Publications by authors named "Frédéric Lamblin"

20 Publications

  • Page 1 of 1

Salivary proteins of Phloeomyzus passerinii, a plant-manipulating aphid, and their impact on early gene responses of susceptible and resistant poplar genotypes.

Plant Sci 2020 May 12;294:110468. Epub 2020 Mar 12.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, INRA, Université d'Orléans, 45067, Orléans, France. Electronic address:

Successful plant colonization by parasites requires the circumvention of host defenses, and sometimes a reprogramming of host metabolism, mediated by effector molecules delivered into the host. Using transcriptomic and enzymatic approaches, we characterized salivary glands and saliva of Phloeomyzus passerinii, an aphid exhibiting an atypical feeding strategy. Plant responses to salivary extracts of P. passerinii and Myzus persicae were assessed with poplar protoplasts of a susceptible and a resistant genotype, and in a heterologous Arabidopsis system. We predict that P. passerinii secretes a highly peculiar saliva containing effectors potentially interfering with host defenses, biotic stress signaling and plant metabolism, notably phosphatidylinositol phosphate kinases which seemed specific to P. passerinii. Gene expression profiles indicated that salivary extracts of M. persicae markedly affected host defenses and biotic stress signaling, while salivary extracts of P. passerinii induced only weak responses. The effector-triggered susceptibility was characterized by downregulations of genes involved in cytokinin signaling and auxin homeostasis. This suggests that P. passerinii induces an intracellular accumulation of auxin in susceptible host genotypes, which is supported by histochemical assays in Arabidopsis. This might in turn affect biotic stress signaling and contribute to host tissue manipulation by the aphid.
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http://dx.doi.org/10.1016/j.plantsci.2020.110468DOI Listing
May 2020

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

Plants (Basel) 2019 Dec 11;8(12). Epub 2019 Dec 11.

LBLGC, University of Orléans, EA1207, INRA, USC1328, rue de Chartres, CEDEX 2, 45067 Orléans, France.

We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, , , , , . These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, , was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors / and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in .
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http://dx.doi.org/10.3390/plants8120591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963366PMC
December 2019

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

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

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

[Interest of lignans in prevention and treatment of cancers].

Med Sci (Paris) 2008 May;24(5):511-9

Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, Centre Universitaire de Chartres, 21, rue de Loigny la Bataille, 28000 Chartres, France.

Lignans are diphenolic compounds widely distributed in the plant kingdom. They are mainly localised in lignified tissues, seeds and roots. These molecules are involved in plant defence mechanisms, but are also interesting for human health. Flax lignans belonging to the phytoestrogens are metabolised after ingestion into enterolignans that may offer a protection against the onset and development of hormono-dependant cancers. In vitro studies based on mammalian cellular models tend to confirm their beneficial effects observed during epidemiological studies and give us insights about their mechanisms of action. The most studied lignan, podophyllotoxin, and its semi-synthetic derivatives (etoposide, teniposide, etoposide phosphate), are particularly interesting at a curative level due to their cytotoxic properties. These semi-synthetic derivatives are used in chemotherapy of lung cancer for example. However, the extensive use of these anticancer drugs will lead to the problem of podophyllotoxin supply. This molecule is currently extracted from the rhizomes and roots of an Indian species Podophyllum hexandrum which has subsequently become endangered. Strategies are investigated to obtain economically viable alternative sources of Podophyllotoxin from plants and in vitro cultures of several species. Among them, north american Podophyllum peltatum, Linum wild species, Hyptis, Anthriscus, Juniperus or Dysosma species which accumulate Podophyllotoxin or closely related derivatives, are good candidates. double dagger.
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http://dx.doi.org/10.1051/medsci/2008245511DOI Listing
May 2008

Molecular characterization of cell death induced by a compatible interaction between Fusarium oxysporum f. sp. linii and flax (Linum usitatissimum) cells.

Plant Physiol Biochem 2008 May-Jun;46(5-6):590-600. Epub 2008 Mar 4.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, Antenne Scientifique Universitaire de Chartres, 21 rue de Loigny la Bataille, 28000 Chartres, France.

The cellular and molecular events associated with cell death during compatible interaction between Fusarium oxysporum sp. linii and a susceptible flax (Linum usitatissimum) cell suspension are reported here. In order to determine the physiological and molecular sequence of cell death of inoculated cells, reactive oxygen species (ROS) production, mitochondrial potential, lipoxygenase, DNase, protease and caspase-3-like activities, lipid peroxidation and secondary metabolite production were monitored. We also used microscopy, in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation assay. Cell death was associated with specific morphological and biochemical changes that are generally noticed in hypersensitive (incompatible) reaction. An oxidative burst as well as a loss of mitochondrial potential of inoculated cells, an activation of lipoxygenase and lipid peroxidation were noted. Enzyme-mediated nuclear DNA degradation was detectable but oligonucleosomal fragmentation was not observed. Caspase-3-like activity was dramatically increased in inoculated cells. Phenylpropanoid metabolism was also affected as demonstrated by activation of PAL and PCBER gene expressions and reduced soluble lignan and neolignan contents. These results obtained in flax suggest that compatible interaction triggers a cell death sequence sharing a number of common features with the hypersensitive response observed in incompatible interaction and in animal apoptosis.
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http://dx.doi.org/10.1016/j.plaphy.2008.02.004DOI Listing
September 2008

Coniferin dimerisation in lignan biosynthesis in flax cells.

