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Cuticle are multifunctional hydrophobic biocomposites that protect aerial organs of plants. Along plant development, plant cuticle must accommodate different mechanical constraints combining extensibility and stiffness, the corresponding structure-function relationships are unknown. Recent data showed a fine architectural tuning of the cuticle architecture and the corresponding chemical clusters along fruit development which raise the question of their impact on the mechanical properties of the cuticle.We investigated the in-depth nanomechanical properties of tomato fruit cuticle from early development to ripening, in relation to chemical and structural heterogeneities by developing a correlative multimodal imaging approach.Unprecedented sharps heterogeneities were evidenced with the highlighting of an in-depth mechanical gradient and a ‘soft’ central furrow that were maintained throughout the plant development despite the overall increase in elastic modulus. In addition, we demonstrated that these local mechanical areas are correlated to chemical and structural gradients.This study shed light on a fine tuning of mechanical properties of cuticle through the modulation of their architecture, providing new insight for our understanding of structure-function relationships of plant cuticle and for the design of biosinpired material.

The cuticle is a complex polymer matrix that protects all aerial organs of plants, fulfills multiple roles in plant–environment interactions, and is critical for plant development. These functions are associated with the structural features of cuticles, and the architectural modeling of cuticles during plant development is crucial for understanding their physical properties and biological functions. In this work, the in-depth architecture of the cutin polymer matrix during fruit development was investigated. Using cherry tomato fruit (Solanum lycopersicum) as a model from the beginning of the cell expansion phase to the red ripe stage, we designed an experimental scheme combining sample pretreatment, Raman mapping, multivariate data analyses, and biochemical analyses. These approaches revealed clear chemical areas with different contributions of cutin, polysaccharides, and phenolics within the cutin polymer matrix. Besides, we demonstrated that these areas are finely tuned during fruit development, including compositional and macromolecular rearrangements. The specific spatiotemporal accumulation of phenolic compounds (p-coumaric acid and flavonoids) suggests that they fulfill distinct functions during fruit development. In addition, we highlighted an unexpected dynamic remodeling of the cutin-embedded polysaccharides pectin, cellulose, and hemicellulose. Such structural tuning enables consistent adaption of the cutin-polysaccharide continuum and the functional performance of the fruit cuticle at the different developmental stages. This study provides insights into the plant cuticle architecture and in particular into the organization of the epidermal cell wall-cuticle.

Agro-industrial residues comprise a rich diversity of plant polymers and bioactive compounds, constituting promising sources for the development of materials, including bioplastics, and food supplements, among other applications. In particular, the polyester cutin is abundant in fruit peel, a plentiful constituent of pomace agro-industrial residues. The potential of diverse fruit pomaces as a source for the development of cutin-derived materials/products has been extensively sought out. This study expands the established knowledge: it sets proof of concept for the production of antimicrobial oligomers from cutin-rich materials isolated in a single step from tomato pomaces generated by two remote agro-industries. Specifically, it first analyzed how the chemical signature (nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS)) of a pomace (and of its major constituents) mirrors that of the corresponding cutin-rich material isolated using an ionic liquid extractant. The cutin-rich materials were then deconstructed (using mild hydrolyses), and the resultant mixtures were chemically characterized and screened for bactericidal activity against

The tomato (Solanum lycopersicum) fruit has a thick, astomatous cuticle that has become a model for the study of cuticle formation, structure, and properties in plants. Tomato is also a major horticultural crop and a long-standing model for research in genetics, fruit development, and disease resistance. As a result, a wealth of genetic resources and genomic tools have been established, including collections of natural and artificially induced genetic diversity, introgression lines of genome fragments from wild relatives, high-quality genome sequences, phenotype and gene expression databases, and efficient methods for genetic transformation and editing of target genes. This mini-review reports the considerable progresses made in recent years in our understanding of cuticle by using and generating genetic diversity for cuticle-associated traits in tomato. These include the synthesis of the main cuticle components (cutin and waxes), their role in the structure and properties of the cuticle, their interaction with other cell wall polymers as well as the regulation of cuticle formation. It also addresses the opportunities offered by the untapped germplasm diversity available in tomato and the current strategies available to exploit them.

