Nathalie Marnet

A Detailed Survey of Seed Coat Flavonoids in Developing Seeds of Brassica napus L.

Proanthocyanidins (PAs) are seed coat flavonoids that impair the digestibility of Brassica napus meal. Development of low-PA lines is associated with a high-quality meal and with increased contents in oil and proteins, but requires better knowledge of seed flavonoids. Flavonoids in Brassica mature seed are mostly insoluble so that very few qualitative and quantitative data are available yet. In the present study, the profiling of seed coat flavonoids was established in eight black-seeded B. napus genotypes, during seed development when soluble flavonoids were present and predominated over the insoluble forms. Thirteen different flavonoids including (-)-epicatechin, five procyanidins (PCs which are PAs composed of epicatechin oligomers only) and seven flavonols (quercetin-3-O-glucoside, quercetin-dihexoside, isorhamnetin-3-O-glucoside, isorhamnetin-hexoside-sulfate, isorhamnetin-dihexoside, isorhamnetin-sinapoyl-trihexoside and kaempferol-sinapoyl-trihexoside) were identified and quantified using liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS(n)). These flavonol derivatives were characterized for the first time in the seed coat of B. napus, and isorhamnetin-hexoside-sulfate and isorhamnetin-sinapoyl-trihexoside were newly identified in Brassica spp. High amounts of PCs accumulated in the seed coat, with solvent-soluble polymers of (-)-epicatechin reaching up to 10% of the seed coat weight during seed maturation. In addition, variability for both PC and flavonol contents was observed within the panel of eight black-seeded genotypes. Our results provide new insights into breeding for low-PC B. napus genotypes.

Both the Jasmonic Acid and the Salicylic Acid Pathways Contribute to Resistance to the Biotrophic Clubroot Agent Plasmodiophora brassicae in Arabidopsis

The role of salicylic acid (SA) and jasmonic acid (JA) signaling in resistance to root pathogens has been poorly documented. We assessed the contribution of SA and JA to basal and partial resistance of Arabidopsis to the biotrophic clubroot agent Plasmodiophora brassicae. SA and JA levels as well as the expression of the SA-responsive genes PR2 and PR5 and the JA-responsive genes ARGAH2 and THI2.1 were monitored in infected roots of the accessions Col-0 (susceptible) and Bur-0 (partially resistant). SA signaling was activated in Bur-0 but not in Col-0. The JA pathway was weakly activated in Bur-0 but was strongly induced in Col-0. The contribution of both pathways to clubroot resistance was then assessed using exogenous phytohormone application and mutants affected in SA or JA signaling. Exogenous SA treatment decreased clubroot symptoms in the two Arabidopsis accessions, whereas JA treatment reduced clubroot symptoms only in Col-0. The cpr5-2 mutant, in which SA responses are constitutively induced, was more resistant to clubroot than the corresponding wild type, and the JA signaling-deficient mutant jar1 was more susceptible. Finally, we showed that the JA-mediated induction of NATA1 drove N(δ)-acetylornithine biosynthesis in infected Col-0 roots. The 35S::NATA1 and nata1 lines displayed reduced or enhanced clubroot symptoms, respectively, thus suggesting that in Col-0 this pathway was involved in the JA-mediated basal clubroot resistance. Overall, our data support the idea that, depending on the Arabidopsis accession, both SA and JA signaling can play a role in partial inhibition of clubroot development in compatible interactions with P. brassicae.

Nicotiflorin, rutin and chlorogenic acid: phenylpropanoids involved differently in quantitative resistance of potato tubers to biotrophic and necrotrophic pathogens

Physiological and molecular mechanisms underlying quantitative resistance of plants to pathogens are still poorly understood, but could depend upon differences in the intensity or timing of general defense responses. This may be the case for the biosynthesis of phenolics which are known to increase after elicitation by pathogens. We thus tested the hypothesis that differences in quantitative resistance were related to differential induction of phenolics by pathogen-derived elicitors. Five potato cultivars (Solanum tuberosum, L.) spanning a range of quantitative resistance were treated with a concentrated culture filtrate (CCF) of Phytophthora infestans or purified lipopolysaccharides (LPS) from Pectobacterium atrosepticum. The kinetic of phenolics accumulation was followed and a set of typical phenolics was identified: chlorogenic acid, phenolamides and flavonols including rutin (quercetin-3-O-rutinoside) and nicotiflorin (kaempferol-3-O-rutinoside). Our results showed that CCF but not LPS induced differential accumulation of major phenolics among cultivars. Total phenolics were related with resistance to P. atrosepticum but not to P. infestans. However, nicotiflorin was inversely related with resistance to both pathogens. Rutin, but not nicotiflorin, inhibited pathogen growth in vitro at physiological concentrations. These data therefore suggest that (i) several phenolics are candidate markers for quantitative resistance in potato, (ii) some of these are pathogen specific although they are produced by a general defense pathway, (iii) resistance marker molecules do not necessarily have antimicrobial activity, and (iv) the final content of these target molecules-either constitutive or induced-is a better predictor of resistance than their inducibility by pathogen elicitors.

