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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.

Reserve starch, the major component of wheat kernel, consists of amylose and amylopectin. The variation of their ratio (about 1:3 in normal wheat) has a potential impact on nutritional and technological characteristics of wheat-derived foods. Previously, mutations in homeologs of three key genes involved in starch biosynthesis (granule-bound starch synthase I, GBSSI; starch syntase IIa, SSIIa; a-glucan-branching, SBEIIa) have been identified and pyramided obtaining three complete null bread wheat lines, respectively named Cad-GBSSI*, Cad-SSIIa* and Cad-SBEIIa*. These genotypes have altered amylose/amylopectin ratios, starch properties, and polysaccharide compositions. In order to refine the characterisation of starch metabolism and to bring out transcriptional divergences underlying pleiotropic effects, the grain transcriptomes of Cad-GBSSI*, Cad-SSIa* and Cad-SBEIIa* mutants were compared to the wild-type at 15 and 25 days post anthesis (dpa). First, the principal component analysis of RNA-seq data scored similar gene expression between replicates and biological variability of the mutants. At 15 dpa, Cad-GBSSI*, Cad-SSIIa* and Cad-SBEIIa* diverged for 1114, 1737 and 335 genes compared to controls, suggesting a stronger and precocious effect of the SSIIa loss of function. The transcriptome of all mutants encompassed over 3000 DEGs at 25 dpa, suggesting a later responsive boost. As for the starch pathway, DEG profiles were consistent with the patterns of over 30 key genes that were targeted in a previous work. DEG analyses also pointed to gene sets that were modified recurrently or specifically in the mutants. The commonly altered genes included ca. 60 sequences related to carbohydrate pathways, while 658, 420 and 1282 were respectively peculiar for the Cad-GBSSI*, Cad-SSIIa* and Cad-SBEIIa* genotypes. Moreover, these mutants shared common enriched GO-terms involved in nitrogen, glutamate/glutamine metabolism and proteolysis processes that further supported the alteration of C/N equilibria. Interestingly, GO-term analysis also pointed at some collateral effects that recurred in the three mutants and regarded the functioning of both chloroplast and photosystems, suggesting some interference with photosynthesis, a well-known process in immature seeds.

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 'Catalogna' chicory ( Cichorium intybus L.) is a typical Italian vegetable cultivated for leaves and stems ('puntarelle'); these latter are novel foods, consumed as fresh or processed, and rapidly moving to global markets. The holistic characterization of stems was carried - out by combining NMR metabolic and RNAseq profiling of 'Galatina' (Gal) and 'Molfettese' (Mol) landraces, grown in Apulia and Lazio, in order to investigat e on the nutritional quality and performance in ex situ cultivation. The content variation of thirty - two compounds was affected by the genotype (G), environment (E) and GxE interaction; PCA explained over 90% of variation, and PC1 and PC2 components neatly discriminated Gal vs Mol based on growth sites and genotypes, respectively. Focusing on carbohydrates and amino acids, the total amount of the former was higher in Gal than Mol in both areas (consistently with predicted sweetness and sensory test), whilst the opposite trend occurred for total amino acid content. The Mol vs Gal gene differential transcription was performed based on a Gal reference transcriptome ; G, E, and GxE effects were found and KEGG analysis showed the enrichment of differentially expre ssed genes within the sucrose and phenylalanine metabolisms, consistently with the metabolic profile variations. Pearson's correlation analyses of gene expression vs metabolite amount variations allowed the selection of gene sets that could explain sugar a nd amino acid content differences between Gal and Mol stems. Overall, these analyses widened knowledge on the pathways that influence nutritive quality traits and pointed at target genes useful for plant breeding.

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 Catalogna chicory (Cichorium intybus L., Catalogna group) is an Italian typical vegetable; Molfettese and Galatina are Apulian landraces consumed for their tender stems (turions or "puntarelle"). Bitterness is an important organoleptic trait, which directs both consumer and breeding choices and is linked to the content of sesquiterpene lactones (STLs). Major STLs (lactucin, 8-deoxylactucin, lactucopicrin) and respective dihydro-derivatives (1,3-dihydrolactucin, 11(s),13-dihydro-8-deoxylactucin, 11(s),13-dihydrolactucopicrin) were quantified in turions of the two landraces (HPLC). Regardless of the cultivation sites, the content of 1,3-dihydrolactucin and 11(s),13-dihydro-8-deoxylactucin was significantly higher in Molfettese than Galatina. consistently with the more bitter taste of the former. The Galatina reference transcriptome contained 79,716 unigenes and 75 mapped into the sesquiterpenoid and triterpenoid (S&T) biosynthesis pathway (KEGG). In order to survey gene transcriptional variations and polymorphism data, the RNA-seq of Molfettese and Galatina stem transcriptomes was performed. Over two thousand differentially expressed genes (DEGs) were scored between the two genotypes independently of the cultivation zone. KEGG enrichment analyses revealed that four DEGs were up-regulated in Molfettese compared to Galatina within the S&T biosynthesis map. Specifically, the expression of the germacrene A-synthase and -oxidase genes of the Costulonide (a key STL precursor) branch was directly correlated with the contents of 1,3-dihydrolactucin and 11(s),13-dihydro-8-deoxylactucin. The sequences of these genes showed several polymorphic events (SNPs) between the two genotypes and together with the transcriptional divergences represent potential markers for assisted breeding.

