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Tomato (Solanum lycopersicum L.) is a high value horticultural crop and an important dietary source of nutrients, vitamins and antioxidants. The cultivation is often subjected to drought, salinity and N-limited conditions. Thus, understanding the responses of tomato to salt and low N conditions will be instrumental to improve yields in stress-prone environments. In this study, physiological and molecular responses of three Italian landraces (TRPO0040, TRPA0130 and TRPO0670) to salt stress and low nitrate, alone or in combination were analysed. The experimental set up allowed four different treatments named: Control (13.5 mM NO3- - 0 mM NaCl), Salt stress (13.5 mM NO3- - 80 mM NaCl), N stress (3.4 mM NO3-- 0 mM NaCl) and Combined stress (3.4 mM NO3- - 80 mM NaCl). The treatments had different effects on plants: low N caused chlorosis, while salt stress inhibited growth causing loss of basal leaves. Leaf relative water content was affected by both salt and low nitrate, depending on the genotype, while proline content was dramatically increased by salt stress but not under combined stress. Growth parameters and fruit yield were reduced by single stress condition, and the highest reduction was observed in the combined stress. TRPO0040 genotype showed high Nitrogen Use Efficiency and Nitrogen Utilization Efficiency under salt, indicating that nitrogen allocation to fruits was not affected by this stress. We evaluated the impact of single and combined stresses on tomato transcriptome by performing RNAseq on roots and leaves of TRPO0040 after long-term exposure to the treatments. In leaves, a more extensive transcriptome reorganization was observed in response to N stress, while salt induced a significant variation in gene expression of a handful of genes. Parallel to the proline levels, expression of P5CS gene, encoding the rate limiting proline biosynthetic enzyme, was induced by salt, while the catabolic proline dehydrogenase was suppressed. Similarly, several genes encoding nitrate transporters were induced in N stress, including NRT2;2 and others. By contrast, in roots several thousands of differentially expressed genes (DEGs) DEGs in salt stress and only few hundreds in N stress were identified. Consistent with results gathered in Arabidopsis, SLAH1, a gene encoding a root specific anion transporter involved in the long distance transport of MOLECULAR MECHANISMS UNDERLYING MORPHO- PHYSIOLOGICAL ADAPTATION TO COMBINED SALT/LOW NUTRIENT STRESS IN TOMATO chloride ions, was upregulated in roots in salt stress condition, while a NRT2;4 like and AMT1 were induced by low nitrate. In the combined stress, a more extensive overlap of the DEGs was observed with the N stress rather than the salt stress for both roots and leaves. This indicates that nutrient availability may have a higher and longer- term impact on gene expression, compared to salt stress, which may induce rapid and transient responses, attenuated, particularly in leaves, when plant adaptation occurs. Altogether, we show that different cultivation regimes affect metabolic and transcriptomic profiles as well as growth in tomato, identifying a set of physiological and molecular targets specifically influenced by single or combined stress.

Tolerance to water deficit as well as extended fruit shelf life are sought-after traits in tomato breeding, very appreciated both by farmers and consumers. Both these features characterize several Mediterranean landraces, including many genotypes adapted to the dry growth seasons typical of Southern Italy. Glucose 6 phosphate dehydrogenase (G6PDH) is the rate-limiting enzyme of the Oxidative Pentose Phosphate Pathway, inducing the oxidation of glucose-6-phosphate and the corresponding reduction of moieties of NADP+ to NADPH. A functional role has been suggested for this enzyme in ROS detoxification, highlighting its contribution in abiotic stress response. Furthermore, G6PDH activity and the corresponding NAPDH availability have been recently associated with red color and ripening of tomato fruits. In order to clarify the G6PDH roles in tomato drought tolerance and fruit ripening, we pursued an integrated approach where biochemical and molecular parameters were evaluated together in the Italian landrace Giallo Beneventano grown under different conditions. This landrace showed interesting yields upon drought and an unusually long shelf life (LSL) of fruits, up to 5-6 months. LSL trait has been related with a mutation in the nor allele Solyc10g006880, suggesting that a change in the ethylene signalling pathway regulating the ripening processes occurs in this particular landrace. Enzyme activity assays as well as gene expression quantifications indicated that G6PDH - and the related ascorbate peroxidase -showed significant changes in Giallo Beneventano compared to commercial varieties under drought stress in leaf tissues and during fruit ripening (from green to fully mature). Based on these data, we suggest that a constitutively high level of specific activities involved in ROS detoxification can play a key role in the enhanced drought tolerance and longer fruit shelf life exhibited by Mediterranean tomato landraces. Acknowledgments - The authors thank the Department of Agriculture (IPA Sector) of Regione Campania for providing seeds of the tomato landrace Giallo Beneventano.