How oilseed rape (Brassica napus) genotype influences pollen beetle (Meligethes aeneus) oviposition

Oviposition of phytophagous insects is determined either by adaptive behaviours allowing evaluation and response to host plant quality and/or by nutritional constraints occurring during oogenesis. Besides differences found among host plant species, plant intraspecific diversity can also affect insect oviposition. However, to date few studies have extensively investigated the factors accounting for the effect of this intraspecific variation. We addressed this question using oilseed rape (Brassica napus) and the pollen beetle (Meligethes aeneus), a phytophagous insect that uses the same plants and plant organs both for feeding and laying eggs. Our objectives were to test for a genotypic effect of oilseed rape on pollen beetle oviposition and identify the origin of the possible intergenotypic differences. We tested three hypotheses: oviposition is directly linked to (1) the amount of food eaten; (2) the nutritional quality of the food eaten; (3) a preference of females for certain plant genotypes. Results showed intergenotypic differences in both the number and the size of eggs laid. The factor that best accounted for most of these differences was the amount of food eaten. Nutritional quality of the pollen was of minor importance and females exhibited no preference among genotypes. These results reveal the importance of adult feeding on subsequent oviposition in phytophagous insects, an often neglected factor which partly determines the amount of energy available for oogenesis. Taking into account this factor may be of crucial importance in studies conducted on synovogenic insect species feeding on the same plant on which they lay eggs.

Effect of alcoholic fermentation in the content of phenolic compounds in cider processing

ABSTRACT The objective of this work was to study the effect of alcoholic fermentation on the content of phenol compounds of five cider apple varieties. The initial content in the apple juice samples, as determined by HPLC, varied from 188.4 to 2776.17 m mg.L -1 . In three of them (DC, PJ, GU), the total phenol compounds remained unaffected by fermentation. However, in two (DM, KE), the final values were lower (55 and 313 mg.L -1 ). In these apple cider, the values of caffeic acid increased from 6.6 mg.L -1 to 41.8 mg.L -1 . The catechin content increased during the process, approximately 13 mg.L -1 independent of the variety. The other phenols class did not present any modifications due to the alcoholic fermentation, maintaining the phenolic compounds of original clarified apple juice in the cider. Keywords : cider, fermentation alcoholic, polyphenols * Author for correspondence INTRODUCTION Polyphenols play important roles in the cider quality as they are related to the color, bitterness and astringency, whose balance defines the overall mouthfeel of the beveradge (Guyot et al., 1998; Alonso-Salces et al., 2001; Lea and Drilleau, 2003; Alonso-Salces, et al., 2004). They may be involved in the fermentative processes, providing the cider aroma, and as inhibitors of the microorganims development, controlling the fermentation rates and avoiding some faults that can develop in cider from the action of lactic acid bacteria such as acidification, mannitol taint, “framboise”, bitterness (Alonso-Salces et al., 2004). Furthermore, the phenolic compounds participate in the formation of sediments during the cider storage, due to their colloidal interaction with the proteins through the van der Waals forces (Siebert et al., 1996; Kawamoto and Nakatsubo, 1997). They can also inhibit the pre-fermentative clarification enzymes (Cowan, 1999). The polyphenols are receiving increasing attention due to their natural antioxidation and health protective properties (Vanzani et al., 2005; Tsao et al., 2005). The polyphenolic composition of a cider depends on the mixture of the apple varieties and the cidermaking procedures. The five main polyphenol classes in the apples are as follows: [1] flavan-3-ols, which includes the monomeric

Manipulating Feeding Stimulation to Protect Crops Against Insect Pests

Enhancing natural mechanisms of plant defense against herbivores is one of the possible strategies to protect cultivated species against insect pests. Host plant feeding stimulation, which results from phagostimulant and phagodeterrent effects of both primary and secondary metabolites, could play a key role in levels of damage caused to crop plants. We tested this hypothesis by comparing the feeding intensity of the pollen beetle Meligethes aeneus on six oilseed rape (Brassica napus) genotypes in a feeding experiment, and by assessing the content of possible phagostimulant and phagodeterrent compounds in tissues targeted by the insect (flower buds). For this purpose, several dozens of primary and secondary metabolites were quantified by a set of chromatographic techniques. Intergenotypic variability was found both in the feeding experiment and in the metabolic profile of plant tissues. Biochemical composition of the perianth was in particular highly correlated with insect damage. Only a few compounds explained this correlation, among which was sucrose, known to be highly phagostimulating. Further testing is needed to validate the suggested impact of the specific compounds we have identified. Nevertheless, our results open the way for a crop protection strategy based on artificial selection of key determinants of insect feeding stimulation.

