Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production

[EN] Volatile compounds are the major determinants of aroma and flavor in both grapes and wine. In this study, we investigated the emission of volatile and non-volatile compounds during berry maturation in two grape varieties (Airen and Tempranillo) throughout 2010 and 2011. HS-SPME coupled to gas chromatography and mass spectrometry was applied for the identification and relative quantitation of these compounds. Principal component analysis was performed to search for variability between the two cultivars and evolution during 10 developmental stages. Results showed that there are distinct differences in volatile compounds between cultivars throughout fruit development. Early stages were characterized in both cultivars by higher levels of some apocarotenoids such as beta-cyclocitral or beta-ionone, terpenoids (E)-linalool oxide and (Z)-linalool oxide and several furans, while the final stages were characterized by the highest amounts of ethanol, benzenoid phenylacetaldehyde and 2-phenylethanol, branched-amino acid-derived 3-methylbutanol and 2-methylbutanol, and a large number of lipid derivatives. Additionally, we measured the levels of the different classes of volatile precursors by using liquid chromatography coupled to high resolution mass spectrometry. In both varieties, higher levels of carotenoid compounds were detected in the earlier stages, zeaxanthin and alpha-carotene were only detected in Airen while neoxanthin was found only in Tempranillo; more variable trends were observed in the case of the other volatile precursors. Furthermore, we monitored the expression of homolog genes of a set of transcripts potentially involved in the biosynthesis of these metabolites, such as some glycosyl hydrolases family 1, lipoxygenases, alcohol dehydrogenases hydroperoxide lyases, O-methyltransferases and carotenoid cleavage dioxygenases during the defined developmental stages. Finally, based on Pearson correlation analyses, we explored the metabolite-metabolite fluctuations within VOCs/precursors during the berry development; as well as tentatively linking the formation of some metabolites detected to the expression of some of these genes. Our data showed that the two varieties displayed a very different pattern of relationships regarding the precursor/volatile metabolite-metabolite fluctuations, being the lipid and the carotenoid metabolism the most distinctive between the two varieties. Correlation analysis showed a higher degree of overall correlation in precursor/volatile metabolite-metabolite levels in Airen, confirming the enriched aroma bouquet characteristic of the white varieties.We thank J. Argandona (Institute Botanico, Universidad de Castilla-La Mancha, Albacete, Spain) for excellent technical support, and K.A. Walsh for language revision. This work was supported by the "Junta de comunidades de Castilla-La Mancha" (JCCM) [PPII10-0062-7718] and benefited from the networking activities within the European Cooperation in Science and Technology Action CA15136 (EUROCAROTEN). GD was supported by short-term fellowships of the Quality Fruit (FA1106) European Cooperation in Science and Technology actions. OA was funded by FPCYTCLM through the INCRECYT Programme.Rambla Nebot, JL.; Trapero-Mozos, A.; Diretto, G.; Rubio-Moraga, A.; Granell Richart, A.; Gomez-Gomez, L.; Ahrazem, O. (2016). Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production. Frontiers in Plant Science. 7(1619):1-23. https://doi.org/10.3389/fpls.2016.01619S1237161

