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.

Light controls stamen elongation via cryptochromes, phytochromes and COP1 through HY5 and HYH.

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 - oppositely to ARF8 - directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far-red light receptors PHYA/PHYB. In conclusion, different light qualities - sequentially perceived by specific photoreceptors - and the downstream COP1-HY5/HYH module finely tune auxin-induced stamen elongation and thus male fertility.

Transcription Factor Networks in Leaves of Cichorium endivia: New Insights into the Relationship Between Photosynthesis and Leaf Development.

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.

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.

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps.

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.

A Newly Identified Flower-Specific Splice Variant of AUXIN RESPONSE FACTOR8 Regulates Stamen Elongation and Endothecium Lignification in Arabidopsis.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26 Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes.

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.

Arabidopsis Motif Scanner.

BACKGROUND: The major mechanism driving cellular differentiation and organism development is the regulation of gene expression. Cis-acting enhancers and silencers have key roles in controlling gene transcription. The genomic era allowed the transition from single gene analysis to the investigation of full transcriptomes. This transition increased the complexity of the analyses and the difficulty in the interpretation of the results. In this context, there is demand for new tools aimed at the creation of gene networks that can facilitate the interpretation of Next Generation Sequencing (NGS) data. RESULTS: Arabidopsis Motif Scanner (AMS) is a Windows application that runs on local computers. It was developed to build gene networks by identifying the positions of cis-regulatory elements in the model plant Arabidopsis thaliana and by providing an easy interface to assess and evaluate gene relationships. Its major innovative feature is to combine the cis-regulatory element positions, NGS and DNA Chip Arrays expression data, Arabidopsis annotations and gene interactions for the identification of gene networks regulated by transcription factors. In studies focused on transcription factors function, the software uses the expression data and binding site motifs in the regulative gene regions to predict direct target genes. Additionally, AMS utilizes DNA-protein and protein-protein interaction data to facilitate the identification of the metabolic pathways regulated by the transcription factor of interest. CONCLUSIONS: Arabidopsis Motif Scanner is a new tool that helps researchers to unravel gene relations and functions. In fact, it facilitates studies focused on the effects and the impact that transcription factors have on the transcriptome by correlating the position of cis-acting elements, gene expression data and interactions.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

The peach (Prunus persica L. Batsch) genome harbours 10 KNOX genes, which are differentially expressed in stem development, and the class 1 KNOPE1 regulates elongation and lignification during primary growth.

The KNOTTED-like (KNOX) genes encode homeodomain transcription factors and regulate several processes of plant organ development. The peach (Prunus persica L. Batsch) genome was found to contain 10 KNOX members (KNOPE genes); six of them were experimentally located on the Prunus reference map and the class 1 KNOPE1 was found to link to a quantitative trait locus (QTL) for the internode length in the peach×Ferganensis population. All the KNOPE genes were differentially transcribed in the internodes of growing shoots; the KNOPE1 mRNA abundance decreased progressively from primary (elongation) to secondary growth (radial expansion). During primary growth, the KNOPE1 mRNA was localized in the cortex and in the procambium/metaphloem zones, whereas it was undetected in incipient phloem and xylem fibres. KNOPE1 overexpression 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 the 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.

Peach [Prunus persica (L.) Batsch] KNOPE1, a class 1 KNOX orthologue to Arabidopsis BREVIPEDICELLUS/KNAT1, is misexpressed during hyperplasia of leaf curl disease.

