Identification of tomato accessions as source of new genes for improving heat tolerance: from controlled experiments to field.

BACKGROUND: Due to global warming, the search for new sources for heat tolerance and the identification of genes involved in this process has become an important challenge as of today. The main objective of the current research was to verify whether the heat tolerance determined in controlled greenhouse experiments could be a good predictor of the agronomic performance in field cultivation under climatic high temperature stress. RESULTS: Tomato accessions were grown in greenhouse under three temperature regimes: control (T1), moderate (T2) and extreme heat stress (T3). Reproductive traits (flower and fruit number and fruit set) were used to define heat tolerance. In a first screening, heat tolerance was evaluated in 219 tomato accessions. A total of 51 accessions were identified as being potentially heat tolerant. Among those, 28 accessions, together with 10 accessions from Italy (7) and Bulgaria (3), selected for their heat tolerance in the field in parallel experiments, were re-evaluated at three temperature treatments. Sixteen tomato accessions showed a significant heat tolerance at T3, including five wild species, two traditional cultivars and four commercial varieties, one accession from Bulgaria and four from Italy. The 15 most promising accessions for heat tolerance were assayed in field trials in Italy and Bulgaria, confirming the good performance of most of them at high temperatures. Finally, a differential gene expression analysis in pre-anthesis (ovary) and post-anthesis (developing fruit) under heat stress among pairs of contrasting genotypes (tolerant and sensitive from traditional and modern groups) showed that the major differential responses were produced in post-anthesis fruit. The response of the sensitive genotypes included the induction of HSP genes, whereas the tolerant genotype response included the induction of genes involved in the regulation of hormones or enzymes such as abscisic acid and transferases. CONCLUSIONS: The high temperature tolerance of fifteen tomato accessions observed in controlled greenhouse experiments were confirmed in agronomic field experiments providing new sources of heat tolerance that could be incorporated into breeding programs. A DEG analysis showed the complex response of tomato to heat and deciphered the different mechanisms activated in sensitive and tolerant tomato accessions under heat stress.

Selection of tomato landraces with high fruit yield and nutritional quality under elevated temperatures.

BACKGROUND: Global warming and extreme or adverse events induced by climatic fluctuations are an important threat for plants growth and agricultural production. Adaptability to environmental changes prevalently derives from a large set of genetic traits affecting physiological and agronomic parameters. Therefore, the identification of genotypes that are good yield performer at high temperatures is becoming increasingly necessary for future breeding programs. Here, we analyzed the performances of different tomato landraces grown under elevated temperatures in terms of yield and nutritional quality of the fruit. Finally, we evaluated the antioxidant and anti-inflammatory activities of fruit extracts from the tomato landraces selected. RESULTS: The tomato landraces analyzed here in a hot climate differed in terms of yield performance, physicochemical parameters of fruit (pH, titratable acidity, degrees Brix, firmness), bioactive compounds (ascorbic acid, carotenoids, and polyphenols), and anti-inflammatory potential. Three of these landraces (named E30, E94, and PDVIT) showed higher fruit quality and nutritional value. An estimated evaluation index allowed identification of PDVIT as the best performer in terms of yield and fruit quality under high temperatures. CONCLUSION: The analyses performed here highlight the possibility to identify new landraces that can combine good yield performances and fruit nutritional quality at high temperatures, information that is useful for future breeding programs. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

New insights in the control of antioxidants accumulation in tomato by transcriptomic analyses of genotypes exhibiting contrasting levels of fruit metabolites.

