Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing

The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts

Selection of superior heat tolerant tomato lines through genetic and genomic strategies

Climate is unequivocally changing. The planet's average surface temperature has risen about 1.62 degrees Fahrenheit (0.9 degrees Celsius) since the late 19th century. Most of the warming climates occurred in the past 35 years, were recorded in the last 10 years. Global warming affects agriculture in a number of ways, including temperate region average temperature changes and climate extremes. Tomato (Solanum lycopersicum) is both an important commercial crop and a model system for genetic studies, due to its diploid, relatively compact, and recently sequenced genome and to the availability of large genetic and genomic resource collections. Tomato species is highly sensitive to high temperature and few degrees above its optimum growth temperature threshold can lead to serious deleterious effects, such as flower abscission, decrease of pollen quality, abnormal growth, reduced fruit set and yield. Therefore, the development of innovative strategies to obtain tomato cultivars with improved yield under high temperature conditions is a main goal for plant molecular science and breeding. In this thesis, different breeding strategies were used to improve tomato cultivars tolerance to heat stress. A F4 segregating population, deriving from the tomato variety JAG8810, previously selected for yield performance under hot stress, was phenotypically investigated under heat stress conditions to evaluate quantitative and qualitative traits. By the means of a PCA analysis, best and worst performers were selected. Extreme individuals for yield were also evaluated for sub-traits, such as pollen viability, to better investigate the basis of heat stress tolerance and correlations among heat tolerance component traits. In addition, the cultivar Moneymaker was tested for a heat treatment on limited areas, suggesting that heat stress response is a local process. The F4 population deriving from the JAG8810 variety, was sequenced by genotyping by sequencing (GBS) approach to identify all possible variants. Genomic prediction models for yield production per plant (YP) and soluble solid content (SSC) under heat stress, were developed. Several parameters, including training population size and composition and marker quality were adjusted to obtain optimized models for assessed traits and population. The predicted GEBVs (genetic breeding values) of F5 offspring were phenotypically validated in field. Furthermore, the most meaningful SNPs selected for model construction were used to conduct a QTL analysis to shed light on the genetic basis of heat tolerant traits in tomato. The analysis permitted the identification of 5 QTLs involved in yield and one in SSC. Two candidate genes putatively involved in heat stress tolerance were discovered in regions underlining QTLs. Finally, with the aim to identify regulatory elements involved in the abiotic stress tolerance, a tomato genome scan and a phylogenetic analysis of Dof proteins was performed identifying SlDof11 as suitable target for CRISPR/Cas9 experiments

Accelerating Tomato Breeding by Exploiting Genomic Selection Approaches

Genomic selection (GS) is a predictive approach that was built up to increase the rate of genetic gain per unit of time and reduce the generation interval by utilizing genome-wide markers in breeding programs. It has emerged as a valuable method for improving complex traits that are controlled by many genes with small effects. GS enables the prediction of the breeding value of candidate genotypes for selection. In this work, we address important issues related to GS and its implementation in the plant context with special emphasis on tomato breeding. Genomic constraints and critical parameters affecting the accuracy of prediction such as the number of markers, statistical model, phenotyping and complexity of trait, training population size and composition should be carefully evaluated. The comparison of GS approaches for facilitating the selection of tomato superior genotypes during breeding programs is also discussed. GS applied to tomato breeding has already been shown to be feasible. We illustrated how GS can improve the rate of gain in elite line selection, and descendent and backcross schemes. The GS schemes have begun to be delineated and computer science can provide support for future selection strategies. A new promising breeding framework is beginning to emerge for optimizing tomato improvement procedures

Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors

Plant abietane diterpenoids (e.g. aethiopinone, 1- oxoaethiopinone, salvipisone and ferruginol), synthesized in the roots of several Salvia spp, have antibacterial, antifungal, sedative and anti-proliferative properties. Recently we have reported that content of these compounds in S. sclarea hairy roots is strongly depending on transcriptional regulation of genes belonging to the plastidial MEP-dependent terpenoid pathway, from which they mostly derive. To boost the synthesis of this interesting class of compounds, heterologous AtWRKY18, AtWRKY40, and AtMYC2 TFs were overexpressed in S. sclarea hairy roots and proved to regulate in a coordinated manner the expression of several genes encoding enzymes of the MEP-dependent pathway, especially DXS, DXR, GGPPS and CPPS. The content of total abietane diterpenes was enhanced in all overexpressing lines, although in a variable manner due to a negative pleiotropic effect on HR growth. Interestingly, in the best performing HR lines overexpressing the AtWRKY40 TF induced a significant 4-fold increase in the final yield of aethiopinone, for which we have reported an interesting anti-proliferative activity against resistant melanoma cells. The present results are also informative and instrumental to enhance the synthesis of abietane diterpenes derived from the plastidial MEP-derived terpenoid pathway in other Salvia species

