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This study evaluates the postharvest performance and responses of the red-fleshed Kirkwood Navel and Ruby Valencia oranges to cold storage (28 d at 2 °C) and shelf-life (5 d at 20 °C) in comparison with the standard varieties Washington Navel and Valencia late, respectively. Fruit of Kirkwood and Ruby exhibited lower chilling injury than the reference varieties while maintained similar weight loss and peel firmness. Moreover, the large concentration and the original composition of carotenoids in the pulp of Kirkwood and Ruby was preserved during the postharvest storage. Similarly, the content of vitamin C, phenolics and flavonoids, and the higher lipophilic antioxidant capacity and the capacity to quench singlet oxygen of the red-fleshed oranges remained unaltered after the whole storage period. The content of other compounds as tocopherols and sugars increased at the end of the storage period in fruit of the red-fleshed varieties. Monoterpenes, norisoprenoids, ethanol, aldehydes and ethyl esters were the compounds that exhibited the major changes during the postharvest storage, especially in the red-fleshed varieties. The profile of volatile compounds of Kirkwood and Ruby fruit was characterized by higher levels of norisoprenoids, monoterpenes, long-chain aldehydes, and variable content of aliphatic esters compared to the reference varieties. Altogether, our results indicated that Kirkwood Navel and Ruby Valencia oranges show good postharvest performance and potential for long-term cold storage, ensuring their quality and retaining their enhanced properties and added value to consumers. Data availability: All data are available in the manuscript or supplementary materials.

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild-type levels), Valine (ninefold) and Isoleucine (threefold) and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

[EN] The invention provides methods and materials for recombinantly producing apocarotenoids from transgenic plants, and more particularly, tomato and potato plants, expressing heterologous genes from Crocus sativus, the saffron plant, under the control of specific promoters. Moreover, the present disclosure also relates to the genetic constructs comprising the heterologous genes, and the use thereof for the expression in plants and to a method for obtaining transgenic plants which express the genetic construct mentioned above, and which have a high concentration of apocarotenoids products, in particular crocins and picrocrocins. Furthermore, the present disclosure also refers to the food products obtained from such transgenic plants, nutraceutical and pharmaceutical compositions comprising thereof and the use of such compositions as medicine, preferably for the treatment of neurodegenerative diseases such as Alzheimer's disease., [FR] L'invention concerne des procédés et des matériaux pour produire par recombinaison des apocaroténoïdes à partir de plantes transgéniques, et plus particulièrement, des plants de tomate et de pomme de terre, exprimant des gènes hétérologues à partir de Crocus sativus, le plant de safran, sous le contrôle de promoteurs spécifiques. De plus, la présente invention concerne également les constructions génétiques comprenant les gènes hétérologues, et leur utilisation pour l'expression dans des plantes et un procédé d'obtention de plantes transgéniques qui expriment la construction génétique mentionnée ci-dessus, et qui ont une concentration élevée en produits apocaroténoïdes, en particulier des crocines et des picrocrocines. En outre, la présente invention concerne également les produits alimentaires obtenus à partir de telles plantes transgéniques, des compositions nutraceutiques et pharmaceutiques les comprenant et l'utilisation de telles compositions en tant que médicament, de préférence pour le traitement de maladies neurodégénératives telles que la maladie d'Alzheimer.

Woodland strawberry (Fragaria vesca subsp. vesca) is a wild relative of cultivated strawberry (F. × ananassa) producing small and typically conical fruits with an intense flavor and aroma. The wild strawberry species, F. vesca, is a rich resource of genetic and metabolic variability, but its diversity remains largely unexplored and unexploited. In this study, we aim for an in-depth characterization of the fruit complex volatilome by GC–MS as well as the fruit size and shape using a European germplasm collection that represents the continental diversity of the species. We report characteristic volatilome footprints and fruit phenotypes of specific geographical areas. Thus, this study uncovers phenotypic variation linked to geographical distribution that will be valuable for further genetic studies to identify candidate genes or develop markers linked to volatile compounds or fruit shape and size traits.

