Publications by authors named "Sullivan Renouard"

23 Publications

  • Page 1 of 1

A Critical Cross-Species Comparison of Pollen from Gaertn. vs. L. for Authentication of Thai Medicinal Herbal Tea.

Plants (Basel) 2020 Jul 21;9(7). Epub 2020 Jul 21.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, INRAE USC1328, University of Orleans, 45067 Orléans CEDEX 2, France.

"Bau Luang" or Gaertn. is an aquatic medicinal herb that has been used as a component of traditional medicines, medicinal products, and herbal tea for good health, particularly in Asia. The stamen of is an important part of this medicinal plant that is used in the form of dried and/or powdered stamens for herbal tea as well as the main ingredient of some traditional remedies. However, there is another aquatic herb called "Bau Sai" or L. that is distributed in similar locations. Living plants of these two aquatic species may be classified according to their morphology, but the dried and powdered stamens of these two medicinal species are difficult to distinguish. The major reason of adulteration is the higher price of Bau Luang stamen. As a result, various methods of authentication, such as pollen micromorphology evaluation using scanning electron microscopy (SEM) analysis, bioinformatics analysis of two nuclear and plastic DNA markers, phytochemical stamen profiling, and Fourier transform infrared (FTIR) analysis of stamen plant material authentication from Bau Luang and Bau Sai, have been used in this present research in order to avoid some adulteration and/or misuse between the dried stamens of Bau Luang and Bau Sai. These results showed that the micro-morphology of pollen (size of pollen grain, number of apertures, and surface ornamentation) from the SEM analysis, some phytochemical compounds and the FTIR sporopollenin-to-protein ratio signal analysis are potential tools for authentication and identification of these two medicinal plants from their dried-stamen materials. This model of investigation may also be used to distinguish dried plant material from other problematic plant groups.
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http://dx.doi.org/10.3390/plants9070921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412456PMC
July 2020

Characterization of LuWRKY36, a flax transcription factor promoting secoisolariciresinol biosynthesis in response to Fusarium oxysporum elicitors in Linum usitatissimum L. hairy roots.

Planta 2019 Jul 29;250(1):347-366. Epub 2019 Apr 29.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, EA 1207, INRA USC 1328, Université d'Orléans, Pôle Universitaire d'Eure et Loir, 21 Rue de Loigny la Bataille, 28000, Chartres, France.

Main Conclusion: The involvement of a WRKY transcription factor in the regulation of lignan biosynthesis in flax using a hairy root system is described. Secoisolariciresinol is the main flax lignan synthesized by action of LuPLR1 (pinoresinol-lariciresinol reductase 1). LuPLR1 gene promoter deletion experiments have revealed a promoter region containing W boxes potentially responsible for the response to Fusarium oxysporum. W boxes are bound by WRKY transcription factors that play a role in the response to stress. A candidate WRKY transcription factor, LuWRKY36, was isolated from both abscisic acid and Fusarium elicitor-treated flax cell cDNA libraries. This transcription factors contains two WRKY DNA-binding domains and is a homolog of AtWRKY33. Different approaches confirmed LuWRKY36 binding to a W box located in the LuPLR1 promoter occurring through a unique direct interaction mediated by its N-terminal WRKY domain. Our results propose that the positive regulator action of LuWRKY36 on the LuPLR1 gene regulation and lignan biosynthesis in response to biotic stress is positively mediated by abscisic acid and inhibited by ethylene. Additionally, we demonstrate a differential Fusarium elicitor response in susceptible and resistant flax cultivars, seen as a faster and stronger LuPLR1 gene expression response accompanied with higher secoisolariciresinol accumulation in HR of the resistant cultivar.
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http://dx.doi.org/10.1007/s00425-019-03172-9DOI Listing
July 2019

The control exerted by ABA on lignan biosynthesis in flax (Linum usitatissimum L.) is modulated by a Ca signal transduction involving the calmodulin-like LuCML15b.

J Plant Physiol 2019 May 23;236:74-87. Epub 2019 Mar 23.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), INRA, USC1328, Université d'Orléans, Pôle Universitaire d'Eure et Loir, 21 rue de Loigny la Bataille, F-28000 Chartres, France; Bioactifs et Cosmétiques, GDR 3711 COSMACTIFS, CNRS Université d'Orléans, rue de Chartres, F-45100 Orléans, France. Electronic address:

