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International audience; Linum usitatissimum is a source of pharmacologically active lignans and neolignans. An effective protocol has been established for the enhanced biosynthesis of lignans and neolignans in cell cultures of Linum usitatissimum by using chitosan addition. Gene expression analysis of monolignols (PAL, CCR and CAD), lignans (DIR, PLR and UGT) and neolignans (PCBER) biosynthetic genes by RT-qPCR as well as monolignol biosynthetic PAL, CCR and CAD enzyme activities evidenced a stimulation following chitosan treatment. Validated reverse phase high-performance liquid chromatography coupled to diode array detection was used to quantify secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG), dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) showed that chitosan treated cell cultures had better accumulation of these metabolites. Maximum enhancements of 7.3-fold (28 mg/g DW) occurred for LDG, 3.5-fold (58.85 mg/g DW) in DCG and while the least enhancement of 2-fold (18.42 mg/g DW) for SDG was observed in 10 mg/l chitosan treated cell cultures than to controls. Furthermore, same concentration of chitosan also resulted in 1.3-fold increase in antioxidant activity. Compared to the lignans and neolignans accumulations observed in wild type and RNAi-PLR transgenic flaxseeds, chitosan-treated cell cultures appeared to be a very effective production system for these compounds.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

International audience; Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

International audience; Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by β-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine β-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks β-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of β-glucosidase multimerization, an organization common to many defensive GH1 members.

Grape canes are under-valued byproducts of viticulture that accumulate large amounts of stilbenoids. These specialized metabolites are phytoalexins of Vitis vinifera that play a keyrole in defence of Vitis vinifera against pathogens. Since in vitro assays demonstrated the antifungal activity of several pure stilbenoids, grape cane extracts (GCE) have been proposed as eco-friendly alternative to classical fungicides in a context of sustainable viticulture. Following the grape pruning during winter, a storage period associated to an efficient extraction procedure, are determinant to obtain GCE with optimal stilbenoid titration. Stilbenoid-enriched extracts have shown promising protection in vitro against various grape pathogens including downy mildew, powdery mildew, gray mold and wood diseases. Additionally, large-scale studies in vineyards reported a significant partial protection against downy mildew. Protection level is relative to the concentration of stilbenoids contained in the grape cane extract. Therefore, the huge varietal diversity of grape could be explored to select high-producing varieties for future use as biocontrol agent.

Ocimum basilicum L. var purpurascens (purple basil) contains medicinally valuable metabolites. Light greatly influences the physiological processes, including biomass accumulation and secondary metabolites production in medicinal plants. Herein, we investigated the influence of different spectral lights on the biosynthesis of phenylpropanoid metabolites in purple basil callus cultures. Growth kinetics was ostudied for a total of 49 days, with 7 days of sampling time. Among the various treatments, blue light resulted in maximum biomass accumulation, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant DPPH, FRAP and ABTS activities, as compared to controls. Moreover, blue light also encouraged higher superoxide dismutase activity while the red light was found effective for enhanced peroxidase activity. HPLC analysis revealed enhanced rosmarinic acid (87.62 mg/g DW) and anthocyanins (cyanidin: 0.15 mg/g DW and peonidin: 0.13 mg/g DW) contents under dark grown callus cultures which were almost 1.55, 1.25 and 1.18–fold greater than controls, respectively. Conversely, red light caused maximum production of cichoric acid (14.65 mg/g DW). Moreover, a positive correlation occurred among the accumulation of phenolic and flavonoids and antioxidant activities. These results suggest that light quality strongly influences medicinally valuable phenylpropanoid metabolites biosynthesis along with antioxidant potential in in vitro cultures of purple basil. Light-enhanced precious metabolites in callus of Basil.

Monoterpene indole alkaloids (MIAs) are specialized metabolites synthesized in many plants of the Apocynaceae family including Catharanthus roseus and Rauvolfia sp. MIAs are part of the chemical arsenal that plants evolved to face pet and herbivore attacks, and their high biological activities also confer pharmaceutical properties exploited in human pharmacopeia. Developing robust and straightforward tools to elucidate each step of MIA biosynthetic pathways thus constitutes a prerequisite to the understanding of Apocynaceae defense mechanisms and to the exploitation of MIA cytotoxicity through their production by metabolic engineering. While protocols of virus-induced gene silencing (VIGS) based on Agrobacterium-based transformation have emerged, the recalcitrance of Apocynaceae to this type of transformation prompted us to develop an universal procedure of VIGS vector inoculation. Such procedure relies on the delivery of the transforming plasmids through a particle bombardment performed using a biolistic device and offers the possibility to overcome host specificity to silence genes in any plant species. Using silencing of geissoschizine oxidase as an example, we described the main steps of this biolistic mediated VIGS in C. roseus and R. tetraphylla.

