Prunus Knotted-like genes. Genome-wide analysis, transcriptional response to Cytokinin in micropropagation, and rootstock transformation

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

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

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

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

NMR-Metabolic Methodology in the Study of GM Foods

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

Phytochemicals in Asteraceae Leafy Vegetables

Knowledge about quantitative and qualitative characterization of phytochemical contents in Asteraceae leafy vegetables is continuously increasing due to the strong interest from scientists and consumers. This chapter deals with leafy vegetables of Lactuca sativa, Cichorium intybus and C. endivia species, given their relevance within the botanical family. It gives an overview of the wide differences occurring in genotype panels, in bioactive compound types and levels, focusing on phenolics, carotenoids and sesquiterpenes lactones. It also provides information on their biosynthesis pathways in plants together with health effects, bioaccessibility and bioavailability of diverse phytonutrient classes. Finally, it outlines the effects of the main pre-harvest and post-harvest factors affecting the amount and status of final products intended for dietary intake, and reports on some genetic aspects and biotechnologies aimed to biofortification of these species

The Chloroplast Genome of Endive (<i>Cichorium endivia</i> 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

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

Nutritive Parameters and Antioxidant Quality of Minimally Processed "Cime di Rapa" (Brassica rapa subsp. sylvestris) Vary as Influenced by Genotype and Storage Time

In order to assess the quality and performance of bagged broccoli-raab, a recently marketed product, several nutritive parameters were determined in novel hybrid and conventional cultivars at pre- and post-packaging stages in the industrial environment. The characterization of shoots and composing organs at post-cut stage included contents of dietary fibre (DF), glycaemic carbohydrates (GC), antioxidant compounds (ACC) and capacity (AOC), which were determined by chromatographic methods and spectrophotometric assays. ACC and AOC were analysed during shelf life of bagged products. Genotype and storage effects were addressed as variability factors at fixed packaging conditions. Contents of DF and GC (39.64-34.57; 7.56-2.21 g/100 g), glucosinolates (37.47-24.63 mg/g SIN), and ACC (total phenolics: 18.64-14.92 mg GAE/g; flavonoids: 34.74-30.96 mg/g CE; flavonols: 14.62-14.08 mg QE/g), and AOC (Oxygen Radical Absorbance Capacity: 354.62-293.25 µmol/g TE; DPPH• scavenging activity: 59.35-46.14) were lower in shoots of the hybrid than marketed cultivar. In both genotypes, AOC was maximal in leaves, followed by florets and stems. The integrated analyses suggested that the hybrid genotype was better suited for fresh consumption and that increased ratio of florets/leaves vs. stem is expected to raise product antioxidant properties. The comparison of unprocessed and bagged products pointed at a value decay of most parameters except for glucosinolates and correlation analyses supported the necessity of performing multiple antioxidant assays to enhance product quality evaluation. As for shelf life, storage time was the major factor affecting antioxidant properties, while genotype and interaction effects were minimal