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. Here we report on the evaluation of the largest traditional tomato collection, which includes 1499 accessions from Southern Europe. Analyses of 70 traits revealed a broad range of phenotypic variability with different distributions among countries, with the culinary end use within each country being the main driver of tomato diversification. Furthermore, eight main tomato types (phenoclusters) were defined by integrating phenotypic data, country of origin, and end use. Genome-wide association study (GWAS) meta-analyses identified associations in 211 loci, 159 of which were novel. The multidimensional integration of phenoclusters and the GWAS meta-analysis identified the molecular signatures for each traditional tomato type and indicated that signatures originated from differential combinations of loci, which in some cases converged in the same tomato phenotype. Our results provide a roadmap for studying and exploiting this untapped tomato diversity., We thank Universitat Illes Balears, the Greek Gene Bank (GGB-NAGREF), Università degli Studi Mediterranea Reggio Calabria, the CRB-Leg (INRA-GAFL)”, the Genebank of CNR-IBBR (Bari, Italy) and ARCA 2010 for seed sharing. CNR-IBBR also acknowledges the seed donors, the Leibniz Institute of Plant Genetics and Crop Plant Research, Maria Cristina Patané (CNR-IBE, Catania, Italy) and La Semiorto Sementi SRL, as well as Mrs. Roberta Nurcato for technical assistance. IBMCP-UPV acknowledges Maurizio CWe thank Universitat Illes Balears, the Greek Gene Bank (GGB-NAGREF), Università degli Studi Mediterranea Reggio Calabria, the CRB-Leg (INRA-GAFL)”, the Genebank of CNR-IBBR (Bari, Italy) and ARCA 2010 for seed sharing. CNR-IBBR also acknowledges the seed donors, the Leibniz Institute of Plant Genetics and Crop Plant Research, Maria Cristina Patané (CNR-IBE, Catania, Italy) and La Semiorto Sementi SRL, as well as Mrs. Roberta Nurcato for technical assistance. IBMCP-UPV acknowledges Maurizio Calduch (ALCALAX) for technical assistance and Mario Fon for English grammar editing. This work was supported by European Commission H2020 research and innovation program through TRADITOM grant agreement No.634561, G2P-SOL, grant agreement No. 677379, and HARNESSTOM grant agreement No. 101000716. Clara Pons and Mariola Plazas are grateful to Spanish Ministerio de Ciencia e Innovación for postdoctoral grants FJCI-2016-29118 and IJC2019-039091-I/AEI/10.13039/501100011033; Joan Casals to a Serra Húnter Fellow at Universistat Politècnica de Catalunya.alduch (ALCALAX) for technical assistance and Mario Fon for English grammar editing. This work was supported by European Commission H2020 research and innovation program through TRADITOM grant agreement No.634561, G2P-SOL, grant agreement No. 677379, and HARNESSTOM grant agreement No. 101000716. Clara Pons and Mariola Plazas are grateful to Spanish Ministerio de Ciencia e Innovación for postdoctoral grants FJCI-2016-29118 and IJC2019-039091-I/AEI/10., Postprint (published version)

The traditional tomato varieties known as de Penjar, Da Serbo, de Ramellet and Invernale, have been cultivated locally in Eastern Iberian Peninsula, Balearic Islands and Southern Italy with no watering in the hot and non-rainy summer conditions of those regions. Fruit of those varieties have a longer shelf life (LSL) and maintain quality characteristics without refrigeration for up to 5-12 months after ripening. Thirty hundred sixty-seven accessions collected in these regions in the frame of TRADITOM project were genotyped by Genotyping-By-Sequencing. Analysis of genetic fixation index (FST) among pairwise sub-populations reveal genetic signatures differentiating each LSL type. A core collection was developed to capture as much of the genotypic, phenotypic diversity and geographic distribution of the collection. This collection was used to study the effect of water stress on ripening and postharvest in different locations. To get some insights underlying molecular mechanisms we 1) phenotypes the core collection during ripening and postharvest up 6 months post-harvest, 2) analyzed the expression levels of key genes involved in the different aspects of ripening and post-harvest and 3) measure metabolites related with flavor and aroma highlights. Our results reveal the existence of substantial cultivar-to-cultivar variation in the ripening and postharvest evolution of the considered traits according to maturity stage at harvest and in response to water stress, Postprint (published version)

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. Here we report on the evaluation of the largest traditional tomato collection, which includes 1499 accessions from Southern Europe. Analyses of 70 traits revealed a broad range of phenotypic variability with different distributions among countries, with the culinary end use within each country being the main driver of tomato diversification. Furthermore, eight main tomato types (phenoclusters) were defined by integrating phenotypic data, country of origin, and end use. Genome-wide association study (GWAS) meta-analyses identified associations in 211 loci, 159 of which were novel. The multidimensional integration of phenoclusters and the GWAS meta-analysis identified the molecular signatures for each traditional tomato type and indicated that signatures originated from differential combinations of loci, which in some cases converged in the same tomato phenotype. Our results provide a roadmap for studying and exploiting this untapped tomato diversity.

