Age-specific allocation of glucosinolates within plant reproductive tissues.

The Optimal Defense Theory (ODT) predicts that the distribution of defenses within a plant should mirror the value and vulnerability of each tissue. Although the ODT has received much experimental support, very few studies have examined defense allocation among reproductive tissues and none assessed simultaneously how these defenses evolve with age. We quantified glucosinolates in perianths, anthers and pistils at different bud maturity stages (i.e., intermediate flower buds, old flower buds and flowers) of undamaged and mechanically damaged plants of an annual brassicaceous species. The youngest leaf was used as a reference for vegetative organs, since it is predicted to be one of the most defended. In line with ODT predictions, reproductive tissues were more defended than vegetative tissues constitutively, and within the former, pistils and anthers more defended than perianths. No change in the overall defense level was found between bud maturity stages, but a significant temporal shift was observed between pistils and anthers. Contrary to ODT predictions, mechanical damage did not induce systemic defenses in leaves but only in pistils. Our results show that defense allocation in plant reproductive tissues occurs at fine spatial and temporal scales, extending the application framework of the ODT. They also demonstrate interactions between space and time in fine-scale defense allocation.

Identification and Quantification of Glucosinolates and Phenolics in a Large Panel of Brassica napus Highlight Valuable Genetic Resources for Chemical Ecology and Breeding.

Glucosinolate (GLS) and phenolic contents in Brassicaceae contribute to biotic and abiotic stress responses. Breeding crop accessions harboring agroecologically relevant metabolic profiles require a characterization of the chemical diversity in Brassica germplasm. This work investigates the diversity of specialized metabolites in 281 accessions of B. napus. First, an LC-HRMS2-based approach allowed the annotation of 32 phenolics and 36 GLSs, revealing 13 branched and linear alkyl-GLSs and 4 isomers of hydroxyphenylalkyl-GLSs, many of which have been rarely reported in Brassica. Then, quantitative UPLC-UV-MS-based profiling was performed in leaves and roots for the whole panel. This revealed striking variations in the content of 1-methylpropyl-GLS (glucocochlearin) and a large variation of tetra- and penta-glucosyl kaempferol derivatives among accessions. It also highlighted two main chemotypes related to sinapoyl-O-hexoside and kaempferol-O-trihexoside contents. By offering an unprecedented overview of the phytochemical diversity in B. napus, this work provides a useful resource for chemical ecology and breeding.

Multi-Omic Investigation of Low-Nitrogen Conditional Resistance to Clubroot Reveals Brassica napus Genes Involved in Nitrate Assimilation.

Nitrogen fertilization has been reported to influence the development of clubroot, a root disease of Brassicaceae species, caused by the obligate protist Plasmodiophora brassicae. Our previous works highlighted that low-nitrogen fertilization induced a strong reduction of clubroot symptoms in some oilseed rape genotypes. To further understand the underlying mechanisms, the response to P. brassicae infection was investigated in two genotypes "Yudal" and HD018 harboring sharply contrasted nitrogen-driven modulation of resistance toward P. brassicae. Targeted hormone and metabolic profiling, as well as RNA-seq analysis, were performed in inoculated and non-inoculated roots at 14 and 27 days post-inoculation, under high and low-nitrogen conditions. Clubroot infection triggered a large increase of SA concentration and an induction of the SA gene markers expression whatever the genotype and nitrogen conditions. Overall, metabolic profiles suggested that N-driven induction of resistance was independent of SA signaling, soluble carbohydrate and amino acid concentrations. Low-nitrogen-driven resistance in "Yudal" was associated with the transcriptional regulation of a small set of genes, among which the induction of NRT2- and NR-encoding genes. Altogether, our results indicate a possible role of nitrate transporters and auxin signaling in the crosstalk between plant nutrition and partial resistance to pathogens.

Dataset for the metabolic and physiological characterization of seeds from oilseed rape (Brassica napus L.) plants grown under single or combined effects of drought and clubroot pathogen Plasmodiophora brassicae.

Faced with the challenges of adapting agriculture to climate change, seed production should have increased resilience to abiotic stress factors and the expected proliferation of pathogens. This concerns both the nutritional quality and seed vigor, two crucial factors in seedling establishment and yield. Both qualities are acquired during seed development, but how environment influences the genetic and physiological determinisms of these qualities remains to be elucidated. With a world production of 71 Mt of seeds per year, oilseed rape (Brassica napus) is the third largest oleaginous crop. But its productivity must cope with several abiotic stresses, among which drought is one of the main constraints in current and future climate scenarios. In addition, clubroot disease, caused by the pathogen Plasmodiophora brassicae, leads to severe yield losses for the Brassica crops worldwide. Clubroot provokes the formation of galls on the infected roots that can restrict the flow of water and nutrients within the plant throughout the growth cycle. In order to get new insights into the impact of single or combined constraints on seed qualities, metabolic profiling assays were run for a collection of 330 seed samples (including developing, mature and imbibed seeds) harvested from plants of two B. napus cultivars ("Express" and "Montego") that were grown under either drought conditions, the presence of P. brassicae, or a combination of both stresses. Metabolites were identified and quantified by UPLC or GC. In addition, monitoring germination traits was conducted for 60 mature seed lots under in vitro conditions using an automated phenotyping platform. The present dataset contains the raw contents for 42 metabolites (nmol.mg-1 of seed dry weight) filtered and analyzed with statistical tests as well as germination speed and percentages. This dataset is available under accession at Data INRAE. These data will contribute to a better understanding of the crosstalk between the plant responses to water deprivation and/or pathogen attack and how it compromises seed quality. A better understanding of the molecular and physiological responses of the seed to (a)biotic stress on a molecular and physiological will be a first step to meet scientific and technological challenges of adapting seeds to their environment.