This article belongs to the Special Issue Biosynthesis and Function of Plant Specialized Metabolites., 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., We acknowledge funding from ARD2020 Biopharmaceutical program of the Région Centre Val de Loire (BioPROPHARM and CatharSIS projects). G.G. acknowledges a research fellowship of Le Studium-Institute for Advanced Studies, Loire Valley, Orléans, France.

International audience; Cell suspension culture of Linum usitatissimum is a great source of the novel and multipurpose medicinal compounds lignans and neolignans. Conventional culturing practices usually result in low yield of plant secondary metabolites; therefore, we conceived a successful mechanism to elicit production of lignans and neolignans in cell suspension cultures, simply, by addition of chemogenic Ag-NPs into the culture medium. A three stage feeding strategy (day 10, 10 and 15, and 10 and 20, respectively, after inoculation) spanning the log growth phase (day 10–20), was implemented to elicit cell suspension cultures of Linum usitatissimum. Though enhancing effects of Ag-NPs were observed at each stage, feeding Ag-NPs at day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Furthermore, a linear trend in accumulation of lignans and neolignans was observed throughout log phase as compared to control, wherein growth non-associated trend in biosynthesis of these metabolites was observed. Optimum production of both lignans and neolignans occurred on day 20 of culture; a ten fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, five fold increase in dehydrodiconiferyl alcohol glucoside and 1.75 fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively.

International audience; Melatonin as plant growth regulator induces differential effects on metabolites that are responsible for reduction, capping and stabilization of zinc oxide nanoparticles. Phytochemical analysis of callus cultures was performed and results were compared with callus cultures supplemented with other plant growth regulators (α-napthalene acetic acid, 2,4-dichlorophenoxy acetic acid and thidiazuron). Highest total phenolic and flavonoid content [42.23 mg of gallic acid equivalent (GAE) g-1 DW and 36.4 mg of (quercetin equivalent) g-1 DW, respectively] were recorded at melatonin (1.0 µM) + NAA (13.5 µM). ZnONPs were synthesized from NAA (13.5 µM) and melatonin (1.0 µM) + NAA (13.5 µM)-induced calli extracts separately and characterized via X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). FTIR analysis confirmed the presence of phenolics and flavonoids that were mainly found responsible for reduction and capping of ZnONPs. SEM analysis showed triangular shaped ZnONPs synthesized from melatonin + NAA callus extract and these NPs were more dispersed as compared to the spherical-agglomerates of ZnONPs synthesized from NAA-mediated callus extract. Melatonin + NAA callus extract-mediated ZnONPs (having smaller size) were more potent against multiple drug resistant bacterial strains, e.g. Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by producing zone of inhibitions 17 ± 0.76 mm,10 ± 0.57 mm and 13 ± 0.54 mm, respectively.

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and horhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

BACKGROUND 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. © 2018 Society of Chemical Industry.

Silybum marianum (L.) Gaertn. is a well-known medicinal herb, primarily used in liver protection. Light strongly affects several physiological processes along with secondary metabolites biosynthesis in plants. Herein, S. marianum was exploited for in vitro potential under different light regimes in the presence of melatonin. The optimal callogenic response occurred in the combination of 1.0 mg/L &alpha, naphthalene acetic acid and 0.5 mg/L 6-benzylaminopurine under photoperiod. Continuous light associated with melatonin treatment increased total flavonoid content (TFC), total phenolic content (TPC) and antioxidant potential, followed by photoperiod and dark treatments. The increased level of melatonin has a synergistic effect on biomass accumulation under continuous light and photoperiod, while an adverse effect was observed under dark conditions. More detailed phytochemical analysis showed maximum total silymarin content (11.92 mg/g dry weight (DW)) when placed under continuous light + 1.0 mg/L melatonin. Individually, the level of silybins (A and B), silydianin, isolsilychristin and silychristin was found highest under continuous light. Anti-inflammatory activities were also studied and highest percent inhibition was recorded against 15-lipoxygenase (15-LOX) for cultures cultivated under continuous light (42.33%). The current study helps us to better understand the influence of melatonin and different light regimes on silymarin production as well as antioxidant and anti-inflammatory activities in S. marianum callus extracts.