BACKGROUND: Tomato is an economically important crop with fruits that are a significant source of bioactive compounds such as ascorbic acid and phenolics. Nowadays, the majority of the enzymes of the biosynthetic pathways and of the structural genes controlling the production and the accumulation of antioxidants in plants are known; however, the mechanisms that regulate the expression of these genes are yet to be investigated. Here, we analyzed the transcriptomic changes occurring during ripening in the fruits of two tomato cultivars (E1 and E115), characterized by a different accumulation of antioxidants, in order to identify candidate genes potentially involved in the biosynthesis of ascorbic acid and phenylpropanoids. RESULTS: RNA sequencing analyses allowed identifying several structural and regulator genes putatively involved in ascorbate and phenylpropanoids biosynthesis in tomato fruits. Furthermore, transcription factors that may control antioxidants biosynthesis were identified through a weighted gene co-expression network analysis (WGCNA). Results obtained by RNA-seq and WGCNA analyses were further confirmed by RT-qPCR carried out at different ripening stages on ten cultivated tomato genotypes that accumulate different amount of bioactive compounds in the fruit. These analyses allowed us to identify one pectin methylesterase, which may affect the release of pectin-derived D-Galacturonic acid as metabolic precursor of ascorbate biosynthesis. Results reported in the present work allowed also identifying one L-ascorbate oxidase, which may favor the accumulation of reduced ascorbate in tomato fruits. Finally, the pivotal role of the enzymes chalcone synthases (CHS) in controlling the accumulation of phenolic compounds in cultivated tomato genotypes and the transcriptional control of the CHS genes exerted by Myb12 were confirmed. CONCLUSIONS: By using transcriptomic analyses, candidate genes encoding transcription factors and structural genes were identified that may be involved in the accumulation of ascorbic acid and phenylpropanoids in tomato fruits of cultivated genotypes. These analyses provided novel insights into the molecular mechanisms controlling antioxidants accumulation in ripening tomato fruits. The structural genes and regulators here identified could also be used as efficient genetic markers for selecting high antioxidants tomato cultivars.

Bioactive Compounds in Brassicaceae Vegetables with a Role in the Prevention of Chronic Diseases.

The beneficial role of the Mediterranean diet in the prevention of chronic diseases, including cardiovascular diseases, diabetes, and obesity, is well-recognized. In this context, Brassicaceae are considered important vegetables due to several evidences of their health promoting effects that are associated to bioactive compounds present in the edible parts of the plants. In this review, the mechanisms of action and the factors regulating the levels of the bioactive compounds in Brassicaceae have been discussed. In addition, the impact of industrial and domestic processing on the amount of these compounds have been considered, in order to identify the best conditions that are able to preserve the functional properties of the Brassicaceae products before consumption. Finally, the main strategies used to increase the content of health-promoting metabolites in Brassica plants through biofortification have been analyzed.

Carotenoids in fresh and processed tomato (Solanum lycopersicum) fruits protect cells from oxidative stress injury.

BACKGROUND: Lipophilic antioxidants in tomato (Solanum lycopersicum) fruits exert important functions in reducing the risk of human diseases. Here the effect of thermal processing on the antioxidant activity of lipophilic extracts from the commercial tomato hybrid 'Zebrino' was analysed. Carotenoid content and lipophilic antioxidant activity were determined and the ability of tomato extracts in rescuing cells from oxidative stress was assessed. RESULTS: Lipophilic antioxidant activity was completely retained after heat treatment and extracts were able to mitigate the detrimental effect induced by oxidative stress on different cell lines. Lycopene alone was able to rescue cells from oxidative stress, even if to a lower extent compared with tomato extracts. These results were probably due to the synergistic effect of tomato compounds in protecting cells from oxidative stress injury. CONCLUSION: The current study provides valuable insights into the health effect of the dietary carotenoids present in fresh and processed tomato fruits. © 2016 Society of Chemical Industry.

Vitamin E Content and Composition in Tomato Fruits: Beneficial Roles and Bio-Fortification.

Several epidemiological studies have demonstrated that high vitamin E intakes are related to a reduced risk of non-communicable diseases, while other dietary antioxidants are not, suggesting that vitamin E exerts specific healthy functions in addition to its antioxidant role. In this regard, tomato (Solanum lycopersicum), one of the most consumed vegetables of the whole world population, is an important source of both tocopherols and tocotrienols. However, vitamin E content may strongly depend on several biotic and abiotic factors. In this review we will debate the elements affecting the synthesis of tocopherols and tocotrienols in tomato fruit, such as environmental conditions, genotype, fruit maturity level, and the impact of classical processing methods, such as pasteurization and lyophilization on the amount of these compounds. In addition we will analyze the specific vitamin E mechanisms of action in humans and the consequent functional effects derived from its dietary intake. Finally, we will examine the currently available molecular techniques used to increase the content of vitamin E in tomato fruit, starting from the identification of genetic determinants and quantitative trait loci that control the accumulation of these metabolites.

