Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene

Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.Peer reviewe

Tagging and Capture of Prenylated CaaX-Proteins from Plant Cell Cultures

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Prenylated Proteins Are Required for Methyl-Jasmonate-Induced Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus roseus

In Catharanthus roseus, monoterpenoid indole alkaloids (MIA) result from the condensation of the indole precursor tryptamine with the terpenoid precursor secologanin, which is derived from the plastidial methyl-d-erythritol 4-phosphate (MEP) pathway. Nevertheless, inhibition of the classical so-called mevalonate pathway leads to inhibition of MIA biosynthesis, suggesting that there is some cross regulation between these two pathways. The purpose of this chapter is to outline a new function for protein prenylation. Our results suggest that prenylated proteins, apparently mevalonate pathway end products, act as part of the regulatory mechanism coordinating the exchange of metabolites between compartmentalized metabolic pathways and that this process is governed by methyl jasmonate. Methyl jasmonate is a major inducer of alkaloid biosynthesis through enhancing MEP pathway gene expression. In C. roseus cells, inhibition of protein prenylation leads to the down-regulation of methyl-jasmonate-induced expression of MEP pathway genes and thus abolishes MIA biosynthesis. Jointly, failure of protein prenylation also inhibits the methyl-jasmonate-induced expression of the transcription factor ORCA3 which acts as a central regulator of MIA biosynthesis. Furthermore, the specific silencing of protein prenyltransferases in C. roseus cells mediated by RNA interference shows that inhibition of type I protein geranylgeranyltransferase down-regulates the expression of ORCA3. These data point to a specific role of protein geranylgeranylation in jasmonate signalling leading to MIA formation

Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

International audienceCell 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

A look inside an alkaloid multisite plant: the Catharanthus logistics

International audienceEnvironmental 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 intracellular 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

Biogenic zinc oxide nanoparticles-enhanced biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L

International audienceZinc oxide nanoparticles (NPs) have emerged as a novel elicitor for enhanced biosynthesis of secondary metabolites in in vitro plant cell cultures. The current study was aimed to explore elicitation abilities of ZnO-NPs for enhanced accumulation of lignans and neolignans in cell cultures of Linum usitatissimum. We optimized concentration of zinc oxide NPs before carrying out a full-fledged experiment. Subsequently, an optimum dose of 100 mg/l was introduced into the culture medium on day 0, days 0 and 15, and finally days 0 and 25. We observed that repeated elicitation stimulated various parameters and physiological responses in Linum usitatissimum cell cultures than one-time elicitation. Repeated elicitation of cell cultures on day 0 and 15 resulted in highest fresh weight (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Contrarily, repeated elicitation on day 0 and 25 resulted in highest DW (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-β-coniferyl alcohol ether glucoside 307.69 mg/l). Enhancement in plant growth and secondary metabolites accumulation was several fold higher than controls. Furthermore, a linear relationship existed between total phenolic and flavonoid contents which in turn was correlated with higher antioxidant activities

Differential Production of Phenylpropanoid Metabolites in Callus Cultures of Ocimum basilicum L. with Distinct In Vitro Antioxidant Activities and In Vivo Protective Effects against UV stress

International audienceOcimum 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

Yeast-extract improved biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

International audienceLignans 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

Light-mediated biosynthesis of phenylpropanoid metabolites and antioxidant potential in callus cultures of purple basil (Ocimum basilicum L. var purpurascens)

International audienceOcimum 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

Interactive Effects of Light and Melatonin on Biosynthesis of Silymarin and Anti-Inflammatory Potential in Callus Cultures of <i>Silybum marianum</i> (L.) Gaertn.

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 &#945;-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