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The biopolyester cutin is ubiquitous in land plants, building the polymeric matrix of the plant's outermost defensive barrier, the cuticle. Cutin influences many biological processes in planta; however, due to its complexity and highly branched nature, the native structure remains partially unresolved. Our aim was to define an original workflow for the purification and systematic characterization of the molecular structure of cutin. To purify cutin we tested the ionic liquids cholinium hexanoate and 1-butyl-3-methyl-imidazolium acetate. The ensuing polymeric materials are highly esterified, amorphous, and have a typical monomeric composition as demonstrated by solid-state NMR, complemented by spectroscopic, thermal, and x-ray scattering analyses. We performed a systematic study by solution-state NMR of cryogenically milled cutins extracted from tomatoes (Solanum lycopersicum 'Micro-Tom'; the wild type and the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE [GPAT6] and CUTIN SYNTHASE [CUS1] mutants). We resolved their molecular structures, relative distribution of ester aliphatics, free acid end-groups and free hydroxyl groups, differentiating between those derived from primary and secondary esters. Our data demonstrate the existence of free hydroxyl groups in cutin and provide insight into how the mutations affect the esterification arrangement of cutin. The usage of ionic liquids for studying plant polyesters has advantages over conventional approaches, since simple modifications can be applied to recover a biopolymer carrying distinct types/degrees of modifications (e.g. preservation of esters or cuticular polysaccharides), which in combination with the solution NMR methodologies developed here, constitutes essential tools to fingerprint the multifunctionality and the structure of cutin in planta.

International audience; Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.

The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-l-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening., Instituto de Fisiología Vegetal

Tomato is a major crop suffering substantial yield losses from diseases, as fruit decay at a postharvest level can claim up to 50% of the total production worldwide. Due to the environmental risks of fungicides, there is an increasing interest in exploiting plant immunity through priming, which is an adaptive strategy that improves plant defensive capacity by stimulating induced mechanisms. Broad-spectrum defence priming can be triggered by the compound ß-aminobutyric acid (BABA). In tomato plants, BABA induces resistance against various fungal and bacterial pathogens and different methods of application result in durable protection. Here, we demonstrate that the treatment of tomato plants with BABA resulted in a durable induced resistance in tomato fruit against Botrytis cinerea, Phytophthora infestans and Pseudomonas syringae. Targeted and untargeted metabolomics were used to investigate the metabolic regulations that underpin the priming of tomato fruit against pathogenic microbes that present different infection strategies. Metabolomic analyses revealed major changes after BABA treatment and after inoculation. Remarkably, primed responses seemed specific to the type of infection, rather than showing a common fingerprint of BABA-induced priming. Furthermore, top-down modelling from the detected metabolic markers allowed for the accurate prediction of the measured resistance to fruit pathogens and demonstrated that soluble sugars are essential to predict resistance to fruit pathogens. Altogether, our results demonstrate that metabolomics is particularly insightful for a better understanding of defence priming in fruit. Further experiments are underway in order to identify key metabolites that mediate broad-spectrum BABA-induced priming in tomato fruit.

Strawberry is a fruit crop species of major horticultural importance, for which fruit quality and the control of flowering (for fruit yield), runnering (for vegetative propagation), and the trade-off between the two are main breeding targets. The octoploid cultivated strawberry has a limited genetic basis. This raises the question of how to identify important gene targets and successfully exploit them for strawberry improvement. In this Opinion article we propose to apply to woodland strawberry, a wild diploid species displaying wide diversity, the strategies successfully employed in recent years for the identification of genetic variations underlying fruit quality and fruit yield traits in solanaceous crops (tomato, potato). Next we propose to use gene editing technologies to translate the findings to cultivated strawberry.

UMR BFP - Equipe Métabolisme; International audience; Central metabolism is the engine of plant biomass, supplying fruit growth with building blocks, energy, and biochemical cofactors. Among metabolic cornerstones, nicotinamide adenine dinucleotide (NAD) is particularly pivotal for electron transfer through reduction–oxidation (redox) reactions, thus participating in a myriad of biochemical processes. Besides redox functions, NAD is now assumed to act as an integral regulator of signaling cascades involved in growth and environmental responses. However, the regulation of NAD metabolism and signaling during fruit development remains poorly studied and understood. Here, we benefit from RNAseq and proteomic data obtained from nine growth stages of tomato fruit (var. Moneymaker) to dissect mRNA and protein profiles that link to NAD metabolism, including de novo biosynthesis, recycling, utilization, and putative transport. As expected for a cofactor synthesis pathway, protein profiles failed to detect enzymes involved in NAD synthesis or utilization, except for nicotinic acid phosphoribosyltransferase (NaPT) and nicotinamidase (NIC), which suggested that most NAD metabolic enzymes were poorly represented quantitatively. Further investigations on transcript data unveiled differential expression patterns during fruit development. Interestingly, among specific NAD metabolism-related genes, early de novo biosynthetic genes were transcriptionally induced in very young fruits, in association with NAD kinase, while later stages of fruit growth rather showed an accumulation of transcripts involved in later stages of de novo synthesis and in NAD recycling, which agreed with augmented NAD(P) levels. In addition, a more global overview of 119 mRNA and 78 protein significant markers for NAD(P)-dependent enzymes revealed differential patterns during tomato growth that evidenced clear regulations of primary metabolism, notably with respect to mitochondrial functions. Overall, we propose that NAD metabolism and signaling are very dynamic in the developing tomato fruit and that its differential regulation is certainly critical to fuel central metabolism linking to growth mechanisms.