Melon Genetic Resources Characterization for Rind Volatile Profile

[EN] A melon core collection was analyzed for rind volatile compounds as, despite the fact that they are scarcely studied, these compounds play an important role in consumer preferences. Gas chromatography coupled to mass spectrometry allowed the detection of 171 volatiles. The high volatile diversity found was analyzed by Hierarchical Cluster Analysis (HCA), giving rise to two major clusters of accessions. The first cluster included climacteric and aromatic types such as Cantalupensis, Ameri, Dudaim and Momordica, rich in esters; the second one mainly included non-climacteric non-aromatic types such as Inodorus, Flexuosus, Acidulus, Conomon and wild Agrestis, with low volatiles content, specifically affecting esters. Many interesting accessions were identified, with different combinations of aroma profiles for rind and flesh, such as Spanish Inodorus landraces with low aroma flesh but rind levels of esters similar to those in climacteric Cantalupensis, exotic accessions sharing high contents of specific compounds responsible for the unique aroma of Dudaim melons or wild Agrestis with unexpected high content of some esters. Sesquiterpenes were present in rinds of some Asian Ameri and Momordica landraces, and discriminate groups of cultivars (sesquiterpene-rich/-poor) within each of the two most commercial melon horticultural groups (Cantalupensis and Inodorus), suggesting that the Asian germplasm is in the origin of specific current varieties or that this feature has been introgressed more recently from Asian sources. This rind characterization will encourage future efforts for breeding melon quality as many of the characterized landraces and wild accessions have been underexploited.This work was supported by ERA-PG project (MELRIP: GEN2006-27773-C2-2-E), Plant KBBE project (SAFQIM: PIM2010PKB-00691), Ministerio de Economia y Competitividad AGL2014-53398-C2-2-R (jointly funded by FEDER), Ministerio de Ciencia, Innovacion y Universidades, cofunded with FEDER funds (Project No. AGL2017-85563-C2-1-R), by PROMETEO project 2017/078 (to promote excellence groups) by the Conselleria d'Educacio, Investigacio, Cultura i Esports (Generalitat Valenciana) and partly by GV/2020/025 by the Conselleria de Innovacion, Universidades, Ciencia y Sociedad digital. J.L. Rambla is supported by the Spanish Ministry of Economy and Competitiveness through a "Juan de la Cierva-Formacion" grant (FJCI-2016-28601).Esteras Gómez, C.; Rambla Nebot, JL.; Sánchez, G.; Granell Richart, A.; Picó Sirvent, MB. (2020). Melon Genetic Resources Characterization for Rind Volatile Profile. Agronomy. 10:1-18. https://doi.org/10.3390/agronomy10101512S11810Burger, Y., Sa’ar, U., Paris, H., Lewinsohn, E., Katzir, N., Tadmor, Y., & Schaffer, A. (2006). Genetic variability for valuable fruit quality traits in Cucumis melo. Israel Journal of Plant Sciences, 54(3), 233-242. doi:10.1560/ijps_54_3_233Moing, A., Allwood, J. W., Aharoni, A., Baker, J., Beale, M. H., Ben-Dor, S., … Schaffer, A. A. (2020). Comparative Metabolomics and Molecular Phylogenetics of Melon (Cucumis melo, Cucurbitaceae) Biodiversity. Metabolites, 10(3), 121. doi:10.3390/metabo10030121Nee, M., & Kirkbride, J. H. (1994). Biosystematic Monograph of the Genus Cucumis (Cucurbitaceae)-Botanical Identification of Cucumbers and Melons. Bulletin of the Torrey Botanical Club, 121(3), 300. doi:10.2307/2997187Bernillon, S., Biais, B., Deborde, C., Maucourt, M., Cabasson, C., Gibon, Y., … Moing, A. (2012). Metabolomic and elemental profiling of melon fruit quality as affected by genotype and environment. Metabolomics, 9(1), 57-77. doi:10.1007/s11306-012-0429-1Aubert, C., & Bourger, N. (2004). Investigation of Volatiles in Charentais Cantaloupe Melons (Cucumis melo Var. cantalupensis). Characterization of Aroma Constituents in Some Cultivars. Journal of Agricultural and Food Chemistry, 52(14), 4522-4528. doi:10.1021/jf049777sObando-Ulloa, J. M., Ruiz, J., Monforte, A. J., & Fernández-Trujillo, J. P. (2010). Aroma profile of a collection of near-isogenic lines of melon (Cucumis melo L.). Food Chemistry, 118(3), 815-822. doi:10.1016/j.foodchem.2009.05.068Verzera, A., Dima, G., Tripodi, G., Ziino, M., Lanza, C. M., & Mazzaglia, A. (2010). Fast Quantitative Determination of Aroma Volatile Constituents in Melon Fruits by Headspace–Solid-Phase Microextraction and Gas Chromatography–Mass Spectrometry. 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The Plant Journal, 74(3), 458-472. doi:10.1111/tpj.12149Freilich, S., Lev, S., Gonda, I., Reuveni, E., Portnoy, V., Oren, E., … Katzir, N. (2015). Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits. BMC Plant Biology, 15(1). doi:10.1186/s12870-015-0449-xGonda, I., Davidovich-Rikanati, R., Bar, E., Lev, S., Jhirad, P., Meshulam, Y., … Lewinsohn, E. (2018). Differential metabolism of L–phenylalanine in the formation of aromatic volatiles in melon (Cucumis melo L.) fruit. Phytochemistry, 148, 122-131. doi:10.1016/j.phytochem.2017.12.018Galpaz, N., Gonda, I., Shem‐Tov, D., Barad, O., Tzuri, G., Lev, S., … Katzir, N. (2018). Deciphering genetic factors that determine melon fruit‐quality traits using RNA ‐Seq‐based high‐resolution QTL and eQTL mapping. The Plant Journal, 94(1), 169-191. doi:10.1111/tpj.13838Feder, A., Jiao, C., Galpaz, N., Vrebalov, J., Xu, Y., Portnoy, V., … Giovannoni, J. J. (2020). Melon ethylene-mediated transcriptome and methylome dynamics provide insights to volatile production. doi:10.1101/2020.01.28.923284El-Sharkawy, I., Manríquez, D., Flores, F. B., Regad, F., Bouzayen, M., Latché, A., & Pech, J.