To move today’s agricultural and urban systems towards tomorrow’s circular economy and respond to climate change, it is imperative to turn organic residues and wastewater into resourceful assets. This article discusses the changes that are needed in research to drive this paradigm shift and to go from a “losses and waste” situation to a “resource and opportunities” ambition. The current lines of research aim to maximise the use and value of biomass or organic residues and wastewater and propose new organisational schemes driven by technical innovations. Exploring the pathways to a sustainable future through many domains let us identify five challenges to structure the research efforts and find circular bioeconomy solutions for organic residues and wastewater: (1) proposing innovative processes and integrated multi-process systems; (2) promoting the emergence of multi-scale and cross-sectoral organisations; (3) developing multi-performance evaluation methods, (4) rethinking research–society intersections, and (5) enhancing research–legislation interactions. We end by outlining prospects for moving forward past current limitations: beyond increasing knowledge, research will continue its own transition. Our responsibility today is not to think about what we could do for a better world but what we should do to make our ever-changing world even better and more sustainable. Graphic Abstract

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

Polyesters, as they exist in planta, are promising materials with which to begin the development of “green” replacements. Cutin and suberin, polyesters found ubiquitously in plants, are prime candidates. Samples enriched for plant polyesters, and in which their native backbones were largely preserved, were studied to identify “natural” structural features; features that influence critical physical properties. Quantitative nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and X-ray scattering methods were used to quantify structure–property relationships in these polymeric materials. The degree of esterification, namely, the presence of acylglycerol linkages in suberin and of secondary esters in cutin, and the existence of mid-chain epoxide groups defining the packing of the aliphatic chains were observed. This packing determines polymer crystallinity, the resulting crystal structure, and the melting temperature. To evaluate the strength of this rule, tomato cutin from the same genotype, studying wild-type plants and two well-characterized mutants, was analyzed. The results show that cutin’s material properties are influenced by the amount of unbound aliphatic hydroxyl groups and by the length of the aliphatic chain. Collectively, the acquired data can be used as a tool to guide the selection of plant polyesters with precise structural features, and hence physicochemical properties., A detailed insight into plant polyesters identifies correlations of structural features with physical properties that are translated into five essential rules for the development of novel green materials.

International audience; Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α-and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr) 8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindolineprolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.

The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin-embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin-deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin-embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross-talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.

By mimicking the cutin natural polyester networks of plant cuticles, we produced hydrophobic elastomers by a sustainable process, i.e., using a catalyst- and solvent-free polycondensation of glycerol and hydroxy fatty acids, two by-products of the agro-food industry. The hydroxy fatty acid fraction was obtained by ethanolic alkaline hydrolysis of cuticle from industrial tomato. This industrial-like fatty acid fraction contained more than 90% wt. of 9(10)-16 dihydroxyhexadecanoic acid (diOHC16). The co-polyesters were designed by increasing the ratio of esterified glycerol/diOHC16 in a range observed in plant cutins (up to 6% wt.). Their structure and functional properties (thermal, mechanical, gas permeability, interaction with bacterial cells) were characterized. Increasing the glycerol contents induced a significant decrease in the crosslink density of the polyesters and the formation of crystalline domains with a hexagonal organization. These structural modifications were related to a marked increase of elastomeric extensibility (up to 217%). While water vapor permeability was not impacted, the increase of glycerol content induced a significant decrease in oxygen permeability. None of the polyesters displayed biocide activity, but an increase of glycerol content significantly reduced the adhesion of bacterial cells, potentially giving rise to antifouling applications.

Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by theHardnesslocus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with thePina-D1aandPinb-D1dalleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 µm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α– and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α–gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.

The main storage components of the maize endosperm are starch, proteins and lipids. Starch and proteins are heterogeneously deposited, leading to the formation of vitreous and floury regions at the periphery and at the centre of the endosperm. The vitreous/floury mass ratio is a key physical parameter of maize end-uses for the food, feed and non-food sectors, as well as for the resistance of seeds to environmental aggressions. To improve maize breeding for vitreousness, one of the main issues is to finely delineate the molecular and physicochemical mechanisms associated with the formation of endosperm texture. In this context, we use scanning transmission X-ray microscopy at the C K-edge on maize endosperm resin-embedded ultrathin sections. The combination of local near edge X-ray absorption fine structure (NEXAFS) spectroscopy and high-resolution images enable us to achieve a quantitative fine description of the spatial distribution of the main components within the endosperm.

