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Vegetable salad shelf life largely depends on leaf senescence and abiotic and biotic stress responses, both under control of the phytohormone cytokinin (CK). Flowering time (FT) diversity has adaptive value in natural populations and plays a major role in leafy crops production. Our studies aim at identifying molecular markers and hub/master gene regulators of these key agronomic traits in the Compositae (Asteraceae) family. This will produce novel genetic tools and strategies to improve leafy crops bel onging to this family. A de novo assembled Cichorium endivia transcriptome obtained in our lab was used for deep annotation and gene prediction. With the aim to dissect the genetic pathways of cytokinins and flowering time in this species, manual curation of the database was carried out to obtain high quality functional annotations based on knowledge acquired in plant model species. CK and FT genetic pathways were subdivided into functional modules for systematic bioinformatic analyses. Correlation analyse s of RNAseq and hormone metabolites data, obtained from edible leaves of four commercial cultivars, identified key genes involved in cytokinin nucleotide - nucleoside interconversion and O - glycosylation, and their relationships with other CK metabolism genes , CK transport and components of the signal transduction pathway. Cis - zeatin (cZ) was identified as the most abundant form of CK in C. endivia leaves, and functional gene modules connected to either cZ or trans - zeatin (tZ) pathways were characterized. Analysis of CK and flowering time gene RNAseq expression profiles over two production cycles allowed the identification of known and novel co - expression networks, giving new insights into CK connection with plant innate immune response and novel information on flowering time control in endive and escarole. A connection of both pathways with the T hree A mino acid L oop E xtension (TALE) superclass of homeobox transcription factors (TF) was revealed. Genome - wide in silico analyses of TALE binding sites in Arabidops is allowed us to predict that several TFs involved in the control of flowering time may be direct targets of TALEs. The 1135 sequenced genomes of A. thaliana natural accessions, available in the "1001 Genomes Project", were screened for SNPs/mutations in the core consensus region of the identified TALE binding sites, which may result in loss of transcriptional regulation. The screen identified several putative lost and some newly evolved TALE binding sites, some of them being excellent candidates for adapt ive mutations and diversity in the flowering time pathway.

The CISIA project (http://www.ibba.mlib.cnr.it/Projects/Cisia/Project.html) provided tools to enhance valorization and traceability of endives (Cichorium endivia), which are consumed as fresh or minimally processed foods. Sesquiterpene lactones (SL) and phenolics complexly regulate the bitter flavor of these vegetables. The SL content of lactucin-like (lactucin, 1,3-dihydrolactucin, 8- deoxylactucin, 11(s),13-dihydro-8-deoxylactucin) and lactucopicrin-like (lactucopicrin, 11(s),13- dihydrolactucopicrin) compounds was measured in seven cultivars. Principal component analysis of SL content variation could mostly discriminate curly- from smooth-leafed (syn. escaroles) types and pointed at a strong positive correlation among curly-leafed genotypes, high amounts of lactucinand lactupicrin-like molecules and bitterness predictive scores. An endive reference transcriptome, previously assembled from a curly-cultivar, allowed the annotation of unigenes in the sesquiterpenoid and triterpenoid biosynthesis pathway. Out of 22 unigenes, the germacrene Asynthase (GAS), which acts in the branch leading to the key SL precursor costulonide, showed significant differential expression in those cultivars with most divergent SL contents ('Myrna' vs 'Domari' in the curly group; 'Confiance' vs 'Flester' in the smooth group; 'Myrna' vs 'Flester'). The very strong positive correlation (r=0.9) between SL contents and the transcriptions of three different GAS genes suggested a key role of these, consistently with the finding that terpene accumulation and synthase gene expression go in parallel.

The switch from plant vegetative growth to reproductive development (transition to flowering), is a critical stage in the life cycle of a plant. As such, flowering is tightly controlled by diverse developmental, hormonal and environmental cues, day length and temperature being the most important of these environmental signals (Posè et al. Curr Opin Plant Biol. 2012; Huijser and Schmid, Development 2011). Flowering time diversity has adaptive value in natural populations and plays a major role in agricultural production. Specifically, flowering delay in different environmental conditions is a crucial breeding trait for yield and nutritive quality of leafy crops and forages. Genetic and molecular analyses in the model plant Arabidopsis thaliana have identified regulatory pathways that control the floral transition (Turck et al. Annu. Rev. Plant Biol. 2008; Amasino Plant J. 2010; Fornara et al. Cell 2010). These pathways act in different tissues of the plant but ultimately converge at the shoot apical meristem (SAM) (Huijser and Schmid, Development 2011). Our studies aim to translate the knowledge acquired in Arabidopsis to leafy vegetables of high commercial value, endive and escarole (Cichorium endivia), whose transcriptome was recently sequenced in our lab in collaboration with the International seed breeding company Enza Zaden (R&D Italia). The final goal is to identify novel functional single nucleotide polymorphisms (SNPs), which may account for flowering time diversity as a tool for molecular breeding of leafy crops. We used a candidate gene approach and focus on well known Arabidopsis regulators of flowering transition and novel potential regulators belonging to the TALE (Three Amino acid Loop Extension, KNOX and BLH genes) family of homeobox transcription factors, which are expressed in the SAM (Hake et al. 2004; Di Giacomo et al. 2013). In silico analysis of Arabidopsis genome identified potential TALE binding sites in the regulatory regions of several flowering time genes. Sequenced genomes of A. thaliana natural accessions were screened for SNPs in the potential TALE binding sites to select Arabidopsis accessions for further studies. Characterization of the flowering time response of mutants and transgenics with altered KNOX expression is in progress. Homologous genes for known Arabidopsis regulators of flowering transition and TALEs were identified in the C. endivia transcriptome. Identification of putative functional SNPs in flowering regulatory genes and TALEs in different C. endivia varieties is in progress. Transcript profiling of adult leaves through RNAseq, in varieties which display different flowering time responses, identified possible gene expression markers of flowering time to be considered for functional validation.