-C. (2005). Functional Characterization of a Melon Alcohol Acyl-transferase Gene Family Involved in the Biosynthesis of Ester Volatiles. Identification of the Crucial Role of a Threonine Residue for Enzyme Activity*. Plant Molecular Biology, 59(2), 345-362. doi:10.1007/s11103-005-8884-yPerry, P. L., Wang, Y., & Lin, J. (2009). Analysis of honeydew melon (Cucumis melovar.inodorus) flavour and GC-MS/MS identification of (E,Z)-2,6-nonadienyl acetate. Flavour and Fragrance Journal, 24(6), 341-347. doi:10.1002/ffj.1947Rodríguez-Pérez, C., Quirantes-Piné, R., Fernández-Gutiérrez, A., & Segura-Carretero, A. (2013). Comparative characterization of phenolic and other polar compounds in Spanish melon cultivars by using high-performance liquid chromatography coupled to electrospray ionization quadrupole-time of flight mass spectrometry. Food Research International, 54(2), 1519-1527. doi:10.1016/j.foodres.2013.09.011Allwood, J. W., Cheung, W., Xu, Y., Mumm, R., De Vos, R. C. H., Deborde, C., … Goodacre, R. (2014). Metabolomics in melon: A new opportunity for aroma analysis. Phytochemistry, 99, 61-72. doi:10.1016/j.phytochem.2013.12.010Portnoy, V., Benyamini, Y., Bar, E., Harel-Beja, R., Gepstein, S., Giovannoni, J. J., … Katzir, N. (2008). The molecular and biochemical basis for varietal variation in sesquiterpene content in melon (Cucumis melo L.) rinds. Plant Molecular Biology, 66(6), 647-661. doi:10.1007/s11103-008-9296-6Esteras, C., Formisano, G., Roig, C., Díaz, A., Blanca, J., Garcia-Mas, J., … Picó, B. (2013). SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theoretical and Applied Genetics, 126(5), 1285-1303. doi:10.1007/s00122-013-2053-5Leida, C., Moser, C., Esteras, C., Sulpice, R., Lunn, J. E., de Langen, F., … Picó, B. (2015). Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.). BMC Genetics, 16(1). doi:10.1186/s12863-015-0183-2Sánchez, G., Martínez, J., Romeu, J., García, J., Monforte, A. J., Badenes, M. L., & Granell, A. (2014). The peach volatilome modularity is reflected at the genetic and environmental response levels in a QTL mapping population. BMC Plant Biology, 14(1), 137. doi:10.1186/1471-2229-14-137Sánchez, G., Besada, C., Badenes, M. L., Monforte, A. J., & Granell, A. (2012). A Non-Targeted Approach Unravels the Volatile Network in Peach Fruit. PLoS ONE, 7(6), e38992. doi:10.1371/journal.pone.0038992Zorrilla-Fontanesi, Y., Rambla, J.-L., Cabeza, A., Medina, J. J., Sánchez-Sevilla, J. F., Valpuesta, V., … Amaya, I. (2012). Genetic Analysis of Strawberry Fruit Aroma and Identification of O-Methyltransferase FaOMT as the Locus Controlling Natural Variation in Mesifurane Content      . Plant Physiology, 159(2), 851-870. doi:10.1104/pp.111.188318Rambla, J. L., Medina, A., Fernández-del-Carmen, A., Barrantes, W., Grandillo, S., Cammareri, M., … Granell, A. (2016). Identification, introgression, and validation of fruit volatile QTLs from a red-fruited wild tomato species. Journal of Experimental Botany, erw455. doi:10.1093/jxb/erw455Verzera, A., Dima, G., Tripodi, G., Condurso, C., Crinò, P., Romano, D., … Paratore, A. (2014). Aroma and sensory quality of honeydew melon fruits (Cucumis melo L. subsp. melo var. inodorus H. Jacq.) in relation to different rootstocks. Scientia Horticulturae, 169, 118-124. doi:10.1016/j.scienta.2014.02.008López, C., Ferriol, M., & Picó, M. B. (2015). Mechanical transmission of Tomato leaf curl New Delhi virus to cucurbit germplasm: selection of tolerance sources in Cucumis melo. Euphytica, 204(3), 679-691. doi:10.1007/s10681-015-1371-xSharon-Asa, L., Shalit, M., Frydman, A., Bar, E., Holland, D., Or, E., … Eyal, Y. (2003). Citrus fruit flavor and aroma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1 , a key gene in the production of the sesquiterpene aroma compound valencene. The Plant Journal, 36(5), 664-674. doi:10.1046/j.1365-313x.2003.01910.xPechous, S. W., & Whitaker, B. D. (2004). Cloning and functional expression of an ( E , E )-a-farnesene synthase cDNA from peel tissue of apple fruit. Planta, 219(1), 84-94. doi:10.1007/s00425-003-1191-4MARUYAMA, T., ITO, M., & HONDA, G. (2001). Molecular Cloning, Functional Expression and Characterization of (E)-.BETA.-Farnesene Synthase from Citrus junos. Biological and Pharmaceutical Bulletin, 24(10), 1171-1175. doi:10.1248/bpb.24.1171Lourenço, A. M., Haddi, K., Ribeiro, B. M., Corrêia, R. F. T., Tomé, H. V. V., Santos-Amaya, O., … Aguiar, R. W. S. (2018). Essential oil of Siparuna guianensis as an alternative tool for improved lepidopteran control and resistance management practices. Scientific Reports, 8(1). doi:10.1038/s41598-018-25721-0Monforte, A. J., Garcia-Mas, J., & Arus, P. (2003). Genetic variability in melon based on microsatellite variation. Plant Breeding, 122(2), 153-157. doi:10.1046/j.1439-0523.2003.00848.xBlanca, J., Esteras, C., Ziarsolo, P., Pérez, D., Fernández-Pedrosa, V., Collado, C., … Picó, B. (2012). Transcriptome sequencing for SNP discovery across Cucumis melo. BMC Genomics, 13(1). doi:10.1186/1471-2164-13-280Zhao, G., Lian, Q., Zhang, Z., Fu, Q., He, Y., Ma, S., … Huang, S. (2019). A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits. Nature Genetics, 51(11), 1607-1615. doi:10.1038/s41588-019-0522-8Gonzalo, M. J., Díaz, A., Dhillon, N. P. S., Reddy, U. K., Picó, B., & Monforte, A. J. (2019). Re-evaluation of the role of Indian germplasm as center of melon diversification based on genotyping-by-sequencing analysis. BMC Genomics, 20(1). doi:10.1186/s12864-019-5784-0Atkinson, R. G. (2016). Phenylpropenes: Occurrence, Distribution, and Biosynthesis in Fruit. Journal of Agricultural and Food Chemistry, 66(10), 2259-2272. doi:10.1021/acs.jafc.6b04696Castro, G., Perpiñá, G., Monforte, A. J., Picó, B., & Esteras, C. (2019). New melon introgression lines in a Piel de Sapo genetic background with desirable agronomical traits from dudaim melons. Euphytica, 215(10). doi:10.1007/s10681-019-2479-

Breeding Tomato Hybrids for Flavour: Comparison of GWAS Results Obtained on Lines and F1 Hybrids

[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.This research was funded by the CIFRE project Qualhytom, grant number 2018/1239, the ANR project TomEpiSet, grant number ANR-16-CE20-0014 and European Union's Horizon 2020 research and innovation programme, HARNESSTOM, grant number No. 101000716.Bineau, E.; Rambla Nebot, JL.; Priego-Cubero, S.; Hereil, A.; Bitton, F.; Plissonneau, C.; Granell Richart, A.... (2021). Breeding Tomato Hybrids for Flavour: Comparison of GWAS Results Obtained on Lines and F1 Hybrids. Genes. 12(9):1-20. https://doi.org/10.3390/genes12091443S12012

Eliciting tomato plant defenses by exposure to herbivore induced plant volatiles

[EN] When zoophytophagous mirids (Hemiptera: Miridae) feed on tomato plants they activate both direct and indirect defense mechanisms, which include the release of herbivore induced plant volatiles (HIPVs). HIPVs are capable of activating defense mechanisms in healthy neighboring plants. In this work, we investigated which of these mirid-induced HIPVs are responsible for inducing plant defenses. Healthy tomato plants were individually exposed to eight HIPVs [1-hexanol, (Z)-3-hexenol, (Z)-3-hexenyl acetate, (Z)-3-hexenyl propanoate, (Z)-3-hexenyl butanoate, hexyl butanoate, methyl jasmonate and methyl salicylate] for 24 hours. Then, the expression level of defensive genes was quantified. All HIPVs led to increased expression of defensive genes by the plant when compared to unexposed tomato plants. In a further step, (Z)-3-hexenyl propanoate and methyl salicylate were selected to study the response of four tomato key pests and one natural enemy to tomato plants previously exposed to both HIPVs relative to unexposed control plants. Plants previously exposed to both HIPVs were repellent to Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), attractive to the parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae) and indifferent to Tetranychus urticae Koch (Acari: Tetranychidae). The volatiles emitted by plants previously exposed to both selected volatiles were also determined. Increased levels of C5 and C6 fatty acid-derived volatile compounds and beta-ionone were detected, confirming that both HIPVs significantly activated the lipoxygenase pathway. These results are the starting point to advance the use of volatile compounds as defense elicitors in tomato crops.The research leading to these results was partially funded by the Spanish Ministry of Economy and Competitiveness MINECO (AGL2014-55616-C3 and RTA201700073-00-00) and the Conselleria d'Agricultura, Pesca i Alimentacio de la Generalitat Valenciana. The authors thank Dr. Alejandro Tena (IVIA) and Alice Mockford (University of Worcester) for helpful comments on earlier versions of the manuscript.Perez-Hedo, M.; Alonso-Valiente, M.; Vacas, S.; Gallego, C.; Rambla Nebot, JL.; Navarro-Llopis, V.; Granell Richart, A.... (2021). Eliciting tomato plant defenses by exposure to herbivore induced plant volatiles. Entomologia Generalis. 41(3):209-218. https://doi.org/10.1127/entomologia/2021/1196S20921841