Sink/Source Balance of Leaves Influences Amino Acid Pools and Their Associated Metabolic Fluxes in Winter Oilseed Rape (Brassica napus L.).

Nitrogen remobilization processes from source to sink tissues in plants are determinant for seed yield and their implementation results in a complete reorganization of the primary metabolism during sink/source transition. Here, we decided to characterize the impact of the sink/source balance on amino acid metabolism in the leaves of winter oilseed rape grown at the vegetative stage. We combined a quantitative metabolomics approach with an instationary 15N-labeling experiment by using [15N]L-glycine as a metabolic probe on leaf ranks with a gradual increase in their source status. We showed that the acquisition of the source status by leaves was specifically accompanied by a decrease in asparagine, glutamine, proline and S-methyl-l-cysteine sulphoxide contents and an increase in valine and threonine contents. Dynamic analysis of 15N enrichment and concentration of amino acids revealed gradual changes in the dynamics of amino acid metabolism with respect to the sink/source status of leaf ranks. Notably, nitrogen assimilation into valine, threonine and proline were all decreased in source leaves compared to sink leaves. Overall, our results suggested a reduction in de novo amino acid biosynthesis during sink/source transition at the vegetative stage.

Leaf status and environmental signals jointly regulate proline metabolism in winter oilseed rape.

Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).

Resolution of quantitative resistance to clubroot into QTL-specific metabolic modules.

Plant disease resistance is often under quantitative genetic control. Thus, in a given interaction, plant cellular responses to infection are influenced by resistance or susceptibility alleles at different loci. In this study, a genetic linkage analysis was used to address the complexity of the metabolic responses of Brassica napus roots to infection by Plasmodiophora brassicae. Metabolome profiling and pathogen quantification in a segregating progeny allowed a comparative mapping of quantitative trait loci (QTLs) involved in resistance and in metabolic adjustments. Distinct metabolic modules were associated with each resistance QTL, suggesting the involvement of different underlying cellular mechanisms. This approach highlighted the possible role of gluconasturtiin and two unknown metabolites in the resistance conferred by two QTLs on chromosomes C03 and C09, respectively. Only two susceptibility biomarkers (glycine and glutathione) were simultaneously linked to the three main resistance QTLs, suggesting the central role of these compounds in the interaction. By contrast, several genotype-specific metabolic responses to infection were genetically unconnected to resistance or susceptibility. Likewise, variations of root sugar profiles, which might have influenced pathogen nutrition, were not found to be related to resistance QTLs. This work illustrates how genetic metabolomics can help to understand plant stress responses and their possible links with disease.

Evolution of DMSP (dimethylsulfoniopropionate) biosynthesis pathway: Origin and phylogenetic distribution in polyploid Spartina (Poaceae, Chloridoideae).

DMSP (dimethylsulfoniopropionate) is an ecologically important sulfur metabolite commonly produced by marine algae and by some higher plant lineages, including the polyploid salt marsh genus Spartina (Poaceae). The molecular mechanisms and genes involved in the DMSP biosynthesis pathways are still unknown. In this study, we performed comparative analyses of DMSP amounts and molecular phylogenetic analyses to decipher the origin of DMSP in Spartina that represents one of the major source of terrestrial DMSP in coastal marshes. DMSP content was explored in 14 Spartina species using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). Putative genes encoding the four enzymatic steps of the DMSP biosynthesis pathway in Spartina were examined and their evolutionary dynamics were studied. We found that the hexaploid lineage containing S. alterniflora, S. foliosa and S. maritima and their derived hybrids and allopolyploids are all able to produce DMSP, in contrast to species in the tetraploid clade. Thus, examination of DMSP synthesis in a phylogenetic context implicated a single origin of this physiological innovation, which occurred in the ancestor of the hexaploid Spartina lineage, 3-6MYA. Candidate genes specific to the Spartina DMSP biosynthesis pathway were also retrieved from Spartina transcriptomes, and provide a framework for future investigations to decipher the molecular mechanisms involved in this plant phenotypic novelty that has major ecological impacts in saltmarsh ecosystems.

Can soil microbial diversity influence plant metabolites and life history traits of a rhizophagous insect? A demonstration in oilseed rape.

