identified as possible resistance-related metabolites. These metabolites included heptadecanoic acid, 9-(Z)-hexadecenoic acid, sophorose, and secolaganin in grains of FHB-resistant varieties at the Osijek location, as well as 2-methylaminomethyltartronic acid, maleamic acid, 4-hydroxyphenylacetonitrile, 1,4-lactonearabinonic acid, secolaganin, and alanine in grains of FHB-resistant varieties at the Tovarnik location. Moreover, on the PCA bi-plot, FHB-susceptible wheat varieties were closer to glycyl proline, decanoic acid, and lactic acid dimer that could have affected other metabolites, and thus, suppressed resistance to FHB. Although defense reactions were genetically conditioned and variety specific, resulting metabolomics changes may give insight into defense-related pathways that could be manipulated to engineer plants with improved resistance to the pathogen.

A New Fluorescence Detection Method for Tryptophan- and Tyrosine-Derived Allelopathic Compounds in Barley and Lupin.

Barley (Hordeum vulgare) is one of the most widely cultivated crops for feedstock and beer production, whereas lupins (Lupinus spp.) are grown as fodder and their seeds are a source of protein. Both species produce the allelopathic alkaloids gramine and hordenine. These plant-specialized metabolites may be of economic interest for crop protection, depending on their tissue distribution. However, in high concentrations they pose a health risk to humans and animals that feed on them. This study was carried out to develop and validate a new method for monitoring these alkaloids and their related metabolites using fluorescence detection. Separation was performed on an HSS T3 column using slightly acidified water-acetonitrile eluents. Calibration plots expressed linearity over the range 0.09-100 pmol/µL for gramine. The accuracy and precision ranged from 97.8 to 123.4%, <7% RSD. The method was successfully applied in a study of the natural range of abundance of gramine, hordenine and their related metabolites, AMI, tryptophan and tyramine, in 22 barley accessions and 10 lupin species. This method provides accurate and highly sensitive chromatographic separation and detection of tryptophan- and tyrosine-derived allelochemicals and is an accessible alternative to LC-MS techniques for routine screening.

BAHD Company: The Ever-Expanding Roles of the BAHD Acyltransferase Gene Family in Plants.

Plants' ability to chemically modify core structures of specialized metabolites is the main reason why the plant kingdom contains such a wide and rich array of diverse compounds. One of the most important types of chemical modifications of small molecules is the addition of an acyl moiety to produce esters and amides. Large-scale phylogenomics analyses have shown that the enzymes that perform acyl transfer reactions on the myriad small molecules synthesized by plants belong to only a few gene families. This review is focused on describing the biochemistry, evolutionary origins, and chemical ecology implications of one of these families-the BAHD acyltransferases. The growth of advanced metabolomic studies coupled with next-generation sequencing of diverse plant species has confirmed that the BAHD family plays critical roles in modifying nearly all known classes of specialized metabolites. The current and future outlook for research on BAHDs includes expanding their roles in synthetic biology and metabolic engineering.

The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases.

Despite the long history of cocaine use among humans and its social and economic significance today, little information is available about the biochemical and molecular aspects of cocaine biosynthesis in coca (Erythroxylum coca) in comparison to what is known about the formation of other pharmacologically-important tropane alkaloids in species of the Solanaceae. In this work, we investigated the site of cocaine biosynthesis in E. coca and the nature of the first step. The two principal tropane alkaloids of E. coca, cocaine and cinnamoyl cocaine, were present in highest concentrations in buds and rolled leaves. These are also the organs in which the rate of alkaloid biosynthesis was the highest based on the incorporation of ¹³CO2. In contrast, tropane alkaloids in the Solanaceae are biosynthesized in the roots and translocated to the leaves. A collection of EST sequences from a cDNA library made from young E. coca leaves was employed to search for genes encoding the first step in tropane alkaloid biosynthesis. Full-length cDNA clones were identified encoding two candidate enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), and the enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli and complementation of a yeast mutant. The transcript levels of both ODC and ADC genes were highest in buds and rolled leaves and lower in other organs. The levels of both ornithine and arginine themselves showed a similar pattern, so it was not possible to assign a preferential role in cocaine biosynthesis to one of these proteins.