Identification of ABA-Mediated Genetic and Metabolic Responses to Soil Flooding in Tomato (Solanum lycopersicum L. Mill)

[EN] Soil flooding is a compound abiotic stress that alters soil properties and limits atmospheric gas diffusion (O-2 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 g(s). 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/O-2-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 data indicate that ABA depletion in waterlogged tissues acts as a positive signal, inducing several specific genetic and metabolic responses to soil flooding.This work was supported by the Spanish Ministerio de Economia y Competitividad, Universitat Jaume I and Generalitat Valenciana/Fondo Europeo de Desarrollo Regional (FEDER), co-funded through grant nos. AGL2016-76574-R, UJI-B201623, UJI-B2016-24, IDIFEDER/2018/010, and UJI-B2019-24 to AG-C, VA, and MG-G, respectively. CD was supported by UJI PICD program. MG-G and JM were supported by Ramon y Cajal contracts from Spanish Ministerio de Economia y Competitividad (RYC-2016-19325 and RYC-201723645, respectively). JR was supported by a Juan de la CiervaFormacion contract from the Spanish Ministerio de Economia y Competitividad (FJCI-2016-28601).De Ollas, C.; González-Guzmán, M.; Pitarch, Z.; Matus, JT.; Candela, H.; Rambla Nebot, JL.; Granell Richart, A.... (2021). Identification of ABA-Mediated Genetic and Metabolic Responses to Soil Flooding in Tomato (Solanum lycopersicum L. Mill). Frontiers in Plant Science. 12:1-20. https://doi.org/10.3389/fpls.2021.613059S1201

A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae

[EN] Volatile organic compounds (VOCs) emitted by plants are secondary metabolites that mediate the plant interaction with pathogens and herbivores. These compounds may perform direct defensive functions, i. e., acting as antioxidant, antibacterial, or antifungal agents, or indirectly by signaling the activation of the plant's defensive responses. Using a non-targeted GC-MS metabolomics approach, we identified the profile of the VOCs associated with the differential immune response of the Rio Grande tomato leaves infected with either virulent or avirulent strains of Pseudomonas syringae DC3000 pv. tomato. The VOC profile of the tomato leaves infected with avirulent bacteria is characterized by esters of (Z)-3-hexenol with acetic, propionic, isobutyric or butyric acids, and several hydroxylated monoterpenes, e. g., linalool, a -terpineol, and 4-terpineol, which defines the profile of an immunized plant response. In contrast, the same tomato cultivar infected with the virulent bacteria strain produced a VOC profile characterized by monoterpenes and SA derivatives. Interestingly, the differential VOCs emission correlated statistically with the induction of the genes involved in their biosynthetic pathway. Our results extend plant defense system knowledge and suggest the possibility for generating plants engineered to over-produce these VOCs as a complementary strategy for resistance.This work was funded by Grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness.López-Gresa, MP.; Lisón, P.; Campos Beneyto, L.; Rodrigo Bravo, I.; Rambla Nebot, JL.; Granell Richart, A.; Conejero Tomás, V.... (2017). A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae. Frontiers in Plant Science. 8. doi:10.3389/fpls.2017.01188S

Fruit flesh volatile and carotenoid profile analysis within the Cucumis melo L. species reveals unexploited variability for future genetic breeding

