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In order to assess the quality and performance of bagged broccoli-raab, a recently marketed product, several nutritive parameters were determined in novel hybrid and conventional cultivars at pre- and post-packaging stages in the industrial environment. The characterization of shoots and composing organs at post-cut stage included contents of dietary fibre (DF), glycaemic carbohydrates (GC), antioxidant compounds (ACC) and capacity (AOC), which were determined by chromatographic methods and spectrophotometric assays. ACC and AOC were analysed during shelf life of bagged products. Genotype and storage effects were addressed as variability factors at fixed packaging conditions. Contents of DF and GC (39.64-34.57; 7.56-2.21 g/100 g), glucosinolates (37.47-24.63 mg/g SIN), and ACC (total phenolics: 18.64-14.92 mg GAE/g; flavonoids: 34.74-30.96 mg/g CE; flavonols: 14.62-14.08 mg QE/g), and AOC (Oxygen Radical Absorbance Capacity: 354.62-293.25 µmol/g TE; DPPH• scavenging activity: 59.35-46.14) were lower in shoots of the hybrid than marketed cultivar. In both genotypes, AOC was maximal in leaves, followed by florets and stems. The integrated analyses suggested that the hybrid genotype was better suited for fresh consumption and that increased ratio of florets/leaves vs. stem is expected to raise product antioxidant properties. The comparison of unprocessed and bagged products pointed at a value decay of most parameters except for glucosinolates and correlation analyses supported the necessity of performing multiple antioxidant assays to enhance product quality evaluation. As for shelf life, storage time was the major factor affecting antioxidant properties, while genotype and interaction effects were minimal.

Cichorium endivia is a typical Mediterranean species that has been cultivated since ancient times in the Mediterranean basin. C. endivia leaves represent an excellent source of health benefiting plant nutrients and are widely consumed as fresh, minimally processed and cooked traditional food. C. endivia commercial varieties show a high degree of intraspecific morphological variation, and two major varieties can be distinguished: var. latifolium with broad leaves (escarole) and var. crispum with narrow curly/crisp leaves (endive). Due to the striking morphological diversity of the leaves in escarole and endive, C. endivia represents a good model to study the relationship between leaf shape and function. A standing question, that is both of fundamental and applied interest, is the adaptive value of leaf shape in relation with its photosynthetic activity. We used transcriptomic data from leaves of two commercial cultivars of endive and two of escarole to explore regulatory networks of transcription factors (TFs) involved in leaf development and leaf function through cluster analysis and construction of Gene Co-expression Networks (GCN). We identified modules of functionally related TF genes acting in C. endivia leaves and regulatory networks that are differential between broad and curly leaves. We then analyzed how genes related to photosynthetic activity, as well as to molecules involved in energy dissipation and reactive oxygen species (ROS) detoxification (flavonoids, carotenoids and ascorbic acid), enter into the GCN networks and relate to the TF modules identified with a guide-gene approach. This allowed to predict the regulatory networks controlling photosynthesis in C. endivia leaves, their relationship with leaf morphological characteristics and the genetic hubs that are at the boundaries between shape and function. Physiological analysis of the photosynthesis properties linked to different leaf shapes were carried out to corroborate predictive models of the regulatory networks identified. Finally, target genes to be used in breeding selection programs and biotechnology were predicted based on network topological properties and node ranking.

The Three Amino acid Loop Extension (TALE) proteins constitute an ancestral superclass of homeodomain transcription factors conserved in animals, plants and fungi, which play major roles in development. In plants they comprise two classes, KNOTTED1-LIKE homeobox (KNOX) and BEL1-like homeobox (BLH or BELL), which form heterodimers with different target affinity. KNOX and BLH genes are key players in morphogenetic processes at the shoot apical meristem (leaf, stem and inflorescence development, transition to flowering) and in legume root nodule organogenesis. TALE transcription factors cooperate to establish boundaries between undifferentiated cells and organ outgrowth through the modulation of multiple hormonal pathways, thus affecting several different plant developmental processes. Due to their pleiotropic effects, strategies to exploit TALE transcription factors potential for crop improvement need to: i) focus on fine tuning of gene expression of specific TALE TFs; ii) to selectively uncouple regulation of TALEs and their specific targets, the latter being mostly unknown. Also, very little is known about how specific KNOX and BLH homo- and heterodimers regulate subsets of target genes in different cells and developmental context. Based on previous knowledge of the developmental processes controlled by TALE transcription factors, we selected tissue-specific or developmental series transcript profiling data (obtained in our lab or publicly available) to construct TALE gene co-expression networks in model and crop species. Different algorithms, "a priori" functional module selection and extensive examination of network properties, like node degree distribution and gene centrality, were used to extract information on TALE gene regulatory networks. Such information was integrated with genome-wide in silico analyses of TALE binding sites to predict tissue/development-specific direct targets and design strategies to modify TALE regulatory networks to increase crop production.