The LuPLR1 gene encodes a pinoresinol lariciresinol reductase responsible for the biosynthesis of (+)-secoisolariciresinol, a cancer chemopreventive lignan, highly accumulated in the seedcoat of flax (Linum usitatissimum L.). Abscisic acid (ABA) plays a key role in the regulation of LuPLR1 gene expression and lignan accumulation in both seeds and cell suspensions, which require two cis-acting elements (ABRE and MYB2) for this regulation. Ca is a universal secondary messenger involved in a wide range of physiological processes including ABA signaling. Therefore, Ca may be involved as a mediator of LuPLR1 gene expression and lignan biosynthesis regulation exerted by ABA. To test the potential implication of Ca signaling, a pharmacological approach was conducted using both flax cell suspensions and maturing seed systems coupled with a ß-glucuronidase reporter gene experiment, RT-qPCR analysis, lignan quantification as well as Ca fluorescence imaging. Exogenous ABA application results in an increase in the intracellular Ca cytosolic concentration, originating mainly from the extracellular medium. Promoter-reporter deletion experiments suggest that the ABRE and MYB2 cis-acting elements of the LuPLR1 gene promoter functioned as Ca-sensitive sequences involved in the ABA-mediated regulation. The use of specific inhibitors pointed the crucial roles of the Ca sensors calmodulin-like proteins and Ca-dependent protein kinases in this regulation. This regulation appeared conserved in the two different studied systems, i.e. cell suspensions and maturing seeds. A calmodulin-like, LuCML15b, identified from gene network analysis is proposed as a key player involved in this signal transduction since RNAi experiments provided direct evidences of this role. Taken together, these results provide new information on the regulation of plant defense and human health-promoting compounds, which could be used to optimize their production.
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http://dx.doi.org/10.1016/j.jplph.2019.03.005DOI Listing
May 2019

Pinoresinol-lariciresinol reductases, key to the lignan synthesis in plants.

Planta 2019 Jun 20;249(6):1695-1714. Epub 2019 Mar 20.

LBLGC, INRA USC 1328 Université d'Orléans, Orléans, France.

Main Conclusion: This paper provides an overview on activity, stereospecificity, expression and regulation of pinoresinol-lariciresinol reductases in plants. These enzymes are shared by the pathways to all 8-8' lignans derived from pinoresinol. Pinoresinol-lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols. They catalyze two successive reduction steps leading to the production of lariciresinol or secoisolariciresinol from pinoresinol. Two secoisolariciresinol enantiomers can be synthetized with different fates. Depending on the plant species, these enantiomers are either final products (e.g., in the flaxseed where it is stored after glycosylation) or are the starting point for the synthesis of a wide range of lignans, among which the aryltetralin type lignans are used to semisynthesize anticancer drugs such as Etoposide. Thus, the regulation of the gene expression of PLRs as well as the possible specificities of these reductases for one reduction step or one enantiomer are key factors to fine-tune the lignan synthesis. Results published in the last decade have shed light on the presence of more than one PLR in each plant and revealed various modes of action. Nevertheless, there are not many results published on the PLRs and most of them were obtained in a limited range of species. Indeed, a number of them deal with wild and cultivated flax belonging to the genus Linum. Despite the occurrence of lignans in bryophytes, pteridophytes and monocots, data on PLRs in these taxa are still missing and indeed the whole diversity of PLRs is still unknown. This review summarizes the data, published mainly in the last decade, on the PLR gene expression, enzymatic activity and biological function.
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http://dx.doi.org/10.1007/s00425-019-03137-yDOI Listing
June 2019

Green Ultrasound Assisted Extraction of Rosmarinic Acid from (L.) R.Br. Leaves.

Plants (Basel) 2019 Feb 27;8(3). Epub 2019 Feb 27.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, INRA USC1328, Orleans University, 45067 Orléans Cedex 2, France.

Painted nettle ( (L.) R.Br.) is an ornamental plant belonging to family, native of Asia. Its leaves constitute one of the richest sources of -rosmarinic acid, a well-known antioxidant and antimicrobial phenolic compound. These biological activities attract interest from the cosmetic industry and the demand for the development of green sustainable extraction processes. Here, we report on the optimization and validation of an ultrasound-assisted extraction (USAE) method using ethanol as solvent. Following preliminary single factor experiments, the identified limiting extraction parameters (i.e., ultrasound frequency, extraction duration, and ethanol concentration) were further optimized using a full factorial design approach. The method was then validated following the recommendations of the association of analytical communities (AOAC) to ensure the precision and accuracy of the method used to quantify -rosmarinic acid. Highest -rosmarinic acid content was obtained using pure ethanol as extraction solvent following a 45-minute extraction in an ultrasound bath operating at an ultrasound frequency of 30 kHz. The antioxidant (in vitro radical scavenging activity) and antimicrobial (directed toward ACTT6538) activities were significantly correlated with the -rosmarinic acid concentration of the extract evidencing that these key biological activities were retained following the extraction using this validated method. Under these conditions, 110.8 mg/g DW of -rosmarinic acid were extracted from lyophilized leaves as starting material evidencing the great potential of this renewable material for cosmetic applications. Comparison to other classical extraction methods evidenced a clear benefit of the present USAE method both in terms of yield and extraction duration.
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http://dx.doi.org/10.3390/plants8030050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473734PMC
February 2019

Insight into the Influence of Cultivar Type, Cultivation Year, and Site on the Lignans and Related Phenolic Profiles, and the Health-Promoting Antioxidant Potential of Flax ( L.) Seeds.