[EN] BACKGROUNDAroma profile and carotenoids content of melon flesh are two important aspects influencing the quality of this fruit that have been characterized using only selected genotypes. However, the extant variability of the whole species remains unknown. RESULTSA complete view of the volatile/carotenoid profiles of melon flesh was obtained analyzing 71 accessions, representing the whole diversity of the species. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used to analyze 200 volatile compounds and five carotenoids. Genotypes were classified into two main clusters (high/low aroma), but with a large diversity of differential profiles within each cluster, consistent with the ripening behavior, flesh color and proposed evolutionary and breeding history of the different horticultural groups. CONCLUSIONOur results highlight the huge amount of untapped aroma diversity of melon germplasm, especially of non-commercial types. Also, landraces with high nutritional value with regard to carotenoids have been identified. All this knowledge will encourage melon breeding, facilitating the selection of the genetic resources more appropriate to develop cultivars with new aromatic profiles or to minimize the impact of breeding on melon quality. The newly characterized sources provide the basis for further investigations into specific genes/alleles contributing to melon flesh quality. (c) 2018 Society of Chemical IndustryWe would like to thank the metabolomics lab at the IBMCP for technical support. This work was supported by ERA-PG project (MELRIP: GEN2006-27773-C2-2-E), Plant KBBE project (SAFQIM: PIM2010PKB-00691), Accion Complementaria ACOMP/2012/173 and ACOMP/2013/141, and Ministerio de Economia y Competitividad AGL2014-53398-C2-2-R & AGL2010-20858 (jointly funded by FEDER).Esteras Gómez, C.; Rambla Nebot, JL.; Sánchez, G.; López-Gresa, MP.; González-Mas, M.; Fernández-Trujillo, J.; Belles Albert, JM.... (2018). Fruit flesh volatile and carotenoid profile analysis within the Cucumis melo L. species reveals unexploited variability for future genetic breeding. Journal of the Science of Food and Agriculture. 98(10):3915-3925. https://doi.org/10.1002/jsfa.8909S391539259810Pitrat, M. (2016). Melon Genetic Resources: Phenotypic Diversity and Horticultural Taxonomy. Plant Genetics and Genomics: Crops and Models, 25-60. doi:10.1007/7397_2016_10Pitrat, M. (s. f.). Melon. Vegetables I, 283-315. doi:10.1007/978-0-387-30443-4_9Esteras, C., Formisano, G., Roig, C., Díaz, A., Blanca, J., Garcia-Mas, J., … Picó, B. (2013). SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theoretical and Applied Genetics, 126(5), 1285-1303. doi:10.1007/s00122-013-2053-5Leida, C., Moser, C., Esteras, C., Sulpice, R., Lunn, J. E., de Langen, F., … Picó, B. (2015). 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Multitrait analysis of fresh-cut cantaloupe melon enables discrimination between storage times and temperatures and identifies potential markers for quality assessments. Food Chemistry, 241, 222-231. doi:10.1016/j.foodchem.2017.08.050Freilich, S., Lev, S., Gonda, I., Reuveni, E., Portnoy, V., Oren, E., … Katzir, N. (2015). Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits. BMC Plant Biology, 15(1). doi:10.1186/s12870-015-0449-xGranell, A., & Rambla, J. L. (2013). Biosynthesis of Volatile Compounds. The Molecular Biology and Biochemistry of Fruit Ripening, 135-161. doi:10.1002/9781118593714.ch6Gur, A., Gonda, I., Portnoy, V., Tzuri, G., Chayut, N., Cohen, S., … Katzir, N. (2016). Genomic Aspects of Melon Fruit Quality. Plant Genetics and Genomics: Crops and Models, 377-408. doi:10.1007/7397_2016_29Ibdah, M., Azulay, Y., Portnoy, V., Wasserman, B., Bar, E., Meir, A., … Katzir, N. (2006). 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European traditional tomatoes galore: a result of farmers' selection of a few diversity-rich loci

[EN] The high phenotypic diversity observed among European traditional tomato varieties was created by traditional farmer-driven selection by inadvertently combining a very few polymorphic loci subjected to balancing selection. 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 Innovacion for a postdoctoral grant (IJC2019-039091-I/AEI/10.13039/501100011033).Blanca Postigo, JM.; Pons Puig, C.; Montero-Pau, J.; Sánchez-Matarredona, D.; Ziarsolo, P.; Fontanet, L.; Fisher, J.... (2022). European traditional tomatoes galore: a result of farmers' selection of a few diversity-rich loci. Journal of Experimental Botany. 73(11):3431-3445. https://doi.org/10.1093/jxb/erac07234313445731

Atlas of phenotypic, genotypic and geographical diversity present in the European traditional tomato

[EN] 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), Universita 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 Patane (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 Innovacion for postdoctoral grants FJCI-2016-29118 and IJC2019-039091I/AEI/10.13039/501100011033; Joan Casals to a Serra Hunter Fellow at Universitat Politècnica de Catalunya.Pons Puig, C.; Casals, J.; Palombieri, S.; Fontanet, L.; Riccini, A.; Rambla Nebot, JL.; Ruggiero, A.... (2022). Atlas of phenotypic, genotypic and geographical diversity present in the European traditional tomato. Horticulture Research. 9:1-16. https://doi.org/10.1093/hr/uhac112116