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.

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

The tender stems (turions) of chicory (Cichorium intybus L. Catalogna group) are widely consumed as fresh or processed food in central and southern Italy. They are also known as 'puntarelle' and considered as elite groceries, cultivated as local varieties in Apulia and Lazio. The project CISIA has developed markers based on metabolic, transcriptional and allelic variations aimed to identity, traceability and valorisation of the Galatina and Molfettese Apulian landraces propagated by local enterprises. A de novo reference transcriptome was assembled using RNA of several organs (borne on young and adult plants of Galatina) and Illumina HiSeq2000 technology. Ca. 170M reads were generated (100bp paired-end) and assembled into 96,514 contigs of 1,168bp average length (N50 = 1,435bp). Among them, 68,260 (70.7%) showed significant similarity (Evalue

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.

The KNOTTED-like genes (KNOX) encode homeodomain transcription factors and regulate several processes of plant organ development. The peach (Prunus persica L. Batsch) KNOPE1 was assessed to link to a QTL for the internode length in the Peach x Ferganensis population. The KNOPE1 expression decreased progressively from stem primary (elongation) to secondary growth (radial expansion) of adult shoots. During primary growth, the KNOPE1 mRNA was localised in the cortex and in the procambium/metaphloem zones, whereas it was undetected in incipient phloem and xylem fibres. KNOPE1 over-expression in the arabidopsis bp4 loss-of-function background (35S:KNOPE1/bp genotype) restored the rachis length, suggesting, together with the QTL association, a role for KNOPE1 in peach shoot elongation. Several lignin biosynthesis genes were up-regulated in the bp4 internodes but repressed in the 35S:KNOPE1/bp lines similarly to the wild type. Moreover, the lignin deposition pattern of 35S:KNOPE1/bp and the wild type internodes were the same. The KNOPE1 protein was found to recognize in vitro one of the typical KNOX-DNA binding sites that recurred in peach and arabidopsis lignin genes. KNOPE1 expression was inversely correlated with that of lignin genes and lignin deposition along the peach shoot stems and was down-regulated in lignifying vascular tissues. These data strongly support that KNOPE1 prevents cell lignification by repressing lignin genes during peach stem primary growth

Knotted1-like homeobox (KNOX) transcription factors belong to the three amino acid loop extension (TALE) superclass of homeodomain proteins in higher plants. KNOXs play a key role in several developmental processes by maintaining a proper balance between undifferentiated and differentiated cell state through the modulation of multiple hormonal pathways. Besides the homeodomain, KNOX proteins are characterized by the presence of a consensus region called MEINOX that defines this subclass of the TALE family. MEINOX domain is important for protein-protein interactions with a second group of TALE proteins, the BEL-like homeodomain (BLH) family. This interaction modulates KNOX nuclear localization and, likely, target selection. The major goal of our laboratory is to define the molecular mechanisms that underlie KNOX function in cell fate determination and organogenesis using model organisms and transfer the acquired knowledge to non-model plant species including important crops. Our research aims at developing strategies and tools for using KNOX genes, or other components of KNOX developmental pathways, in the selection of plant traits relevant to agriculture production. The traits we are interested in are: leaf morphology and development, vascular differentiation, leaf/stem ratio, lignin content, vegetative-to-reproductive transition, stem architecture, hormone homeostasis, organogenesis, maintenance of indeterminate cell fate, optimization of nitrogen fixation in legumes. Our main research projects regard: post-transcriptional regulation of KNOX1 genes in Arabidopsis thaliana and Medicago truncatula - the role of KNOX1 in the determination of M. truncatula compound leaves - the identification of novel developmental pathways which involve different KNOX transcription factors - the identification of Arabidopsis KNOX functional orthologs in Medicago truncatula for forage legumes, Prunus persica for fruit tree species and Cichorium intybus for cultivated salad species of commercial interest. We will present our latest data on KNOX transcription factors coming from a comparative scientific approach. To gain new insights into the role of alternative splicing in KNOX genes, we will present the functional characterization of a novel isoform of the KNAT1/BP transcription factor (BPhox) that encodes the homeodomain but lacks the MEINOX domain. We will also show data regarding an alternative spliced STM-like gene of M. truncatula (MtKNOX6) that induces novel phenotypes when overexpressed in Arabidopsis thaliana. We will explore the possibility to use the structural polymorphism of KNOX proteins from different species to identify key amino acid residues that are important for functional diversification.