Linum usitatissimum biosynthesizes lignans and neolignans that are diet and medicinally valuable metabolites. In recent years, zinc oxide nanoparticles (ZnONPs) have emerged as potential elicitors for the enhanced biosynthesis of commercial secondary metabolites. Herein, we investigated the influence of biogenic ZnONPs on both seedlings and stem-derived callus of L. usitatissimum. Seedlings of L. usitatissimum grown on Murashige and Skoog (MS) medium supplemented with ZnONPs (1&ndash, 1000 mg/L) presented the highest antioxidant activity, total phenolic content, total flavonoid content, peroxidase and superoxide dismutase activities at 500 mg/L, while the maximum plantlet length was achieved with 10 mg/L. Likewise, the high-performance liquid chromatography (HPLC) analysis revealed the enhanced production of secoisolariciresinol diglucoside, lariciresinol diglucoside, dehydrodiconiferyl alcohol glucoside and guaiacylglycerol-&beta, coniferyl alcohol ether glucoside in the plantlets grown on the 500 mg/L ZnONPs. On the other hand, the stem explants were cultured on MS media comprising 1-naphthaleneacetic acid (1 mg/L) and ZnONPs (1&ndash, 50 mg/L). The highest antioxidant and other activities with an enhanced rooting effect were noted in 25 mg/L ZnONP-treated callus. Similarly, the maximum metabolites were also accumulated in 25 mg/L ZnONP-treated callus. In both systems, the dose-dependent production of reactive oxygen species (ROS) was recorded, resulting in oxidative damage with a more pronounced toxic effect on in vitro cultures. Altogether, the results from this study constitute a first comprehensive view of the impact of ZnONPs on the oxidative stress and antioxidant responses in seedlings vs. in vitro cultures.

International audience; Ocimum basilicum L. (Purple basil) is a source of biologically active antioxidant compounds, particularly phenolic acids and anthocyanins. In this study, we have developed a valuable protocol for the establishment of in vitro callus cultures of O. basilicum and culture conditions for the enhanced production of distinct classes of phenylpropanoid metabolites such as hydroxycinnamic acid derivatives (caffeic acid, chicoric acid, rosmarinic acid) and anthocyanins (cyanidin and peonidin). Callus cultures were established by culturing leaf explants on Murashige and Skoog medium augmented with different concentrations of plant growth regulators (PGRs) [thidiazuron (TDZ), α-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP)] either alone or in combination with 1.0 mg/L NAA. Among all the above-mentioned PGRs, NAA at 2.5 mg/L led to the highest biomass accumulation (23.2 g/L DW), along with total phenolic (TPP; 210.7 mg/L) and flavonoid (TFP; 196.4 mg/L) production, respectively. HPLC analysis confirmed the differential accumulation of phenolic acid [caffeic acid (44.67 mg/g DW), rosmarinic acid (52.22 mg/g DW), and chicoric acid (43.89 mg/g DW)] and anthocyanins [cyanidin (16.39 mg/g DW) and peonidin (10.77 mg/g DW)] as a function of the PGRs treatment. The highest in vitro antioxidant activity was determined with the ORAC assay as compared to the FRAP assay, suggesting the prominence of the HAT over the ET-based mechanism for the antioxidant action of callus extracts. Furthermore, in vivo results illustrated the protective action of the callus extract to limit the deleterious effects of UV-induced oxidative stress, ROS/RNS production, and membrane integrity in yeast cell culture. Altogether, these results clearly demonstrated the great potential of in vitro callus of O. basilicum as a source of human health-promoting antioxidant phytochemicals.

International audience; Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.

International audience; Lignans and neolignans are important biologically active ingredients (BAIs) biosynthesized by Linum usitatissimum. These BAIs have multi-dimensional effects against cancer, diabetes and cardio vascular diseases. In this study, yeast extract (YE) was employed as an elicitor to evaluate its effects on dynamics of biomass, BAIs and antioxidant activities in L. usitatissimum cell cultures. During preliminary experiments, flax cultures were grown on different concentrations of YE (0–1000 mg/L), and 200 mg/L YE was found to be optimum to enhance several biochemical parameters in these cell cultures. A two-fold increase in fresh (FW) and dry weight (DW) over the control was observed in cultures grown on MS medium supplemented with 200 mg/L YE. Similarly, total phenolic (TPC; 16 mg/g DW) and flavonoids content (TFC; 5.1 mg/g DW) were also positively affected by YE (200 mg/L). Stimulatory effects of YE on biosynthesis of lignans and neolignans was also noted. Thus, 200 mg/L of YE enhanced biosynthesis of secoisolariciresinol diglucoside (SDG; 3.36-fold or 10.1 mg/g DW), lariciresinol diglucoside (LDG; 1.3-fold or 11.0 mg/g DW) and dehydrodiconiferyl alcohol glucoside (DCG; 4.26-fold or 21.3 mg/g DW) in L. usitatissimum cell cultures with respect to controls. This elicitation strategy could be scaled up for production of commercially feasible levels of these precious metabolites by cell cultures of Linum.