GENES AND GENOMIC REGIONS RELATED TO THE PRODUCTION OF VOLATILE COMPOUNDS IN THE TOMATO FRUIT

Tesis por compendio[EN] Fruits both produce and emit volatile chemical compounds. These are short-chained low polarity molecules involved in many processes, and they are responsible of our perception of fruit aroma and of most of their flavour. This thesis is focused on the study of volatile compounds in the tomato fruit, which is one of the most important horticultural worldwide and is a model system for the study of fruit development and ripening. Some of the analytical methods more frequently used for the analysis of tomato fruit volatiles were systematically compared. Results revealed that the observed volatile profile is highly dependent on the precise analytical method used, both for sample processing and for the technique used for volatile acquisition. It was concluded that the method of election for the comparison of large sets of samples from a multi-omics approach consists on flash freezing the biological material with liquid nitrogen at the selected ripening stage and the use of headspace solid phase microextraction coupled to gas chromatography and mass spectrometry for the subsequent analysis. This method was implemented and was used for the determination of volatile compounds in selected NILs harbouring QTLs for characters related to flavour and aroma introgressed in different genetic backgrounds. The results allowed the association of several of the organoleptic characters previously identified with modified levels of several volatiles. It was also observed that the genetic background has a major effect on the production of such metabolites. Correlation analysis between the levels of volatiles and primary metabolites led to the conclusion that the production of volatile compounds is generally not determined by the levels of their precursors. Its regulation is most likely to be due to downstream processes such as the availability of either precursors or intermediate metabolites, the variability in specific processes leading to the concersion of precursors into volatiles, or to other still unknown regulatory mechanisms. Volatile compounds were also studied in a RIL population derived from a cross between Solanum pimpinellifolium accession TO-937, the closest species to cultivated, which produces red fruits, and S. lycopersicum cv. 'Moneymaker', a tomato variety for the fresh market. This allowed the identification of 102 QTLs for 39 different volatile compounds, 76 of which had not been previously described. All these QTLs were mapped along the 12 tomato chromosomes by means of the SOLCAP SNPs molecular market map. Most of the QTLs identified were subsequently evaluated on introgression lines (ILs) generated from the same original genotypes. It was observed that almost half of the QTLs previously identified retained their effect after introgression in the 'Moneymaker' genetic background. Additionally, 12 new QTLs were identified in this IL population. Based on the existing knowledge about the effect of volatile compounds on our perception of flavour and aroma and also on their ability to maintain their effect after introgression in the cultivated tomato, some of the QTLs identified are good candidates to be used in tomato flavour breeding programs. Eventually, the comparison of the localization in the genome of the QTLs identified in the different populations studied with those already described in the literature revealed a very low degree of co-localization between the different QTLs. This implies that there exists a wide range of variability in the wild species related to tomato available for breeding tomato flavour and aroma.[ES] Los frutos producen y emiten compuestos químicos volátiles. Estos son moléculas en general poco polares y de cadena corta que cumplen diversas funciones, y son las responsables de que percibamos el aroma y buena parte del sabor de los frutos. Esta tesis está centrada en el estudio de los volátiles del fruto del tomate, que es uno de los cultivos hortícolas más importantes y un sistema modelo para el estudio del desarrollo y maduración del fruto. Se compararon de forma sistemática los métodos analíticos más comúnmente utilizados para el análisis de volátiles en fruto de tomate, y se observó que el perfil de volátiles detectado está fuertemente condicionado por el método analítico utilizado, tanto por el proceso de preparación de la muestra como por la técnica de adquisición de los volátiles. Finalmente se concluyó que la técnica más adecuada para la comparación de grandes grupos de muestras desde una aproximación multi-ómica consiste en congelar con nitrógeno líquido el material vegetal una vez alcanzado el momento idóneo de recolección, y su análisis posterior mediante microextracción en fase sólida (SPME) acoplada a cromatografía de gases y espectrometría de masas. Se puso a punto esta técnica y se utilizó para la determinación de los compuestos volátiles en varias líneas NILs portadoras de QTLs de caracteres relacionados con el sabor y el aroma en distintos fondos genéticos. Los resultados permitieron asociar varios de los caracteres organolépticos identificados con alteraciones en los niveles de algunos volátiles. Igualmente se observó que el fondo genético tiene un efecto importante sobre la producción de estos metabolitos. Los análisis de correlación entre los niveles de volátiles y metabolitos primarios permitieron concluir que la producción de compuestos volátiles, en general, no está determinada por los niveles de sus precursores, sino que su regulación debe encontrarse más bien en procesos posteriores, tales como la disponibilidad de los precursores o de metabolitos intermedios, variabilidad en procesos específicos relacionados con la conversión de los precursores