Molecules 2018 Oct 14;23(10). Epub 2018 Oct 14.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC) EA1207 INRA USC1328, Plant LIGNANS Team, Université d'Orléans, 28000 Chartres, France.

Flaxseeds are a functional food representing, by far, the richest natural grain source of lignans, and accumulate substantial amounts of other health beneficial phenolic compounds (i.e., flavonols, hydroxycinnamic acids). This specific accumulation pattern is related to their numerous beneficial effects on human health. However, to date, little data is available concerning the relative impact of genetic and geographic parameters on the phytochemical yield and composition. Here, the major influence of the cultivar over geographic parameters on the flaxseed phytochemical accumulation yield and composition is evidenced. The importance of genetic parameters on the lignan accumulation was further confirmed by gene expression analysis monitored by RT-qPCR. The corresponding antioxidant activity of these flaxseed extracts was evaluated, both in vitro, using ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and iron chelating assays, as well as in vivo, by monitoring the impact of UV-induced oxidative stress on the lipid membrane peroxidation of yeast cells. Our results, both the in vitro and in vivo studies, confirm that flaxseed extracts are an effective protector against oxidative stress. The results point out that secoisolariciresinol diglucoside, caffeic acid glucoside, and -coumaric acid glucoside are the main contributors to the antioxidant capacity. Considering the health benefits of these compounds, the present study demonstrates that the flaxseed cultivar type could greatly influence the phytochemical intakes and, therefore, the associated biological activities. We recommend that this crucial parameter be considered in epidemiological studies dealing with flaxseeds.
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http://dx.doi.org/10.3390/molecules23102636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222607PMC
October 2018

Effect of Fe-functionalized biochar on toxicity of a technosol contaminated by Pb and As: sorption and phytotoxicity tests.

Environ Sci Pollut Res Int 2018 Nov 1;25(33):33678-33690. Epub 2018 Oct 1.

University of Orleans, INRA USC1328, LBLGC EA 1207, rue de Chartres, BP 6759, 45067, Orléans Cedex 2, France.

Biochar, produced by the pyrolysis of biomass under low oxygen conditions, has gathered attention in the last few years due to its capability to reduce metal(loid)s bioavailability and mobility in soils, as well as its beneficial effects on soil fertility. Indeed, biochar amendment to polluted soil induced usually an increase of pH, water holding capacity, and nutrient contents, associated with a decrease of metal(loid)s concentrations in soil pore water, through sorption. However, biochar has been shown efficient in sorbing cation pollutants, like Pb, but present a low sorption capacity towards anions like As. This contrasted behavior poses a problem, as most polluted soils are multi-contaminated, with both cation and anion pollutants. One of the solutions to overcome such problem is to functionalize biochar, by modifying its surface. However, most studies actually focused on functionalization effect on metal(loid)s sorption towards batch experiments, and only a few dealt with modified biochar incorporation to the soil. Therefore, this study aimed (i) to assess the sorption capacity of hardwood biochars, harboring different particle sizes, towards Pb and As; (ii) to evaluate the effect of a Fe-functionalization on Pb and As sorption; and (iii) to validate the results, in a phytotoxicity test using Phaseolus vulgaris as bioindicator plant. The batch experiments showed that all four biochars were able to efficiently sorb Pb, the fine biochars showing higher sorption values than the coarse biochars. As sorption was very low. Fe-coating increased As sorption value, while having no effect on Pb sorption. However, when incorporated in the soil, Fe-coated biochar did not improve soil physico-chemical properties compared to the pristine biochar; especially, it did not reduce As soil pore water concentrations. Finally, bean plant did not show differences in terms of biomass production between the two biochars incorporated into polluted soil, demonstrating that Fe-functionalization did not improve biochar capacity to decrease soil toxicity.
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http://dx.doi.org/10.1007/s11356-018-3247-9DOI Listing
November 2018

Exploiting potential and characterization of surface modified Zinc oxide nanoparticles of extract: Their clinical potential towards HepG2 cell line and human pathogenic bacteria.

EXCLI J 2018 16;17:671-687. Epub 2018 Jul 16.

Department of Biotechnology, Quaid-i-Azam University, Islamabad-45320, Pakistan.

