Glandular Trichomes and Isoprenoid Biosynthesis

A bottleneck in the development of new anti‐cancer drugs is the recognition of their mode of action (MoA). Metabolomics combined with machine learning allowed to predict MoAs of novel anti‐proliferative drug candidates, focusing on human prostate cancer cells (PC‐3). As proof of concept, 38 drugs are studied with known effects on 16 key processes of cancer metabolism, profiling low molecular weight intermediates of the central carbon and cellular energy metabolism (CCEM) by LC‐MS/MS. These metabolic patterns unveiled distinct MoAs, enabling accurate MoA predictions for novel agents by machine learning. The transferability of MoA predictions based on PC‐3 cell treatments is validated with two other cancer cell models, i.e., breast cancer and Ewing\'s sarcoma, and show that correct MoA predictions for alternative cancer cells are possible, but still at some expense of prediction quality. Furthermore, metabolic profiles of treated cells yield insights into intracellular processes, exemplified for drugs inducing different types of mitochondrial dysfunction. Specifically, it is predicted that pentacyclic triterpenes inhibit oxidative phosphorylation and affect phospholipid biosynthesis, as confirmed by respiration parameters, lipidomics, and molecular docking. Using biochemical insights from individual drug treatments, this approach offers new opportunities, including the optimization of combinatorial drug applications.

Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses. These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence. Here, we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana (Bs) and Fusarium graminearum (Fg) at the metabolite level. We identify hordedanes, a previously undescribed set of labdane-related diterpenoids with antimicrobial properties, as critical players in these interactions. Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes, including one of the most functionally decorated products 19-b-hydroxy-hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. By contrast, colonization by Fusarium graminearum, another fungal pathogen of barley and wheat, is 4-fold higher in the mutants completely lacking hordedanes. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, whereas it inhibits other pathogenic fungi, including Fg. Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs. Taken together, these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.

The intracellular colonization of plant roots by the beneficial fungal endophyte Serendipita indica follows a biphasic strategy, including a host cell death phase that enables successful colonization of Arabidopsis thaliana roots. How host cell death is initiated and controlled is largely unknown. Here, we show that two fungal enzymes, the ecto-50-nucleotidase SiE5NT and the nuclease SiNucA, act synergistically in the apoplast at the onset of cell death to produce deoxyadenosine (dAdo). The uptake of extracellular dAdo but not the structurally related adenosine activates cell death via the equilibrative nucleoside transporter ENT3. We identified a previously uncharacterized Toll-like interleukin 1 receptor (TIR)-nucleotide-binding leucine-rich repeat receptor (NLR) protein, ISI (induced by S. indica), as an intracellular factor that affects host cell death, fungal colonization, and growth promotion. Our data show that the combined activity of two fungal apoplastic enzymes promotes the production of a metabolite that engages TIR-NLR-modulated pathways to induce plant cell death, providing a link to immunometabolism in plants.Graphical abstract

Methylerythritol cyclodiphosphate (MEcPP) is an intermediate in the biosynthesis of isoprenoids in plant plastids and in bacteria, and acts as a stress signal in plants. Here, we show that MEcPP regulates biofilm formation in Escherichia coli K-12 MG1655. Increased MEcPP levels, triggered by genetic manipulation or oxidative stress, inhibit biofilm development and production of fimbriae. Deletion of fimE, encoding a protein known to downregulate production of adhesive fimbriae, restores biofilm formation in cells with elevated MEcPP levels. Limited proteolysis-coupled mass spectrometry (LiP-MS) reveals that MEcPP interacts with the global regulatory protein H-NS, which is known to repress transcription of fimE. MEcPP prevents the binding of H-NS to the fimE promoter. Therefore, our results indicate that MEcPP can regulate biofilm formation by modulating H-NS activity and thus reducing fimbriae production. Further research is needed to test whether MEcPP plays similar regulatory roles in other bacteria.

