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In this study we investigated the transcriptome and epigenome dynamics of the tomato fruit during post-harvest in a landrace belonging to a group of tomatoes (Solanum lycopersicum L.) collectively known as “Piennolo del Vesuvio”, all characterized by a long shelf-life. Expression of protein-coding genes and microRNAs as well as DNA methylation patterns and histone modifications were analysed in distinct post-harvest phases. Multi-omics data integration contributed to the elucidation of the molecular mechanisms underlying processes leading to long shelf-life. We unveiled global changes in transcriptome and epigenome. DNA methylation increased and the repressive histone mark H3K27me3 was lost as the fruit progressed from red ripe to 150 days post-harvest. Thousands of genes were differentially expressed, about half of which were potentially epi-regulated as they were engaged in at least one epi-mark change in addition to being microRNA targets in ~5% of cases. Down-regulation of the ripening regulator MADS-RIN and of genes involved in ethylene response and cell wall degradation was consistent with the delayed fruit softening. Large-scale epigenome reprogramming that occurred in the fruit during post-harvest likely contributed to delayed fruit senescence.

Litter decomposition releases nutrients beneficial to plants but also induces phytotoxicity. Phytotoxicity can result from either labile allelopathic compounds or species specific and caused by conspecific DNA. Aquatic plants in flowing water generally do not suffer phytotoxicity because litter is regularly removed. In stagnant water or in litter packs an impact on root functionality can occur. So far, studies on water plant roots have been carried out in laboratory and never in field conditions. The effect of conspecific vs heterospecific litter and purified DNA were assessed on aquatic roots of the riparian woody species Alnus glutinosa L. using a novel method, using closed and open plastic tubes fixed to single roots in the field with closed tubes analogous to stagnant water. Four fresh and four decomposed litter types were used and analysed on extractable C, cellulose, lignin, N content and using 13 C-CPMAS NMR spectroscopy. Inhibitory effects were observed with fresh litter in closed systems, with a positive correlation with extractable C and negative with lignin and lignin : N ratio. Alnus self-DNA, but not heterologous one, caused acute toxic effects in the closed system. Our results demonstrate the first field-based evidence for self-DNA inhibition as causal factor of negative feedback between plants and substrate.

Extracellular DNA (exDNA) widely occurs in the environment due to release by either cell lysis or active secretion. The role of exDNA in plant-soil interactions has been investigated and inhibitory effects on the growth of conspecific individuals by their self-DNA have been reported. Transcriptome analysis in the model plant Arabidopsis thaliana showed a clear recognition by the plant roots of self- and nonself-exDNA, with inhibition occurring only after exposure to the former. In this study, an untargeted metabolomics approach was used to assess at molecular level the plant reactions to exDNA exposure. Thus, the effects on the metabolites profile of A. thaliana after exposure to self- and nonself-exDNA from plants and fish, were studied by NMR, LC-MS, chemometrics and molecular networking analyses. Results show that self-DNA significantly induces the accumulation of RNA constituents (nucleobases, ribonucleosides, dinucleotide and trinucleotide oligomers). Interestingly, AMP and GMP are found along with their cyclic analogues cAMP and cGMP, and in form of cyclic dimers (c-di-AMP and c-di-GMP). Also methylated adenosine monophosphate (m6AMP) and the dimeric dinucleotide N-methyladenylyl-(3'→5') cytidine (m6ApC) increased only in the self-DNA treatment. Such striking evidence of self-DNA effects highlights a major role of exDNA in plant sensing of its environment.