Little is known about biogenically synthesized Zinc oxide nanoparticles (ZnONPs) from . Synthesis of metal oxide NPs from essential oil producing medicinal plants results in less harmful side effects to the human population as compared to chemically synthesized NPs. In this article, we report biogenic synthesis of ZnONPs from derived plantlets and thidiazuron (TDZ) induced callus culture of . Synthesized NPs were characterized using UV-spectra, XRD, FTIR, SEM and EDX. Furthermore, the NPs were evaluated for their potential cytotoxic (against HepG2 cell line) and antimicrobial (against drug resistant , , and ) activities. Pure crystalline ZnONPs with hexagonal and triangular shapes were obtained as a result of callus extract (CE) and whole plant extract (WPE), respectively. ZnONPs showed potent cytotoxic and antimicrobial potential. The antimicrobial and cytotoxic activities of ZnONPs were found to be shape and surface bound phytochemicals dependent. CE mediated hexagonal ZnONPs showed superior anti-cancer and antimicrobial activities as compared to WPE mediated triangular shaped ZnONPs. It is concluded that biogenic ZnONPs have incredible potential as theranostic agents and can be adopted as useful drug delivery system in next generation treatment strategies.
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http://dx.doi.org/10.17179/excli2018-1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6088224PMC
July 2018

Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans.

Int J Mol Sci 2018 Mar 26;19(4). Epub 2018 Mar 26.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, LBLGC EA1207/INRA USC1328, Team Plant Lignans, Université d'Orléans, 28000 Chartres, France.

hairy root lines were established from hypocotyl pieces using strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of as potential sources for these valuable metabolites as an alternative, or as a complement to collected from wild stands.
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http://dx.doi.org/10.3390/ijms19040990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979590PMC
March 2018

Functional characterization of the pinoresinol-lariciresinol reductase-2 gene reveals its roles in yatein biosynthesis and flax defense response.

Planta 2017 Sep 27;246(3):405-420. Epub 2017 Apr 27.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), INRA USC1328, Université d'Orléans, 21 rue de Loigny la Bataille, 28000, Chartres, France.

Main Conclusion: This study provides new insights into the biosynthesis regulation and in planta function of the lignan yatein in flax leaves. Pinoresinol-lariciresinol reductases (PLR) catalyze the conversion of pinoresinol into secoisolariciresinol (SECO) in lignan biosynthesis. Several lignans are accumulated in high concentrations, such as SECO accumulated as secoisolariciresinol diglucoside (SDG) in seeds and yatein in aerial parts, in the flax plant (Linum usitatissimum L.) from which two PLR enzymes of opposite enantioselectivity have been isolated. While LuPLR1 catalyzes the biosynthesis of (+)-SECO leading to (+)-SDG in seeds, the role(s) of the second PLR (LuPLR2) is not completely elucidated. This study provides new insights into the in planta regulation and function of the lignan yatein in flax leaves: its biosynthesis relies on a different PLR with opposite stereospecificity but also on a distinct expression regulation. RNAi technology provided evidence for the in vivo involvement of the LuPLR2 gene in the biosynthesis of (-)-yatein accumulated in flax leaves. LuPLR2 expression in different tissues and in response to stress was studied by RT-qPCR and promoter-reporter transgenesis showing that the spatio-temporal expression of the LuPLR2 gene in leaves perfectly matches the (-)-yatein accumulation and that LuPLR2 expression and yatein production are increased by methyl jasmonate and wounding. A promoter deletion approach yielded putative regulatory elements. This expression pattern in relation to a possible role for this lignan in flax defense is discussed.
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http://dx.doi.org/10.1007/s00425-017-2701-0DOI Listing
September 2017

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats.

Front Plant Sci 2016 21;7:1743. Epub 2016 Nov 21.

Max Planck Institute for Chemical Ecology Jena, Germany.

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed ( L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of (), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase () reporter gene, and the spatio-temporal regulation of gene expression in flaxseed was determined by histochemical and activity assays of . The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.
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http://dx.doi.org/10.3389/fpls.2016.01743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116464PMC
November 2016

Investigation of the Lignan Content in Extracts from Linum, Callitris and Juniperus Species in Relation to Their In Vitro Antiproliferative Activities.

Planta Med 2017 Apr 24;83(6):574-581. Epub 2016 Oct 24.

LBLGC, Université d'Orléans, Orléans, France.

