Sugarloaf chicory (Cichorium intybus var. porphyreum) represents a valuable crop for investigating metabolic responses to environmental stress. This study applied quantitative 1H-NMR-based metabolomics to characterize the water-soluble metabolome and evaluate root metabolic adaptations under water-deficit (WD) conditions compared to well-watered (WW) conditions. A total of 44 compounds were identified across roots and leaves, with inulin being root-specific. To address the lack of aqueous NMR data for chicory sesquiterpene lactones (STLs), a solid-phase extraction and fractionation protocol was implemented. Comparison of 1H-NMR and 13C chemical shifts with data from the literature, 2D NMR experiments (HSQC, HMBC), and spiking with standards confirmed that the major root STLs (lactucin, 8-deoxylactucin, and lactucopicrin) are 15-oxalate conjugates with enhanced water solubility. Under water deficit, root profiles revealed significant stress-induced alterations: sucrose, alanine, threonine and phospho-choline increased, whereas asparagine, glutamic acid, chiro-inositol, myo-inositol, and all three STL conjugates decreased markedly (−39% to −50%). These shifts reflect adaptive osmotic adjustments and carbon reallocation strategies under stress. As roots represent a remarkable source of bioactive STLs, these findings support their potential valorization as functional ingredients. This study establishes quantitative NMR metabolomics as a robust tool for assessing physiological responses to water deficit, providing insights into stress adaptation mechanisms and identifying roots as promising targets for alternative applications.

Industrial hemp (Cannabis sativa L.) is valued for its industrial, food, medical and recreational applications due to its bioactive secondary metabolites, including phenols, cannabinoids and terpenes, which accumulate at low concentrations (<1 % dry weight) in female inflorescences and increase under stress. This study aimed to enhance these compounds by applying methyl jasmonate (MeJA), either alone or encapsulated in chitosan nanoparticles, to female plants of the dioecious cultivar Tiborszallasi grown under field conditions. Treatments included 1 mM and 10 mM MeJA, chitosan nanoparticles (CHT) alone and nanoparticles loaded with 1 mM MeJA. Inflorescences were sampled at early and mature developmental stages, at two time-points per stage: 6 h and 6 days post-treatment. Overall, treatment with 10 mM MeJA significantly increased phenols (+21.2 %), flavonoids (+49.5 %), antioxidant activity (+43.7 %), CBDA (+78.4 %) and terpenes (+94.7 %) in mature inflorescences harvested 6 days after treatment. RNA-Seq analysis scored MeJA marker responses and revealed dose-dependent transcriptional changes. Weighted correlation network and gene co-expression network analyses revealed key regulatory and metabolic genes associated with secondary metabolism, including cannabinoid, phenylpropanoid and terpene pathways. These analyses underscored the central role of the fatty acid biosynthetic pathway in cannabinoid accumulation and highlighted a TALE- and SQUAMOSA-driven transcriptional network as pivotal in the developmental transition from meristem-rich to highly differentiated inflorescences specialized in secondary metabolite production. Overall, this study provides insights into MeJA elicitation of secondary metabolism and identifies regulatory hubs and candidates not previously associated with secondary metabolism in Cannabis sativa for enhancing the therapeutic and commercial potential of industrial hemp.

Rising temperatures due to climate change may affect the quality of open-field cultivated processing tomatoes by altering the nutrient content. Bioinoculants are growing in popularity as a nature-based strategy to mitigate these environmental stresses. Untargeted quantitative NMR spectroscopy was leveraged to characterize the metabolome of tomato fruits exposed to abiotic stress during the year 2022, which was marked by unexpected high temperatures and low rainfall compared to the year 2021 with average conditions. This study was conducted at growing sites in Tarquinia and Viterbo, comparing untreated plants to ones treated with a Trichoderma-based bioinoculant. The hotter year affected the water-soluble fraction (28 compounds), causing an increase in amino acids, citrate, and formate contents while decreasing carbohydrates together with a significant drop in β-sitosterol + campesterol in the organic fraction (11 compounds). The site mainly affected the linolenic acid levels, which were more abundant in Tarquinia than Viterbo in the hotter year, whereas ascorbate and myo-inositol were higher in Tarquinia in both years. The year × site interaction significantly affected the content of several amino acids, glucose, sucrose, and trigonelline. The bioinoculant effect was significant only for sucrose, while its interactions with the other factors showed little to no significance across all the measured metabolites.