Bioactive Compound Content and Cytotoxic Effect on Human Cancer Cells of Fresh and Processed Yellow Tomatoes.

Tomato, as a fresh or processed product, has a high nutritional value due to its content of bioactive components such as phenolic compounds. Few studies describe the effect of processing on antioxidant content and the cancer cell growth inhibition activity. In this study we determined the phenolic and ascorbic acid content of three yellow tomato varieties, before and after thermal processing. Moreover, we determined the antioxidative power and tested the effects of tomato extracts on three human cancer cell lines. We found that the amount of phenolic acids (chlorogenic acid and caffeic acid) decreased in all the samples after processing, whereas the flavonoid content increased after the heat treatment in two samples. A cytotoxic effect of tomato extracts was observed only after processing. This result well correlates with the flavonoid content after processing and clearly indicates that processed yellow tomatoes have a high content of bioactive compounds endowed with cytotoxicity towards cancer cells, thus opening the way to obtain tomato-based functional foods.

An association mapping approach to identify favourable alleles for tomato fruit quality breeding.

BACKGROUND: Genome Wide Association Studies (GWAS) have been recently used to dissect complex quantitative traits and identify candidate genes affecting phenotype variation of polygenic traits. In order to map loci controlling variation in tomato marketable and nutritional fruit traits, we used a collection of 96 cultivated genotypes, including Italian, Latin American, and other worldwide-spread landraces and varieties. Phenotyping was carried out by measuring ten quality traits and metabolites in red ripe fruits. In parallel, genotyping was carried out by using the Illumina Infinium SolCAP array, which allows data to be collected from 7,720 single nucleotide polymorphism (SNP) markers. RESULTS: The Mixed Linear Model used to detect associations between markers and traits allowed population structure and relatedness to be evidenced within our collection, which have been taken into consideration for association analysis. GWAS identified 20 SNPs that were significantly associated with seven out of ten traits considered. In particular, our analysis revealed two markers associated with phenolic compounds, three with ascorbic acid, ß-carotene and trans-lycopene, six with titratable acidity, and only one with pH and fresh weight. Co-localization of a group of associated loci with candidate genes/QTLs previously reported in other studies validated the approach. Moreover, 19 putative genes in linkage disequilibrium with markers were found. These genes might be involved in the biosynthetic pathways of the traits analyzed or might be implied in their transcriptional regulation. Finally, favourable allelic combinations between associated loci were identified that could be pyramided to obtain new improved genotypes. CONCLUSIONS: Our results led to the identification of promising candidate loci controlling fruit quality that, in the future, might be transferred into tomato genotypes by Marker Assisted Selection or genetic engineering, and highlighted that intraspecific variability might be still exploited for enhancing tomato fruit quality.

Enhancing the health-promoting effects of tomato fruit for biofortified food.

Consumption of tomato fruits, like those of many other plant species that are part of the human diet, is considered to be associated with several positive effects on health. Indeed, tomato fruits are an important source of bioactive compounds with known beneficial effects including vitamins, antioxidants, and anticancer substances. In particular, antioxidant metabolites are a group of vitamins, carotenoids, phenolic compounds, and phenolic acid that can provide effective protection by neutralizing free radicals, which are unstable molecules linked to the development of a number of degenerative diseases and conditions. In this review, we will summarize the recent progress on tomatoes nutritional importance and mechanisms of action of different phytochemicals against inflammation processes and prevention of chronic noncommunicable diseases (e.g., obesity, diabetes, coronary heart disease, and hypertension). In addition, we will summarize the significant progress recently made to improve the nutritional quality of tomato fruits through metabolic engineering and/or breeding.

Cistus incanus: a natural source of antimicrobial metabolites.