Interactions between plants and phytophagous insects play an important part in shaping the biochemical composition of plants. Reciprocally plant metabolites can influence major life history traits in these insects and largely contribute to their fitness. Plant rhizospheric microorganisms are an important biotic factor modulating plant metabolites and adaptation to stress. While plant-insects or plant-microorganisms interactions and their consequences on the plant metabolite signature are well-documented, the impact of soil microbial communities on plant defenses against phytophagous insects remains poorly known. In this study, we used oilseed rape (Brassica napus) and the cabbage root fly (Delia radicum) as biological models to tackle this question. Even though D. radicum is a belowground herbivore as a larva, its adult life history traits depend on aboveground signals. We therefore tested whether soil microbial diversity influenced emergence rate and fitness but also fly oviposition behavior, and tried to link possible effects to modifications in leaf and root metabolites. Through a removal-recolonization experiment, 3 soil microbial modalities ("high," "medium," "low") were established and assessed through amplicon sequencing of 16S and 18S ribosomal RNA genes. The "medium" modality in the rhizosphere significantly improved insect development traits. Plant-microorganism interactions were marginally associated to modulations of root metabolites profiles, which could partly explain these results. We highlighted the potential role of plant-microbial interaction in plant defenses against Delia radicum. Rhizospheric microbial communities must be taken into account when analyzing plant defenses against herbivores, being either below or aboveground.

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.

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.

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.

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.

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.

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.

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.

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

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.

O objetivo deste trabalho foi estudar o efeito da fermentação alcoólica no teor de compostos fenólicos de cinco mostos de maçãs industriais. Os compostos fenólicos foram analisados por HPLC. Os mostos apresentaram fenóis totais entre 188,4 a 2776,17 mg.L-1. Os teores de fenóis durante a fermentação permaneceram os mesmos para as variedades DC, PJ e GU, entretanto, em DM e KE foi observada uma diminuição dos teores de fenóis (55 e 313 mg.L-1, respectivamente). Em KE o teor do ácido caféico aumentou de 6,6 mg.L-1 para 41,8 mg.L-1. O teor de catequinas aumentou cerca de 13 mg.L-1 durante o processo, independente da variedade. As outras classes de fenóis não apresentaram modificações com a fermentação alcoólica, permanecendo na sidra os compostos fenólicos do suco de maçã clarificado.

Characterization of plum procyanidins by thiolytic depolymerization.

The phenolic compounds of 'Green Gage' (GG) plums ( Prunus domestica L.), "Rainha Claudia Verde", from a 'protected designation of origin' (PDO), in Portugal, were quantified in both flesh and skin tissues of plums collected in two different orchards (GG-V and GG-C). Analyzes of phenolic compounds were also performed on another GG European plum obtained in France (GG-F) and two other French plums, 'Mirabelle' (M) and 'Golden Japan' (GJ). Thiolysis was used for the first time in the analysis of plum phenolic compounds. This methodology showed that the flesh and skin contain a large proportion of flavan-3-ols, which account, respectively, for 92 and 85% in GJ, 61 and 44% in GG-V, 62 and 48% in GG-C, 54 and 27% in M, and 45 and 37% in GG-F. Terminal units of procyanidins observed in plums are mainly (+)-catechin (54-77% of all terminal units in flesh and 57-81% in skin). The GJ plums showed a phenolic composition different from all of the others, with a lower content of chlorogenic acid isomers and the presence of A-type procyanidins as dimers and terminal residues of polymerized forms. The average degree of polymerization (DPn) of plum procyanidins was higher in the flesh (5-9 units) than in the skin (4-6 units). Procyanidin B7 was observed in the flesh of all GG plums and in the skin of the Portuguese ones. Principal component analysis of the phenolic composition of the flesh and skin of these plums obtained after thiolysis allowed their distinction according to the variety and origin, opening the possibility of the use of phenolic composition for variety/origin identification.

Degradation of procyanidins by Aspergillus fumigatus: identification of a novel aromatic ring cleavage product.

Aspergillus fumigatus was able to grow on apple-purified procyanidins (PCs). PCs concentration decreased 30% over the first 60 h of liquid fermentation. The mean degree of polymerization (DPn) of apple-purified PCs increased from 8 to 15 during the fermentation. A fungal enzyme extract from the liquid fermentation was used to study procyanidin B2 [(-)-epicatechin-(4beta-8)-(-)-epicatechin] degradation. The major degradation product (PB2-X) had a retention time of 10.5 min and a molecular mass at m/z 609. High-performance liquid chromatography/multiple fragment mass spectrometry (HPLC/MS(n)) was used for the structural characterization of PB2-X as well as that of thiolysis-treated PB2-X. Twelve fragment ions at m/z 565, 547, 457, 439 (two fragment ions), 421, 413, 377, 395, 351, 287 and 277 were completely identified. It was therefore deduced that the terminal unit of procyanidin B2 dimer was modified by an oxygenase from A. fumigatus leaving the extension unit intact. In addition, FT-IR analysis confirmed a lactone formation in (-)-epicatechin moiety involved in oxidative degradation. Two reaction schemes were postulated for the interpretation of the results.

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products.

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.