Podophyllotoxin, a lignan still extracted from the rhizomes of (Berberidaceae), is the starting molecule for the semisynthesis of widely used anticancer drugs such as etoposide. However, this source is threatened by the over-collection of . Plants belonging to the Linaceae and Cupressaceae families could be attractive alternative sources with species that contain the lignan podophyllotoxin or its precursors and derivatives. Wild flax species, such as , as well as some and species were investigated for their lignan content, and the antiproliferative capacity of their extracts was assayed on four tumor cell lines. Some of the lignans were detected by LC-HRMS for the first time in these extracts.In addition, lignans purified from these plants and compounds semisynthesized from commercially available podophyllotoxin were tested in terms of their antiproliferative activity. The genus was the most promising given its antiproliferative effects, which were also observed with extracts from and species.The antiproliferative effect of the plant extracts studied here appears to correlate well with the contents of the aryltetralin lignan podophyllotoxin and its glycoside as well as with deoxypodophyllotoxin and 6-methoxypodophyllotoxin. The strongest correlation between the lignan content of the extracts and the antiproliferative activity was observed for 6-methoxypodophyllotoxin. Regarding the possibility of producing large renewable amounts of 6-methoxypodophyllotoxin, this molecule could be of interest to produce new anticancer drugs and to bypass the resistance mechanisms against podophyllotoxin-derived drugs.
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http://dx.doi.org/10.1055/s-0042-118650DOI Listing
April 2017

Development and validation of an efficient ultrasound assisted extraction of phenolic compounds from flax (Linum usitatissimum L.) seeds.

Ultrason Sonochem 2015 Sep 28;26:176-185. Epub 2015 Feb 28.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), UPRES EA 1207, Université d'Orléans, Chartres, France. Electronic address:

Flaxseed accumulates in its seedcoat a macromolecular complex composed of lignan (secoisolariciresinol diglucoside, SDG), flavonol (herbacetin diglucoside, HDG) and hydroxycinnamic acids (p-couramic, caffeic and ferulic acid glucosides). Their antioxidant and/or cancer chemopreventive properties support their interest in human health and therefore, the demand for their extraction. In the present study, ultrasound-assisted extraction (UAE) of flaxseed phenolic compounds was investigated. Scanning Electron Microscopy imaging and histochemical analysis revealed the deep alteration of the seedcoat ultrastructure and the release of the mucilage following ultrasound treatment. Therefore, this method was found to be very efficient for the reduction of mucilage entrapment of flaxseed phenolics. The optimal conditions for UAE phenolic compounds extraction from flaxseeds were found to be: water as solvent supplemented with 0.2N of sodium hydroxide for alkaline hydrolysis of the SDG-HMG complex, an extraction time of 60 min at a temperature of 25°C and an ultrasound frequency of 30 kHz. Under these optimized and validated conditions, highest yields of SDG, HDG and hydroxycinnamic acid glucosides were detected in comparison to other published methods. Therefore, the procedure presented herein is a valuable method for efficient extraction and quantification of the main flaxseed phenolics. Moreover, this UAE is of particular interest within the context of green chemistry in terms of reducing energy consumption and valuation of flaxseed cakes as by-products resulting from the production of flax oil.
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http://dx.doi.org/10.1016/j.ultsonch.2015.02.008DOI Listing
September 2015

Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum hairy root cultures.

Phytochemistry 2015 Jul 16;115:70-8. Epub 2015 Feb 16.

Unité de Recherche Biologie des Plantes et Innovation (BIOPI-EA 3900), Université de Picardie Jules Verne, 33 rue Saint Leu, 80039 Amiens Cedex, France. Electronic address:

Due to their pronounced cytotoxic activity, a number of aryltetralin lignans (ATLs), such as podophyllotoxin (PTOX), are used as antitumor compounds. The production of such molecules from entire plants or plant cell-tissue-organ cultures is thus of interest to the pharmaceutical industry. Hairy root cultures constitute a good tool not only for phytochemical production but also for investigating plant secondary metabolism. This work reports on the growth and ATL biosynthesis in two hairy root cultures of Linum album Kotschy ex Boiss. and Linum flavum. The kinetics of accumulation of the intermediates of MPTOX biosynthesis and of their glucosylated forms are described over a 21-day period of growth. An accumulation of non-glucosylated forms of the ATLs during the exponential phase of the cultures is followed by an accumulation of the glucosylated forms during the stationary phase. Our results show a strong coordination of the biosynthetic paths derived from deoxypodophyllotoxin via deoxypodophyllotoxin 6-hydroxylase and deoxypodophyllotoxin 7-hydroxylase, and a coordinated glucosylation of podophyllotoxin, methoxypodophyllotoxin, and 5'-demethoxymethoxypodophyllotoxin. Furthermore, our results suggest an important role of β-peltatin-6-glucoside formation in the control of ATL accumulation in Linum hairy root cultures.
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http://dx.doi.org/10.1016/j.phytochem.2015.01.001DOI Listing
July 2015

RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: consequences on lignans and neolignans accumulation.

J Plant Physiol 2014 Sep 23;171(15):1372-7. Epub 2014 Jun 23.

Laboratoire LBLGC EA 1207, Antenne Scientifique Universitaire de Chartres Univ Orleans, 21 rue de Loigny la Bataille, 28000 Chartres, France.