Assembly and development of Cichorium endivia transcriptome to assess identity and traceability of endives and escaroles. Endives (Cichorium endivia var. crispum) and escaroles (C. e. latifolium) are widely consumed as fresh, minimally processed and cooked food. Italy is a major world consumer and producer. The project CISIA (www.ibba.mlib.cnr.it/Projects/Cisia/Project.html) has been developing markers (metabolic, transcriptional and allelic variants) used for identity, traceability and valorisation of curly- and broad- leafed cultivars in collaboration with the Enza Zaden Group. A de novo transcriptome was assembled (reference cultivar 'Domari') using RNA of several organs (from young and adult plants) and Illumina HiSeq2000 technology (100bp paired-end), which generated ca. 240M reads. The filtered reads were assembled into 91,784 contigs (77,360 unigenes) with an average length of ca. 1,202bp (N50 = 1,545bp). As for the unigenes, 68,858 (75.0%) showed significant similarity (E-value

Endives (Cichorium endivia var. crispum) and escaroles (C. endivia var. latifolium) are leafy crops belonging to the Cichorieae tribe which leaves are widely consumed as fresh, minimally processed and cooked food. Similar to lettuce, endives and escarole leaves display a wide range of leaf morphology diversity, ranging from smooth (escaroles) to extremely curly leaves (endives). Cytokinins (CK) are plant hormones that occur naturally as N6-substituted adenine derivatives. CKs regulate several important aspects of plant development and growth such as cell division and differentiation, shoot development and organogenesis (Kieber and Shaller, The Arabidopsis book, 2014). Besides this, cytokinins control leaf shape determination and development (Frugis et al. Plant Physiol. 2001; Shani et al. Plant Cell 2010) and have long been known to inhibit leaf senescence (Gun and Amasino, Science 1995) in model and crop species (Ori at al. Plant Cell 1999; McCabe et al. Plant Physiol. 2001). Being potent general antagonists of senescence, cytokinins might be exploited to extend the shelf life properties of leafy vegetables. As for leaf patterning, cytokinin homeostasis and responses may contribute to leaf morphology diversity among Cichorium species and C. endivia cultivars. We reconstructed the genetic pathway of cytokinins in endives (reference cultivar Domari) which transcriptome was assembled de novo using RNA of several organs and Illumina HiSeq2000 technology. C. endivia genes involved in cytokinin metabolism, perception, signaling and response were identified, classified and annotated based on homology with the plant model species Arabidopsis thaliana. Transcript profiling of CK genes were analyzed in edible leaves of endive and escarole commercial cultivars and compared to CK metabolites content. Putative cultivar-specific transcriptional markers and SNPs were identified and characterized.

Italy is leader in the production of chicory (Cichorium spp.) foods. Typical vegetables such as endive and escarole (C. endivia var. latifolium and var. chrispum) and "puntarelle" (C. intybus subsp. foliosum) are mainly consumed as fresh or minimally processed food. The project CISIA (Integrated Knowledge for the Agro-food Sustainability and Innovation for the made in Italy produce) is sustained by governmental and SMEs and aims at assessing the identity and traceability of local varieties (Lazio and Puglia shires) and at enhancing their nutritive and economic value by "olistic" approaches targeted to specific metabolites and gene pathways. The profiles of quality metabolite, related gene transcription and allelic variability will be used to fingerprint and valorise escarole, endive and "puntarelle" cultivars. In this context, the brassinosteroid (BR) pathway was widely investigated, considering that anti-cancer/anti-cholesterol properties of BR can impact on food quality and cultivar selection/marketing. The BR content of edible products (leaves) was quantified by Mass spectrometry in a set of patented and local cultivars and used to rank for BR accumulation. The BR biosynthetic and catabolic genes of C. intybus and C. endivia were identified by de novo transcriptome assembly of sequences obtained by Next Generation Sequencing. The comparison of BR gene expression pattern among the different cultivars will be combined with BR metabolic profile changes. Finally, variations of structural gene (SNP) and metabolite contents will be tested as markers for discrimination and traceability.

[This corrects the article on p. 1676 in vol. 7, PMID: 27877190.].