Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

<p>Abstract</p> <p>Background</p> <p>The 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 <it>Catharanthus roseus </it>or ajmaline in <it>Rauvolfia serpentina</it>. However, very few reports focused on the MIA physiological functions.</p> <p>Results</p> <p>In this study we showed that a strictosidine pool existed <it>in planta </it>and that the strictosidine deglucosylation product(s) was (were) specifically responsible for <it>in vitro </it>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 <it>in planta</it>. On the one hand, <it>in situ </it>hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of <it>C. roseus </it>aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both <it>C. roseus </it>and <it>R. serpentina </it>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.</p> <p>Conclusions</p> <p>A 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.</p

Field-Based Metabolomics of Vitis vinifera L. Stems Provides New Insights for Genotype Discrimination and Polyphenol Metabolism Structuring

Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that in planta act as key components of the plant defense system against diseases. Considerable advances have been made in the chemical characterization of wine metabolites particularly volatile and polyphenolic compounds. However, the metabotyping (metabolite-phenotype characterization) of grape varieties, from polyphenolic-rich vineyard by-product is unprecedented. As this composition might result from the complex interaction between genotype, environment and viticultural practices, a field experiment was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. As a result, UPLC-MS-based targeted metabolomic analyses of grape stems from 8 Vitis vinifera L. cultivars allowed the determination of 42 polyphenols related to phenolic acids, flavonoids, procyanidins, and stilbenoids as resveratrol oligomers (degree of oligomerization 1–4). Using a partial least-square discriminant analysis approach, grape stem chemical profiles were discriminated according to their genotypic origin showing that polyphenol profile express a varietal signature. Furthermore, hierarchical clustering highlights various degree of polyphenol similarity between grape varieties that were in agreement with the genetic distance using clustering analyses of 22 microsatellite DNA markers. Metabolite correlation network suggested that several polyphenol subclasses were differently controlled. The present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with both health-benefit potential and plant defense traits

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis

Mechanistic evaluation of phytochemicals in breast cancer remedy: current understanding and future perspectives

International audienceBreast cancer is one of the most commonly diagnosed cancers around the globe and accounts for a large proportion of fatalities in women. Despite the advancement in therapeutic and diagnostic procedures, breast cancer still represents a major challenge. Current anti-breast cancer approaches include surgical removal, radiotherapy, hormonal therapy and the use of various chemotherapeutic drugs. However, drug resistance, associated serious adverse effects, metastasis and recurrence complications still need to be resolved which demand safe and alternative strategies. In this scenario, phytochemicals have recently gained huge attention due to their safety profile and cost-effectiveness. These phytochemicals modulate various genes, gene products and signalling pathways, thereby inhibiting breast cancer cell proliferation, invasion, angiogenesis and metastasis and inducing apoptosis. Moreover, they also target breast cancer stem cells and overcome drug resistance problems in breast carcinomas. Phytochemicals as adjuvants with chemotherapeutic drugs have greatly enhanced their therapeutic efficacy. This review focuses on the recently recognized molecular mechanisms underlying breast cancer chemoprevention with the use of phytochemicals such as curcumin, resveratrol, silibinin, genistein, epigallocatechin gallate, secoisolariciresinol, thymoquinone, kaempferol, quercetin, parthenolide, sulforaphane, ginsenosides, naringenin, isoliquiritigenin, luteolin, benzyl isothiocyanate, α-mangostin, 3,3′-diindolylmethane, pterostilbene, vinca alkaloids and apigenin

The Arabidopsis DUF239 gene family encodes Neprosin‐like proteins that are widely expressed in seed endosperm

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Differential accumulation of silymarin induced by exposure of Silybum marianum L. callus cultures to several spectres of monochromatic lights