Class 1 KNOTTED-like (KNOX) transcription factors control cell meristematic identity. An investigation was carried out to determine whether they maintain this function in peach plants and might act in leaf curliness caused by the ascomycete Taphrina deformans. KNOPE1 function was assessed by overexpression in Arabidopsis and by yeast two-hybrid assays with Arabidopsis BELL proteins. Subsequently, KNOPE1 mRNA and zeatin localization was monitored during leaf curl disease. KNOPE1 and Arabidopsis BREVIPEDICELLUS (BP) proteins fell into the same phyletic group and recognized the same BELL factors. 35S:KNOPE1 Arabidopsis lines exhibited altered traits resembling those of BP-overexpressing lines. In peach shoot apical meristem, KNOPE1 was expressed in the peripheral and central zones but not in leaf primordia, identically to the BP expression pattern. These results strongly suggest that KNOPE1 must be down-regulated for leaf initiation and that it can control cell meristem identity equally as well as all class 1 KNOX genes. Leaves attacked by T. deformans share histological alterations with class 1 KNOX-overexpressing leaves, including cell proliferation and loss of cell differentiation. Both KNOPE1 and a cytokinin synthesis ISOPENTENYLTRANSFERASE gene were found to be up-regulated in infected curled leaves. At early disease stages, KNOPE1 was uniquely triggered in the palisade cells interacting with subepidermal mycelium, while zeatin vascular localization was unaltered compared with healthy leaves. Subsequently, when mycelium colonization and asci development occurred, both KNOPE1 and zeatin signals were scattered in sectors of cell disorders. These results suggest that KNOPE1 misexpression and de novo zeatin synthesis of host origin might participate in hyperplasia of leaf curl disease.

The role of knox genes in plant development.

knox genes encode homeodomain-containing transcription factors that are required for meristem maintenance and proper patterning of organ initiation. In plants with simple leaves, knox genes are expressed exclusively in the meristem and stem, but in dissected leaves, they are also expressed in leaf primordia, suggesting that they may play a role in the diversity of leaf form. This hypothesis is supported by the intriguing phenotypes found in gain-of-function mutations where knox gene misexpression affects leaf and petal shape. Similar phenotypes are also found in recessive mutations of genes that function to negatively regulate knox genes. KNOX proteins function as heterodimers with other homeodomains in the TALE superclass. The gibberellin and lignin biosynthetic pathways are known to be negatively regulated by KNOX proteins, which results in indeterminate cell fates.

Isolation and characterization of a maintenance DNA-methyltransferase gene from peach (Prunus persica [L.] Batsch): transcript localization in vegetative and reproductive meristems of triple buds.

A cDNA coding for a DNA (cytosine-5)-methyltransferase (METase) was isolated from peach (Prunus persica [L.] Batsch) and the corresponding gene designated as PpMETI. The latter encoded a predicted polypeptide of 1564 amino acid residues and harboured all the functional domains conserved in the maintenance METases group type I. PpMETI was a single copy in the cultivar Chiripa which was used as a model in the present study. Expression analyses revealed that PpMETI transcripts were more abundant in tissues with actively proliferating cells such as apical tips, uncurled leaves, elongating herbaceous stems, and small immature fruits. Peach plants bear bud clusters (triads or triple buds), consisting of two lateral and one central bud with floral and vegetative fates, respectively. PpMETI in situ hybridization was performed in triple buds during their entire developmental cycle. High and low levels of PpMETI transcript were related to burst and quiescence of vegetative growth, respectively. Message localization distinguished lateral from central buds during the meristem switch to the floral phase. In fact, the PpMETI message was abundant in the L1 layer of protruding domes, a morphological trait marking the beginning of floral transition. The PpMETI transcript was also monitored during organ flower formation. Altogether, these data suggest a relationship between DNA replication and PpMETI gene expression.

The knotted1-like homeobox gene BREVIPEDICELLUS regulates cell differentiation by modulating metabolic pathways.

Members of the KNOX gene family have important roles in plant meristems by regulating cell division and differentiation. BREVIPEDICELLUS (BP), one of seven KNOX genes in Arabidopsis, has a primary role in internode patterning. We carried out a comparison of RNA expression profiles between wild-type seedlings and bp mutants at a developmental stage prior to a visible phenotypic difference. Transcript differences were found for a number of genes in cell wall biosynthesis, especially genes in the lignin pathway. The regulation of lignin biosynthesis by BP was demonstrated by observing increased lignin deposition in bp mutants following bolting, decreased lignification in plants overexpressing BP, and aberrant lignin deposition in discrete regions of the bp stem. Furthermore, we showed that BP binds promoters of some genes in the lignin pathway. Our results provide a metabolic fingerprint for BP and identify the lignin pathway as one of the coordinate processes that BP regulates.

Expression profiling of plant development.

Microarrays have been used to study the response of plants to many signals, including light, hormones and transcription factors. The results in each case can give an overall view of the global response to the signal or identify direct targets of the signal, and can reveal new links between different signaling pathways.