Sesquiterpene lactones (STLs) are bitter tasting plant specialized metabolites derived from farnesyl pyrophosphate (FPP) that contain a characteristic lactone ring. STLs can be found in many plant families that are distantly related to each other and outside the plant kingdom. They are especially prevalent in the plant families Apiaceae and Asteraceae, the latter being one of the largest plant families besides the Orchidaceae. The STL diversity is especially large in the Asteraceae, which made them an ideal object for chemosystematic studies in these species. Many STLs show a high bioactivity, for example as protective compounds against herbivory. STLs are also relevant for pharmaceutical applications, such as the treatment of malaria with artemisinin. Recent findings have dramatically changed our knowledge about the biosynthesis of STLs, as well as their developmental, spatial, and environmental regulation. This review intents to update the currently achieved progress in these aspects. With the advancement of genome editing tools such as CRISPR/Cas and the rapid acceleration of the speed of genome sequencing, even deeper insights into the biosynthesis, regulation, and enzyme evolution of STL can be expected in the future. Apart from their role as protective compounds, there may be a more subtle role of STL in regulatory processes of plants that will be discussed as well.

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyldiphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.

RNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4–HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.

In plants, exposure to high light irradiation induces various stress responses, which entail complex metabolic rearrangements. To explore these dynamics, we conducted time‐course experiments spanning 2 min to 72 h with Arabidopsis thaliana under high and control light. Comparative metabolomics, transcriptomics, redox proteomics, and stable isotope labeling on leaf rosettes identified a series of synchronous and successive responses that provide a deeper insight into well‐orchestrated mechanisms contributing to high‐light acclimation. We observed transient transcriptome downregulation related to light harvesting and electron flow before the profound remodeling of the photosynthetic apparatus. Throughout the entire time course, redox homeostasis is tightly balanced between downregulation of production and enhanced transformation of NADPH accompanied by redistribution of reducing equivalents across several subcellular compartments. In both light conditions, C4 acids such as malate and fumarate are produced via anaplerosis. In carbon units, their accumulation in vacuoles surpasses plastidic levels of starch and intensifies notably under high light. In parallel, citrate synthesis from pyruvate is significantly hindered diurnally. Isotopic labeling in 2‐oxoglutarate and glutamate suggests a moderate de novo synthesis of C5 acids from a vacuolar citrate reservoir during the light phase while they are largely renewed during the night. In the absence of a diurnal clockwise flow through the tricarboxylic acid (TCA) cycle, increased oxidation of photorespiratory glycine takes over as a source of reductants to fuel mitochondrial ATP production. These findings, along with previous research, contribute to a model integrating redox balance and linking increased carbon assimilation and nitrogen metabolism, especially in the context of an incomplete TCA cycle.

Several commercially important secondary metabolites are produced and accumulated in high amounts by glandular trichomes, giving the prospect of using them as metabolic cell factories. Due to extremely high metabolic fluxes through glandular trichomes, previous research focused on how such flows are achieved. The question regarding their bioenergetics became even more interesting with the discovery of photosynthetic activity in some glandular trichomes. Despite recent advances, how primary metabolism contributes to the high metabolic fluxes in glandular trichomes is still not fully elucidated. Using computational methods and available multi-omics data, we first developed a quantitative framework to investigate the possible role of photosynthetic energy supply in terpenoid production and next tested experimentally the simulation-driven hypothesis. With this work, we provide the first reconstruction of specialised metabolism in Type-VI photosynthetic glandular trichomes of Solanum lycopersicum. Our model predicted that increasing light intensities results in a shift of carbon partitioning from catabolic to anabolic reactions driven by the energy availability of the cell. Moreover, we show the benefit of shifting between isoprenoid pathways under different light regimes, leading to a production of different classes of terpenes. Our computational predictions were confirmed in vivo, demonstrating a significant increase in production of monoterpenoids while the sesquiterpenes remained unchanged under higher light intensities. The outcomes of this research provide quantitative measures to assess the beneficial role of chloroplast in glandular trichomes for enhanced production of secondary metabolites and can guide the design of new experiments that aim at modulating terpenoid production.

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