Plant Extracellular Vesicles: Current Landscape and Future Directions

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell–cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant–pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production

Cultivated cardoon (Cynara cardunculus L. var altilis) is a Mediterranean traditional food crop. It is adapted to xerothermic conditions and also grows in marginal lands, producing a large biomass rich in phenolic bioactive metabolites and has therefore received attention for pharmaceutical, cosmetic and innovative materials applications. Cardoon cell cultures can be used for the biotechnological production of valuable molecules in accordance with the principles of cellular agriculture. In the current study, we developed an elicitation strategy on leaf-derived cardoon calli for boosting the production of bioactive extracts for cosmetics. We tested elicitation conditions that trigger hyper-accumulation of bioactive phenolic metabolites without compromising calli growth through the application of chilling and salt stresses. We monitored changes in growth, polyphenol accumulation, and antioxidant capability, along with transcriptional variations of key chlorogenic acid and flavonoids biosynthetic genes. At moderate stress intensity and duration (14 days at 50–100 mM NaCl) salt exerted the best eliciting effect by stimulating total phenols and antioxidant power without impairing growth. Hydroalcoholic extracts from elicited cardoon calli with optimal growth and bioactive metabolite accumulation were demonstrated to lack cytotoxicity by MTT assay and were able to stimulate pro-collagen and aquaporin production in dermal cells. In conclusion, we propose a “natural” elicitation system that can be easily and safely employed to boost bioactive metabolite accumulation in cardoon cell cultures and also in pilot-scale cell culture production

Transcriptional, metabolic and DNA methylation changes underpinning the response of Arundo donax ecotypes to NaCl excess

Arundo donax ecotypes react differently to salinity, partly due to differences in constitutive defences and methylome plasticity. Arundo donax L. is a C3 fast-growing grass that yields high biomass under stress. To elucidate its ability to produce biomass under high salinity, we investigated short/long-term NaCl responses of three ecotypes through transcriptional, metabolic and DNA methylation profiling of leaves and roots. Prolonged salt treatment discriminated the sensitive ecotype 'Cercola' from the tolerant 'Domitiana' and 'Canneto' in terms of biomass. Transcriptional and metabolic responses to NaCl differed between the ecotypes. In roots, constitutive expression of ion transporter and stress-related transcription factors' genes was higher in 'Canneto' and 'Domitiana' than 'Cercola' and 21-day NaCl drove strong up-regulation in all ecotypes. In leaves, unstressed 'Domitiana' confirmed higher expression of the above genes, whose transcription was repressed in 'Domitiana' but induced in 'Cercola' following NaCl treatment. In all ecotypes, salinity increased proline, ABA and leaf antioxidants, paralleled by up-regulation of antioxidant genes in 'Canneto' and 'Cercola' but not in 'Domitiana', which tolerated a higher level of oxidative damage. Changes in DNA methylation patterns highlighted a marked capacity of the tolerant 'Domitiana' ecotype to adjust DNA methylation to salt stress. The reduced salt sensitivity of 'Domitiana' and, to a lesser extent, 'Canneto' appears to rely on a complex set of constitutively activated defences, possibly due to the environmental conditions of the site of origin, and on higher plasticity of the methylome. Our findings provide insights into the mechanisms of adaptability of A. donax ecotypes to salinity, offering new perspectives for the improvement of this species for cultivation in limiting environments

Screening of giant reed (<i>Arundo donax</i> L.) ecotypes for biomass production under salt stress

<p>Given its high biomass and plasticity, <i>Arundo donax</i> L. is a promising ligno-cellulosic crop for cultivation in marginal lands in temperate climates. In order to test for adaptation to salinity, growth parameters of several <i>A. donax</i> clones were evaluated under two salt regimes in hydroponics. Mild NaCl stress (50 mM NaCl, 5.6 mS cm⁻¹ EC, for 10 days) failed to discriminate between ecotypes, while a more severe NaCl treatment (150 mM, 18.8 mS cm⁻¹ EC, for 21 days) enabled the identification of ecotypes maintaining plant growth under high salinity. Among several biometric parameters, 4th leaf width, and shoot and root DW consistently highlighted differences between ecotypes. Gas-exchange parameters also responded to severe NaCl treatment, while the photosystem efficiency was good, regardless of treatment. The results confirm that <i>A. donax</i> can be considered moderately tolerant to NaCl stress, with variation between ecotypes. Our screening protocol identified ecotypes with higher biomass production under severe NaCl treatment and can be useful for preliminary evaluation of NaCl tolerant clones for increasing productivity under salinity. The detected inter-ecotype variability could also be investigated to identify suitable clones for different environments.</p