International audience; Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.

International audience; Puroindolines (PINs) A and B were purified from soft (Paledor) and hard (Recital, Courtot) wheat cultivars. Their purity and heterogeneity due to post-translational processing were characterized by SDS- and acid-PAGE, reversed-phase HPLC and mass spectrometry. By using dynamic light scattering (DLS), asymmetrical flow field-flow fractionation (AF4) and size exclusion chromatography (SEC), we showed that the size distributions of PINA are similar for the three varieties and that, in solution, they self-assembled into small aggregates, mainly dimers. Conversely, PINB isolated from hard varieties (PINB-D1b and PINB-D1d) are assembled into large aggregates while PINB-D1a formed small aggregates, mainly monomers. Mixed solutions of PINA and PINB formed heteromeric aggregates. The large PINB-D1b aggregates were retained even at a high (4:1) PINA/PINB weight ratio. Reversible dissociation of large aggregates into small aggregates suggested that weak interactions control the self-assembly of PINs. The aggregative properties of PINs have now to be taken into account when studying their interactions with other components to decipher the causal relationships between these proteins and grain hardness. (C) 2015 Elsevier Ltd. All rights reserved.

National audience; Additive Manufacturing (AM) opens new perspectives for biopolymers to obtain functional parts, like biomedical devices, by exploiting their biocompatibility and resorbability. Plasticized materials from zein, a storage protein from maize seed endosperm, display thermomechanical properties that could match with Fused Deposition Modeling (FDM). The objective of this work was to evaluate their thenno-rheological behavior and their structural modifications during processing. 20% glycerol was added to conunercial zein, containing naturally about 4% lipids and 5% water. After storage at intermediate relative humidity (RH=59%). its glass transition temperature, measured by DSC, was T(g)=42°C. The main mechanical relaxation, measured by DMA, was found at Tα=50°C, leading to a drop of the elastic modulus from E'=1.1GPa, at ambient temperature, to E'=0.6MPa at Tα+100°C. These values are in a similar range as those of standard polymers used for AM-FDM processing, such as PLA and ABS. The structure of zein was characterized at different scales by SDS-PAGE, reversed-phase HPLC, FTIR and WAXS, at each processing stage: (i) the initial formulation of the plasticized powdery material, (ii) after extrusion at 130°C for shaping printable filaments, and (iii) after deposition through the 3D printer nozzle (Ø(nozzle)=0.5mm, T(printing)=130°C, v(printing)≈10mm/s). The presence of disulfide bond cross-links was evidenced in extruded filaments and the level after printing. WAXS showed that, in these conditions, no molecular orientation was obtained in the deposited material. By tuning zein-based printable compositions, these results open the field of their processing as resorbable printed parts, with a controlled geometry and a designed tridimensional structure.

International audience; Cuticular matrices covering aerial plant organs or delimiting compartments in these organs are composed of an insoluble hydrophobic polymer of high molecular mass, i.e., cutin, that encompass some cell wall polysaccharides and is filled by waxes. Cutin is a polyester of hydroxy and-or epoxy fatty acids including a low amount of glycerol. Screening of Arabidopsis and more recently of tomato (Solanum lycopersicum) mutants allowed the delineation of the metabolic pathway involved in the formation of cutin monomers, as well as their translocation in the apoplast. Furthermore, these studies identified an extracellular enzyme involved in the polymerization of these monomers, i.e., cutin synthase 1 (CUS1), an acyl transferase of the GDSL lipase protein family. By comparing the structure of tomato fruit cutins from wild type and down-regulated CUS1 mutants, as well as with the CUS1-catalyzed formation of oligomers in vitro, hypothetical models can be elaborated on the polymerization of cutins. The polymorphism of the GDSL-lipase family raises a number of questions concerning the function of the different isoforms in relation with the formation of a composite material, the cuticle, containing entangled hydrophilic and hydrophobic polymers, i.e., polysaccharides and cutin, and plasticizers, i.e., waxes.

Improving crop productivity and quality while promoting sustainable agriculture have become major goals in plant breeding. The cuticle is a natural film covering the aerial organs of plants and consists of lipid polyesters covered and embedded with wax. The cuticle protects plants against water loss and pathogens and affects traits with strong impacts on crop quality such as, for horticultural crops, fruit brightness, cracking, russeting, netting, and shelf life. Here we provide an overview of the most important cuticle-associated traits that can be targeted for crop improvement. To date, most studies on cuticle-associated traits aimed at crop breeding have been done on fleshy fruits. Less information is available for staple crops such as rice, wheat or maize. Here we present new insights into cuticle formation and properties resulting from the study of genetic resources available for the various crop species. Our review also covers the current strategies and tools aimed at exploiting available natural and artificially induced genetic diversity and the technologies used to transfer the beneficial alleles affecting cuticle-associated traits to commercial varieties.

International audience; Relationships between kernel vitreousness and proteins and starch partitioning to the floury and vitreous regions of the endosperm were monitored in a set of 13 maize inbred lines. Decrease of protein contents from the vitreous to the floury endosperms were mainly assigned to a-zeins. Using Raman microspectroscopy, we observed a protein gradient from the periphery to the center of endosperms that well fitted with the inverse relationships between vitreousness and protein content of the vitreous and floury regions. In addition, Raman microspectroscopy highlighted an increase of starch crystallinity from the periphery to the center of the maize endosperms. This agrees with the higher amylose and associated lipid contents within starches of vitreous than in those of floury endosperms. Finally, starch granules from vitreous regions displayed more channels than the floury ones. These channels contain proteins that might favor adhesion of proteins to starch granules or granule granule contacts to form the close packing of the vitreous endosperm. Therefore transition from vitreous to floury endosperm is at least the result of both protein and starch gradients. These gradients are probably associated with metabolic gradients that have been observed during endosperm development. (C) 2016 Elsevier Ltd. All rights reserved.

Summary Background At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. Objective To characterize the epitopes of different wheat kernel allergens: α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. Methods Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. Results Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit. Conclusion and Clinical Relevance The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations. Cite this as: S. Denery-Papini, M. Bodinier, F. Pineau, S. Triballeau, O. Tranquet, K. Adel-Patient, D.A. Moneret-Vautrin, B. Bakan, D. Marion, T. Mothes, H. Mameri and D. Kasarda, Clinical & Experimental Allergy, 2011 (41) 1478–1492.

International audience; One of the objectives of the malting industry is to reduce the energy cost during kilning without major effect on malt quality. In this study, the impact of a low hydration steeping process on lipid transfer protein (LTP1) modifications and beta-glucan breakdown was evaluated in low (LH) and high (HH) hydrated malts. LTP1 modifications analyzed by MS/MS revealed acylation, glycation, and disulfide bond breakage in both LH and HH malts. LTP1 free amine content measurement and fluorescence of Maillard protein adducts revealed no significant difference between LH and HH malts. Immunolabeling of LTP1 during malting highlighted the diffusion of the protein from the aleurone layer to the endosperm at the end of steeping in both LH and HH malts. By contrast, a significant higher amount of beta-glucans was measured in LH malts after five days of germination, whereas no significant difference between LH and HH malts was revealed through immunostaining of beta-glucans or evaluation of the endosperm integrity after seven days of germination. The possibility to reduce the effects of a low hydration steeping process on beta-glucan hydrolysis by increasing germination time was discussed.

International audience; Growth and production of type-B trichothecenes mycotoxins by the Fusarium graminearum strain CBS 185.32 were compared in GYEP medium supplemented with Mg2+ at different concentrations (0–4 mM). Mg2+ led to a strong decrease in toxin accumulation without affecting the mycelial growth, suggesting a specific Mg2+ effect on fungal secondary metabolism. Expression of Tri5, Tri6, and Tri12 genes was followed throughout the time courses of type-B trichothecenes (TCTB) yield in standard and 2 mM Mg2+-supplemented GYEP media. Mg2+ addition significantly decreased Tri5, Tri6, and Tri12 expression. The inhibition of toxin production by Mg2+ was shown to be highly correlated with inhibition of Tri5 transcription and, to a lesser extend, of Tri6 and Tri12. This is the first report of a transcriptional control of TCTB production by Mg2+.