en volátiles, o algún otro mecanismo regulador aún desconocido. También se estudiaron los volátiles en una población de RILs derivada de un cruce entre Solanum pimpinellifolium entrada TO-937, la especie más próxima al tomate cultivado, la cual produce frutos rojos, y S. lycopersicum cv. "Moneymaker", una variedad de tomate para el mercado en fresco. Esto permitió identificar 102 QTLs para 39 volátiles diferentes, 76 de las cuales no se habían descrito previamente, las cuales se mapearon a lo largo de los 12 cromosomas del tomate utilizando el mapa de marcadores moleculares de SNPs SOLCAP. Posteriormente se evaluaron la mayoría de las QTLs identificadas mediante la determinación de los volátiles en líneas ILs generadas a partir de los mismos materiales. Se observó que casi la mitad de estas QTLs mantuvieron su efecto al ser introgresadas en el fondo genético "Moneymaker", al tiempo que 12 nuevas QTLs se identificaron en esta población de ILs. Algunas de las QTLs identificadas, en base al conocimiento existente sobre el efecto de los compuestos volátiles en nuestra percepción del sabor y el aroma, y en base a su capacidad para mantener su efecto tras su introgresión en el tomate cultivado, resultan ser candidatos prometedores para su utilización en la mejora genética del sabor del tomate. Finalmente, el análisis de la localización en el genoma de las QTLs analizadas en las distintas poblaciones objeto de estudio en esta tesis, junto con las descritas en la bibliografía disponible, puso de relieve el bajo grado de co-localización existente entre las distintas QTLs, lo cual implica que en las especies silvestres relacionadas con el tomate existe un amplio rango de variabilidad genética susceptible de ser utilizado para la mejora de su sabor y su aroma.[CA] Els fruits produixen i emitixen compostos químics volàtils. Estos són molècules en general de baixa polarita i cadena curta que tenen diverses funcions, i són les responsables de la nostra percepció de l'aroma i de bona part del sabor dels fruits. Esta tesi està centrada a l'estudi dels volàtils del fruit de la tomata, que és un dels cultius hortícoles més importants i un sistema model per a l'estudi del desenvolupament i la maduració del fruit. Es van comparar de forma sistemática els mètodes analítics més habituals per a l'anàlisi de volàtils en fruits de tomata, i es va observar que el perfil de volàtils detectat està fortament condicionat per el mètode analític utilitzat, tant per el procés de preparació de la mostra com per la técnica d'adquisició dels volàtils. Es va concluir que la t`cnica més adequada per a la comparació de grans grups de mostres desde una aproximació multi-òmica consistix en congelar en nitrògen líquid el material vegetal en el momento idoni de recolecció, i analitzar-lo posteriorment per microextracció en fase sòlida (SPME) acoplada a cromatografía de gasos i espectrometría de masses. Es va posar a punt esta técnica i es va utilitzar per a la determinació dels composteos volàtils en varies línies NILs portadores de QTLs de caràcters relacionats en el sabor i l'aroma en fons genètics diversos. Els resultats van permetre associar alguns dels caràcters organolèptics identificats en alteracions en els nivells d'alguns compostos volàtils. També es va observar que el fons genètic té un efecte important sobre la producción d'estos metabolits. Els anàlisi de correlació entre els nivells de volàtils i els metabolits primaris ens van permetre concluir que la producción de compostos volàtils, en general, no està determinada per els nivells dels seus precursors. La seua regulació és deguda a procesos posteriors, com la disponibilitat dels precursors o de metabolits intermediaris, la variabilitat en processos específics relacionats en la conversió dels precursors en volàtils, o en algún altre mecanisme regulador encara desconegut. També es van estudiar els volàtils en una población de RILs rerivada d'un creuament entre Solanum pimpinellifolium entrada TO-937, la espècie silvestre més próxima a la tomatera cultivada i que produix fruits rojos, i S. lycopersicum cv. Moneymaker, una varietat de tomata per al mercat en fresc. Açò va permetre l'identificació de 102 QTLs de 39 volàtils diferents, 76 de les quals no s'havien descrit prèviament, i que es van mapejar al llarg dels 12 cromosomes de la tomatera mitjançant el mapa de marcadors moleculars de SNPs SOLCAP. Posteriorment es van evaluar la majoria de les QTLs identificades mitjançant la determinació dels volàtils en línies ILs generades a partir dels mateixos materials. Es va observar que quasi la meitat de estes QTLs van mantindre el seu efecte al ser introgressades en el fons genètic "Moneymaker". Adicionalment, 12 noves QTLs es van identificar en esta población d'ILs. Algunes de les QTLs identificades, en base al coneiximent existent respecte a l'efecte dels volàtils en la nostra percepció del sabor i l'aroma, i tenin en cónter la seua capacitat de mantindre el seu efecte al ser introgressats en la tomata cultivada, són candidats prometedors per a ser utilitzats en la millora genética del sabor de les tomates. Finalment, l'anàlisi de la localització en el genoma de les QTLs analitzades en les distintes poblacions objecte d'aquesta tesi, junt a les descrites en la bibliografía disponible, va evidenciar que existix una baixa freqüència de co-localització entre les distintes QTLs. Açò implica que existix molta variabilitat genética en les espècies silvestres relacionades en la tomatera, que pot ser utilitzada per a la millora del sabor i l'aroma dels seus fruits.Rambla Nebot, JL. (2016). GENES AND GENOMIC REGIONS RELATED TO THE PRODUCTION OF VOLATILE COMPOUNDS IN THE TOMATO FRUIT [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61768TESISPremios Extraordinarios de tesis doctoralesCompendi