Vitis vinifera represents an important and renowned source of compounds with significant biological activity. Wines and winery bioproducts, such as grape pomace, skins, and seeds, are rich in bioactive compounds against a wide range of human pathogens, including bacteria, fungi, and viruses. However, little is known about the biological properties of vine leaves. The aim of this study was the evaluation of phenolic composition and antiviral activity of Vitis vinifera leaf extract against two human viruses: the Herpes simplex virus type 1 (HSV-1) and the pandemic and currently widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). About 40 phenolic compounds were identified in the extract by HPLC-MS/MS analysis: most of them were quercetin derivatives, others included derivatives of luteolin, kaempferol, apigenin, isorhamnetin, myricetin, chrysoeriol, biochanin, isookanin, and scutellarein. Leaf extract was able to inhibit both HSV-1 and SARS-CoV-2 replication in the early stages of infection by directly blocking the proteins enriched on the viral surface, at a very low concentration of 10 μg/mL. These results are very promising and highlight how natural extracts could be used in the design of antiviral drugs and the development of future vaccines.

RNA interference (RNAi) is a posttranscriptional gene silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in many organisms. In tomato, RNAi technology has widely been used as a reverse genetic tool for functional genomics study. Generally, RNAi is often achieved through transgenes producing hairpin RNA molecules. RNAi lines have the advantage with respect to more modern CRISPR/Cas9 mutants of different levels of downregulation of target gene, and allow the characterization of life-essential genes that cannot be knocked out without killing the organism. Also, RNAi allows to suppress gene expression in multigene families in a regulated manner. In this chapter, an efficient approach to create RNAi stable knockdown-transformed tomato lines is reported. In order, it describes the choice of the target silencing fragment, a highly efficient cloning strategy for the hairpin RNA construct production, a relatively easy procedure to transform and regenerate tomato plants using Agrobacterium tumefaciens and a methodology to test the goodness of the transformation procedure.

RNA interference (RNAi) is a posttranscriptional gene silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in many organisms. In tomato, RNAi technology has widely been used as a reverse genetic tool for functional genomics study. Generally, RNAi is often achieved through transgenes producing hairpin RNA molecules. RNAi lines have the advantage with respect to more modern CRISPR/Cas9 mutants of different levels of downregulation of target gene, and allow the characterization of life-essential genes that cannot be knocked out without killing the organism. Also, RNAi allows to suppress gene expression in multigene families in a regulated manner. In this chapter, an efficient approach to create RNAi stable knockdown-transformed tomato lines is reported. In order, it describes the choice of the target silencing fragment, a highly efficient cloning strategy for the hairpin RNA construct production, a relatively easy procedure to transform and regenerate tomato plants using Agrobacterium tumefaciens and a methodology to test the goodness of the transformation procedure.

Parallel Spindle1 of Arabidopsis thaliana (AtPS1) is implicated in the formation of 2n pollen generated by parallel orientation of the spindles at 2nd meiotic division. Since decades, 2n pollen is known in potato, its wild relatives and interspecific hybrids, as a noteworthy reproductive anomaly with important breeding applications. For this reason, we investigated AtPS1 orthologs in potato. In our previous work, sequence-homology-based strategy combined with a bioinformatic approach revealed three Parallel Spindle Like loci (PSL1-3) in potato genome thus showing a greater PSL complexity respect to Arabidopsis. PSL1 and PSL3 encode predicted proteins characterized by both FHA and PINc domains such as found in Arabidopsis. FHA domain is implicated in protein-protein interactions while PINc domain has RNA-binding properties often associated with RNAse activity. In this work, to establish the function of PSLs, their silencing was performed in S. tuberosum and S. commersonii through short interfering RNA target sequence common to PSLs. Down-regulated expression of PSL1 and PSL3 was found in several independent transformants. Interestingly, a strong reduction of pollen viability, estimated by Alexander staining, was observed in lines with the lowest PSL expression, whereas the expected 2n pollen formation was lacking. Microsporogenesis and microgametogenesis analysis pointed out irregularities during pollen development at the first mitotic division after meiosis. These results, together with the evidence that PSL3 failed to functionally complement Atps1 mutation, suggest that a functional diversification of PSLs occur in potato respect to Arabidopsis.

Summary During meiosis, recombination ensures allelic exchanges through crossovers (COs) between the homologous chromosomes. Advances in our understanding of the rules of COs have come from studies of mutations including structural chromosomal rearrangements that, when heterozygous, are known to impair COs in various organisms. In this work, we investigate the effect of a large heterozygous pericentric inversion on male and female recombination in Arabidopsis. The inversion was discovered in the Atmcc1 mutant background and was characterized through genetic and next‐generation sequencing analysis. Reciprocal backcross populations, each consisting of over 400 individuals, obtained from the mutant and the wild type, both crossed with Landsberg erecta, were analyzed genome‐wide by 143 single‐nucleotide polymorphisms. The negative impact of inversion became evident in terms of CO loss in the rearranged chromosome in both male and female meiosis. No single‐CO event was detected within the inversion, consistent with a post‐meiotic selection operating against unbalanced gametes. Cytological analysis of chiasmata in F1 plants confirmed that COs were reduced in male meiosis in the chromosome with inversion. Crossover suppression on the rearranged chromosome is associated with a significant increase of COs in the other chromosomes, thereby maintaining unchanged the number of COs per cell. The CO pattern observed in our study is consistent with the interchromosomal (IC) effect as first described in Drosophila. In contrast to male meiosis, in female meiosis no IC effect is visible. This may be related to the greater strength of interference that constrains the CO number in excess of the minimum value imposed by CO assurance in Arabidopsis female meiosis., Significance Statement This work provides further information into the poorly described phenomenon of the interchromosomal (IC) effect in plants. Indeed, the few studies on this phenomenon in maize date back to the 1960s and 1970s and have low resolution, being limited to a few specific regions and genetic markers. In this paper we underscore the significance for plant scientists of the IC effect during meiosis and provide more detailed data and analyses.

During meiosis, recombination ensures the allele exchange through crossovers (COs) between the homologous chromosomes and, additionally, their proper segregation. CO events are under a strict control but molecular mechanisms underlying CO regulation are still elusive. Some advances in this field were made by structural chromosomal rearrangements that are known at heterozygous state to impair COs in various organisms. In this paper, we have investigated the effect that a large pericentric inversion involving chromosome 3 ofArabidopsis thalianahas on male and female recombination. The inversion associated to a T-DNA dependent mutation likely resulted from a side effect of the T-DNA integration. Reciprocal backcross populations, each consisting of over 400 individuals, obtained from the T-DNA mutant and the wild type, both crossed withLandsberg, have been analyzed at genome-wide level by 143 SNPs. We found a strong suppression of COs in the rearranged region in both male and female meiosis. As expected, we did not detect single COs in the inverted region consistently with the post-meiotic selection operating against unbalanced gametes. Cytological analysis of chiasmata in F1 plants confirmed that COs are effectively dropping in chromosome 3 pair. Indeed, CO failure within the inversion is not altogether counterbalanced by CO increase in the regions outside the inversion on chromosome 3. Strikingly, this CO suppression induces a significant increase of COs in chromosome pairs 1, 2 and 5 in male meiosis. We conclude that these chromosomes acquire additional COs thereby compensating the recombination suppression occurring in chromosome 3, similarly to what has been described as interchromosomal (IC) effect in other organisms. In female meiosis, IC effect is not evident. This may be related to the fact that CO number in female is close to the minimum value imposed by the obligatory CO rule.Author SummaryIt is well known that chromosome structure changes in heterozygous condition influence the pattern of meiotic recombination at broad scale. In natural populations, inversions are recognized as the most effective force to reduce COs. In this way, adaptive allele combinations which otherwise would be broken by recombination are maintained. In the present work, we studied the effect on recombination of a large pericentric inversion involving Arabidopsis chromosome 3. The analysis on heterozygous populations provided evidence of strong recombination suppression in chromosome 3. However, the most striking aspect of this study is the finding that the failure of chromosome 3 to recombine is coupled to increased CO frequencies on the other chromosome pairs in male meiosis. These CO compensatory increases are strictly an interchromosomal (IC) effect as was first described inDrosophila. As far as we know, it is the first time IC effect has been reported in plants. Unfortunately, the molecular mechanisms underlying IC effect in the other organisms are still elusive. To understand how a CO change on just one chromosome triggers the global response of the meiocyte to obtain the adequate CO number/cell remains a fascinating question in sexually reproducing species.

Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8 M and 56.4 M of 30–150 bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC.

The mechanistic bases of saprotrophic fungal dynamics in soil are not fully clarified. By assessing hyphal density and radial expansion of Aspergillus niger on extracts 45 plant litter types (15 species at 3 decomposition stages), encompassing a broad range of organic quality, we investigated how changes in litter biochemistry affected fungal growth. Plant litter were characterized by classic proximate chemical analyses (total C and N, labile C, cellulose and lignin content, C/N and lignin/N ratios) and, at molecular level, by solid-state 13 C-CPMAS NMR. The growth of A. niger decreased during the decomposition process over all organic matter types, consistently with the well-known disappearance of this species during the early successional stages. The litter suitability as a substrate to A. niger progressively decreased during decomposition, both considering proximate parameters and C types corresponding to spectral regions, with the latter being also invariably predictive of fungal growth over the 45 substrates. A. niger growth was positively associated with the content of labile C, and with di- O -alkyl C and O -alkyl C spectral regions, but negatively with lignin content and with methoxyl C region. Our results suggest that organic matter quality may control saprotrophic fungal dynamics, at least for the tested species.

Histone modifications are involved in the regulation of many processes in eukaryotic development. In this work, we provide evidence that AtHDA7, a HISTONE DEACETYLASE (HDAC) of the Reduced Potassium Dependency3 (RPD3) superfamily, is crucial for female gametophyte development and embryogenesis in Arabidopsis (Arabidopsis thaliana). Silencing of AtHDA7 causes degeneration of micropylar nuclei at the stage of four-nucleate embryo sac and delay in the progression of embryo development, thereby bringing the seed set down in the Athda7-2 mutant. Furthermore, AtHDA7 down- and up-regulation lead to a delay of growth in postgermination and later developmental stages. The Athda7-2 mutation that induces histone hyperacetylation significantly increases the transcription of other HDACs (AtHDA6 and AtHDA9). Moreover, silencing of AtHDA7 affects the expression of ARABIDOPSIS HOMOLOG OF SEPARASE (AtAESP), previously demonstrated to be involved in female gametophyte and embryo development. However, chromatin immunoprecipitation analysis with acetylated H3 antibody provided evidence that the acetylation levels of H3 at AtAESP and HDACs does not change in the mutant. Further investigations are essential to ascertain the mechanism by which AtHDA7 affects female gametophyte and embryo development.

This study aimed at providing further insights into the positive and negative drivers of tomato liking. For this purpose, 13 tomato cultivars representing different typologies were characterized for physicochemical parameters and aroma volatiles, and were assessed by a trained panel for sensory descriptors, and by Italian consumers for liking. The relationships among the different parameters and their effects on consumer liking were studied by Partial Least Squares (PLS) analysis. Among physicochemical traits and sensory descriptors, seeds, reducing sugars, firmness, thick epicarp, soluble solids, sour taste, total acidity, citrate, herbaceous aroma and brightness were found to be drivers of liking, whereas pulp thickness, humidity, fruit weight, diacetyl-like odor and mealiness showed an opposite influence. For the aroma volatiles, 2-isobutylthiazole played a key role on liking and its positive contribution seemed to be supported by (Z)-3-hexen-1-ol, but suppressed by 6-methyl-5-hepten-2-ol, especially when tomatoes had a poor volatile fraction. These results represent a contribution to the knowledge that could lead to more effective breeding strategies aimed at improving tomato sensory quality. (c) 2012 Elsevier Ltd. All rights reserved.

Polyploidy is an important factor in plant evolution that may trigger drastic genome reorganization and phenotypic differentiation. In the last decade, extensive studies have been carried to understand the consequences of allopolyploidization, where the effects of ploidy change may be confounded by interspecific hybridization. By contrast, less is known on autopolyploidization, which only involves doubling of homologous chromosomes. This study was undertaken to assess leaf anatomical modifications and gene expression changes occurring after doubling the somatic chromosome complement of diploid (2n = 2x = 24) potato species Solanum commersonii Dunal and S. bulbocastanum Dunal. Polyploidization did not induce qualitative changes in leaf structure and, for several leaf traits, anatomic modifications were stochastic. In addition, in both species a diploid superiority was generally observed, suggesting the occurrence of a high-ploidy syndrome. Expression change study was carried out on eight important cell cycle-regulatory genes in plant. It revealed a strong alteration of the expression patterns in the 4x genotypes with respect to the 2x parents. Changes often exceed the twofold, with no consistent trend towards up- or down regulation when comparing 2x vis-a-vis 4x. We discuss the possible relevance of epigenetic changes in controlling the expression of duplicated genes.

Arabidopsis is characterized by heterochiasmy: indeed, it is reported that crossover (CO) rate and distribution vary between male and female meiosis. Chromosomes recombine at a higher rate in male than female meiosis (CO per cell: 11.1 vs 6.6). Furthermore, CO rates are high at the telomeric ends of the chromosomes in male meiosis and at proximal regions in female meiosis. Our research team investigated whether histone acetylation was determinant in heterochiasmy. To address this question, we analyzed chromosome-wide recombination frequency through SNP genotyping in populations derived from a T-DNA mutant (Atmcc1) characterized by increase in histone acetylation. Firstly, two F1 populations were generated by crossing Atmcc1 as male and female parent with Ler genotype as well as two F1 control populations (reciprocal crosses C24 x Ler). Afterwards, four BC1 populations (1680 plants in all), obtained as following [F1(Ler x C24) x Ler], [Ler x F1(Ler x C24)], [F1(Ler x Atmcc1) x Ler] and [Ler x F1(Ler x Atmcc1)], were genotyped by 143 SNPs homogenously distributed on the whole genome with a mean interval length of 800 kb. Genotyping was performed using the patented KASP SNP genotyping system that uses Fluorescent Resonance Energy Transfer (FRET) probes on an arrayed 96 well plate resulting in high throughput and very efficient SNPs detection. Wide analysis revealed that histone acetylation is not determinant for heterochiasmy. Furthermore, genotyping of Atmcc1 evidenced that T-DNA insertion was associated to a reciprocal translocation involving chromosomes 3 and 4. Interestingly, as a consequence of chromosome rearrangements a global redistribution of crossovers along chromosomes occurred.

In crop plants many important traits such as yield, quality and stress tolerance show continuous variation, and result from the combined action of multiple, segregating genes interacting with each other and the environment. In early investigations, before molecular markers were available, the genetics of these complex traits was studied in general terms by ‘quantitative genetics’ using statistical techniques based on means, variances and covariances of relatives, rather than in terms of individual gene effects (Mather and Jinks 1982; Falconer 1989). These analyses provided general estimates including the approximate number of loci affecting a character, the average gene action (e.g., dominance, recessiveness) and the degree to which the various polygenes interacted with each other and the environment. However, they did not provide any information about the number and location of the genes underlying quantitative traits, termed polygenes by Mather (1949).