International audience; Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amyloselysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness.

International audience; The cutin polymers of different fruit cuticles (tomato, apple, nectarine) were examined using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) after in situ release of the lipid monomers by alkaline hydrolysis. The mass spectra were acquired from each coordinate with a lateral spatial resolution of approximately 100m. Specific monomers were released at their original location in the tissue, suggesting that post-hydrolysis diffusion can be neglected. Relative quantification of the species was achieved by introducing an internal standard, and the collection of data was subjected to non-supervised and supervised statistical treatments. The molecular images obtained showed a specific distribution of ions that could unambiguously be ascribed to cutinized and suberized regions observed at the surface of fruit cuticles, thus demonstrating that the method is able to probe some structural changes that affect hydrophobic cuticle polymers. Subsequent chemical assignment of the differentiating ions was performed, and all of these ions could be matched to cutin and suberin molecular markers. Therefore, this MALDI-MSI procedure provides a powerful tool for probing the surface heterogeneity of plant lipid polymers. This method should facilitate rapid investigation of the relationships between cuticle phenotypes and the structure of cutin within a large population of mutants.

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

L'article original est publié par The American Society of Plant Biologists Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

The barley lipid transfer protein (LTP1) adducted by an alpha-ketol, (9-hydroxy-10-oxo-12(Z)-octadecenoic acid) exhibits an unexpected high lipid transfer activity. The crystal structure of this oxylipin-adducted LTP1, (LTP1b) was determined at 1.8A resolution. The covalently bound oxylipin was partly exposed at the surface of the protein and partly buried within the hydrophobic cavity. The structure of the oxylipidated LTP1 emphasizes the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins when compared to the other members of the family. The plasticity of the hydrophobic cavity and increase of its surface hydrophobicity induced by the oxylipin account for the improvement of the lipid transfer activity of LTP1b. These observations open new perspectives to explore the different biological functions of LTPs, including their allergenic properties.

Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1-1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1–lipid complex were both determined by fluorescence and X-ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side-by-side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways.

International audience; Lipid transfer proteins (LTP) and puroindolines are abundant lipid binding proteins of plant seeds. While LTP are ubiquitous plant proteins, puroindolines are only found in the seeds of plants from the Triticae and Avenae tribes. These proteins display a similar overall folding pattern but different lipid binding properties. The unique and diverse biological and technological functions of LTPs and puroindolines are closely related to their structural and lipid binding properties. These proteins are attractive to improve the agronomic performances and food quality of crops. Heterologous expression and genetic engineering should allow industrial production and enlarge applications of these lipid binding proteins.

DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P212121 with one molecule per asymmetric unit., DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P212121 with one molecule per asymmetric unit. The crystals diffract to a resolution of 1.6 Å.

International audience; Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the {alpha}-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development.

cited By 37; International audience; Four trichothecene-producing strains of Fusarium graminearum were grown on three maize grain fractions, whole grain, degermed grain, and the germ, to determine the effect of natural substrates on mycotoxin production. Monitoring the ergosterol content after 25 days of incubation indicated that fungal growth on all grain fractions was comparable. Trichothecene (TCT) production was highest on degermed grain, less on whole grain, and very low or nondetectable on the germ; similar results were found with four different strains. It was concluded that inhibitor(s) of TCT biosynthesis were present in maize germ. The presence of phenolic compounds was investigated in the different fractions. The hydroxamate 4-acetylbenzoxazolin-2-one (4-ABOA), a known inhibitor of mycotoxin production, was found in the degermed and whole grain fractions but not in the germ. Therefore, the TCT inhibition observed on the maize germ fraction used in our study is clearly not linked to 4-ABOA. Other soluble phenolic compounds were found at a much higher concentration in the germ than in the two other fractions. The inhibition property of the soluble ester-bound extracts was tested in liquid culture. A possible role for these compounds is discussed.

