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

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

A Novel Role of <i>Medicago truncatula</i> KNAT3/4/5-like Class 2 KNOX Transcription Factors in Drought Stress Tolerance

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

Strong increase of foliar inulin occurs in transgenic lettuce plants (Lactuca sativa L.) overexpressing the Asparagine Synthetase A gene from Escherichia coli

Transgenic lettuce (Lactuca sativa L. cv. 'Cortina') lines expressing the asparagine synthetase A gene from Escherichia coli were produced to alter the plant nitrogen status and eventually enhance growth. The relative molecular abundance of water-soluble metabolites was measured by H-1 NMR in transgenic and conventional plants at early developmental stages and grown under the same conditions. NMR metabolic profiles assessed that a transgenic, line and the wild-type counterpart shared the same compounds, but it also revealed side effects on the carbon metabolism following genetic modification. Concerning the nitrogen status, the amino acid content did not vary significantly, except for glutamic acid and gamma-aminobutyric acid, which diminished in the transgenics. As for the carbon metabolism, in transgenic leaves the contents of sucrose, glucose, and fructose decreased, whereas that of inulin increased up to 30 times, accompanied by the alteration of most Krebs's cycle organic acids and the rise of tartaric acid compared to nontransformed controls. Lettuce leaf inulins consisted of short oligomeric chains made of one glucose unit bound to two/four fructose units. Inulins are beneficial for human health, and they are extracted from plants and commercialized as long-chain types, whereas the short forms are synthesized chemically. Hence, lettuce genotypes with high content of foliar short-chain inulin represent useful materials for breeding strategies and a potential source for low molecular weight inulin

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

The KNOTTED-like genes of peach (Prunus persica L. Batsch) are differentially expressed during drupe growth and the class 1 KNOPE1 contributes to mesocarp development

none10noThe KNOTTED-like transcription factors (KNOX) contribute to plant organ development. The expression patterns of peach KNOX genes showed that the class 1 members act precociously (S1-S2 stages) and differentially during drupe growth. Specifically, the transcription of KNOPE1 and 6 decreased from early (cell division) to late (cell expansion) S1 sub-stages, whilst that of STMlike1, 2, KNOPE2, 2.1 ceased at early S1. The KNOPE1 role in mesocarp was further addressed by studying the mRNA localization in the pulp cells and vascular net at early and late Si. The message signal was first diffuse in parenchymatous cells and then confined to hypodermal cell layers, showing that the gene down-tuning accompanied cell expansion. As for bundles, the mRNA mainly featured in the procambium/phloem of collateral open types and subsequently in the phloem side of complex structures (converging bundles, ducts). The KNOPE1 overexpression in Arabidopsis caused fruit shortening, decrease of mesocarp cell size, diminution of vascular lignification together with the repression of the major gibberellin synthesis genes AtGA20ox1 and AtGA3ox1. Negative correlation between the expression of KNOPE1 and PpGA3ox1 was observed in four cultivars at S1, suggesting that the KNOPE1 repression of PpGA3ox1 may regulate mesocarp differentiation by acting on gibberellin homeostasis.noneTestone, Giulio; Condello, Emiliano; Di Giacomo, Elisabetta; Nicolodi, Chiara; Caboni, Emilia; Rasori, Angela; Bonghi, Claudio; Bruno, Leonardo; Bitonti, Maria Beatrice; Giannino, DonatoTestone, Giulio; Condello, Emiliano; Di Giacomo, Elisabetta; Nicolodi, Chiara; Caboni, Emilia; Rasori, Angela; Bonghi, Claudio; Bruno, Leonardo; Bitonti, Maria Beatrice; Giannino, Donat

Quality Traits of Conventional and Transgenic Lettuce (Lactuca Sativa L.) at Harvesting by NMR Metabolic Profing

Metabolism of genetically modified (GM) lettuce (Lactuca sativa L.) leaves was investigated by comparing NMR metabolic profiles of three lines (T3B12, T7B7, and T7B14) overexpressing the E. coli asparagine synthetase A gene with those of the wild type (WT) at 24, 56, and 64 days after sowing (DAS). Statistical analyses based on hydro-soluble compound profiles significantly and maximally discriminated the WT from GM-lines at optimal harvest time (56 DAS). The T7B14 metabolic variations were opposite to those of both T3B12/T7B7 lines, suggesting that unexpected effects of transgenesis had occurred. Compared to controls, the T3B12/T7B7 plants shared the leaf mass increase, higher amino acid (asparagine, glutamine, valine, and isoleucine) and protein levels, and lower nitrate contents, accompanied by a modest sink of organic acids (R-chetoglutarate, succinate, fumarate, and malate), sucrose, fructose, and inulins. Incongruously, the T7B14 butter heads were less leafy than the controls and showed lowered amino acid/protein contents and overstored inulin. To further investigate the metabolic discrepancies among the GM-lines, a set of key nitrogen and inulin genes was monitored. The T3B12/T7B7 lines shared comparable gene expression changes, including the induction of the endogenous ASPARAGINE SYNTHETASE1 and NITRATE REDUCTASE1 that supported the targeted enhancement of nitrogen status. Transgene product malfunctioning and T-DNA rearrangements throughout generations were proposed to explain the decreased asparagine content and the complex expression pattern of N genes in T7B14 leaves. In the latter, the inulin accumulation was associated with the upregulation of fructan biosynthesis genes and the intense repression of fructan hydrolase

Overexpression of KNAT1 in Lettuce Shifts Leaf Determinate Growth to a Shoot-Like Indeterminate Growth Associated with an Accumulation of Isopentenyl-Type Cytokinins

Leaves are specialized organs characterized by defined developmental destiny and determinate growth. The overexpression of Knotted1-like homeobox genes in different species has been shown to alter leaf shape and development, but a definite role for this class of genes remains to be established. Transgenics that overexpress Knotted1-like genes present some traits that are characteristic of altered cytokinin physiology. Here we show that lettuce (Lactuca sativa) leaves that overexpress KNAT1, an Arabidopsis kn1-like gene, acquire characteristics of indeterminate growth typical of the shoot and that this cell fate change is associated with the accumulation of specific types of cytokinins. The possibility that the phenotypic effects of KNAT1 overexpression may arise primarily from the modulation of local ratios of different cytokinins is discussed