International audience; The 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.

International audience; Silybum 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.

International audience; The UDP-glycosyltransferase UGT88F subfamily has been described first in Malus x domestica with the characterization of UGT88F1. Up to now UGT88F1 was one of the most active UGT glycosylating dihydrochalcones in vitro. The involvement of UGT88F1 in phloridzin (phloretin 2'-O-glucoside) synthesis, the main apple tree dihydrochalcone, was further confirmed in planta. Since the characterization of UGT88F1, this new UGT subfamily has been poorly studied probably because it seemed restricted to Maloideae. In the present study, we investigate the apple tree genome to identify and biochemically characterize the whole UGT88F subfamily. The apple tree genome contains five full-length UGT88F genes out of which three newly identified members (UGT88F6, UGT88F7 and UGT88F8) and a pseudogene. These genes are organized into two genomic clusters resulting from the recent global genomic duplication event in the apple tree. We show that recombinant UGT88F8 protein specifically glycosylates phloretin in the 2'OH position to synthetize phloridzin in vitro and was therefore named UDP-glucose: phloretin 2'-O-glycosyltransferase. The Km values of UGT88F8 are 7.72 μM and 10.84 μM for phloretin and UDP-glucose respectively and are in the same range as UGT88F1 catalytic parameters thus constituting two isoforms. Co-expression patterns of both UGT88F1 and UGT88F8 argue for a redundant function in phloridzin biosynthesis in planta. Contrastingly, recombinant UGT88F6 protein is able to glycosylate in vitro a wide range of flavonoids including flavonols, flavones, flavanones, chalcones and dihydrochalcones, although flavonols are the preferred substrates, e.g. Km value for kaempferol is 2.1 μM. Depending on the flavonoid, glycosylation occurs at least on the 3-OH and 7-OH positions. Therefore UGT88F6 corresponds to an UDP-glucose: flavonoid 3/7-O-glycosyltransferase. Finally, a molecular modeling study highlights a very high substitution rate of residues in the acceptor binding pocket between UGT88F8 and UGT88F6 which is responsible for the enzymes divergence in substrate and regiospecificity, despite an overall high protein homology.

International audience; Co-expression networks are essential tools to infer biological associations between gene products and predict gene annotation. Global networks can be analyzed at the transcriptome-wide scale or after querying them with a set of guide genes to capture the transcriptional landscape of a given pathway in a process named Pathway Level Coexpression (PLC). A critical step in network construction remains the definition of gene co-expression. In the present work, we compared how Pearson Correlation Coefficient (PCC), Spearman Correlation Coefficient (SCC), their respective ranked values (Highest Reciprocal Rank (HRR)), Mutual Information (MI) and Partial Correlations (PC) performed on global networks and PLCs. This evaluation was conducted on the model plant Arabidopsis thaliana using microarray and differently pre-processed RNA-seq datasets. We particularly evaluated how dataset × distance measurement combinations performed in 5 PLCs corresponding to 4 well described plant metabolic pathways (phenylpropanoid, carbohydrate, fatty acid and terpene metabolisms) and the cytokinin signaling pathway. Our present work highlights how PCC ranked with HRR is better suited for global network construction and PLC with microarray and RNA-seq data than other distance methods, especially to cluster genes in partitions similar to biological subpathways. Constructing global gene co-expression networks is a popular approach to highlight transcriptional relationships (edges) between genes (vertices). The 'Guilt-by-Association' (GBA) principle supposes that genes sharing similar functions are preferentially connected and aims at predicting new functions for proteins by determining how their respective encoding genes are co-expressed with others using a reference dataset containing known gene functions such as the Gene Ontology (GO) 1. Defining edges connecting genes remains a critical step in global co-expression network construction. Expression data (microarray or RNA-seq) are used to construct expression matrices (genes × samples) and to calculate a distance or a similarity for each possible gene pair. The resulting pairwise distance matrix is then thresholded to obtain an adjacency matrix that discriminates relevant edges. Only edges with a distance below (or a similarity above) the set threshold are considered significant and retained for network construction. The procedure is expected to remove non biologically relevant gene associations while retaining the relevant ones and can be assessed with any reference dataset. Alternatively, guide gene sets may be used to extract more human-readable information from large networks in a process named Pathway-Level Coexpression (PLC) 2–7. This approach aims at capturing the best transcriptional associations of a gene set and at highlighting functional gene groups such as known subpathways in this set. There are two types of approaches to determine transcriptional associations of genes: those that are supervised and those that are unsupervised. Supervised approaches such as regression and machine learning based methods require a prior knowledge which is used as a training dataset to recover biologically relevant gene associations and are used to infer regulatory networks, i.e. to uncover preferential and sequential interactions of a gene over the others. The superiority of

Grape 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. Mechanical wounding of freshly-pruned canes drastically shortens the time required to reach maximal stilbenoid accumulation from 6 to 2weeks.

