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The present work describes the development of a headspace solid-phase microextraction (HS-SPME) followed by gas chromatography - mass spectrometry (GC-MS) method for the qualitative analysis of compounds in seized ecstasy tablets that can be easily implemented in regular laboratories. HS-SPME with a DVB/CAR/PDMS 50/30 µm fiber was used to extract the ecstasy pills' components, including major and minor ones, in a single extraction/chromatographic run. For HS-SPME, the incubation time (0 min to 30 min), the extraction time (10 min to 40 min) and temperature (40 °C to 80 ºC), the buffer volume (3 mL to 8 mL), the buffer pH (6 to 9) and the NaCl concentration (0 mol/L to 6 mol/L) were evaluated using fractional factorial design. Different split ratios and detector voltages were also evaluated. The optimal compromise between sensitivity and peak resolution was found to be incubation and extraction at 65 ºC for 10 min and 25 min, respectively, 3 mL of pH 9 buffer containing 3 mol/L NaCl, using 40.0 mg of the powdered samples in a 15-mL amber glass vial, and an injection with a split ratio of 1:10 at 260 ºC for 10 min. Under optimal conditions, 44 samples from different seizures were analyzed. Seventy-five compounds were tentatively identified by the proposed method, including active substances, medicines, caffeine, safrole derivatives, synthesis intermediates and solvent residues. The number of tentatively identified compounds per sample varied from 8 to 24, with a mean of 15. Important findings in ecstasy samples, such as norcinamolaurin, α-methyl-1,3-benzodioxole-5-propanamide, α-methyl-3,4-methylenedioxyphenylpropionitrile, acetylsalicylic acid, piperonylonitrile, methyl isobutyl ketone, mesitylene, and 4-[3-(dimethylamino)propyl]- 2,6-dimethylphenol, identified with a frequency higher than 10%, are not found in the literature so far. The method precision, based on relative standard deviation of peak areas, ranged from 5% to 15%, depending on the compound. The method was shown to be simple, relatively fast, precise and a powerful tool for the identification of major and minor components in ecstasy tablets in a single analytical cycle, being useful for screening or quantitative purposes, if authentic standards are available., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Class 2 KNOX homeobox transcription factors (KNOX2) play a role in promoting cell differentiation in several plant developmental processes. In Arabidopsis , they antagonize the meristematic KNOX1 function during leaf development through the modulation of phytohormones. In Medicago truncatula , three KNOX2 genes belonging to the KNAT3/4/5-like subclass ( Mt KNAT3/4/5-like or MtKNOX3-like ) redundantly works upstream of a cytokinin-signaling module to control the symbiotic root nodule formation. Their possible role in the response to abiotic stress is as-of-yet unknown. We produced transgenic M. truncatula lines, in which the expression of four MtKNOX3-like genes was knocked down by RNA interference. When tested for response to water withdrawal in the soil, RNAi lines displayed a lower tolerance to drought conditions compared to the control lines, measured as increased leaf water loss, accelerated leaf wilting time, and faster chlorophyll loss. Reanalysis of a transcriptomic M. truncatula drought stress experiment via cluster analysis and gene co-expression networks pointed to a possible role of MtKNOX3-like transcription factors in repressing a proline dehydrogenase gene ( MtPDH ), specifically at 4 days after water withdrawal. Proline measurement and gene expression analysis of transgenic RNAi plants compared to the controls confirmed the role of KNOX3-like genes in inhibiting proline degradation through the regulation of the MtPDH gene.

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.

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., Competing Interests: The authors declare no conflict of interest.

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.

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., Competing Interests: The authors declare no conflict of interest.

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.

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.

The 1H-NMR methodology used in the study of genetically modified (GM) foods is discussed. Transgenic lettuce (Lactuca sativa cv "Luxor") over-expressing the ArabidopsisKNAT1 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.