RNAi technology was applied to down regulate LuPLR1 gene expression in flax (Linum usitatissimum L.) seeds. This gene encodes a pinoresinol lariciresinol reductase responsible for the synthesis of (+)-secoisolariciresinol diglucoside (SDG), the major lignan accumulated in the seed coat. If flax lignans biological properties and health benefits are well documented their roles in planta remain unclear. This loss of function strategy was developed to better understand the implication of the PLR1 enzyme in the lignan biosynthetic pathway and to provide new insights on the functions of these compounds. RNAi plants generated exhibited LuPLR1 gene silencing as demonstrated by quantitative RT-PCR experiments and the failed to accumulate SDG. The accumulation of pinoresinol the substrate of the PLR1 enzyme under its diglucosylated form (PDG) was increased in transgenic seeds but did not compensate the overall loss of SDG. The monolignol flux was also deviated through the synthesis of 8-5' linked neolignans dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) which were observed for the first time in flax seeds.
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http://dx.doi.org/10.1016/j.jplph.2014.06.005DOI Listing
September 2014

Microwave-assisted extraction of herbacetin diglucoside from flax (Linum usitatissimum L.) seed cakes and its quantification using an RP-HPLC-UV system.

Molecules 2014 Mar 10;19(3):3025-37. Epub 2014 Mar 10.

Laboratoire de Biologie des Ligneux et des Grandes Cultures UPRES EA 1207, Equipe Lignanes des Linacées, Université d'Orléans - Antenne Scientifique Universitaire de Chartres, 21 rue de Loigny la Bataille, 28000 Chartres, France.

Flax (Linum usitatissimum L.) seeds are widely used for oil extraction and the cold-pressed flaxseed (or linseed) cakes obtained during this process constitute a valuable by-product. The flavonol herbacetin diglucoside (HDG) has been previously reported as a constituent of the flaxseed lignan macromolecule linked through ester bonds to the linker molecule hydroxymethylglutaric acid. In this context, the development and validation of a new approach using microwave-assisted extraction (MAE) of HDG from flaxseed cakes followed by quantification with a reverse-phase HPLC system with UV detection was purposed. The experimental parameters affecting the HDG extraction yield, such as microwave power, extraction time and sodium hydroxide concentration, from the lignan macromolecule were optimized. A maximum HDG concentration of 5.76 mg/g DW in flaxseed cakes was measured following an irradiation time of 6 min, for a microwave power of 150 W using a direct extraction in 0.1 M NaOH in 70% (v/v) aqueous methanol. The optimized method was proven to be rapid and reliable in terms of precision, repeatability, stability and accuracy for the extraction of HDG. Comparison with a conventional extraction method demonstrated that MAE is more effective and less time-consuming.
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http://dx.doi.org/10.3390/molecules19033025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6270660PMC
March 2014

Role of protein farnesylation events in the ABA-mediated regulation of the Pinoresinol-Lariciresinol Reductase 1 (LuPLR1) gene expression and lignan biosynthesis in flax (Linum usitatissimum L.).

Plant Physiol Biochem 2013 Nov 15;72:96-111. Epub 2013 Jun 15.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), UPRES EA 1207, Antenne Scientifique Universitaire de Chartres (ASUC), Université d'Orléans, 21 rue de Loigny la Bataille, F28000 Chartres, France.

A Linum usitatissimum LuERA1 gene encoding a putative ortholog of the ERA1 (Enhanced Response to ABA 1) gene of Arabidopsis thaliana (encoding the beta subunit of a farnesyltransferase) was analyzed in silico and for its expression in flax. The gene and the protein sequences are highly similar to other sequences already characterized in plants and all the features of a farnesyltransferase were detected. Molecular modeling of LuERA1 protein confirmed its farnesyltransferase nature. LuERA1 is expressed in the vegetative organs and also in the outer seedcoat of the flaxseed, where it could modulate the previously observed regulation operated by ABA on lignan synthesis. This effect could be mediated by the regulation of the transcription of a key gene for lignan synthesis in flax, the LuPLR1 gene, encoding a pinoresinol lariciresinol reductase. The positive effect of manumycin A, a specific inhibitor of farnesyltransferase, on lignan biosynthesis in flax cell suspension systems supports the hypothesis of the involvement of such an enzyme in the negative regulation of ABA action. In Arabidopsis, ERA1 is able to negatively regulate the ABA effects and the mutant era1 has an enhanced sensitivity to ABA. When expressed in an Arabidopsis cell suspension (heterologous system) LuERA1 is able to reverse the effect of the era1 mutation. RNAi experiments in flax targeting the farnesyltransferase β-subunit encoded by the LuERA1 gene led to an increase LuPLR1 expression level associated with an increased content of lignan in transgenic calli. Altogether these results strongly suggest a role of the product of this LuERA1 gene in the ABA-mediated upregulation of lignan biosynthesis in flax cells through the activation of LuPLR1 promoter. This ABA signaling pathway involving ERA1 probably acts through the ABRE box found in the promoter sequence of LuPLR1, a key gene for lignan synthesis in flax, as demonstrated by LuPLR1 gene promoter-reporter experiments in flax cells using wild type and mutated promoter sequences.
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http://dx.doi.org/10.1016/j.plaphy.2013.06.001DOI Listing
November 2013

Flaxseed (Linum usitatissimum L.) extract as well as (+)-secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α-amylase activity.

