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.

An Integrated Multilevel Approach Unveils Complex Seed-Nanoparticle Interactions and Their Implications for Seed Priming.

Nanotechnology has the potential to revolutionize agriculture with the introduction of engineered nanomaterials. However, their use is hindered by high cost, marginal knowledge of their interactions with plants, and unpredictable effects related to massive use in crop cultivation. Nanopriming is an innovative seed priming technology able to match economic, agronomic, and environmental needs in agriculture. The present study was focused on unveiling, by a multilevel integrated approach, undisclosed aspects of seed priming mediated by iron oxide magnetic nanoparticles in pepper seeds (Capsicum annuum), one of the most economically important crops worldwide. Inductively coupled plasma atomic emission mass spectrometry and scanning electron microscopy were used to quantify the MNP uptake and assess seed surface changes. Magnetic resonance imaging mapped the distribution of MNPs prevalently in the seed coat. The application of MNPs significantly enhanced the root and vegetative growth of pepper plants, whereas seed priming with equivalent Fe concentrations supplied as FeCl3 did not yield these positive effects. Finally, global gene expression by RNA-sequencing identified more than 2,200 differentially expressed genes, most of them involved in plant developmental processes and defense mechanisms. Collectively, these data provide evidence on the link between structural seed changes and an extensive transcriptional reprogramming, which boosts the plant growth and primes the embryo to cope with environmental challenges that might occur during the subsequent developmental and growth stages.

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.

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.

Tomato genomic prediction for good performance under high-temperature and identification of loci involved in thermotolerance response.

Many studies showed that few degrees above tomato optimum growth temperature threshold can lead to serious loss in production. Therefore, the development of innovative strategies to obtain tomato cultivars with improved yield under high temperature conditions is a main goal both for basic genetic studies and breeding activities. In this paper, a F4 segregating population was phenotypically evaluated for quantitative and qualitative traits under heat stress conditions. Moreover, a genotyping by sequencing (GBS) approach has been employed for building up genomic selection (GS) models both for yield and soluble solid content (SCC). Several parameters, including training population size, composition and marker quality were tested to predict genotype performance under heat stress conditions. A good prediction accuracy for the two analyzed traits (0.729 for yield production and 0.715 for SCC) was obtained. The predicted models improved the genetic gain of selection in the next breeding cycles, suggesting that GS approach is a promising strategy to accelerate breeding for heat tolerance in tomato. Finally, the annotation of SNPs located in gene body regions combined with QTL analysis allowed the identification of five candidates putatively involved in high temperatures response, and the building up of a GS model based on calibrated panel of SNP markers.

Practical Workflow from High-Throughput Genotyping to Genomic Estimated Breeding Values (GEBVs).

The global climate is changing, resulting in significant economic losses worldwide. It is thus necessary to speed up the plant selection process, especially for complex traits such as biotic and abiotic stresses. Nowadays, genomic selection (GS) is paving new ways to boost plant breeding, facilitating the rapid selection of superior genotypes based on the genomic estimated breeding value (GEBV). GEBVs consider all markers positioned throughout the genome, including those with minor effects. Indeed, although the effect of each marker may be very small, a large number of genome-wide markers retrieved by high-throughput genotyping (HTG) systems (mainly genotyping-by-sequencing, GBS) have the potential to explain all the genetic variance for a particular trait under selection. Although several workflows for GBS and GS data have been described, it is still hard for researchers without a bioinformatics background to carry out these analyses. This chapter has outlined some of the recently available bioinformatics resources that enable researchers to establish GBS applications for GS analysis in laboratories. Moreover, we provide useful scripts that could be used for this purpose and a description of key factors that need to be considered in these approaches.

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.

Physiological, Biochemical, and Metabolic Responses to Short and Prolonged Saline Stress in Two Cultivated Cardoon Genotypes.

Cultivated cardoon is a multipurpose crop with adaptability to limiting environments. Two genotypes ("Bianco Avorio" and "Spagnolo") were comparatively characterized in response to short and prolonged 100 mM NaCl stress in hydroponics. Salt induced no growth variations between genotypes or symptoms of NaCl toxicity, but boosted ABA accumulation in roots and leaves. Both genotypes had high constitutive phenol content, whose major components were depleted upon 2 days of stress only in "Bianco Avorio". Prolonged stress stimulated accumulation of proline, phenylpropanoids, and related transcripts, and non-enzymatic antioxidant activity. Decreased antioxidant enzymes activities upon short stress did not occur for APX in "Spagnolo", indicating a stronger impairment of enzymatic defenses in "Bianco Avorio". Nonetheless, H2O2 and lipid peroxidation did not increase under short and prolonged stress in both genotypes. Overall, the two genotypes appear to share similar defense mechanisms but, in the short term, "Bianco Avorio" depends mainly on non-enzymatic antioxidant phenylpropanoids for ROS scavenging, while "Spagnolo" maintains a larger arsenal of defenses. Upon prolonged stress, proline could have contributed to protection of metabolic functions in both genotypes. Our results provide cues that can be exploited for cardoon genetic improvement and highlight genotypic differences for breeding salinity tolerant varieties.

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

MAIN CONCLUSION: 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.

Deciphering the biological processes underlying tomato biomass production and composition.

The huge amounts of biomass residues, remaining in the field after tomato fruits harvesting, can be utilized to produce bioenergy. A multiple level approach aimed to characterize two Solanum pennellii introgression lines (ILs), with contrasting phenotypes for plant architecture and biomass was carried out. The study of gene expression dynamics, microscopy cell traits and qualitative and quantitative cell wall chemical compounds variation enabled the discovery of key genes and cell processes involved biomass accumulation and composition. Enhanced biomass production observed in IL2-6 line is due to a more effective coordination of chloroplasts and mitochondria energy fluxes. Microscopy analysis revealed a higher number of cells and chloroplasts in leaf epidermis in the high biomass line whilst chemical measurements on the two lines pointed out striking differences in the cell wall composition and organization. Taken together, our findings shed light on the mechanisms underlying the tomato biomass production and processability.

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.