Methods for Determining the Degree of Polymerization of Condensed Tannins: A New 1H-NMR Procedure Applied to Cider Apple Procyanidins

The degree of polymerization (DP) that corresponds to the number of flavan-3-ol units is one of the most important features that characterize condensed tannins (proanthocyanidins) because of its direct link to the various properties of this kind of phenolic compound. In their definition of vegetable tanning substances, Bate-Smith and Swain1 referred to the molecular weight that must range from 500 to 3,000. By considering their extraction, their biological activities, their sensory effects, condensed tannins often behave according to their molecular weight, although this single feature is quite insufficient to show evidence of all their properties. On the whole, the molecular weight of condensed tannins is related to their ability to associate with proteins and polysaccharides; this “tanning capacity” varies in an increasing order with the DP.2–6 This property is also related to other applications. For instance, the work of Lea and Arnold7 pointed out the influence of the DP of procyanidins in relation to bitterness and astringency of cider. Proanthocyanidins are also partly involved in haze formation in beers: the capacity of beer tannins to precipitate proteins increases with the DPn.4,5,8 Many studies dealing with the biological activities of proanthocyanidins also show that antioxidant,9 antifungal,10 anti-enzymic,11 antisecretory,12 or antitumor13 activities may correlate with the DP.

Camalexin contributes to the partial resistance of Arabidopsis thaliana to the biotrophic soilborne protist Plasmodiophora brassicae

Camalexin has been reported to play defensive functions against several pathogens in Arabidopsis. In this study, we investigated the possible role of camalexin accumulation in two Arabidopsis genotypes with different levels of basal resistance to the compatible eH strain of the clubroot agent Plasmodiophora brassicae. Camalexin biosynthesis was induced in infected roots of both Col-0 (susceptible) and Bur-0 (partially resistant) accessions during the secondary phase of infection. However, the level of accumulation was four-to-seven times higher in Bur-0 than Col-0. This was associated with the enhanced transcription of a set of camalexin biosynthetic P450 genes in Bur-0: CYP71A13, CYP71A12, and CYP79B2. This induction correlated with slower P. brassicae growth in Bur-0 compared to Col-0, thus suggesting a relationship between the levels of camalexin biosynthesis and the different levels of resistance. Clubroot-triggered biosynthesis of camalexin may also participate in basal defense in Col-0, as gall symptoms and pathogen development were enhanced in the pad3 mutant (Col-0 genetic background), which is defective in camalexin biosynthesis. Clubroot and camalexin responses were then studied in Heterogeneous Inbred Families (HIF) lines derived from a cross between Bur-0 and Col-0. The Bur/Col allelic substitution in the region of the previously identified clubroot resistance QTL PbAt5.2 (Chromosome 5) was associated with both the enhanced clubroot-triggered induction of camalexin biosynthesis and the reduced P. brassicae development. Altogether, our results suggest that high levels of clubroot-triggered camalexin biosynthesis play a role in the quantitative control of partial resistance of Arabidopsis to clubroot.

Robust Method for Investigating Nitrogen Metabolism of 15N Labeled Amino Acids Using AccQ•Tag Ultra Performance Liquid Chromatography-Photodiode Array-Electrospray Ionization-Mass Spectrometry: Application to a Parasitic Plant–Plant Interaction

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 (15)N 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 (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-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.

Polyphenol variability in the fruits and juices of a cider apple progeny.

BACKGROUND Polyphenols have a favourable antioxidant potential on human health, suggesting that their high content in apple is responsible for the beneficial effects of apple consumption. They are also linked to the quality of apple juices and ciders since they are predominantly responsible for astringency, bitterness and colour. Major phenolic compounds were quantified by liquid chromatography in fruits and juices from a cider apple progeny harvested for 3 years. The total content of procyanidins and their average degree of polymerisation (DPn) were also determined in fruits by phloroglucinolysis. Variability and extraction yield of these compounds were determined. RESULTS The variability observed in the progeny was representative of the variability observed in many cider apple varieties. Hydroxycinnamic acids were the most extractable group, with an average extraction yield of 67%, whereas flavonols and anthocyanins were the least. CONCLUSION This study is the first to introduce variability and extraction yields of the main phenolic compounds in both fruits and juices of a cider apple progeny. This dataset will be used for an upcoming QTL mapping study, an original approach that has never been undertaken for cider apple.