Bioorg Med Chem Lett 2013 May 16;23(10):3007-12. Epub 2013 Mar 16.

Laboratoire de Biologie des Ligneux et des Grandes Cultures UPRES EA 1207, Equipe Lignanes des Linacées, Antenne Scientifique Universitaire de Chartres, 21 rue de Loigny la Bataille, F28000 Chartres, France.

Type 2 diabetes mellitus (T2DM) is one of the common global diseases. Flaxseed is by far the richest source of the dietary lignans (i.e., secoisolariciresinol diglucoside) which have been shown to delay the development of T2DM in animal models. Herein, we propose the first evidences for a mechanism of action involving the inhibition of the pancreatic α-amylase (EC 3.2.1.1) by flaxseed-derived lignans that could therefore constitute a promising nutraceutical for the prevention and the treatment of T2DM.
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http://dx.doi.org/10.1016/j.bmcl.2013.03.029DOI Listing
May 2013

Identification and characterization of cis-acting elements involved in the regulation of ABA- and/or GA-mediated LuPLR1 gene expression and lignan biosynthesis in flax (Linum usitatissimum L.) cell cultures.

J Plant Physiol 2013 Mar 27;170(5):516-22. Epub 2012 Dec 27.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), EA 1207, Antenne Scientifique Universitaire de Chartres (ASUC), Université d'Orléans, 21 rue de Loigny la Bataille, F28000, Chartres, France.

Pinoresinol lariciresinol reductase 1, encoded by the LuPLR1 gene in flax (Linum usitatissimum L.), is responsible for the biosynthesis of (+)-secoisolariciresinol, a cancer chemopreventive phytoestrogenic lignan accumulated in high amount in the hull of flaxseed. Our recent studies have demonstrated a key role of abscisic acid (ABA) in the regulation of LuPLR1 gene expression and thus of the (+)-secoisolariciresinol synthesis during the flax seedcoat development. It is well accepted that gibberellins (GA) and ABA play antagonistic roles in the regulation of numerous developmental processes; therefore it is of interest to clarify their respective effects on lignan biosynthesis. Herein, using flax cell suspension cultures, we demonstrate that LuPLR1 gene expression and (+)-secoisolariciresinol synthesis are up-regulated by ABA and down-regulated by GA. The LuPLR1 gene promoter analysis and mutation experiments allow us to identify and characterize two important cis-acting sequences (ABRE and MYB2) required for these regulations. These results imply that a cross-talk between ABA and GA signaling orchestrated by transcription factors is involved in the regulation of lignan biosynthesis. This is particularly evidenced in the case of the ABRE cis-regulatory sequence of LuPLR1 gene promoter that appears to be a common target sequence of GA and ABA signals.
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http://dx.doi.org/10.1016/j.jplph.2012.11.003DOI Listing
March 2013

Concentration kinetics of secoisolariciresinol diglucoside and its biosynthetic precursor coniferin in developing flaxseed.

Phytochem Anal 2013 Jan-Feb;24(1):41-6. Epub 2012 Jun 12.

Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, Beutenberg Campus, 07745 Jena, Germany.

Introduction: In the plant kingdom, flaxseed (Linum usitatissimum L.) is the richest source of secoisolariciresinol diglucoside (SDG), which is of great interest because of its potential health benefits for human beings. The information about the kinetics of SDG formation during flaxseed development is rare and incomplete.

Objective: In this study, a reversed-phase high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed to quantify SDG and coniferin, a key biosynthetic precursor of SDG in flaxseed.

Methodology: Seeds from different developmental stages, which were scaled by days after flowering (DAF), were harvested. After alkaline hydrolysis, the validated HPLC method was applied to determine SDG and coniferin concentrations of flaxseed from different developing stages.

Results: Coniferin was found in the entire capsule as soon as flowering started and became undetectable 20 DAF. SDG was detected 6 DAF, and the concentration increased until maturity. On the other hand, the SDG amount in a single flaxseed approached the maximum around 25 DAF, before desiccation started. Concentration increase between 25 DAF and 35 DAF can be attributed to corresponding seed weight decrease.