The discovery of natural molecules with antimicrobial properties has become an urgent need for the global treatment of bacterium and virus infections. Cistus incanus, a Mediterranean shrub species, represents a valuable source of phytochemicals with an interesting wide-spectrum antimicrobial potential. In this study, we analysed the spectrum of molecules composing a commercial hydroalcoholic extract of C. incanus finding ellagitannins as the most abundant. The effect of the extract and its main constituents (gallic acid, ellagic acid and punicalin) was assessed as co-treatment during viral (HSV-1, HCoV-229E, SARS-CoV-2) and bacterial infection (Staphylococcus aureus and Escherichia coli) of cells and as pre-treatment before virus infections. The results indicated a remarkable antiviral activity of punicalin against SARS-CoV-2 by pre-treating both the viral and the host cells, and a major sensitivity of S. aureus to the C. incanus extract compared to E. coli. The present study highlights broad antimicrobial potential of C. incanus extract.

Production of pharmaceutical proteins in solanaceae food crops.

The benefits of increased safety and cost-effectiveness make vegetable crops appropriate systems for the production and delivery of pharmaceutical proteins. In particular, Solanaceae edible crops could be inexpensive biofactories for oral vaccines and other pharmaceutical proteins that can be ingested as minimally processed extracts or as partially purified products. The field of crop plant biotechnology is advancing rapidly due to novel developments in genetic and genomic tools being made available today for the scientific community. In this review, we briefly summarize data now available regarding genomic resources for the Solanaceae family. In addition, we describe novel strategies developed for the expression of foreign proteins in vegetable crops and the utilization of these techniques to manufacture pharmaceutical proteins.

Genomic Insights into the Origin of a Thermotolerant Tomato Line and Identification of Candidate Genes for Heat Stress.

Climate change represents the main problem for agricultural crops, and the constitution of heat-tolerant genotypes is an important breeder's strategy to reduce yield losses. The aim of the present study was to investigate the whole genome of a heat-tolerant tomato genotype (E42), in order to identify candidate genes involved in its response to high temperature. E42 presented a high variability for chromosomes 1, 4, 7 and 12, and phylogenetic analysis highlighted its relationship with the wild S. pimpinellifolium species. Variants with high (18) and moderate (139) impact on protein function were retrieved from two lists of genes related to heat tolerance and reproduction. This analysis permitted us to prioritize a subset of 35 candidate gene mapping in polymorphic regions, some colocalizing in QTLs controlling flowering in tomato. Among these genes, we identified 23 HSPs, one HSF, six involved in flowering and five in pollen activity. Interestingly, one gene coded for a flowering locus T1 and mapping on chromosome 11 resides in a QTL region controlling flowering and also showed 100% identity with an S. pimpinellifolium allele. This study provides useful information on both the E42 genetic background and heat stress response, and further studies will be conducted to validate these genes.

The hydrophilic extract from a new tomato genotype (named DHO) kills cancer cell lines through the modulation of the DNA damage response induced by Campthotecin treatment.

Introduction: DNA double-strand breaks are the most toxic lesions repaired through the non-homologous and joining (NHEJ) or the homologous recombination (HR), which is dependent on the generation of single-strand tails, by the DNA end resection mechanism. The resolution of the HR intermediates leads to error-free repair (Gene Conversion) or the mutagenic pathways (Single Strand Annealing and Alternative End-Joining); the regulation of processes leading to the resolution of the HR intermediates is not fully understood. Methods: Here, we used a hydrophilic extract of a new tomato genotype (named DHO) in order to modulate the Camptothecin (CPT) DNA damage response. Results: We demonstrated increased phosphorylation of Replication Protein A 32 Serine 4/8 (RPA32 S4/8) protein in HeLa cells treated with the CPT in combination with DHO extract with respect to CPT alone. Moreover, we pointed out a change in HR intermediates resolution from Gene Conversion to Single Strand Annealing through the modified DNA repair protein RAD52 homolog (RAD52), DNA excision repair protein ERCC-1 (ERCC1) chromatin loading in response to DHO extract, and CPT co-treatment, with respect to the vehicle. Finally, we showed an increased sensitivity of HeLa cell lines to DHO extract and CPT co-treatment suggesting a possible mechanism for increasing the efficiency of cancer therapy. Discussion: We described the potential role of DHO extract in the modulation of DNA repair, in response to Camptothecin treatment (CPT), favoring an increased sensitivity of HeLa cell lines to topoisomerase inhibitor therapy.

Phenolics diversity among wild populations of Salvia multicaulis: as a precious source for antimicrobial and antioxidant applications.

The genus Salvia L. belongs to the Lamiaceae family including several known species rich in natural compounds that are extensively used in pharmaceutical, food, and cosmetic industries. Salvia multicaulis populations contain a broad diversity of flavonoids and phenolic acids. The present study aimed to explore biological and pharmacological effects including antimicrobial and antioxidant activities of nineteen S. multicaulis populations (SMPs) grown in Iran for the first time. High content of rosmarinic acid (RA) in SMP12 (Gazan) (5.65 ± 0.33 mg/g DW) caused high antimicrobial activity against two bacteria (Staphylococcus aureus, Escherichia coli) and the fungus Candida albicans, while methanolic extract of SMP1 (Taleghan) showed high antioxidant activity due to high content of salvianolic acid A (SAA) and quercetin (0.53 ± 0.04 and 0.49 ± 0.12 mg/g DW, respectively). Altogether these results can be considered for further commercial exploitations to meet the demands of the food and pharmaceutical industries.[Formula: see text].

Phenotyping to dissect the biostimulant action of a protein hydrolysate in tomato plants under combined abiotic stress.

Drought and heat stresses are the main constrains to agricultural crop production worldwide. Precise and efficient phenotyping is essential to understand the complexity of plant responses to abiotic stresses and to identify the best management strategies to increase plant tolerance. In the present study, two phenotyping platforms were used to investigate the effects of a protein hydrolysate-based biostimulant on the physiological response of two tomato genotypes ('E42' and 'LA3120') subjected to heat, drought, or combined stress. The free amino acids in the biostimulant, or other molecules, stimulated growth in treated plants subjected to combined stress, probably promoting endogenous phytohormonal biosynthesis. Moreover, biostimulant application increased the net photosynthetic rate and maximal efficiency of PSII photochemistry under drought, possibly related to the presence of glycine betaine and aspartic acid in the protein hydrolysate. Increased antioxidant content and a decreased accumulation of hydrogen peroxide, proline, and soluble sugars in treated plants under drought and combined stress further demonstrated that the biostimulant application mitigated the negative effects of abiotic stresses. Generally, the response to biostimulant in plants had a genotype-dependent effect, with 'E42' showing a stronger response to protein hydrolysate application than 'LA3120'. Altogether, in this study a fine and multilevel phenotyping revealed increased plant performances under water-limited conditions and elevated temperatures induced by a protein hydrolysate, thus highlighting the great potential biostimulants have in improving plant resilience to abiotic stresses.

Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.

Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.

Specialized Metabolites and Valuable Molecules in Crop and Medicinal Plants: The Evolution of Their Use and Strategies for Their Production.

Plants naturally produce a terrific diversity of molecules, which we exploit for promoting our overall well-being. Plants are also green factories. Indeed, they may be exploited to biosynthesize bioactive molecules, proteins, carbohydrates and biopolymers for sustainable and large-scale production. These molecules are easily converted into commodities such as pharmaceuticals, antioxidants, food, feed and biofuels for multiple industrial processes. Novel plant biotechnological, genetics and metabolic insights ensure and increase the applicability of plant-derived compounds in several industrial sectors. In particular, synergy between disciplines, including apparently distant ones such as plant physiology, pharmacology, 'omics sciences, bioinformatics and nanotechnology paves the path to novel applications of the so-called molecular farming. We present an overview of the novel studies recently published regarding these issues in the hope to have brought out all the interesting aspects of these published studies.

Accelerating the Development of Heat Tolerant Tomato Hybrids through a Multi-Traits Evaluation of Parental Lines Combining Phenotypic and Genotypic Analysis.

The constitution of heat tolerant F1 hybrids is a challenge to ensure high yield and good fruit quality in the global climate. In the present work, we evaluated 15 genotypes for yield-related traits highly affected by high temperatures (HT). This phenotypic analysis allowed to identify four parental genotypes showing promising yield performances under HT conditions. Two of these genotypes also exhibited good fruit quality traits. A molecular marker analysis was carried out for six resistance genes to pathogens mostly affecting tomatoes. This analysis evidenced the presence of a maximum of three resistant alleles in parental genotypes. Exploring single nucleotide polymorphisms (SNPs) revealed by two high-throughput genotyping platforms allowed identifying additional 12 genes potentially involved in resistance to biotic stress, to be further investigated. Following these considerations, 13 F1 hybrids were constituted combining the parental genotypes and then evaluated for multiple traits under HT conditions. By estimating a hybrid index based on yield performances, desirable quality and resistance gene, we identified seven hybrids showing the best performances. The promising results obtained in the present work should be confirmed by evaluating the best hybrids selected for additional years and environments before proposing them as novel commercial hybrids that could maintain high performances under HT conditions.

A Novel Protein Hydrolysate-Based Biostimulant Improves Tomato Performances under Drought Stress.

Abiotic stresses adversely affect crop production causing yield reductions in important crops, including tomato (Solanum lycopersicum L.). Among the different abiotic stresses, drought is considered to be the most critical one, since limited water availability negatively impacts plant growth and development, especially in arid and semi-arid areas. The aim of this study was to understand how biostimulants may interact with critical physiological response mechanisms in tomato under limited water availability and to define strategies to improve tomato performances under drought stress. We investigated the physiological responses of the tomato genotype 'E42' grown in open fields under optimal conditions (100% irrigation) and limited water availability (50% irrigation) treated or not with a novel protein hydrolysate-based biostimulant (CycoFlow, Agriges, BN, Italy). Plants treated with the protein hydrolysate showed a better water status and pollen viability, which also resulted in higher yield under drought stress compared to untreated plants. The treatment with the biostimulant had also an effect on antioxidant contents and activity in leaves and fruits depending on the level of irrigation provided. Altogether, these results indicate that the application of protein hydrolysates on tomato improved plant performances under limited water availability and in different experimental fields.

Triterpenic and phenolic acids production changed in Salvia officinalis via in vitro and in vivo polyploidization: A consequence of altered genes expression.

The induction of polyploidy is an efficient technique for creating a diversity of genetic, phenotypic, and phytochemical novelties in plant taxa. Sage (Salvia officinalis L.) is a well-known medicinal plant rich of valuable bioactive molecules such as triterpenic and phenolic acids. In the present study, the effect of in vitro and in vivo polyploidization on morphological characteristics, anatomical structures, phytochemical traits, and expression level of the genes involved in the biosynthesis of major triterpenic acids (ursolic, betulinic, and oleanolic acids) of the plant was studied. The sterile seeds treated with different concentrations (0, 0.05, 0.1, and 0.2%) of colchicine for 24 and 48 h were considered for polyploidy induction. Flow cytometry and chromosome counting were used to confirm the ploidy level of diploid (2n = 2x = 14, 2C DNA = 1.10 pg) and tetraploid (2n = 4x = 28, 2C DNA = 2.12 pg) plants after seven months. The highest polyploidy induction was obtained by applying 0.1% (w/v) colchicine for 48 h with an efficiency of 19.05% in vitro tetraploidy. Polyploids showed differences in leaf shape and color, leaf and stem thickness, trichrome density, root length, plant height, and number of leaves compared to diploid plants. There was also a significant decrease in rosmarinic acid content in polyploid (plants) as compared to diploid plants. Although a significant decrease in ursolic acid content was observed in polyploids, betulinic acid content associated with the expression levels of genes encoding enzymes being active in triterpene biosynthesis such as squalene epoxidase (SQE) and lupeol synthase (LUS). The expression of SQE and LUS was significantly increased in in vitro tertaploids (2.9-fold) and in vivo mixoploids (2.4-fold). The results confirm the idea that induced polyploidy can randomly alter breeding traits of plants as well as the content of bioactive compounds.