Tomato (Solanum lycopersicum L.) is an important horticultural crop and a significant source of antioxidants in our diet. Yields suffer losses due to environmental stresses, including drought and salinity, which are among the most significant challenges for the future of global food production. Reduction of fertilizer inputs, including nitrate is also a priority in plant research, and it is aimed at minimizing pollution caused by agricultural activities, while obtaining fruits of high quality and long storability. In this study, we analysed vegetative and molecular responses of three tomato landraces (TRPO0040, TRPA0130 and TRPO0670) challenged with salt stress and low nitrate, alone or in combination, and their effects on fruit yield, quality and storability. The experiment was setup in a closed soilless system, in greenhouse, using four different conditions: N1S0: 13.5 mM NO3- - 0 mM NaCl; N1S1: 13.5 mM NO3- - 80 mM NaCl; N0S0: 3.4 mM NO3-- 0 mM NaCl; N0S1: 3.4 mM NO3- - 80 mM NaCl. Several parameters were monitored during the vegetative phase, including stomatal conductance, leaf relative water and proline content, expression of selected ion transporter genes. Relative water content was affected by both salt and nitrate concentration, while proline content was dramatically increased by salt stress in high nitrate condition only. Growth parameters as well as fruit yield were reduced in the presence of stress, with the highest reduction observed in plants exposed to the combined stress. Highest yields were obtained in N1S0, however fruits harvested from low nutrient-treated plants were virtually all marketable, regardless the presence of NaCl. Soluble solids content was highly affected by salt, as expected. The cultivation regime also affected fruit shelf life of the three genotypes over the 90-day period monitored. High salt in the nutrient solution improved fruit shelf life regardless of genotype and nitrate concentration. In the absence of NaCl, NO3- supply in the nutrient solution did not affect fruit shelf life in TRPO0670 and TRPA0130, while TRPO0040 fruits showed an extended shelf life at low nitrate compared to high nitrate supply. Fruit firmness and weight declined in all genotypes and treatments during post-harvest; hence, they were not ?? RUGGIERO A.*, BATELLI G.*, VENEZIA A. ??NURCATO R.*, COSTA A.*, ?? discriminating parameters between treatments. Nitrogen Use Efficiency (NUE) evaluation in all treatments and landraces revealed that TRPO0040 maintained high NUE under salt stress, indicating that N allocation to fruits was not inhibited by salt in this genotype. Altogether, we have verified that different cultivation regimes affect fruit shelf life and quality in tomato, and identified a set of vegetative parameters specifically influenced by single or combined stress. In addition, RNAseq and qRT-PCRs analyses are being pursued in order to get further insights into the molecular basis of fruit shelf life under different cultivation regimes.

Considered as one of the most important horticultural crops worldwide, tomato (Solanum lycopersicum L.) has had a huge agronomical success in the Mediterranean area, where numerous local landraces which show great phenotypic diversity were selected. Among the several landraces grown in Southern Italy and analyzed by our research group, two of these, Lucariello (LUC) and Crovarese (CRO) showed interesting phenotypes in field trials repeated over several years. LUC and CRO displayed a high tolerance to drought stress, and produced fruits of high quality and long shelf life despite the low water input regimes, making them very attractive economically and scientifically. To identify the components involved in drought stress responses, we pursued a transcriptome study on CRO and LUC plants subjected to drought stress by water withholding for 13 days. Our trascriptome sequencing was done in conjunction with a whole-genome resequencing project of CRO and LUC, which allowed us first to use the specific genomes of CRO and LUC as reference for the bioinformatic pipeline, and to merge the genomic data with drought stress-induced gene expression variations. Using RNA sequencing on leaf samples, we identified 2954 and 2015 genes up-regulated upon stress in CRO and LUC, respectively. By contrast, 3912 genes were down- regulated in CRO and 2671 in LUC. Gene Onthology (GO) enrichment analysis on up-regulated and down-regulated genes shared between CRO and LUC revealed that several of the enriched categories were stress-related. Comparison of the RNA-seq results with polymorphic genes identified within the resequencing project revealed 300 drought-responsive genes common to CRO and LUC which carried sequence polymorphisms with a potentially disruptive impact on their function. Among these, 101 were up- and 199 down-regulated genes in drought stress conditions. The polymorphisms identified included nonsense mutations generating premature stop codons in a number of expressed genes. The pectinase family involved in cell wall organization showed a striking number of members, 42 whose expression was repressed by water deprivation. Among these, 3 genes were selected (Solyc01g079180, Solyc05g014000, Solyc09g075460) for expression analysis in CRO, LUC and in a commercial genotype (Red Setter, RS) during two cycles of stress and a recovery phase. Results highlighted an opposite behavior of these genes between the traditional landraces and RS during the second cycle of stress, where an expression inhibition was observed in CRO and LUC compared to an upregulation in RS. The observed downregulation of the pectinase genes could be part of a genotype-specific response to drought, leading to reduced degradation of pectin structure in the cell wall to respond to water scarcity. Altogether, the gene expression data presented here contribute to an understanding of the drought tolerance displayed in ? the field by CRO and LUC and to the identification of genes putatively involved in the stress adaptation features of these traditional landraces.

