CNRS Nantes Laboratory of Vegetal Biology and Pathology

Nantes, France

CNRS Nantes Laboratory of Vegetal Biology and Pathology

Nantes, France
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Normand J.,French National Institute for Agricultural Research | Normand J.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Bonnin E.,French National Institute for Agricultural Research | Delavault P.,CNRS Nantes Laboratory of Vegetal Biology and Pathology
Applied Microbiology and Biotechnology | Year: 2012

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5' and 3' rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme. © Springer-Verlag 2012.

Boyer F.,CNRS Natural Product Chemistry Institute | Germain A.S.,French National Institute for Agricultural Research | Pillot J.-P.,French National Institute for Agricultural Research | Pouvreau J.-B.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | And 7 more authors.
Plant Physiology | Year: 2012

Initially known for their role in the rhizosphere in stimulating the seed germination of parasitic weeds such as the Striga and Orobanche species, and later as host recognition signals for arbuscular mycorrhizal fungi, strigolactones (SLs) were recently rediscovered as a new class of plant hormones involved in the control of shoot branching in plants. Herein, we report the synthesis of new SL analogs and, to our knowledge, the first study of SL structure-activity relationships for their hormonal activity in garden pea (Pisum sativum). Comparisons with their action for the germination of broomrape (Phelipanche ramosa) are also presented. The pea rms1 SL-deficient mutant was used in a SL bioassay based on axillary bud length after direct SL application on the bud. This assay was compared with an assay where SLs were fed via the roots using hydroponics and with a molecular assay in which transcript levels of BRANCHED1, the pea homolog of the maize TEOSINTE BRANCHED1 gene were quantified in axillary buds only 6 h after application of SLs. We have demonstrated that the presence of a Michael acceptor and a methylbutenolide or dimethylbutenolide motif in the same molecule is essential. It was established that the more active analog 23 with a dimethylbutenolide as the D-ring could be used to control the plant architecture without strongly favoring the germination of P. ramosa seeds. Bold numerals refer to numbers of compounds. © 2012 American Society of Plant Biologists. All Rights Reserved.

Gaudin Z.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Cerveau D.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Marnet N.,French National Institute for Agricultural Research | Bouchereau A.,CNRS Institute of Genetics, Environment and Plant Protection | And 3 more authors.
Analytical Chemistry | Year: 2014

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of 15N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing 15N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of 15N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as 15N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to 15N2-glutamine. 15N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa. © 2013 American Chemical Society.

Boyer F.-D.,French National Institute for Agricultural Research | Boyer F.-D.,CNRS Natural Product Chemistry Institute | De Saint Germain A.,French National Institute for Agricultural Research | Pouvreau J.-B.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | And 15 more authors.
Molecular Plant | Year: 2014

Strigolactones (SLs) are known not only as plant hormones, but also as rhizosphere signals for establishing symbiotic and parasitic interactions. The design of new specific SL analogs is a challenging goal in understanding the basic plant biology and is also useful to control plant architectures without favoring the development of parasitic plants. Two different molecules (23 (3′-methyl-GR24), 31 (thia-3′-methyl-debranone-like molecule)) already described, and a new one (AR36), for which the synthesis is presented, are biologically compared with the well-known GR24 and the recently identified CISA-1. These different structures emphasize the wide range of parts attached to the D-ring for the bioactivity as a plant hormone. These new compounds possess a common dimethylbutenolide motif but their structure varies in the ABC part of the molecules: 23 has the same ABC part as GR24, while 31 and AR36 carry, respectively, an aromatic ring and an acyclic carbon chain. Detailed information is given for the bioactivity of such derivatives in strigolactone synthesis or in perception mutant plants (pea rms1 and rms4, Arabidopsis max2 and, max4) for different hormonal functions along with their action in the rhizosphere on arbuscular mycorrhizal hyphal growth and parasitic weed germination. © 2013 The Author.

Gastineau R.,University of Maine, France | Pouvreau J.-B.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Hellio C.,University of Portsmouth | Morancais M.,University of Nantes | And 4 more authors.
Journal of Agricultural and Food Chemistry | Year: 2012

Marennine, the blue pigment produced by the diatom Haslea ostrearia, exists in two different forms, the intra- and extracellular forms. We investigated the antibacterial, antiviral, and antiproliferative properties of both of these forms. Both forms of marennine inhibited the development of marine bacteria, in particular the pathogenic organism Vibrio aesturianus, at concentrations as low as 1 μg/mL, but they did not display any effect on a wide range of pathogenic bacteria that are relevant for food safety. Both forms of the pigment produced by H. ostrearia also exhibited antiviral activity against the HSV1 herpes virus, with intra- and extracellular marennine having EC 50 values of 24.0 and 27.0 μg/mL, respectively. These values are 2 orders of magnitude higher than the value for the reference drug, Zovirax. Moreover, both forms of marennine were effective in slowing or inhibiting the proliferation of cancer cells. This study confirms the potential of marennine as a biologically active organic molecule, which could have a protective effect on bivalves, which filter seawater and fix the pigment on their gills. Moreover, marennine could be used in food engineering and chemistry as a natural blue pigment. However, despite that it is eaten and possibly assimilated by green oyster consumers, it also deserves in depth evaluation before being considered for use as a nutraceutical. © 2012 American Chemical Society.

