The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.

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SummaryIn higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR ) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H ), a cytochrome P450 monooxygenase involved in the ®rst committed step in the formation of iridoid monoterpenoids display identical cell-speci®c expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.

The first understanding of regiochemical and stereochemical control of monolignol coupling in lignan biosynthesis has been established via the participation of a new class of dirigent proteins. Immunological studies have also implicated the involvement of potential corresponding arrays of dirigent protein sites in controlling lignin biopolymer assembly.

Summary• Despite recent advances in elucidation of natural products in root exudates, there are significant gaps in our understanding of the ecological significance of products in the rhizosphere.• Here, we investigated the potential of barley (Hordeum vulgare) to secrete defense root exudates when challenged by the soilborne pathogen Fusarium graminearum.• Liquid chromatography with photodiode array detection (LC-DAD) was used to profile induced small-molecular-weight exudates. Thus, t-cinnamic, p-coumaric, ferulic, syringic and vanillic acids were assigned to plant metabolism and were induced within 2 d after Fusarium inoculation. Biological tests demonstrated the ability of those induced root exudates to inhibit the germination of F. graminearum macroconidia. In vivo labeling experiments with 13 CO 2 revealed that the secreted t-cinnamic acid was synthesized de novo within 2 d of fungal infection. Simultaneously to its root exudation, t-cinnamic acid was accumulated in the roots. Microscopic analysis showed that nonlignin cell wall phenolics were induced not only in necrosed zones but in all root tissues.• Results suggest that barley plants under attack respond by de novo biosynthesis and secretion of compounds with antimicrobial functions that may mediate natural disease resistance.

BackgroundThe first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions.ResultsIn this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences.ConclusionsA spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.

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