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

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.

Knotted1-like homeobox (KNOX) transcription factors belong to the three amino acid loop extension (TALE) superclass of homeodomain proteins in higher plants (Hay and Tsiantis, 2010; Hamant and Pautot, 2010; Wu and Smith 2012; Di Giacomo et al., 2013). KNOX1 proteins 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 . Mutations in KNAT1/BREVIPEDICELLUS (KNAT1/BP), one of the four Arabidopsis KNOX1 genes, result in plants with shorter internodes, downward pointing siliques, altered intra-bundle cell identity and altered lignin deposition (Mele et al., 2004). Recently, KNATM, a novel Arabidopsis KNOX1 gene that encodes a MEINOX domain but lacks the homeodomain was identified and shown to regulate KNAT1/BP (Magnani and Hake, 2008). We identified and characterized a novel isoform of KNAT1/BP transcription factor that arises from an event of alternative splicing and may encode a novel protein (BPhox) that contains the homeodomain but lacks the MEINOX domain. Phenotype and molecular analysis of Arabidopsis lines that overexpress each isoforms under the control of constitutive/inducible promoters and complementation analyses were carried out to unravel the function of BPhox in inflorescence architecture, vascular development and lignin deposition.

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.

For ages, humans have developed technologies to exploit living organisms and their metabo- ism to produce food (e.g., bread, cheese and wine). Modern biotechnology implies the ndustrial use of scientific knowledge of cellular and molecular processes to make or modify ts, to improve plants and animals, or to develop microorganism for specific uses. Nowadays, the term biotechnology is associated with techniques such as genetic engineering, cell fusion, novel bioprocessing, bioremediation etc. The introduction of genetic manipulations in large-scale processing has raised several questions regarding health risks for consumers and environ- ment. Moreover, the scenario regarding biotechnology perception and acceptance is rather complex (for a review: Plant Genetic Engineering. Towards the Third Millennium. Proceedings of the Inter- national Symposium on Plant Genetic Engineering edited by A.D. Arencibia, Elsevier Press, 2000). Among Europeans, there has been great suspicion and reluctance to accept genetically modified food on a wide scale, though medical and environmental applications of biotechnology have been recognised as useful and subsequently have been favoured.2 Over the past ten years, biotech devices (biosensors) have been developed to monitor polluting molecules. Biosensors employ natural or genetically engineered living organisms, or part of them, whose use is tightly controlled so as to prevent their release into the environment. Moreover, the lack of ethical and moral implications have contributed to generate their approval from the public.