Flavour and nutritional quality are important goals for tomato breeders. This study aimed to shed light upon transgressive behaviors for fruit metabolic content. We studied the metabolic contents of 44 volatile organic compounds (VOCs), 18 polyphenolics, together with transcriptome profiles in a factorial design comprising six parental lines and their 14 F1 hybrids (HF1) among which were five pairs of reciprocal HF1. After cluster analyses of the metabolome dataset and co-expression network construction of the transcriptome dataset, we characterized the mode of inheritance of each component. Both overall and per-cross mode of inheritance analyses revealed as many additive and non-additive modes of inheritance with few reciprocal effects. Up to 66% of metabolites displayed transgressions in a HF1 relative to parental values. Analysis of the modes of inheritance of metabolites revealed that: (i) transgressions were mostly of a single type whichever the cross and poorly correlated to the genetic distance between parental lines; (ii) modes of inheritance were scarcely consistent between the 14 crosses but metabolites belonging to the same cluster displayed similar modes of inheritance for a given cross. Integrating metabolome, transcriptome and modes of inheritance analyses suggested a few candidate genes that may drive important changes in fruit VOC contents.

A comprehensive collection of 1254 tomato accessions, corresponding to European traditional and modern varieties, early domesticated varieties, and wild relatives, was analyzed by genotyping by sequencing. A continuous genetic gradient between the traditional and modern varieties was observed. European traditional tomatoes displayed very low genetic diversity, with only 298 polymorphic loci (95% threshold) out of 64 943 total variants. European traditional tomatoes could be classified into several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed higher genetic diversity than the remaining varieties, suggesting that these regions might be independent secondary centers of diversity with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a genome-wide association study with fruit morphological traits in the European traditional collection. The corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise a low-diversity population suggests a history of balancing selection, in which tomato farmers likely maintained the morphological variation by inadvertently applying a high selective pressure within different varietal types.

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing ta

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. To unveil the molecular basis of Southern European tomato phenotypic diversity, we carried out the most comprehensive phenotypic and genomic variability analysis on traditional European tomato by using 1) a collection of 1,499 traditional European tomatoes (TRADITOM collection) and 2) a multipurpose core collection (TCC), comprising 227 European traditional tomato accessions from the original TRADITOM collection, that captured most of genotypic and phenotypic variation and geographical origin present in traditional tomato, Postprint (published version)

A comprehensive collection of 1254 tomato accessions, corresponding to European traditional and modern varieties, early domesticated varieties, and wild relatives, was analyzed by genotyping by sequencing. A continuous genetic gradient between the traditional and modern varieties was observed. European traditional tomatoes displayed very low genetic diversity, with only 298 polymorphic loci (95% threshold) out of 64 943 total variants. European traditional tomatoes could be classified into several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed higher genetic diversity than the remaining varieties, suggesting that these regions might be independent secondary centers of diversity with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a genome-wide association study with fruit morphological traits in the European traditional collection. The corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise a low-diversity population suggests a history of balancing selection, in which tomato farmers likely maintained the morphological variation by inadvertently applying a high selective pressure within different varietal types., This work was supported by the European Commission H2020 research and innovation program through TRADITOM grant agreement no. 634561, G2P-SOL, grant agreement no. 677379, and HARNESSTOM grant agreement no. 101000716. MP is grateful to the Spanish Ministerio de Ciencia e Innovación for a postdoctoral grant (IJC2019-039091-I/AEI/10.13039/501100011033)., Postprint (published version)

La presente invención se refiere al uso de los ésteres acetato, propionato y butirato de (Z)-3-hexenüo, o de una composición que comprende dichos compuestos, para proteger plantas, en particular de cultivos agrícolas, frente a plagas, mediante la estimulación de los mecanismos de defensa que tienen las propias plantas. La invención también se refiere a un método para proteger plantas frente a plagas poniendo los mencionados ésteres en contacto con las plantas. [ES], The present invention relates to the use of the esters acetate, propionate and (Z)-3-hexenyl butyrate, or of a composition comprising these compounds, to protect plants, in particular agricultural crops, against pests, by stimulating the defense mechanisms of the plants themselves. The invention also relates to a method for protecting plants against pests by bringing said esters into contact with the plants. [EN]