Metabolomics is the comprehensive and simultaneous identification and quantification of metabolites in living cells. The term metabolome is used to describe the observable chemical profile or fingerprint of the metabolites in a whole tissue. Although being a new approach to study natural compounds, metabolomics uses traditional analytical techniques, including extraction methods, which can be followed by nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Although metabolomics has been successfully applied to quality control issues, the examples of its use for chemosystematics are few. Thus, the analysis of four taxa of Rosa x damascena (R. damascena Mill., R. damascena semperflorens, R. damascena trigintipetala and R. duchesse of Portland) was carried out by NMR spectroscopy as a tool for their classification. A principal component analysis of the 1H NMR spectra, based on the metabolites found in organic and aqueous fractions, showed a clear similarity of the samples. In particular, the major contributions from the aqueous fraction, preliminarily considered as a biomarker of R. x damascena group, are the flavonoids kaempferol and quercetin, glycosilated with glucose and rhamnose units. Our analysis demonstrated a close chemotaxonomic correlation among the four taxa, making this method a reliable tool for chemosystematics.

In vascular plants, at least three SMT isoforms are present to control the pattern of phytosterols. In the present study, we report the isolation of a full-length coding SMT1 cDNA from S. lycopersicum. The SMT1 transcript has an open reading frame of 1038 bp nucleotides and encodes a putative protein of 346 amino acids with theoretical pI of 5.10 and calculated molecular mass of 84.18 KDa. The comparative analysis and phylogenetic analysis showed that the SMT1 sequence was closely related to other sterol methyl transferases. The predicted protein sequence showed four substrate binding regions including regions I (Y81 to F91), III (Y192 to H199) and IV (K215 to W235) that are sterol binding sites and region II (L124 to F133) that is the AdoMet binding site. No transmembrane and hydrophobic domains were predicted in tomato SMT1 protein; this suggested that tomato sterol 24-C-methyltransferase cloned in this work belongs to SMT1 class that catalyses the first committed step of sterol biosynthesis.

In this review, we examine the plant group Solanum sect. Lycopersicon – a clade of 13 species, including the domesticated tomato (Solanum lycopersicum L.) and its wild relatives – along with four allied species in the immediate outgroups Solanum sects. Juglandifolia and Lycopersicoides. We summarize the geographic distribution and morphological characters of these plant groups, describing their evolutionary relationships in the context of a new taxonomic revision at the species level of all these groups. We provide an overview of the role that wild tomato species have played in the development of cytogenetic stocks, in classical and molecular genetic studies as well as in crop improvement through traditional and advanced tools. We discuss how the very narrow genetic basis of cultivated tomato germplasm has forced tomato geneticists and breeders to rely on the wealth of genetic variation present in the wild relatives to address the many breeding challenges. The numerous molecular mapping studies conducted using interspecific crosses have clearly demonstrated that the breeding value of exotic (wild) tomato germplasm goes far beyond its phenotype. These studies also show that we are still far from being able to fully exploit the breeding potential of the thousands of accessions stored in seed banks around the world, in addition to those that may still be found in natural habitats. Over the past decades, tomato breeders have been at the forefront of establishing new principles for crop breeding based on the use of wild species to improve modern cultivars. In this respect, among all model systems, the wild and domesticated species of the tomato clade have pioneered development of novel populations such as “exotic libraries.” These genetic resources, combined with the increasing knowledge deriving from the many “omics” tools, including the tomato genome sequence, are expected to further improve the efficiency with which wild tomato relatives will contribute to the improvement of this important crop.

Genome architecture is shaped by gene-rich and repeat-rich regions also known as euchromatin and heterochromatin, respectively. Under normal conditions, the repeat-containing regions undergo little or no meiotic crossover (CO) recombination. COs within repeats are risky for the genome integrity. Indeed, they can promote non-allelic homologous recombination (NAHR) resulting in deleterious genomic rearrangements associated with diseases in humans. The assembly of heterochromatin is driven by the combinatorial action of many factors including histones, their modifications, and DNA methylation. In this review, we discuss current knowledge dealing with the epigenetic signatures of the major repeat regions where COs are suppressed. Then we describe mutants for epiregulators of heterochromatin in different organisms to find out how chromatin structure influences the CO rate and distribution.