Relazione del progetto "BEEP": Assembly and application of photosystem II-based biosensors for large scale environmental screening of specific herbicides and heavy metals (QLK3-CT-2001-01629) coordinatore Dr. Maria Teresa Giardi Overexpression of D1 Chlamydomonas reinhardtii in E. coli strains Wild type and mutagenised psbA genes were cloned from Chlamydomonas reinhardtii, which were previously provided by partner 2. The polymerase chain reaction (PCR) was performed on total DNA by proof reading Taq (Promega) to avoid any copy mistake. The forward primer container: a site-specific recombination stretch from ? phage (attB1), a Shine-Dalgarno signal and a sequence matching with the 5' coding region of psbA gene. The reverse primer spanned the psbA 3' region, comprising the stop codon and a trailer, followed by a terminal ? phage site-specific recombination sequence (attB2). The attB sequences are necessary to clone the gene in the correct orientation and efficiently in the Gateway technology (Invitrogen). All amplified sequences were checked for correct DNA copying and subsequently cloned into the Gateway expression vector driven by a salt inducible promoter. As a mandatory step, D1 protein overexpression of the intron-less wild type psbA was attempted. The gene expression was salt induced in recombinant E. coli strains which were sampled after 2,3,4 hrs from the induction. Total proteins were extracted from bacterial broth, bacterial lisates, cell inclusive bodies and their membranes. They were electrophoresed, blotted onto nitrocellulose membrane and hybridised to anti-rabbit D1 protein and revealed a chromatic reaction using by anti-rabbit phosphatase conjugated secondary antibody. The strongest signal was detected in all the fractions containing soap washed membranes of inclusive bodies. Isolation and characterization of psbA genes from distinct Medicago sativa ecotypes The DNA of three distinct ecotypes (T, K and R see materials and methods) was isolated and full length gene cloning was performed by PCR, using primers designed on alfalfa psbA gene (accession number: X04973). Proof reading Taq polymerase was used to carry out the experiments to avoid any copy mistake. DNA sequencing and alignment analyses indicated that 5 distinct point mutations distinguished K and R lines, however two of them were silent mutations (positions: 42 and 126). At position 145 an alanine residue appears in K line, while a valine residue is in R line. At positions 166 and 333 glycine residues are found in K, while glutamic acid in R line. PCR products from T lines is being currently sequenced and analysed. Materials and Methods Chlamydomonas reinhardtii strains and culture Strains containing the wild type and mutagenised psbA genes (IL, wild type; Epi1, Epi2, D170E, G178S, D170E/G178S and SK, mutants) were provided and maintained on solid and liquid media according to the provider (partner 2) Plant material and growth conditions Seeds of three alfalfa ecotypes were provided by the Bulgarian University...... a high-yielding line (T), a selected drought tolerant line (R) derived from water stressed T populations selection and the drought tolerant commercial variety Krasnovodopadskaya (K). Seeds were germinated and grown in peat under controlled conditions (continuous light with fluorescent lamps, Philips TLD 25,000 lux) as reported in Ivanova et al. J. Plant Physiol. 150: 224-227, 1997. Leaves were sampled and immediately frost into liquid nitrogen and stored at -80°C. Gene cloning and overexpression PCR experiments were performed on total DNA isolated from alfalfa using Plant DNAzol Kit (Invitrogen) according to the manufacturer's instructions. PCR primers were designed from functional conserved domains of psbA genes. Final PCR conditions were: 100 ng of DNA, 1?M of each primer, 0.5 mM dNTPs, Taq proof reading (Promega) 2.5 U, 1/10 of 10X Taq Buffer, 2.5 mM MgCl2, in a final volume of 50 ?l. Cycling conditions included an initial cycle of 95°C for 5 min followed by 35 cycles of 95°C for 1 min, 60°C for 30 sec and 72°C for 1 min, followed by a final extension cycle at 72°C for 5 min. The 1110 bp fragment was cloned into pGEM-T easy vectors, sequenced and aligned for polymorphisms analysis by ClustalW (European Bio-Informatics Service). Chloroplast DNA was isolated from algae as previously described (Newman et al., 1990. Genetics 126: 875-888). Details on PCR primers are in the results. PCR physical conditions were: 5 min at 94°C followed by 94°C for 30 sec, 50°C for 1 min, 90 sec at 68°C (35 cycles) and a final elongation of 5 min at 68°C using a Platinum Pfx Taq (Invitrogen). Amplified fragments were cloned into pDONR?201 by site-specific recombination according to the manufacturer's instructions . After kanamycin selection, psbA containing vectors were transferred in a pDEST14 vector which allows gene expression in ampicillin selected- recombinant BL21-SI E. coli. We produced E.coli lines overxpressing pbsA genes from psbA from Chlamidomonas IL (wild type), Epi1, Epi2, D170E, G178S, D170E/G178S ed SK mutants. Western analysis Strains harbouring the wild type psbA gene (derived from IL line) were induced by salt treatment following instruction and protein content analysed. Isolation of total protein and purification from distinct cell fractions and components (bodies of inclusion) were carried out according to Sambrook and Maniatis 1984. Protein content was quantified by standard procedures (BIORAD). 50?g of total proteins was loaded on gels containing a urea gradient, 12% Acrilamide, 0,4M Tris-HCl pH 8.8, 0,05% ammonium persulfate (APS), 5?l Temed for the running gel, and 0,05M TrisHCl pH 6.8, 0,04% APS, 3,1?l temed for stacking gel. Running conditions were: 200V for about 45 min. To check for equal loading, one control electrophoresis gel was stained with Comassie Blu, while another equal has been transferred to nitrocelloluse membrane and hybridised with the anti-D1 polyclonal antibody.