Plant KNOTTED-like homeobox (KNOX) transcription factors play important roles in the development of several organs, including diverse fruit types such as tomato berries and Arabidopsis siliques. The peach genome contains ten KNOX genes (KNOPEs), six of class1, three of class 2 and one of class M, which maintain the exon-intron organisation compared to the respective Arabidopsis orthologues. Seven members were positioned on the genetic Prunus reference map by means of co-dominant markers and they were scattered on distinct linkage groups. Putative association between KNOPEs and drupe quantitative trait loci (QTLs) were inferred based on synteny/co-linearity of diverse Prunus spp. genetic maps. The expression of KNOPEs was monitored by qPCR in the drupe mesocarp of three cultivars ('Chiripa', 'Fantasia' and 'Bolero') during the S1-S4 developmental stages. As for class 1 genes, the KNOPE1 and KNOPE6 mRNA levels decreased progressively from S1 to S4, whilst those of KNOPE2, KNOPE2.1, STMlike1, STMlike2 were below the threshold levels. Interestingly, KNOPE1 and KNOPE6 genetic positions were compatible with QTLs regulating drupe size, supporting they may play roles in controlling this trait. Concerning class 2 members, in S1-S2 the KNOPE3 and 4 showed unvaried mRNA abundance, which declined later on, whereas KNOPE7 transcription diminished from S1 to S4. All the above said patterns were maintained in three cultivars. KNOPE3 showed the highest expression levels among the KNOPEs and could be detected by in situ hybridisation in late S1 drupes. The transcript occurred in the parenchyma cells and vascular bundles of mesocarp, in the nucellus of embryo sac and in the seed coat sieves. The KNOPE3 expression was previously shown to be regulated by transport sugar, hence the phloem associated expression might imply a role in fruit sugar metabolism and seed nourishment.

The 1H-NMR methodology used in the study of genetically modified (GM) foods is discussed. Transgenic lettuce (Lactuca sativa cv "Luxor") over-expressing the Arabidopsis KNAT1 gene is presented as a case study. Twenty-two water-soluble metabolites (amino acids, organic acids, sugars) present in leaves of conventional and GM lettuce were monitored by NMR and quantified at two developmental stages. The NMR spectra did not reveal any difference in metabolite composition between the GM lettuce and the wild type counterpart. Statistical analyses of metabolite variables highlighted metabolism variation as a function of leaf development as well as the transgene. A main effect of the transgene was in altering sugar metabolism.

Genetic transformation of lettuce by A. tumefaciens has been standardised and manipulation of traits such as leaf shape and nitrogen metabolism has been carried out. Lettuce (cv Luxor) overexpressing the homeobox knotted-like gene KNAT1 of A. thaliana under the pea plastocyanin promoter featured leaves with increased vascular net, lobes at margins and higher content of cytokinin hormones, which delayed senescence, as compared to wild types. Lettuce overexpressing the asparagine synthetase A from E. coli under a chimaeric promoter derived from 35S and mannopine synthase promoters exhibited accelerated growth, higher fresh and dry weight and reduced content of nitrates compared to untransformed plants. Finally, from open field trials, pollen transmission was estimated very poor (1%) even when donor and recipient plants were in close proximity

Di "Gigliola", opera incompiuta da "La fiaccola sotto il moggio", viene ripercorsa la lunga gestazione attraverso i documenti superstiti, indagata la causa dell'incompiutezza, valutati i tratti stilistici delle pagine composte.

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