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.

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.

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.

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.

Learning from nature: new approaches to the metabolic engineering of plant defense pathways.

Biotechnological manipulation of plant defense pathways can increase crop resistance to herbivores and pathogens while also increasing yields of medicinal, industrial, flavor and fragrance compounds. The most successful achievements in engineering defense pathways can be attributed to researchers striving to imitate natural plant regulatory mechanisms. For example, the introduction of transcription factors that control several genes in one pathway is often a valuable strategy to increase flux in that pathway. The use of multi-gene cassettes which mimic natural gene clusters can facilitate coordinated regulation of a pathway and speed transformation efforts. The targeting of defense pathway genes to organs and tissues in which the defensive products are typically made and stored can also increase yield as well as defensive potential.

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.