Dongo A.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Leflon M.,Center Technique Interprofessionnel des Oleagineux Metropolitains | Simier P.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Delavault P.,CNRS Nantes Laboratory of Vegetal Biology and Pathology
Weed Research | Year: 2012

The objective of this study was to develop a simple and robust quantitative PCR method for the detection of seeds from two parasitic plants, Phelipanche ramosa and Orobanche cumana, as well as their closely related species, in seed harvests of oilseed rape and sunflower. The method was based on the design of primers/probe sets specific to both parasitic plants and targeting internal transcribed spacer sequences for quantitative real-time PCR (TaqMan). Together with the proposed DNA extraction protocol, this diagnostic method allows rapid, high-throughput and accurate assessment of contamination with broomrape seeds, without the requirement of tedious purification steps and identification under a binocular microscope. The TaqMan assay is highly specific, because it did not detect any possible plant contaminants that are likely to be present in harvested crop seeds. The results of this assay can be expressed in terms of the number of parasite seeds per kilogram of crop seeds, a metric that could be utilised to help decisions regarding crop seed lot utilisation and commercialisation. © 2011 The Authors. Weed Research © 2011 European Weed Research Society.

Draie R.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Draie R.,Tichrine University | Peron T.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Peron T.,Tichrine University | And 6 more authors.
Molecular Plant Pathology | Year: 2011

Phelipanche ramosa L. parasitizes major crops, acting as a competitive sink for host photoassimilates, especially sucrose. An understanding of the mechanisms of sucrose utilization in parasites is an important step in the development of new control methods. Therefore, in this study, we characterized the invertase gene family in P. ramosa and analysed its involvement in plant development. Invertase-encoded cDNAs were isolated using degenerate primers corresponding to highly conserved regions of invertases. In addition to enzyme assays, gene expression was analysed using real-time quantitative reverse transcriptase-polymerase chain reaction during overall plant development. The dominant isoform was purified and sequenced using electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS). Five invertase-encoded cDNAs were thus characterized, including PrSai1 which encodes a soluble acid invertase (SAI). Of the five invertases, PrSai1 transcripts and SAI activity were dominant in growing organs. The most active invertase corresponded to the PrSai1 gene product. The purified PrSAI1 displayed low pI and optimal pH values, specificity for β-fructofuranosides and inhibition by metallic ions and competitive inhibition by fructose. PrSAI1 is a typical vacuolar SAI that is actively involved in growth following both germination and attachment to host roots. In addition, germinated seeds displayed enhanced cell wall invertase activity (PrCWI) in comparison with preconditioned seeds, suggesting the contribution of this activity in the sink strength of infected roots during the subsequent step of root penetration. Our results show that PrSAI1 and, possibly, PrCWI constitute good targets for the development of new transgenic resistance in host plants using proteinaceous inhibitors or silencing strategies. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd. No Claim tO Original US Government Works.

Pouvreau J.-B.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Gaudin Z.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Auger B.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | Lechat M.-M.,CNRS Nantes Laboratory of Vegetal Biology and Pathology | And 3 more authors.
Plant Methods | Year: 2013

Background: Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope.Results: We developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann's protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose-response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves.Conclusion: The developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination. © 2013 Pouvreau et al.; licensee BioMed Central Ltd.

PubMed | PHENOTIC, University of Angers, CNRS Research Institute on Horticulture and Seeds and CNRS Nantes Laboratory of Vegetal Biology and Pathology
Type: | Journal: Plant methods | Year: 2015

Image analysis is increasingly used in plant phenotyping. Among the various imaging techniques that can be used in plant phenotyping, chlorophyll fluorescence imaging allows imaging of the impact of biotic or abiotic stresses on leaves. Numerous chlorophyll fluorescence parameters may be measured or calculated, but only a few can produce a contrast in a given condition. Therefore, automated procedures that help screening chlorophyll fluorescence image datasets are needed, especially in the perspective of high-throughput plant phenotyping.We developed an automatic procedure aiming at facilitating the identification of chlorophyll fluorescence parameters impacted on leaves by a stress. First, for each chlorophyll fluorescence parameter, the procedure provides an overview of the data by automatically creating contact sheets of images and/or histograms. Such contact sheets enable a fast comparison of the impact on leaves of various treatments, or of the contrast dynamics during the experiments. Second, based on the global intensity of each chlorophyll fluorescence parameter, the procedure automatically produces radial plots and box plots allowing the user to identify chlorophyll fluorescence parameters that discriminate between treatments. Moreover, basic statistical analysis is automatically generated. Third, for each chlorophyll fluorescence parameter the procedure automatically performs a clustering analysis based on the histograms. This analysis clusters images of plants according to their health status. We applied this procedure to monitor the impact of the inoculation of the root parasitic plant Phelipanche ramosa on Arabidopsis thaliana ecotypes Col-0 and Ler.Using this automatic procedure, we identified eight chlorophyll fluorescence parameters discriminating between the two ecotypes of A. thaliana, and five impacted by the infection of Arabidopsis thaliana by P. ramosa. More generally, this procedure may help to identify chlorophyll fluorescence parameters impacted by various types of stresses. We implemented this procedure at freely accessible to users of the plant phenotyping community.

PubMed | CNRS Nantes Laboratory of Vegetal Biology and Pathology and Agrocampus Ouest
Type: | Journal: Frontiers in plant science | Year: 2017

The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant

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