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 germacrene A synthase, germacrene A oxidase and costunolide synthase 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.

We investigated the role of KNOX genes in legume root nodule organogenesis. Class 1 KNOX homeodomain transcription factors (TFs) are involved in plant shoot development and leaf shape diversity. Class 2 KNOX genes are less characterized, even though an antagonistic function relative to class 1 KNOXs was recently proposed. In silico expression data and further experimental validation identified in the Medicago truncatula model legume three class 2 KNOX genes, belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like), as expressed during nodulation from early stages. RNA interference (RNAi)-mediated silencing and overexpression studies were used to unravel a function for KNOX TFs in nodule development. Mt KNAT3/4/5-like genes encoded four highly homologous proteins showing overlapping expression patterns during nodule organogenesis, suggesting functional redundancy. Simultaneous reduction of Mt KNAT3/4/5-like genes indeed led to an increased formation of fused nodule organs, and decreased the expression of the MtEFD (Ethylene response Factor required for nodule Differentiation) TF and its direct target MtRR4, a cytokinin response gene. Class 2 KNOX TFs therefore regulate legume nodule development, potentially through the MtEFD/MtRR4 cytokinin-related regulatory module, and may control nodule organ boundaries and shape like class 2 KNOX function in leaf development.

We isolated three class I and three class II KNOX genes in Medicago truncatula. The predicted amino acid sequences suggested a possible orthology to the Arabidopsis homeodomain proteins STM, KNAT1/BP, KNAT3 and KNAT7 that was confirmed by phylogenetic and conserved structural domain analyses. Moreover, the STM-like MtKNOX1 and MtKNOX6 proteins were shown to retain the capability to interact with the Arabidopsis BELL protein partners of STM and KNAT1/BP. Amino acid residues that characterize the different classes of KNOX proteins were identified. Gene expression studies revealed organ-specificity, possible cytokinin-dependent transcriptional activation of two MtKNOXs and expression of one STM-like and a BP/KNAT1-like MtKNOX in roots. Interestingly, mRNA localization studies carried out on class I MtKNOX genes revealed important differences with previously characterised legume KNOXs. M. truncatula transcripts were not down-regulated in leaf primordia and early stages of leaf development, features shared with the more distant compound-leaved species Solanum lycopersicum.

The Three Amino acid Loop Extension (TALE) proteins constitute an ancestral superclass of homeodomain transcription factors conserved in animals, plants and fungi. In plants they comprise two classes, KNOTTED1-LIKE homeobox (KNOX) and BEL1-like homeobox (BLH or BELL, hereafter referred to as BLH), which are involved in shoot apical meristem (SAM) function, as well as in the determination and morphological development of leaves, stems and inflorescences. Selective protein-protein interactions between KNOXs and BLHs affect heterodimer subcellular localization and target affinity. KNOXs exert their roles by maintaining a proper balance between undifferentiated and differentiated cell state through the modulation of multiple hormonal pathways. A pivotal function of KNOX in evolutionary diversification of leaf morphology has been assessed. In the SAM of both simple- and compound-leafed seed species, downregulation of most class 1 KNOX (KNOX1) genes marks the sites of leaf primordia initiation. However, KNOX1 expression is re-established during leaf primordia development of compound-leafed species to maintain transient indeterminacy and morphogenetic activity at the leaf margins. Despite the increasing knowledge available about KNOX1 protein function in plant development, a comprehensive view on their downstream effectors remains elusive. This review highlights the role of TALE proteins in leaf initiation and morphological plasticity with a focus on recent advances in the identification of downstream target genes and pathways.

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.

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.

Transgene escape risks are evaluated by assessing the frequency of transgenic hybrids within populations sexually compatible with those genetically modified. We examined the pollen-mediated transgene flow among lettuce varieties in Italy. Transgenic ÔLuxorÕ lines harboured the arabidopsis KNAT1 gene, which caused leaf trait alterations, and the bacterial NPTII gene for resistance to kanamycin. Donor homozygous lines (KNAT1:NPTII/KNAT1:NPTII) were used in two trials: in glasshouse, ÔLuxorÕ transgenic and conventional plants were spaced at 15 cm; in field, a transgenic ÔLuxorÕ donor island was set up and surrounded by 10 pollen receiver varieties sited at 0.5, 11 and 22 m. Progenies of recipient cultivars were screened by kanamycin assays, leaf alterations, transgene transcription and integrity. The out-crossing rate (OCR) was calculated as the frequency of KNAT1:NPTII/) and dKNAT1:NPTII/) genotypes because KNAT1 impair (indicated with d) was observed to vary from the 0.03% to 0.36%. Indoor, ÔLuxorÕ intra-varietal OCR was 0.41 ± 0.11%; outdoor, the average intervarietal OCR was 0.49%, 0.071% and 0.035% for plants at 0.5, 11 and 22 m, respectively.

The shoot apical meristem (SAM) is a population of undifferentiated cells at the tip of the shoot axis that establishes early during plant embryogenesis and gives rise to all shoot organs throughout the plant's life. A plethora of different families of transcription factors (TFs) play a key role in establishing the equilibrium between cell differentiation and stem cell maintenance in the SAM. Fine tuning of these regulatory proteins is crucial for a proper and fast SAM response to environmental and hormonal cues, and for development progression. One effective way to rapidly inactivate TFs involves regulated proteolysis by the ubiquitin/26S proteasome system (UPS). However, a possible role of UPS-dependent protein degradation in the regulation of key SAM TFs has not been thoroughly investigated. Here, we summarize recent evidence supporting a role for the UPS in SAM maintenance and function. We integrate this survey with an in silico analysis of publicly-available microarray databases which identified ubiquitin ligases that are expressed in specific areas within the SAM, suggesting that they may regulate or act downstream of meristem-specific factors.