Objective Free radicals and oxidative stress are important factors in the biology of aging and responsible for the development of age-related diseases. One way to reduce the formation of free radicals is to boost the antioxidative system by nutrition. Heat treatment of food promote the Maillard reaction which is responsible for their characteristic color and taste. During the Maillard reaction reducing sugars react with proteins in a non-enzymatic way leading to the formation of advanced glycation end products (AGEs). As an AGE-rich source our group used bread crust (BCE) to investigate the effect of AGEs on the antioxidant defense. Methods It is well known that the NF-kB pathway is activated by treatment of cells with AGEs. Therefore for stimulation with the BCE we used the macrophage reporter cell line RAW/NF-kB/SEAPorter™. Amino acid analysis and LC-MS/MS by Orbitrap Velo was used to determine the bioactive compounds in the soluble BCE. The radical scavenging effect was conducted by the DPPH-assay. Results BCE induced the NF-kB pathway in RAW/NF-kB/SEAPorter™ cells and also showed a concentration-dependent antioxidative capacity by the DPPH-assay. With the LC/MS and amino acid analyses, we identified the presence of gliadin in BCE confirmed by using specific gliadin antibodies. By immunoprecipitation (IP) with an antibody against γ-gliadin and western blot probing against the AGE carboxymethyllysine (CML) the presence of AGE-gliadin in BCE was confirmed. Stimulation of the RAW/NF-kB/SEAPorter™ cells with the γ-gliadin depleted fractions did not activate the NF-kB pathway. Conclusion CML-modified gliadin in the BCE is a bioactive compound of the bread crust which is responsible for the antioxidative capacity and for the induction of the NF-kB pathway in mouse macrophages.

Sixty F. culmorum strains were isolated from wheat grains collected from different wheat-growing areas in France and from different cultivars. The isolates were grown on autoclaved wheat grain to assess their ability to produce trichothecenes and zearalenone. Fungal biomass was evaluated through the ergosterol grain content. All the isolates produced zearalenone (0.39-1660 mg kg(-1)). Thirty-five of the 60 F. culmorum produced nivalenol (0.11-11.7 mg kg(-1)), 12 of 60 produced fusarenone X (0.05-8.42 mg kg(-1)), five of 60 produced 15-acetyldeoxynivalenol (0.48-27.7 mg kg(-1)), 13 of 60 produced 3-acetyldeoxynivalenol (0.07-21.0 mg kg(-1) and 24 of 60 produced deoxynivalenol (0.92-51.9 mg kg(-1)). According to the results, the distribution of the different chemotypes as well as the high and the low mycotoxin-producing Fusarium strains could not be associated to geographical origin.

The cuticle, an extracellular lipid matrix consisting of waxes and of a cutin skeleton is the outermost plants protection barrier against their environment. The cuticle is involved in many agronomic traits such as post-harvest storage, biomechanical or fruit appearance properties like surface brightness. In order to isolate cuticle mutants, the screening of an EMS tomato mutants collection has been undertaken, based on fruit brightness, leading to the selection of 24 mutants. Biochemical analyzes have shown wide variations in cuticle loads and compositions, especially in cutin mutants. The characterization of 4 remarkable mutants was undertaken to identify the mutations responsible for brightness phenotypes. The most affected mutant shows a cutin load reduced by 85% compared to the wild type, and is due to a mutation in the SlGDSL2 gene, encoding a GDSL-motive acylhydrolase enzyme, responsible for the cutin polymerization. In order to further study the cutin formation and regulation, the next work was to obtain and characterize single and double mutants affected in cutin monomer synthesis, apoplastic transport and cutin polymerization., La cuticule, une matrice lipidique extracellulaire constituée de cires et d’un squelette de cutine, est la barrière de défense la plus externe des plantes face à leur environnement. Elle intervient dans de nombreuses propriétés agronomiques comme la conservation post récolte, les propriétés mécaniques ou bien l’aspect du fruit, dont la brillance. Afin d’isoler des mutants de cuticule, le criblage d’une collection de mutants EMS de tomate a été entrepris, en se basant sur la brillance des fruits, conduisant à la sélection de 24 mutants. Chez ceux-ci, des analyses biochimiques ont montré de fortes variations de charge et de composition de la cuticule, notamment chez les mutants de cutine. La caractérisation de 4 mutants remarquables a été entreprise afin d’identifier les mutations responsables des phénotypes de brillance. Le mutant le plus affecté, présentant une charge en cutine réduite de 85% par rapport au type sauvage, a révélé une mutation du gène SlGDSL2 codant pour une acylhydrolase à motif GDSL, responsable de la polymérisation de la cutine. Afin d’étudier la formation et la régulation de la cutine, la suite du travail a consisté à obtenir et à caractériser des simples et des doubles mutants affectés dans la synthèse des monomères de cutine, le transport apoplastique et la polymérisation de la cutine.

