The Chloroplast Genome of Endive (Cichorium endivia L.): Cultivar Structural Variants and Transcriptome Responses to Stress Due to Rain Extreme Events.

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.

Leaf nutrient content and transcriptomic analyses of endive (Cichorium endivia) stressed by downpour-induced waterlog reveal a gene network regulating kestose and inulin contents.

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.

Insights into sucrose pathway of chicory stems by integrative transcriptomic and metabolic analyses.

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.

Transcriptome driven characterization of curly- and smooth-leafed endives reveals molecular differences in the sesquiterpenoid pathway.

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.

Corrigendum: Insights into the Sesquiterpenoid Pathway by Metabolic Profiling and De novo Transcriptome Assembly of Stem-Chicory (Cichorium intybus Cultigroup "Catalogna").

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

Influence of cultivation sites on sterol, nitrate, total phenolic contents and antioxidant activity in endive and stem chicory edible products.

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.

Insights into the Sesquiterpenoid Pathway by Metabolic Profiling and De novo Transcriptome Assembly of Stem-Chicory (Cichorium intybus Cultigroup "Catalogna").

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.