In the context of organic farming, the introduction of a local product to wider markets and an evaluation of storage effects, metabolic and transcriptomic variations in two broccoli rabe genotypes from production cycles of two different years were studied by comparing florets of stored fresh (SF) and packaged (P) for 4 days with those harvested fresh from the field (H). Twenty-five hydrosoluble compounds, including amino acids, carbohydrates, and organic acids, were quantified by untargeted nuclear magnetic resonance (NMR). Principal component analysis produced a neat separation among the three commodity statuses with P being the most divergent and SF closer to H. In the packaged florets, carbohydrate levels dropped significantly (over -52%), while the levels of amino acids and organic acids varied. There was an increase in stress-responsive phenylalanine and valine (over 30%) and succinic and alpha-ketoglutaric acids (over 75%). Compound correlation analyses indicated a carbohydrate sink towards gamma-aminobutyric acid (GABA) and lactic acid (LA) metabolism under hypoxic conditions in packaged florets. RNA-seq analysis revealed that over 4000 genes were differentially expressed in SF vs H and 8000 in P vs H. Several CAR and AA pathways were significantly enriched in S and even more significantly in P, when compared to H. A map of gene expression (175 genes) and metabolite contents (14 compounds) was constructed to elucidate the gene routes that lead to accumulation of GABA and LA, known for healthy properties, in P. WGCNA and promoter binding site analyses enabled the identification of transcription factors (bZIP, WRKY, ERF types), interactions, and targeted genes encoding key enzymes in GABA and LA accumulation.

Starch bioengineering in cereals has produced a plethora of genotypes with new nutritional and technological functionalities. Modulation of amylose content from 0 to 100% was inversely correlated with starch digestibility and promoted a lower glycemic index in food products. In wheat, starch mutants have been reported to exhibit various side effects, mainly related to the seed phenotype. However, little is known about the impact of altered amylose content and starch structure on plant metabolism. Here, three bread wheat starch mutant lines with extreme phenotypes in starch branching and amylose content were used to study plant responses to starch structural changes. Omics profiling of gene expression and metabolic patterns supported changes, confirmed by ultrastructural analysis in the chloroplast of the immature seeds. In detail, the identification of differentially expressed genes belonging to functional categories related to photosynthesis, chloroplast and thylakoid (e.g. CURT1), the alteration in the accumulation of photosynthesis-related compounds, and the chloroplast alterations (aberrant shape, grana stacking alteration, and increased number of plastoglobules) suggested that the modification of starch structure greatly affects starch turnover in the chloroplast, triggering oxidative stress (ROS accumulation) and premature tissue senescence. In conclusion, this study highlighted a correlation between starch structure and chloroplast functionality in the wheat kernel.

Knotted1-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As Prunus species include important fruit tree crops of Italy, an exhaustive investigation of KNOX genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated KNOX transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in Prunus spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach KNOX transgenes. Based on ten Prunus spp., KNOX proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of Prunus from Cerasus subgenera, and that of Armeniaca from the other sections within Prunus. Cytokinin (CK) cis-elements occurred in peach and almond KNOX promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by KNOX expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified KNOX transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6–0.6%) with the genetic construct, with 35S:GFP being more stable than 35S:KNOPE1 lines, which showed leaf modification typical of KNOX overexpression.