International audience; MADS-box transcription factors are key elements of the genetic networks controlling flower and fruit development. Among these, the class D clade gathers AGAMOUS-like genes which are involved in seed, ovule, and funiculus development. The tomato genome comprises two class D genes, Sl-AGL11 and Sl-MBP3 , both displaying high expression levels in seeds and in central tissues of young fruits. The potential effects of Sl-AGL11 on fruit development were addressed through RNAi silencing and ectopic expression strategies. Sl-AGL11-down-regulated tomato lines failed to show obvious phenotypes except a slight reduction in seed size. In contrast, Sl-AGL11 overexpression triggered dramatic modifications of flower and fruit structure that include: the conversion of sepals into fleshy organs undergoing ethylene-dependent ripening, a placenta hypertrophy to the detriment of locular space, starch and sugar accumulation, and an extreme softening that occurs well before the onset of ripening. RNA-Seq transcriptomic profiling high-lighted substantial metabolic reprogramming occurring in sepals and fruits, with major impacts on cell wall-related genes. While several Sl-AGL11-related phenotypes are reminiscent of class C MADS-box genes (TAG1 and TAGL1), the modifications observed on the placenta and cell wall and the Sl-AGL11 expression pattern suggest an action of this class D MADS-box factor on early fleshy fruit development.

Editorial: Molecular and Metabolic Mechanisms Associated with Fleshy Fruit Quality

The flesh and peel of 19 pear cultivars (8 Tunisian dessert cultivars, 8 European dessert cultivars and 3 French perry pear cultivars) were studied for their phenolic composition. Phenolic compounds were identified by HPLC/ESI-MS2 and individually quantified by HPLC-DAD. Five classes of polyphenols were present: flavan-3-ols, phenolic acids, flavonols, anthocyanins and simple phenolics (hydroquinones). The total phenolic content ranged between 0.1 g/kg Fresh Weight (FW) ('Conference' cultivar) and 8.6 g/kg FW ('Plant De Blanc' cultivar) in the flesh and between 1.6 g/kg FW ('William vert' cultivar) and 40.4 g/kg FW ('Arbi Chiheb' cultivar) in the peel. Procyanidins, analyzed after thioacidolysis, were the main phenolic compounds in all pear cultivars either in the pulp or the peel, their constitutive units being essentially (-)-epicatechin. Tunisian dessert pears and French perry pears are richer in procyanidins with very high degree of polymerization (> 100) for Tunisian pears. Peel procyanidins were less polymerized (from 4 to 20). Pear peel phenolic profile was more complex especially for Tunisian cultivars, with flavonols and in some cultivars anthocyanins.

UMR 1332 - Equipe GFDF/OrFE/Meta; Key mechanisms controlling fruit weight and shape at the levels of meristem, ovary or very young fruit have already been identified using natural tomato diversity. We reasoned that new developmental modules prominent at later stages of fruit growth could be discovered by using new genetic and phenotypic diversity generated by saturated mutagenesis. Twelve fruit weight and tissue morphology mutants likely affected in late fruit growth were selected among thousands of fruit size and shape EMS mutants available in our tomato EMS mutant collection. Their thorough characterization at organ, tissue and cellular levels revealed two major clusters controlling fruit growth and tissue morphogenesis either through (i) the growth of all fruit tissues through isotropic cell expansion or (ii) only the growth of the pericarp through anisotropic cell expansion. These likely correspond to new cell expansion modules controlling fruit growth and tissue morphogenesis in tomato. Our study therefore opens the way for the identification of new gene regulatory networks controlling tomato fruit growth and morphology.