Tomato flavour is an important goal for breeders. Volatile organic compounds (VOCs) are major determinants of tomato flavour. Although most tomato varieties for fresh market are F1 hybrids, most studies on the genetic control of flavour-related traits are performed on lines. We quantified 46 VOCs in a panel of 121 small fruited lines and in a test cross panel of 165 hybrids (the previous panel plus 44 elite cherry tomato lines crossed with a common line). High and consistent heritabilities were assessed for most VOCs in the two panels, and 65% of VOC contents were strongly correlated between lines and hybrids. Additivity was observed for most VOCs. We performed genome wide association studies (GWAS) on the two panels separately, along with a third GWAS on the test cross subset carrying only F1 hybrids corresponding to the line panel. We identified 205, 183 and 138 associations, respectively. We identified numerous overlapping associations for VOCs belonging to the same metabolic pathway within each panel; we focused on seven chromosome regions with clusters of associations simultaneously involved in several key VOCs for tomato aroma. The study highlighted the benefit of testcross panels to create tasty F1 hybrid varieties.

Chemical characterization of clementine varieties (Citrus clementina Hort. ex Tan.) essential oils (EO) can lead to variety identification and valorization of their potential use in food and aroma industries. The goal of this study was the chemometric discrimination between two very closely related and morphologically identical clementine varieties, Clemenules (NL) and Clemen-pons (PO), based on their rind EO, to identify the differential volatile organic compounds (VOCs) and to determine their antioxidant capacity. EO rind volatile profile was determined by gas chro-matography coupled to mass spectrometry in Citrus fruit at different ripening stages grown two independent years in two different locations. Untargeted metabolomics and multivariate data analysis showed an evolution of EO volatile profiles markedly parallel in both varieties. Although EO qualitative composition was identical in both varieties, PLS‐DA allowed the identification of char-acteristic VOCs, quantitatively discriminating them along all the ripening process. PO showed higher accumulation of several mono‐ and sesquiterpene compounds such as trans‐carveol, while NL showed higher levels of aldehyde and alcohol non‐terpenoids like dodecanal. Both varieties evinced identical EO antioxidant activities, indicating a similar value for food preservation. Hence, untargeted metabolomics approach based on rind EO volatiles was revealed as a powerful tech-nique able to differentiate between morphologically undistinguishable Citrus varieties.

Soil flooding is a compound abiotic stress that alters soil properties and limits atmospheric gas diffusion (O2 and CO2) to the roots. The involvement of abscisic acid (ABA) in the regulation of soil flooding-specific genetic and metabolic responses has been scarcely studied despite its key importance as regulator in other abiotic stress conditions. To attain this objective, wild type and ABA-deficient tomatoes were subjected to short-term (24 h) soil waterlogging. After this period, gas exchange parameters were reduced in the wild type but not in ABA-deficient plants that always had higher E and gs. Transcript and metabolite alterations were more intense in waterlogged tissues, with genotype-specific variations. Waterlogging reduced the ABA levels in the roots while inducing PYR/PYL/RCAR ABA receptors and ABA-dependent transcription factor transcripts, of which induction was less pronounced in the ABA-deficient genotype. Ethylene/O2-dependent genetic responses (ERFVIIs, plant anoxia survival responses, and genes involved in the N-degron pathway) were induced in hypoxic tissues independently of the genotype. Interestingly, genes encoding a nitrate reductase and a phytoglobin involved in NO biosynthesis and scavenging and ERFVII stability were induced in waterlogged tissues, but to a lower extent in ABA-deficient tomato. At the metabolic level, flooding-induced accumulation of Ala was enhanced in ABA-deficient lines following a differential accumulation of Glu and Asp in both hypoxic and aerated tissues, supporting their involvement as sources of oxalacetate to feed the tricarboxylic acid cycle in waterlogged tissues and constituting a potential advantage upon long periods of soil waterlogging. The promoter analysis of upregulated genes indicated that the production of oxalacetate from Asp via Asp oxidase, energy processes such as acetyl-CoA, ATP, and starch biosynthesis, and the lignification process were likely subjected to ABA regulation. Taken together, these dat