The chloroplast (cp) genome diversity has been used in phylogeny studies, breeding, and variety protection, and its expression has been shown to play a role in stress response. Smooth- and curly-leafed endives (Cichorium endivia var. latifolium and var. crispum) are of nutritional and economic importance and are the target of ever-changing breeding programmes. A reference cp genome sequence was assembled and annotated (cultivar 'Confiance'), which was 152,809 base pairs long, organized into the angiosperm-typical quadripartite structure, harboring two inverted repeats separated by the large- and short- single copy regions. The annotation included 136 genes, 90 protein-coding genes, 38 transfer, and 8 ribosomal RNAs and the sequence generated a distinct phyletic group within Asteraceae with the well-separated C. endivia and intybus species. SSR variants within the reference genome were mostly of tri-nucleotide type, and the cytosine to uracil (C/U) RNA editing recurred. The cp genome was nearly fully transcribed, hence sequence polymorphism was investigated by RNA-Seq of seven cultivars, and the SNP number was higher in smooth- than curly-leafed ones. All cultivars maintained C/U changes in identical positions, suggesting that RNA editing patterns were conserved; most cultivars shared SNPs of moderate impact on protein changes in the ndhD, ndhA, and psbF genes, suggesting that their variability may have a potential role in adaptive response. The cp transcriptome expression was investigated in leaves of plants affected by pre-harvest rainfall and rainfall excess plus waterlogging events characterized by production loss, compared to those of a cycle not affected by extreme rainfall. Overall, the analyses evidenced stress- and cultivar-specific responses, and further revealed that genes of the Cytochrome b6/f, and PSI-PSII systems were commonly affected and likely to be among major targets of extreme rain-related stress.

Endive ( Cichorium endivia L.), a vegetable consumed as fresh or packaged salads, is mostly cultivated outdoors and known to be sensitive to waterlogging in terms of yield and quality. Phenotypic, metabolic and transcriptomic analyses were used to study variations in curly- (‘Domari’, ‘Myrna’) and smooth-leafed (‘Flester’, ‘Confiance’) cultivars grown in short-term waterlog due to rainfall excess before harvest. After recording loss of head weights in all cultivars (6-35%), which was minimal in ‘Flester’, NMR untargeted profiling revealed variations as influenced by genotype, environment and interactions, and included drop of total carbohydrates (6–50%) and polyols (3–37%), gain of organic acids (2–30%) and phenylpropanoids (98–560%), and cultivar-specific fluctuations of amino acids (−37 to +15%). The analysis of differentially expressed genes showed GO term enrichment consistent with waterlog stress and included the carbohydrate metabolic process. The loss of sucrose, kestose and inulin recurred in all cultivars and the sucrose-inulin route was investigated by covering over 50 genes of sucrose branch and key inulin synthesis ( fructosyltransferases ) and catabolism ( fructan exohydrolases ) genes. The lowered expression of a sucrose gene subset together with that of SUCROSE:SUCROSE-1-FRUCTOSYLTRANSFERASE ( 1-SST ) may have accounted for sucrose and kestose contents drop in the leaves of waterlogged plants. Two anti-correlated modules harbouring candidate hub-genes, including 1-SST , were identified by weighted gene correlation network analysis, and proposed to control positively and negatively kestose levels. In silico analysis further pointed at transcription factors of GATA, DOF, WRKY types as putative regulators of 1-SST .

* Corresponding Author: Tel.: +39 0690672210; E-mail: donato.giannino@cnr.it (Dr. D. Giannino) # Authors have equally contributed to this work cal traits [Bonnema et al., 2011] and molecular phylogeny. It includes “cime di rapa” and “friariello” vegetables from south Italy [Elia & Santamaria, 2013], also known as broccoli-raab or rapini [PlantUse, 2020] and consumed as cooked or raw (salads or sauces). Botanically, broccoli-raab falls into the subspecies sylvestris Janch var. esculenta Hort. [Romani et al., 2006], and the edible products are shoots bearing leaves, stems, and corymb-inflorescences (florets). Local farmers usually select parental lines phenotypically, aiming at progenies with superior traits and minimal inbreeding depression. Seed companies exploit these local selections to develop synthetic varieties or to create inbred lines for F1 hybrids with new traits such as regrowth attitude (for multiple cutting), product tenderness and antioxidant content. Landraces cultivation spans fall-winter cycles of 50–90 days in open field [Elia & SantaNutritive Parameters and Antioxidant Quality of Minimally Processed “Cime di Rapa” (Brassica rapa subsp. sylvestris) Vary as Influenced by Genotype and Storage Time

