Jean-Bernard Pouvreau

Structure-Activity Relationship Studies of Strigolactone-Related Molecules for Branching Inhibition in Garden Pea: Molecule Design for Shoot Branching

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.

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway.

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.

Antioxidant and Free Radical Scavenging Properties of Marennine, a Blue-Green Polyphenolic Pigment from the Diatom Haslea ostrearia (Gaillon/Bory) Simonsen Responsible for the Natural Greening of Cultured Oysters

Among microalgae, the marine diatom Haslea ostrearia has the distinctive feature of synthesizing and releasing, into the surrounding environment, a blue-green polyphenolic pigment called marennine. The oyster-breeding industry commonly makes use of this natural phenomenon for the greening of oysters grown in the ponds of the French Atlantic coast. This article reports the in vitro antioxidant properties of pure marennine. Two kinds of evaluation systems were adopted to test the antioxidative activity of marennine: antioxidant capacity assays (beta-carotene and thymidine protection assays and iron reducing power assay) and free radical scavenging assays (DPPH*, O2*-, and HO*). In almost all cases, marennine exhibited significantly higher antioxidative and free radical scavenging activities than natural and synthetic antioxidants commonly used in food, as shown by comparing median effective concentration (EC 50) values, for each test independently. This medium molecular weight polyphenol (around 10 kDa) from microalgae is thus a potentially useful natural antioxidant. Because of its blue-coloring property and water solubility, it could also be used as a natural food-coloring additive.

Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.

Biological activities of purified marennine, the blue pigment responsible for the greening of oysters

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.

Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel

Highlight This study demonstrates for the first time that DNA demethylation, an epigenetic mechanism, can control parasitic plant seed response to the strigolactones, a new class of plant hormone.

Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.

Invertases involved in the development of the parasitic plant Phelipanche ramosa: characterization of the dominant soluble acid isoform, PrSAI1

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.

Role of the Sucrose Synthase Encoding PrSus1 Gene in the Development of the Parasitic Plant Phelipanche ramosa L. (Pomel)

Phelipanche ramosa L. (Pomel) is a major root-parasitic weed attacking many important crops. Success in controlling this parasite is rare and a better understanding of its unique biology is needed to develop new specific control strategies. In the present study, quantitative polymerase chain reaction experiments showed that sucrose synthase encoding PrSus1 transcripts accumulate at their highest level once the parasite is connected to the host (tomato) vascular system, mainly in the parasite tubercles, which bear numerous adventitious roots. In situ hybridization experiments revealed strong PrSus1 expression in both shoot and root apices, especially in shoot apical meristems and in the vascular tissues of scale leaves and stems, and in the apical meristems and developing xylem in roots. In addition, immunolocalization experiments showed that a sucrose synthase protein co-localized with cell-wall thickening in xylem elements. These findings highlight the role of PrSus1 in the utilization of host-derived sucrose in meristematic areas and in cellulose biosynthesis in differentiating vascular elements. We also demonstrate that PrSus1 is downregulated in response to 2,3,5-triiodobenzoic acid-induced inhibition of polar auxin transport in the host stem, suggesting that PrSus1 activity in xylem maturation is controlled by host-derived auxin.

A high-throughput seed germination assay for root parasitic plants

BackgroundSome 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.ResultsWe 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.ConclusionThe 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.