Phytochemistry 2007 Nov-Dec;68(22-24):2744-52. Epub 2007 Nov 7.

Laboratoire de Phytotechnologie EA 3900, Faculté de Pharmacie, Université de Picardie Jules Verne, 1 Rue des Louvels, 80037 Amiens, France.

[(13)C(2)]-Coniferin was provided to a flax (Linum usitatissimum L.) cell suspension to monitor subsequent dimerisation by MS and NMR. The label was mainly incorporated into a 8-8'-linked lignan, lariciresinol diglucoside, a 8-5'-linked neolignan, dehydrodiconiferyl alcohol glucoside and a diastereoisomeric mixture of a 8-O-4'-linked neolignan, guaiacylglycerol-beta-coniferyl alcohol ether glucoside. This latter compound is reported for the first time in flax. The strong and transient increase in these compounds in fed cells was concomitant with the observed peak in coniferin content. These results suggest (i) a rapid metabolisation of coniferin into lignans and neolignans and indicate the capacity of flax cells to operate different types of couplings, and (ii) a continuous synthesis and subsequent metabolisation of coniferin-derived dimers all over the culture period.
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http://dx.doi.org/10.1016/j.phytochem.2007.09.016DOI Listing
February 2008

Microwave-assisted extraction of the main phenolic compounds in flaxseed.

Phytochem Anal 2007 Jul-Aug;18(4):275-82

Laboratoire de Phytotechnologie EA 3900, Faculté de Pharmacie, Université de Picardie Jules Verne, 1 rue des Louvels, 80037 Amiens, France.

A microwave-assisted extraction (MAE) method has been applied for the first time to the extraction of the main lignan, secoisolariciresinol diglucoside (SDG), and the two most concentrated hydroxycinnamic acid glucosides in flaxseed. The effects of microwave power, extraction time and alkaline treatment were investigated. It was shown that a 3 min MAE resulted in an SDG content of 16.1+/-0.4 mg/g, a p-coumaric acid glucoside content of 3.7+/-0.2 mg/g and a ferulic acid glucoside content of 4.1+/-0.2 mg/g. These values were compared with those obtained using conventional extraction methods and the results demonstrated that MAE was more effective in terms of both yield and time consumption.
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http://dx.doi.org/10.1002/pca.973DOI Listing
September 2007

The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum).

Plant Cell Rep 2007 Jun 5;26(6):765-72. Epub 2007 Jan 5.

Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, Antenne Scientifique Universitaire de Chartres, Chartres, France.

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L(-1) sucrose and 10 g L(-1) mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.
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http://dx.doi.org/10.1007/s00299-006-0280-9DOI Listing
June 2007

Overexpression of a heterologous sam gene encoding S-adenosylmethionine synthetase in flax (Linum usitatissimum) cells: Consequences on methylation of lignin precursors and pectins.

Physiol Plant 2001 Jun;112(2):223-232

Laboratoire de Biotechnologie et Physiologie Végétales, Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, F-80039 Amiens cedex, France Equipe Parois et Polymères Pariétaux, SCUEOR ESA CNRS 6037, F-76821 Mt Saint Aignan cedex, France Laboratoire de Physiologie des Parois Végétales, UPRES 2702, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq cedex, France Equipe Biochimie des Macromolécules Végétales, UPBP, INRA, BP 286, F-51686 Reims cedex, France Institut Supérieur Agricole de Beauvais, BP 30313, F-60026 Beauvais cedex, France.

The Arabidopsis thaliana sam1 gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) was transferred to flax (Linum usitatissimum) cells via Agrobacterium tumefaciens. This enzyme catalyses the conversion of methionine to S-adenosylmethionine (SAM), the major methyl group donor in living cells. The aim of this work was to study the consequences of an increased SAM-synthetase (SAM-S) activity in transgenic cell lines on both the production of mono- and dimethoxylated lignin monomers and the degree of methylesterification of pectins. Hypocotyls were cocultivated with Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the pO35SSAM binary vector carrying the sam1 gene under the control of the 35S promoter and the nptII gene for selection of putative transformed cells. Most of the transgenic cell lines exhibited a significant (up to 3.2-fold) increase in SAM-S activity compared to the controls. The results showed that for the cell lines analysed this transformation had no effect on caffeic acid O-methyltransferase (COMT, EC 2.1.1.68) in vitro activity, degree of methoxylation of lignin precursors or lignin deposition, pectin methyltransferase (PMT, EC 2.1.1) in vitro activity, but led to an increase of pectin methylesterification in friable and fast-growing transgenic cell lines.
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http://dx.doi.org/10.1034/j.1399-3054.2001.1120211.xDOI Listing
June 2001