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

The worldwide-cultivated chicory (Cichorium intybus L.) produces food and beneficial compounds, and young pre-flowering inflorescence stems are newly marketed vegetables. These sink-organs undergo growth by metabolizing sugars of leaf origin; the carbohydrate content and sweetness are crucial aspects for consumers' nutrition and acceptance. NMR profiling of 31 hydrosoluble phytochemicals showed that stem contents varied as influenced by genotype, environment and interaction, and that higher sucrose levels were associated with the sweeter of two landraces. Integrative analyses of metabolic and transcriptomic profile variations allowed the dissection of sucrose pathway. Overall, 427 and 23 unigenes respectively fell into the categories of sucrose metabolism and sugar carriers. Among 10 differentially expressed genes, the 11474/sucrose synthase, 53458/fructokinase, 9306 and 17035/hexokinases, and 20171/SWEET-type genes significantly associated to sugar content variation, and deduced proteins were characterised in silico. Correlation analyses encompassing sugar level variation, expressions of the former genes and of computationally assigned transcription factors (10938/NAC, 14712/bHLH, 40133/TALE and 17846/MIKC) revealed a gene network. The latter was minimally affected by the environment and accomplished with markers, representing a resource for biological studies and breeding.

Cichorium endivia is a leafy crop closely related to Lactuca sativa that comprises two major botanical varieties characterized by a high degree of intraspecific morphological variation: var. latifolium with broad leaves (escarole) and var. crispum with narrow crisp curly leaves (endive). To investigate the relationship between leaf morphology and photosynthetic activity, escaroles and endives were used as a crop model due to the striking morphological diversity of their leaves. We constructed a leaf database for transcription factors (TFs) and photosynthesis-related genes from a refined C. endivia transcriptome and used RNA-seq transcriptomic data from leaves of four commercial endive and escarole cultivars to explore transcription factor regulatory networks. Cluster and gene co-expression network (GCN) analyses identified two main anticorrelated modules that control photosynthesis. Analysis of the GCN network topological properties identified known and novel hub genes controlling photosynthesis, and candidate developmental genes at the boundaries between shape and function. Differential expression analysis between broad and curly leaves suggested three novel TFs putatively involved in leaf shape diversity. Physiological analysis of the photosynthesis properties and gene expression studies on broad and curly leaves provided new insights into the relationship between leaf shape and function.

Endives (Cichorium endivia L.) are popular vegetables, diversified into curly/frisée- and smooth/broad-leafed (escaroles) cultivar types (cultigroups), and consumed as fresh and bagged salads. They are rich in sesquiterpene lactones (STL) that exert proven function on bitter taste and human health. The assembly of a reference transcriptome of 77,022 unigenes and RNA-sequencing experiments were carried out to characterize the differences between endives and escaroles at the gene structural and expression levels. A set of 3177 SNPs distinguished smooth from curly cultivars, and an SNP-supported phylogenetic tree separated the cultigroups into two distinct clades, consistently with the botanical varieties of origin (crispum and latifolium, respectively). A pool of 699 genes maintained differential expression pattern (core-DEGs) in pairwise comparisons between curly vs smooth cultivars grown in the same environment. Accurate annotation allowed the identification of 26 genes in the sesquiterpenoid biosynthesis pathway, which included several g ermacrene A s ynthase, g ermacrene A o xidase and co stunolide s ynthase members (GAS/GAO/COS module), required for the synthesis of costunolide, a key precursor of lactucopicrin- and lactucin-like sesquiterpene lactones. The core-DEGs contained a GAS gene (contig83192) that was positively correlated with STL levels and recurrently more expressed in curly than smooth endives, suggesting a cultigroup-specific behavior. The significant positive correlation of GAS/GAO/COS transcription and STL abundance (2.4-fold higher in frisée endives) suggested that sesquiterpenoid pathway control occurs at the transcriptional level. Based on correlation analyses, five transcription factors (MYB, MYB-related and WRKY) were inferred to act on contig83192/GAS and specific STL, suggesting the occurrence of two distinct routes in STL biosynthesis.