International audienceSilybum marianum L. (Milk thistle) is one of the most extensively studied medicinal herbs with well-known hepatoprotective activity. Light is considered as a key abiotic elicitor influencing several physiological processes in plants, including the biosynthesis of secondary metabolites. In this study, we investigated the influence of light quality on morphological and biochemical aspects in in vitro grown leaf-derived callus cultures of S. marianum. Combination of 6-benzylaminopurine (BAP 2.5 mg/L) and α-naphthalene acetic acid (NAA 1.0 mg/L) resulted in optimum callogenic response (97%) when placed under cool-white light with 16 h light and 8 h dark. Red light significantly increased the total phenolic content (TPC), total flavonoid content (TFC), antioxidant and superoxide dismutase (SOD) activities while highest peroxidase (POD) activity was recorded for the dark grown cultures, followed by green light grown cultures. HPLC analysis revealed enhanced total silymarin content under red light (18.67 mg/g DW), which was almost double than control (9.17 mg/g DW). Individually, the level of silychristin, isosilychristin, silydianin, silybin A and silybin B were found greatest under red light, whereas green spectrum resulted in highest accumulation of isosilybin A and isosilybin B. Conversely, the amount of taxifolin was found maximum under continuous white light (0.480 mg/g DW) which was almost 8-fold greater than control (0.063 mg/g DW). A positive correlation was found between the TPC, TFC and antioxidant activities. This study will assist in comprehending the influence of light quality on production of valuable secondary metabolites in in vitro cultures of S. marianum L

In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

International audienceThe multipurpose plant species Linum usitatissimum famous for producing linen fibre and containing valuable pharmacologically active polyphenols, has rarely been tested for it's in vitro biosynthesis potential of lignans and neolignans. The current study aims at the synergistic effects of mineral nutrients variation and different photoperiod treatments on growth kinetics and biomass accumulation in in vitro cultures of Linum usitatissimum. Both nutrient quality and quantity affected growth patterns, as cultures established on Gamborg B5 medium had comparatively long exponential phase compared to Murashige and Skoog medium, while growth was slow but steady until last phases of the culture on Schenk and Hildebrandt medium. Similarly, we observed that boron deficiency and nitrogen limitation in culture medium (Gamborg B5 medium) enhanced callus biomass (fresh weight 413 g/l and dry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside 6.33 and lariciresinol diglucoside 5.22 mg/g dry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans (dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) in continuous dark after 4th week of culture. Conversely, maximum flavonoids production occurred at both Murashige and Skoog, Schenk and Hildebrandt media (both media types contain comparatively higher boron and nitrogen content) in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter. This study opens new dimension for optimizing growing conditions and evaluating underlying mechanisms in biosynthesis of lignans and neolignans in in vitro cultures of Linum usitatissimum

A synthetic construct for genetic engineering of the emerging pathogenic yeast Candida auris

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Mechanical stress rapidly induces E-resveratrol and E-piceatannol biosynthesis in grape canes stored as a freshly-pruned byproduct

International audienceGrape canes represent a promising source of bioactive phytochemicals. However the stabilization of the raw material after pruning remains challenging. We recently reported the induction of stilbenoid metabolism after winter pruning including a strong accumulation of E-resveratrol and E-piceatannol during the first six weeks of storage. In the present study, the effect of mechanical wounding on freshly-pruned canes was tested to increase the induction of stilbenoid metabolism. Cutting the grape canes in short segments immediately after pruning triggered a transient expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes, followed by a rapid accumulation of E-resveratrol and E-piceatannol. The degree of stilbenoid induction was related to the intensity of mechanical wounding. Data suggest that a global defense response is triggered involving jasmonate signaling, PR proteins and stilbenoid metabolism. Mechanic

Vineyard evaluation of stilbenoid‐rich grape cane extracts against downy mildew: a large‐scale study

International audienceBACKGROUND:Plasmopara viticola control in organic viticulture requires copper-based fungicides with harmful effects on health and the environment. Plant extracts represent a biorational eco-friendly alternative to copper. The aim of this study was to evaluate the potential of stilbenoid-rich grape cane extract (GCE) against downy mildew on three cultivars over 3 years following natural downy mildew infection.RESULTS:Over all field trials, GCE treatments showed an average reduction in disease incidence of -35% and -38% on leaves and clusters, respectively. The average reduction in disease severity was -35% and -43% on leaves and clusters, respectively. Under artificial downy mildew infection, GCE efficacy corresponded to 1 g L-1 of copper. Neither phytotoxicity nor adverse effects on auxiliary fauna were observed after treatment with GCE.CONCLUSION:Because few or no biocontrol agents are active alone against P. viticola, GCE is a promising alternative to copper-based fungicides. Grape canes, an abundant by-product of viticulture, have great potential for valorization as a biocontrol agent for sustainable viticulture