Conclusion: The biosynthesis of coniferin is not synchronous with that of SDG. Hence, the concentrations of SDG and coniferin change during flaxseed development.
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http://dx.doi.org/10.1002/pca.2377DOI Listing
June 2013

Isolation of nuclear proteins from flax (Linum usitatissimum L.) seed coats for gene expression regulation studies.

BMC Res Notes 2012 Jan 9;5:15. Epub 2012 Jan 9.

Laboratoire de Biologie des Ligneux et des Grandes Cultures UPRES EA 1207, Université d'Orléans, Equipe Lignanes des Linacées, Antenne Scientifique Universitaire de Chartres, 21 rue de Loigny la Bataille F-28000 Chartres, France.

Background: While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds.

Findings: In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments.

Conclusions: Routinely, around 250 μg of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies.
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http://dx.doi.org/10.1186/1756-0500-5-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285032PMC
January 2012

Abscisic acid regulates pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol accumulation in developing flax (Linum usitatissimum L.) seeds.

Planta 2012 Jan 12;235(1):85-98. Epub 2011 Aug 12.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), UPRES EA 1207, Antenne Scientifique Universitaire de Chartres (ASUC), Université d'Orléans, 21 rue de Loigny la Bataille, 28000, Chartres, France.

Secoisolariciresinol diglucoside (SDG), the main phytoestrogenic lignan of Linum usitatissimum, is accumulated in the seed coat of flax during its development and pinoresinol-lariciresinol reductase (PLR) is a key enzyme in flax for its synthesis. The promoter of LuPLR1, a flax gene encoding a pinoresinol lariciresinol reductase, contains putative regulatory boxes related to transcription activation by abscisic acid (ABA). Gel mobility shift experiments evidenced an interaction of nuclear proteins extracted from immature flax seed coat with a putative cis-acting element involved in ABA response. As ABA regulates a number of physiological events during seed development and maturation we have investigated its involvement in the regulation of this lignan synthesis by different means. ABA and SDG accumulation time courses in the seed as well as LuPLR1 expression were first determined in natural conditions. These results showed that ABA timing and localization of accumulation in the flax seed coat could be correlated with the LuPLR1 gene expression and SDG biosynthesis. Experimental modulations of ABA levels were performed by exogenous application of ABA or fluridone, an inhibitor of ABA synthesis. When submitted to exogenous ABA, immature seeds synthesized 3-times more SDG, whereas synthesis of SDG was reduced in immature seeds treated with fluridone. Similarly, the expression of LuPLR1 gene in the seed coat was up-regulated by exogenous ABA and down-regulated when fluridone was applied. These results demonstrate that SDG biosynthesis in the flax seed coat is positively controlled by ABA through the transcriptional regulation of LuPLR1 gene.
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http://dx.doi.org/10.1007/s00425-011-1492-yDOI Listing
January 2012

Podophyllotoxin and deoxypodophyllotoxin in Juniperus bermudiana and 12 other Juniperus species: optimization of extraction, method validation, and quantification.

J Agric Food Chem 2011 Aug 15;59(15):8101-7. Epub 2011 Jul 15.

Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), UPRES EA 1207, Antenne Scientifique Universitaire de Chartres (ASUC), Université d'Orléans, Chartres, France.

The lignans podophyllotoxin and deoxypodophyllotoxin are secondary metabolites with potent pharmaceutical applications in cancer therapy. However, the supply of podophyllotoxin from its current natural source, Podophyllum hexandrum, is becoming increasingly problematic, and alternative sources are therefore urgently needed. So far, podophyllotoxin and deoxypodophyllotoxin have been found in some Juniperus species, although at low levels in most cases. Moreover, extraction protocols deserve optimization. This study aimed at developing and validating an efficient extraction protocol of podophyllotoxin and deoxypodophyllotoxin from Juniperus species and applying it to 13 Juniperus species, among which some had never been previously analyzed. Juniperus bermudiana was used for the development and validation of an extraction protocol for podophyllotoxin and deoxypodophyllotoxin allowing extraction yields of up to 22.6 mg/g DW of podophyllotoxin and 4.4 mg/g DW deoxypodophyllotoxin, the highest values found in leaf extract of Juniperus. The optimized extraction protocol and HPLC separation from DAD or MS detections were established and validated to investigate podophyllotoxin and deoxypodophyllotoxin contents in aerial parts of 12 other Juniperus species. This allowed either higher yields to be obtained in some species reported to contain these two compounds or the occurrence of these compounds in some other species to be reported for the first time. This efficient protocol allows effective extraction of podophyllotoxin and deoxypodophyllotoxin from aerial parts of Juniperus species, which could therefore constitute interesting alternative sources of these valuable metabolites.
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http://dx.doi.org/10.1021/jf201410pDOI Listing
August 2011