•Antioxidant (AO) properties (AP) of packaged endives were evaluated during storage.•AP were assessed by combining AO compound quantification, chemical and biological assays.•Cultivar type and growth site majorly affected antioxidant properties.•Curly-leafed cultivars offered an overall better quality performance than smooth ones.

Stem-chicory of the “Catalogna” group is a vegetable consumed for bitter-flavored stems. Type and levels of bitter sesquiterpene lactones (STLs) participate in conferring bitterness in vegetables. The content of lactucin—and lactucopocrin-like STLs was higher in “Molfettese” than “Galatina” landrace stalks, regardless of the cultivation sites, consistently with bitterness scores and gustative differences. The “Galatina” transcriptome assembly resulted in 58,872 unigenes, 77% of which were annotated, paving the way to molecular investigation of the STL pathway. Comparative transcriptome analysis allowed the identification of 69,352 SNPs and of 1640 differentially expressed genes that maintained the pattern independently of the site. Enrichment analyses revealed that 4 out of 29 unigenes were up-regulated in “Molfettese” vs “Galatina” within the sesquiterpenoid pathway. The expression of two germacrene A -synthase (GAS) and one -oxidase (GAO) genes of the costunolide branch correlated positively with the contents of lactucin-like molecules, supporting that STL biosynthesis regulation occurs at the transcriptional level. Finally, 46 genes encoding transcription factors (TFs) maintained a differential expression pattern between the two varieties regardless of the growth site; correlation analyses among TFs, GAS, GAO gene expressions and STLs contents suggest that one MYB and one bHLH may act in the pathway.

Chicories produce a wide range of vegetables with important nutritional value. We determined the variation of sterol, total polyphenol, nitrate contents and antioxidant capacity (SC, TPC, NC, AC) in endive leaves and stem-chicory novel vegetables, cultivated in two Italian regions. Within a given area, the SC was similar in smooth- and curly leafed endives (106.3-176.0 mg/kg FW); sitosterol and stigmasterol were major fractions (45-56 versus 38-43%). The stem SC was independent of landrace (101.5-118.6 mg/kg FW); sitosterol prevailed on stigmasterol and fucosterol (73-76 versus 12-14% versus 8-9%); the latter reached 15.7 mg/kg FW, conferring value as potential antidiabetes food. The planting site affected the AC and TPC of endives (893.1-1571.4 μmTE/100 g FW, 30.8-76.1 GAE100/g FW) and chicory stems (729.8-1152.5 μmTE/100 g FW; 56.2-124.4 GAE100/g FW), while the NC was recurrently below dangerous thresholds. PCA showed that environment was the major cause of variation, though it modestly affected these parameters.

The Knotted-like transcription factors (KNOX) contribute to plant organ development. The expression patterns of peach KNOX genes showed that the class 1 members act precociously (S1-S2 stages) and differentially during drupe growth. Specifically, the transcription of KNOPE1 and 6 decreased from early (cell division) to late (cell expansion) S1 sub-stages, whilst that of STMlike1, 2, KNOPE2, 2.1 ceased at early S1. The KNOPE1 role in mesocarp was further addressed by studying the mRNA localization in the pulp cells and vascular net at early and late S1. The message signal was first diffuse in parenchymatous cells and then confined to hypodermal cell layers, showing that the gene down-tuning accompanied cell expansion. As for bundles, the mRNA mainly featured in the procambium/phloem of collateral open types and subsequently in the phloem side of complex structures (converging bundles, ducts). The KNOPE1 overexpression in Arabidopsis caused fruit shortening, decrease of mesocarp cell size, diminution of vascular lignification together with the repression of the major gibberellin synthesis genes AtGA20ox1 and AtGA3ox1. Negative correlation between the expression of KNOPE1 and PpGA3ox1 was observed in four cultivars at S1, suggesting that the KNOPE1 repression of PpGA3ox1 may regulate mesocarp differentiation by acting on gibberellin homeostasis.