Study of Transcriptional Regulatory Network Controlling Strawberry Fruit Ripening and Quality

Ponencia invitadaRipening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. Also, we are using a collection of around two hundred wild strawberry (Fragaria vesca) accessions to identify loci involved in important traits such as aroma, size or resistance to pathogens. Finally, we are implementing the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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 (Airén 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 β-cyclocitral or β-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 α-carotene were only detected in Airén 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 Airén, confirming the enriched aroma bouquet characteristic of the white varieties.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.Peer reviewedPeer Reviewe

Deciphering Strawberry Ripening by Tissue Specific Gene Regulatory Networks

During ripening, fruits undergo a number of metabolic and physiological changes leading to softening and improvement of characters such as flavor and palatability. Insights into transcriptome changes during strawberry fruit ripening have been reported, but always using either complete fruits in the analysis or separating achenes and the fleshy part (receptacle). However, the receptacle is composed of heterogeneous cell types, each of them with different characteristics and functions. Hence, transcriptomic studies performed so far may have lost important regulatory elements which expression is low but important in a specific cell-type specific. In our study, we use Laser Capture Microdissection (LCM) technique for the isolation of cells from specific tissue types such as the epidermis, vascular bundles, cortex, and pith. Transcriptome profiling of these tissue types was performed by RNAseq. A gene co-expression analysis was performed by Weighted Correlation Network Analysis (WGCNA). Ontology analysis of each module showed wax biosynthesis as the main biological pathway enriched at the red epidermis specific module. In order to elucidate the putative regulatory elements that control the synthesis of waxes in this tissue, a Gene Regulatory Network (GRN) was generated using GENIST (de Luis Balaguer, 2017). As a result, we have identified a set of transcription factors that might regulate the expression of eceriferum genes and a fatty acid elongase necessary for wax biosynthesis in ripe epidermis. Ultimately, our results open the possibility of implementing novel targeted breeding approaches. Moreover, this work shows that LCM followed by RNAseq is a powerful tool that can be used to clarify the regulatory scenario of tissue-specific biological processes during strawberry ripening.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

Identification and functional validation of methyl ketone synthase 2 in woodland strawberry

Methyl ketones are compounds with demonstrated insect repellent effects. They are highly abundant in the glandular trichomes of wild tomato (Solanum habrochaites), where their pathway was first described, but not in the cultivated species (S. lycopersicum). Their synthesis derives from fatty acids in a two-step process mediated by a thioesterase (ShMKS2) and a decarboxylase (ShMKS1). Higher diversity and quantity of methyl ketones are present in the volatilome of woodland strawberry ripe fruits than in those of F. × ananassa. The aim of this study is to reveal the genetic basis of methyl ketone production in strawberry fruit. We quantified methyl ketones (2-heptanone, 2-nonanone, 2-undecanone), their secondary alcohols (2-heptanol, 2-nonanol, 2-undecanol) and the methyl esters of their fatty acid precursors (methyl octanoate, methyl decanoate, methyl dodecanoate) by GC-MS in a natural collection of European woodland strawberry, that comprises 199 accessions fully genotyped with >1.8 M SNPs representing the continental diversity. Conducting a Genome-Wide Association Study (GWAS), we identified a candidate region linked to methyl ketones accumulation harbouring three homologues of ShMKS2: FvMKS2A, FvMKS2B and FvMKS2C. Interestingly, FvMKS2A, which presented two alleles in the European collection (FvMKS2A-1 and FvMKS2A-2), is the only FvMKS2 paralog expressed in woodland strawberry fruit, being up-regulated during ripening. Functional validation of all candidate genes and alleles by transient over-expression and silencing in both Nicotiana benthamiana leaves and F. vesca fruits has revealed that FvMKS2A and FvMKS2B, but not FvMKS2C, are capable of synthesizing methyl ketones, and point to a single SNP in FvMKS2A as responsible for the enzymatic substrate specificity, supporting FvMKS2A as the main MKS2 paralog responsible for methyl ketones in woodland strawberries.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Identificación y caracterización de genes implicados en la maduración y la calidad de la fresa

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project (TRANSFR-Q, Starting Grant-ERC), we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size, and resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance.ERC Starting Grant ERC-2014-StG 63813

Transcriptional regulatory network controlling strawberry fruit ripening and quality

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size or resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Study of Transcriptional Regulatory Network Controlling Strawberry Fruit Ripening and Quality

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size, and resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Strawberry GRN forever: insights into the transcriptional regulatory network controlling strawberry fruit ripening and quality

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors (TF) involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. Among them, we have identified two TFs belonging to the NAC (FaRIF) and the BLH9 (FaRPL) family. Functional analyses establishing stable silencing and overexpression lines support that both TFs play a critical role in the regulation of fruit ripening and development. Furthermore, using a stage- and tissue-specific transcriptome analysis, we have identified TFs specifically expressed in the external layer of ripe receptacles of F. vesca fruits, which are involved in the regulation of wax and cuticle formation. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance