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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.

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.

Induced polyploidy is nowadays an important strategy in plant breeding and for the development of new crops. Salvia multicaulis Vahl is a valuable medicinal plant that produce precious bioactive metabolites including triterpenic acids (TAs) and phenolic compounds. Hence, at first, for selecting elite lines, both HPLC and GC-MC analyses were performed on fourteen S. multicaulis lines. Then, seeds of selected lines of S. multicaulis were exposed to different concentrations (0.00, 0.05, 0.1, and 0.2 %) of colchicine for 24 or 48 h. The flow cytometric analysis and chromosome counting were used to confirm ploidy level of tetraploid control (2n = 4x = 28, 2C DNA = 1.36 pg) and hexaploid (2n = 6x = 42, 2C DNA = 1.97 pg) plants after seven-month. For the first time, the effects of in vitro polyploidization on morphological characteristics, TAs and phenolic acid contents as well as on the expression of six TAs biosynthesis related genes were investigated. The highest efficiency of hexaploidy (12.76 %) was achieved 48 h after exposure to 0.1 % colchicine concentration. The hexaploid plants showed different growth traits compared with those of tetraploid control plant; indeed, hexaploid plants had leaves with a darker green color, a lower trichome density, and lower plant height and root length. Moreover, there was a significant increase in rosmarinic acid and caffeic acid content in hexaploid plants compared with tetraploid control plants. Also, the increase of oleanolic acid (1.33 fold) content in hexaploids was associated with a significant increased expression of squalene synthase (SQS) and β-amyrin synthase (BAS) genes in hexaploid plants. Nevertheless, a significant decreased expression of squalene epoxidase (SQE), mixed-function amyrin synthase (MFAS), and lupeol synthase (LUS) was observed in hexaploid plants, that led to a reduced content of ursolic acid and betulinic acid compared with tetraploid control plants. These results confirmed that polyploidization is a breeding method with stochastic results in secondary metabolites production and gene expression related to biosynthetic pathways.

Climate change is increasing the frequency of high temperature shocks and water shortage, pointing to the need for developing novel tolerant varieties and to understand the mechanisms engaged to withstand combined abiotic stresses. Two tomato genotypes, a heat-tolerant S. lycopersicum accession (LA3120) and a novel genotype (E42), previously selected as a stable yielding genotype under high temperatures, were exposed to single and combined water and heat stress. Plant functional traits, pollen viability and physiological (leaf gas exchanges and chlorophyll a fluorescence emission measurements), and biochemical (antioxidants contents and antioxidant enzymes activity) measurements were carried out. A Reduced Representation Sequencing (RRS) approach allowed to explore the genetic variability of both genotypes to identify candidate genes that could regulate stress responses. Both abiotic stresses had a severe impact on plant growth parameters and on the reproductive phase of development. Growth parameters and leaf gas exchange measurements revealed that the two genotypes used different physiological strategies to overcome individual and combined stresses, with E42 having a more efficient capability to utilise the limiting water resources. Activation of antioxidant defence mechanisms seemed to be critical for both genotypes to counteract combined abiotic stresses. Candidate genes were identified that could explain the different physiological response to stress observed in E42 compared with LA3120. Results here obtained have shown how new tomato genetic resources can be a valuable source of traits for adaptation to combined abiotic stresses and should be used in breeding programs to improve stress tolerance in commercial varieties.

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.

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.

[EN] 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., This work was supported by the European Commission H2020 Research and Innovation Programme through the TomGEM project, grant agreement No. 679796, and HARNESSTOM, grant agreement No. 101000716.

Periodic epidemics of black rot disease occur worldwide causing substantial yield losses. Xanthomonas campestris pv. campestris (Xcc) represents one of the most common bacteria able to cause the above disease in cruciferous plants such as broccoli, cabbage, cauliflower, and Arabidopsis thaliana. In agriculture, several strategies are being developed to contain the Xanthomonas infection. The use of bacteriophages could represent a valid and efficient approach to overcome this widespread phenomenon. Several studies have highlighted the potential usefulness of implementing phage therapy to control plant diseases as well as Xcc infection. In the present study, we characterized the effect of a lytic phage on the plant Brassica oleracea var. gongylodes infected with Xcc and, for the first time, the correlated plant metabolic response. The results highlighted the potential benefits of bacteriophages: reduction of bacterium proliferation, alteration of the biofilm structure and/or modulation of the plant metabolism and defense response.

Abiotic stresses can cause a substantial decline in fruit quality due to negative impacts on plant growth, physiology and reproduction. The objective of this study was to verify if the use of a biostimulant based on plant and yeast extracts, rich in amino acids and that contains microelements (boron, zinc and manganese) can ensure good crop yield and quality in tomato plants grown at elevated temperatures (up to 42 &deg, C). We investigated physiological responses of four different tomato landraces that were cultivated under plastic tunnel and treated with the biostimulant CycoFlow. The application of the biostimulant stimulated growth (plants up to 48.5% taller) and number of fruits (up to 105.3%). In plants treated with the biostimulant, antioxidants contents were higher compared to non-treated plants, both in leaves and in fruits. In particular, the content of ascorbic acid increased after treatments with CycoFlow. For almost all the traits studied, the effect of the biostimulant depended on the genotype it was applied on. Altogether, the use of the biostimulant on tomato plants led to better plant performances at elevated temperatures, that could be attributed also to a stronger antioxidant defence system, and to a better fruit nutritional quality.

The selection of tolerant varieties is a powerful strategy to ensure highly stable yield under elevated temperatures. In this paper, we report the phenotypic and genotypic characterization of 10 tomato landraces to identify the best performing under high temperatures. The phenotyping of five yield-related traits allowed us to select one genotype that exhibits highly stable yield performances in different environmental conditions. Moreover, a Genotyping-by-Sequencing approach allowed us to explore the genetic variability of the tested genotypes. The high and stable yielding landrace E42 was the most polymorphic one, with ~49% and ~47% private SNPs and InDels, respectively. The effect of 26,113 mutations on proteins&rsquo, structure was investigated and it was discovered that 37 had a high impact on the structure of 34 proteins of which some are putatively involved in responses to high temperatures. Additionally, 129 polymorphic sequences aligned against tomato wild species genomes revealed the presence in the genotype E42 of several introgressed regions deriving from S. pimpinellifolium. The position on the tomato map of genes affected by moderate and high impact mutations was also compared with that of known markers/QTLs (Quantitative Trait Loci) associated with reproductive and yield-related traits. The candidate genes/QTLs regulating heat tolerance in the selected landrace E42 could be further investigated to better understand the genetic mechanisms controlling traits for high and stable yield trait under high 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.

High temperatures represent a limitation for growth and development of many crop species. Several studies have demonstrated that the yield reduction of tomato under high temperatures and drought is mainly due to a photosynthetic decline. In this paper, a set of 15 tomato genotypes were screened for tolerance to elevated temperatures by cultivating plants under plastic walk-in tunnels. To assess the potential tolerance of tomato genotypes to high temperatures, measurements of chlorophyll fluorescence, pigments content and leaf functional traits have been carried out together with the evaluation of the final yields. Based on the greenhouse trials, a group of eight putative heat-sensitive and heat-tolerant tomato genotypes was selected for laboratory experiments aimed at investigating the effects of short-term high temperatures treatments in controlled conditions. The chlorophyll fluorescence induction kinetics were recorded on detached leaves treated for 60 min at 35 &deg, C or at 45 &deg, C. The last treatment significantly affected the photosystem II (PSII) photochemical efficiency (namely maximum PSII quantum efficiency, Fv/Fm, and quantum yield of PSII electron transport, &Phi, PSII) and the non-photochemical quenching (NPQ) in the majority of genotypes. The short-term heat shock treatments also led to significant differences in the shape of the slow Kautsky kinetics and its significant time points (chlorophyll fluorescence levels minimum O, peak P, semi-steady state S, maximum M, terminal steady state T) compared to the control, demonstrating heat shock-induced changes in PSII functionality. Genotypes potentially tolerant to high temperatures have been identified. Our findings support the idea that chlorophyll fluorescence parameters (i.e., &Phi, PSII or NPQ) and some leaf functional traits may be used as a tool to detect high temperatures-tolerant tomato cultivars.

Tomato consumption is considered fundamental for human health for its high nutritional value, mainly ascribed to antioxidant metabolites. In the last few years, we have focused our research on deciphering the genetic control of antioxidant compounds in tomato fruits and to produce superior genetic materials with an increased amount of bioactive compounds in the red ripe berry. For this purpose, we used introgression lines (ILs) obtained from the wild species Solanum pennellii, and we selected lines IL7-3 and IL12-4 for their high content of hydrophilic antioxidants in the fruit. These lines were crossed to combine the identified quantitative trait loci (QTLs) and obtain superior hybrids. A set of species-specific molecular markers was used to clearly distinguish the wild S. pennellii from the cultivated Solanum lycopersicum genome. In the present work, we describe the selection of one double homozygous hybrid (DHO22) and its multilevel characterization in comparison with its parental genotypes and with the cultivated genotype M82. The selected hybrid DHO22 exhibits high contents of ascorbic acid and phenolics, combined with good performances in terms of yield and other fruit quality traits, and clearly resembles the cultivated genotype M82. In future, this line could be used as pre-breeding material to obtain a new cultivar improved for nutritional traits.

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.

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.

The Solanum pennellii Introgression Line (IL) population can be exploited to identify favorable alleles that can improve yield and fruit quality traits in commercial tomato varieties. Over the past few years, we have selected ILs that exhibit increased content of antioxidant compounds in the fruit compared to the cultivar M82, which represents the genetic background in which the different wild regions of the S. pennellii ILs were included. Recently, we have identified seven sub-lines of the IL7-3 accumulating different amounts of antioxidants in the ripe fruit. Since the wild region carried on chromosome 7 induces a low fruit production in IL7-3, the first aim of the present work was to evaluate yield performances of the selected sub-lines in three experimental fields located in the South of Italy. Another aim was to confirm in the same lines the high levels of antioxidants and evaluate other fruit quality traits. On red ripe fruit, the levels of soluble solids content, firmness, and ascorbic acid (AsA) were highly variable among the sub-lines grown in three environmental conditions, evidencing a significant genotype by environment interaction for soluble solids and AsA content. Only one sub-line (coded R182) exhibited a significantly higher firmness, even though no differences were observed for this trait between the parental lines M82 and IL7-3. The same sub-line showed significantly higher AsA content compared to M82, thus resembling IL7-3. Even though IL7-3 always exhibited a significantly lower yield, all the sub-lines showed yield variability over the three trials. Interestingly, the sub-line R182, selected for its better performances in terms of fruit quality, in all the trials showed a production comparable to that of the control line M82. A group of species-specific molecular markers was tested on R182 and on the parental genotypes in order to better define the wild genomic regions carried by the elite line R182. In these regions three candidate genes that could increase the level of AsA in the fruit were identified. In the future, the line R182 could be used as pre-breeding material in order to obtain new varieties improved for nutritional traits., This research was supported by the Italian Ministry of University and Research (MIUR) (Grant MIUR-514 PON02-GenoPOMpro) and by MIUR project PON03PE-0060.

The isoforms of lycopene, carotenoids, and their derivatives including precursors of vitamin A are compounds relevant for preventing chronic degenerative diseases such as cardiovascular diseases and cancer. Tomatoes are a major source of these compounds. However, cooking and successive metabolic processes determine the bioavailability of tomatoes in human nutrition. To evaluate the effect of acute/chronic cooking procedures on the bioavailability of lycopene and carotene isoforms in human plasma, we measured the blood levels of these compounds and of the serum antioxidant potential in volunteers after a meal containing two different types of tomato sauce (rustic or strained). Using a randomized cross-over administration design, healthy volunteers were studied, and the above indicated compounds were determined by HPLC. The results indicate an increased bioavailability of the estimated compounds and of the serum antioxidant potential with both types of tomato purée and the subsequently derived sauces (the increase was greater with strained purée). This study sheds light on the content of nutrient precursors of vitamin A and other antioxidant compounds derived from tomatoes cooked with different strategies. Lastly, our study indicates that strained purée should be preferred over rustic purée.

The Solanum pennellii introgression lines (ILs) have been exploited to map quantitative trait loci (QTLs) and identify favorable alleles that could improve fruit quality traits in tomato varieties. Over the past few years, ILs exhibiting increased content of ascorbic acid in the fruit have been selected, among which the sub-line R182. The aims of this work were to identify the genes of the wild donor S. pennellii harbored by the sub-line and to detect genes controlling ascorbic acid accumulation by using genomics tools. A Genotyping-By-Sequencing (GBS) approach confirmed that no wild introgressions were present in the sub-line besides one region on chromosome 7. By using a dense single nucleotide polymorphism (SNP) map obtained by RNA sequencing (RNA-Seq), the wild region of the sub-line was finely identified, thus, defining 39 wild genes that replaced 33 genes of the ILs genetic background (cv. M82). The differentially expressed genes mapping in the region and the variants detected among the cultivated and the wild alleles evidenced the potential role of the novel genes present in the wild region. Interestingly, one upregulated gene, annotated as a major facilitator superfamily protein, showed a novel structure in R182, with respect to the parental lines. These genes will be further investigated using gene editing strategies.