International audience; Grape canes are byproducts of viticulture containing valuable bioactive stilbenoids including monomers and oligomers of E-resveratrol. Although effective contents in stilbenoids are known to be highly variable, the determining factors influencing this composition remain poorly understood. As stilbenoids are locally induced defense compounds in response to phytopathogens, this study assessed the impact of downy mildew infection during the growing season on the stilbenoid composition of winter-harvested grape canes. The spatial distribution between pith, conducting tissues, and cortex of E-piceatannol, E-resveratrol, E-ε-viniferin, ampelopsin A, E-miyabenol C, Z/E-vitisin B, hopeaphenol, and isohopeaphenol in grape canes from infected vineyards was strongly altered. In conducting tissues, representing the main site of stilbenoid accumulation, E-ε-viniferin content was higher and E-resveratrol content was lower. These findings suppose that the health status in vineyards could modify the composition of stilbenoids in winter-harvested grape canes and subsequently the potential biological properties of the valuable extracts.

The tagging-via-substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide-modified farnesyl moiety and captured thanks to biotin alkyne Click-iT® chemistry with further streptavidin-affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C-terminal CaaX-box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.

International audience; Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

Ducos et al. report that the farnesylated protein ASG2 is the Arabidopsis ortholog of human WDCT1, which controls fat accumulation. Both proteins harbor.. ASG2 (Altered Seed Germination 2) is a prenylated protein in Arabidopsis thaliana that participates to abscisic acid signaling and is proposed to act as a substrate adaptor for the DDB1 (DNA damage-binding protein 1)-CUL4 (Cullin 4) E3 ubiquitin ligase complex. ASG2 harbors WD40 and TetratricoPeptide Repeat (TPR) domains, and resembles the well-conserved animal gene called ADP (antiobesity factor ADIPOSE) in fly and WDTC1 (WD40 and TPR 1) in humans. Loss of function of WDTC1 results in an increase in adipocytes, fat accumulation, and obesity. Antiadipogenic functions of WDTC1 involve regulation of fat-related gene transcription, notably through its binding to histone deacetylases (HDACs). Our sequence and phylogenetic analysis reveals that ASG2 belongs to the ADP/WDTC1 cluster. ASG2 and WDTC1 share a highly conserved organization that encompasses structural and functional motifs: seven WD40 domains and WD40 hotspot-related residues, three TPR protein–protein interaction domains, DDB1-binding elements [H-box and DWD (DDB1-binding WD40 protein)-box], and a prenylatable C-terminus. Furthermore, ASG2 involvement in fat metabolism was confirmed by reverse genetic approaches using asg2 knockout Arabidopsis plants. Under limited irradiance, asg2 mutants produce “obese” seeds characterized by increased weight, oil body density, and higher fatty acid contents. In addition, considering some ASG2- and WDTC1-peculiar properties, we show that the WDTC1 C-terminus is prenylated in vitro and HDAC-binding capability is conserved in ASG2, suggesting that the regulation mechanism and targets of ADP/WDTC1-like proteins may be conserved features. Our findings reveal the remarkable evolutionary conservation of the structure and the physiological role of ADIPOSE homologs in animals and plants.

Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.

Environmental pressures forced plants to diversify specialized metabolisms to accumulate noxious molecules such as alkaloids constituting one of the largest classes of defense metabolites. Catharanthus roseus produces monoterpene indole alkaloids via a highly elaborated biosynthetic pathway whose characterization greatly progressed with the recent expansion of transcriptomic resources. The complex architecture of this pathway, sequentially distributed in at least four cell types and further compartmentalized into several organelles, involves partially identified inter-cellular and intra-cellular translocation events acting as potential key-regulators of metabolic fluxes. The description of this spatial organization and the inherent secretion and sequestration of metabolites not only provide new insight into alkaloid cell biology and its involvement in plant defense processes but also present new biotechnological challenges for synthetic biology.

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a K(m) value of 58.5 μM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.