Contact: pauline.ezanno@oniris-nantes.fr Diplôme : Dr. d'Université; Epidemiological models enable to better understand the dynamics of infectious diseases and to assess ex-ante control strategies. For Mycobacterium avium subsp. paratuberculosis (Map), possible transmission routes have been described, but Map spread in a herd and the relative importance of the routes are currently insufficiently understood to prioritize control measures. We aim to predict early after Map introduction in a dairy cattle herd whether infection is likely to fade out or persist, when no control measures are implemented, using a modelling approach. Both vertical transmission and horizontal transmission via the ingestion of colostrum, milk, or faeces present in the contaminated environment were modelled. Calf-to-calf indirect transmission was possible. Six health states were represented: susceptible, transiently infectious, latently infected, subclinically infected, clinically affected, and resistant. The model was partially validated by comparing the simulated prevalence with field data. Housing facilities and contacts between animals were specifically considered for calves and heifers. After the introduction of one infected animal in a naive herd, fadeout occurred in 66% of the runs. When Map persisted, the prevalence of infected animals increased to 88% in 25 years. The two main transmission routes were via the farm’s environment and in utero transmission. Calf-to-calf transmission was minor. Fadeout versus Map persistence could be differentiated with the number of clinically affected animals, which was rarely above one when fadeout occurred. Therefore, early detection of affected animals is crucial in preventing Map persistence in dairy herds

L'industrie du malt tente actuellement de réduire les coûts énergétiques (touraillage) sans affecter la qualité de malt obtenu. Cette économie d’énergie passe nécessairement par une diminution de la teneur en eau du grain d’orge en fin de trempe et durant la germination.Nous avons choisi d'étudier des paramètres clés de la qualité brassicole des malts concernant leurs capacités diastasiques et pro-mousse. Ainsi la diffusion et les modifications de la TP1, protéine pro-mousse du malt, ont montré des résultats équivalents pour un malt faiblement hydraté comparé à un malt fortement hydraté.Par contre, le taux d’hydratation du grain affecte la diffusion et l'activité des β-glucanases et en conséquence la dégradation des β-glucanes. Ces effets sont liés à une différence dans la cinétique de diffusion et de synthèse de ces enzymes et peuvent être fortement réduits en augmentant la durée de germination.La migration de l’eau dans le grain suit un cheminement particulier lié au contournement des barrières hydrophobes du grain situées en surface du péricarpe et à l’interface entre aleurone et péricarpe (testa). Sur des variétés produisant, au niveau pilote industriel, des malts qualitativement différents suivant le taux d’hydratation,nous avons remarqué de faibles différences de composition et d’épaisseur entre les assises cuticulaires. En revanche, nous avons mis en évidence une très grande variabilité de composition des cuticules dans les différentes parties du grain, par exemple le scutellum, le sillon ou les glumelles, During kilning, reduction of energy costs without impacting significantly the malt quality, represents a major challenge of the malting industry. This energy reduction requires a reduction of barley grain water content at the end of steeping and during germination.In the present work, we focused on key malting quality parameters involved in diastatic power and foam promoting capacities of malt. When analysed in a low hydrated malt and a high hydrated malt, the LTP1 (a foam promoting protein) modifications and its diffusion from the aleurone layer to the starchy endosperm were similar. The hydration level of the grain affects however β-glucanase diffusion and activity, and consequently the β-glucan degradation. These effects are linked to a delay in the synthesis and the diffusion of these enzymes.An increase of the time of germination allows to sharply reduce these effects.According to grain hydrophobic barriers localized at the pericarp surface and at the interface of aleurone and pericarp (testa), water diffusion inside the grain follows a specific pathway. Only weak, although significant,differences in cuticular thickness and composition were observed between cultivars with different malt qualities according to their hydration levels. On the contrary, we observed a great variability in the cuticular composition of the various tissues of the grain e.g. scutellum, chalaza or husk