International audience

Water deficit is known to improve fleshy fruit quality at the expense of yield. However in tornato, recentstudies showed that such effects are highly dependent on genotypes. Thus, breeding tomato plants adaptedto grow under limited water supply requires a better understanding of the effects of WD on fruit quality andtheir genetic variability. ln the present work, four experiments have been performed under greenhouseconditions on the 8 parents of the Multi-Parent Advanced Generation Inter-Cross population which offers thelargest allelic variability observed in tomato. Moderate and intensive WDs have been applied during thewhole period of fruit development The experiments were performed either in spring or in autumn toanalyze the interactions between WD and season. Moreover, interaction between moderate WD and plantfruit load was measured in spring. Fruit quality was assessed through fresh and dry masses, and fruitcomposition (soluble sugars, organic acids, vitamin C, carotenoids). Results show that fruit fresh mass isreduced p,roportionally to WD intensity, whereas the % dry mass is increased. Big fruit size genotypes aremuch more sensitive to WD than small fruit size genotypes. The improvement of fruit taste, based on theincrease of the sugar:acid ratio, resulted more from concentration effects than from metabolic trigger effects.Trade-offs between fruit size decrease and quality improvement allowed discriminating the 8 accessions fortheir genetic interest under water limited conditions.

The fruit is a specialized organ which results from the development of the ovary after successful flower pollination and fertilization and provides a suitable environment for seed maturation and seed dispersal mechanisms. Due to their importance in human nutrition and their economic inference, fleshy fruit species have been the subject of developmental studies, especially using tomato (Solanum lycopersicum Mill.) as a model species due to its favourable biological cycle and all the available genetic and molecular resources. Tomato fruit growth results from the interplay between cell division and cell expansion. These two developmental processes are under the control of complex interactions between internal signals (due to hormones) and external factors (carbon partitioning, environmental influences), which altogether contribute to the establishment of crucial determinants for fruit quality such as the final size, weight and shape of fruits as well as the organoleptic and nutritional traits of the ripe fruit. This chapter aims at describing recent advances in the knowledge of molecular breeding applied to modify tomato fruit growth.

National High-tech R & D Program of China 2013AA102602 National Natural Science Foundation of China 31371645 Program for Changjiang Scholars and Innovative Research Team in University PCSIRT13073 MOE 111 Project B08025; Drought stress is a major abiotic stress threatening plant and crop productivity. In case of fleshy fruits, understanding mechanisms governing water and carbon accumulations and identifying genes, QTLs and phenotypes, that will enable trade-offs between fruit growth and quality under Water Deficit (WD) condition is a crucial challenge for breeders and growers. In the present work, 117 recombinant inbred lines of a population of Solanum lycopersicum were phenotyped under control and WD conditions. Plant waterstatus, fruit growth and composition were measured and data were used to calibrate a process-based model describing water and carbon fluxes in a growing fruit as a function of plant and environment. Eight genotype-dependent model parameters were estimated using a multiobjective evolutionary algorithm in order to minimize the prediction errors of fruit dry and fresh mass throughout fruit development. WD increased the fruit dry matter content (up to 85%) and decreased its fresh weight (up to 60%), big fruit sizegenotypes being the most sensitive. The mean normalized root mean squared errors of the predictions ranged between 16–18% in the population. Variability in model genotypic parameters allowed us to explore diverse genetic strategies in response to WD. An interesting group of genotypes could be discriminated in which (i) the low loss of fresh mass under WD was associated with high active uptake of sugars and low value of the maximum cell wall extensibility, and (ii) the high dry matter content in control treatment (C) was associated with a slow decrease of mass flow. Using 501 SNP markers genotyped across the genome, a QTL analysis of model parameters allowed to detect three main QTLs related to xylem and phloem conductivities, on chromosomes 2, 4, and 8. The model was then applied to design ideotypes with high dry matter content in C condition and low fresh mass loss in WD condition. The ideotypes outperformed the RILs especially for large and medium fruit-size genotypes, by combining high pedicel conductance and high active uptake of sugars. Interestingly, five small fruit-size RILs were close to the selected ideotypes, and likely bear interesting traits and alleles for adaptation to WD. Drought stress is a major abiotic stress threatening plant and crop productivity. In case of fleshy fruits, understanding mechanisms governing water and carbon accumulations and identifying genes, QTLs and phenotypes, that will enable trade-offs between fruit growth and quality under Water Deficit (WD) condition is a crucial challenge for breeders and growers. In the present work, 117 recombinant inbred lines of a population ofSolanum lycopersicum were phenotyped under control and WD conditions. Plant water status, fruit growth and composition were measured and data were used to calibrate a process-based model describing water and carbon fluxes in a growing fruit as a function of plant and environment. Eight genotype-dependent model parameters were estimated using a multiobjective evolutionary algorithm in order to minimize the prediction errors of fruit dry and fresh mass throughout fruit development. WD increased the fruit dry matter content (up to 85%) and decreased its fresh weight (up to 60%), big fruit size genotypes being the most sensitive. The mean normalized root mean squared errors of the predictions ranged between 16–18% in the population. Variability in model genotypic parameters allowed us to explore diverse genetic strategies in response to WD. An interesting group of genotypes could be discriminated in which (i) the low loss of fresh mass under WD was associated with high active uptake of sugars and low value of the maximum cell wall extensibility, and (ii) the high dry matter content in control treatment (C)was associated with a slow decrease of mass flow. Using 501 SNP markers genotyped across the genome, a QTL analysis of model parameters allowed to detect three main QTLs related to xylem and phloem conductivities, on chromosomes 2, 4, and 8. The model was then applied to design ideotypes with high dry matter content in C condition and low fresh mass loss in WD condition. The ideotypes outperformed the RILs especially for large and medium fruit-size genotypes, by combining high pedicel conductance and high active uptake of sugars. Interestingly, five small fruit-size RILs were close to theselected ideotypes, and likely bear interesting traits and alleles for adaptation to WD.

UMR BFP - Equipe OrFE; Functional characterization of IMA/MIF2, a new adaptor peptide involved in floral development. 12. Solanaceae Conference SOL 2015

UMR BFP - Equipe GFDF; The glycero-3-phosphate acyl transferase GPAT6-like from tomato plays a crucial role in fruitcutin biosynthesis. PaDiBa 2015. International Symposium on Plant Apoplastic Diffusion Barriers

A multi-scale pipeline for reproducible analyses of tomato leaf expansion and its plasticity in response to drought. 10. International Symposium on Modelling in Fruit Research and Orchard Management

Absolute quantitative metabolomics of plant extracts by fast 2D NMR. 56. Experimental Nuclear Magnetic Resonance Conference

Fruit growth in tomato: how important is the endoreduplication process?. Plant Organ Growth Symposium

UMR BFP - Equipe Métabolisme; Assessment of metabolome compartmentation changes during tomato leaf development using nonaqueousfractionation. 12. Solanaceae Conference SOL 2015

International audience

Combining MS and proton NMR metabolomic profiling during tomato fruit development to studyenvironment effect on metabolism. 12. Solanaceae Conference SOL 2015

Consumer perception and industrial processing of fleshy fruits greatly depend on the morphology and spatial organization of cells within the tissues. To study the relations between the structure and the mechanical properties of fleshy fruits, it is necessary to quantify their cellular morphology. A generic approach is proposed to build cartographies of cellular morphology at the fruit scale, depicting regions corresponding to different cell morphologies. The approach is based on sampling the whole fruit at known positions, imaging and quantifying the local cellular morphology, pooling measurements to take into account the biological variability and projecting results in a 3D model of the whole fruit. The result is a synthetic representation of cell morphology variations within the whole fruit. Tomato pericarp was used as a model for fleshy fruits. Two different scales were investigated through 3D imaging using confocal microscopy, and macroscopy imaging of pericarp slices. The morphology of the tomato pericarp was assessed (1) from 3D microscopy images through stereological estimation of cell wall surface area and (2) from greyscale granulometry computed with mathematical morphology. Spatial heterogeneity within pericarp was described by measuring the morphology for different regions of interest within the whole fruit and for each characterisation method. This resulted in cartography of the cellular structure at the scale of the fruit; either by cell wall surface area estimate or by grey level mean size. Both methods presented complementary results, and made it possible to compare results obtained at different scales.

International audience; Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

BackgroundAcidity is an essential component of the organoleptic quality of fleshy fruits. However, in these fruits, the physiological and molecular mechanisms that control fruit acidity remain unclear. In peach theDlocus controls fruit acidity; low-acidity is determined by the dominant allele. Using a peach progeny of 208 F2trees, theDlocus was mapped to the proximal end of linkage group 5 and co-localized with major QTLs involved in the control of fruit pH, titratable acidity and organic acid concentration and small QTLs for sugar concentration. To investigate the molecular basis of fruit acidity in peach we initiated the map-based cloning of theDlocus.ResultsIn order to generate a high-resolution linkage map in the vicinity of theDlocus, 1,024 AFLP primer combinations were screened using DNA of bulked acid and low-acid segregants. We also screened a segregating population of 1,718 individuals for chromosomal recombination events linked to theDlocus and identified 308 individuals with recombination events close toD. Using these recombinant individuals we delimited theDlocus to a genetic interval of 0.4 cM. We also constructed a peach BAC library of 52,000 clones with a mean insert size of 90 kb. The screening of the BAC library with markers tightly linked toDlocus indicated that 1 cM corresponds to 250 kb at the vicinity of theDlocus.ConclusionIn the present work we presented the first high-resolution genetic map ofDlocus in peach. We also constructed a peach BAC library of approximately 15× genome equivalent. This fine genetic and physical characterization of theDlocus is the first step towards the isolation of the gene(s) underlying fruit acidity in peach.