Abscisic acid (ABA) plays an important role in various aspects of plant growth and development, such as adaptation to stresses (drought and high salinity), seed maturation and germination, and fruit development and ripening. The early stages of ABA signal transduction pathway have been established by identification of its core components: PYR/PYL/RCARs (PYLs), PP2Cs, and SnRK2s. Upon binding to ABA, the PYL receptors undergo a conformational change and become able to bind to and inhibit PP2C phosphatases. SnRK2 kinases are thus released from PP2C inhibition, become active and can phosphorylate downstream effectors, thus inducing transcription of ABA-responsive genes and, in guard cells, loss of turgor which results in closure of the stomata. Different studies have shown the potential applications of ABA signal core components to enhance plant drought resistance, through either genetic engineering or chemical approaches in crops. Here, we identify components of the C. annuum PYL, PP2C, and SnRK2 gene families. Using annotated tomato and Arabidopsis protein sequences, we isolated putative orthologs in pepper genome (C. annuum L Zunla-1). A dendrogram created through maximum likelihood method clusterized each PYL, PP2C, and SnRK2 gene family in subclasses. For further analyses we selected one PYL for each subfamily, three PP2Cs from different subclades and two SnRK2s from subclass III and one from subclass I. We measured the expression of these genes through qRT-PCR in seeds (cv Quadrato D'Asti giallo) incubated for four days with different concentrations of ABA or NaCl and observed an upregulation of most of the tested genes at high concentrations, including PYLs. Interestingly, the presence of NaCl or ABA had little or no effect on seed germination, measured in terms of radical emergence or cotyledon expansion. However, when seedling root growth was measured, after transfer from control media, sensitivity to ABA and salt was restored. Based on these results, our hypothesis is that pepper is insensitive to high concentrations of salt and ABA during the germination stage, due to the low expression of perception and signaling components. Further experiments will analyze in detail the mechanisms implicated in this phenomenon.

Adaptation to osmotic stress requires an extensive alteration of gene expression. Previously, we identified several genes regulated in cells adapted to polyethylene glycol (PEG). Here, the functional role of fifty of these genes was verified. Using a large-scale phenotype screening, we have identified two genes: the splicing factor IAG1 (INSENSITIVE TO ABA IN GERMINATION1) and the putative TOR-pathway component XSA1(EXTRA SENSITIVE TO ABA1). IAG1 is induced upon long-term exposure to abscisic acid (ABA) and PEG and is mainly expressed in trichomes and stomata, organs controlling transpiration. Germination analysis of plants with altered expression of IAG1 and protein interaction with the splicing factor SUA, suggest that IAG1 may be involved in premRNA splicing of effectors of ABA response leading to germination inhibition. XSA1 possibly affects pathways in ABA-mediated response to stress. XSA1 is expressed in vascular tissues and is up-regulated by long-term exposure to NaCl and ABA. xsa1-1 is ABA hypersensitive, indicating alteration in ABA biosynthesis and/or perception. Taken together, our results reveal promising mechanisms of plant adaptation to osmotic stress.

Drought is one of the major abiotic stresses affecting plant growth and productivity. Among horticultural crops, tomato (Solanum lycopersicum) is generally classified as susceptible to drought stress. However, several local landraces adapted to arid Mediterranean conditions are successfully cultivated in low water input regimes. In order to identify the components involved in drought stress responses, we pursued an integrated approach where several biochemical and physiological parameters were evaluated together with transcriptome sequencing in two landraces ( Lucariello and Crovarese) from the Campania Region. Plants were grown in greenhouse and subjected to drought stress by water withhold for 13 days. Stomatal conductance and photosynthetic efficiency parameters showed similar profiles in both genotypes, with values greatly reduced after prolonged water withhold, indicative of a severe water stress condition. Similarly, ABA and proline levels greatly increased due to the stress treatment. Using RNA sequencing on leaf samples, we identified 2851 and 1867 genes upregulated upon stress in Crovarese and Lucariello, respectively. Furthermore, 4000 genes were downregulated in Crovarese and 2642 in Lucariello. Extensive overlapping was observed, with 1419 up- and 2297 down-regulated genes common to both genotypes. Gene Onthology (GO) enrichment analysis carried out on up-regulated and down-regulated genes revealed that several of the enriched categories were stress-related. A k-means clusterization was carried out. We identified in both genotypes 8 clusters (4 in upregulated dataset and 4 in downregulated dataset) of genes with similar expression profile. A new GO enrichment aimed at understanding the fuction of the clusters identified classes, which were peculiar for specific clusters. Among the genes showing the highest up-regulation in the stress treatment several members of the Late Embryogenesis Abundant protein (LEA) family were identified. This family was therefore selected for further studies. LEA proteins are a group of proteins that accumulate in response to cellular dehydration in many organism. Using multiple approaches for the discovery of LEA genes, we could identify 37 LEA proteins encoded in the tomato genome. We then built a dendrogram, which allowed the assignment of each gene to one of the known 5 LEA sub-family. Comparison of the dendrogram structure with transcriptome results highlighted that the 2 subfamilies "LEA 4" and "Dehydrin" in particular were upregulated following drought treatments in leaves. Altogether, our results contribute a broad understanding of the physiological, biochemical and molecular mechanisms employed by tomato to respond to water deprivation.

The similarity to of its genetic make-up, morphology and development together with the recent release of the genome sequence make it an ideal species to study mechanisms of tolerance to extreme environments. To generate a mutagenized population for both forward and reverse genetic studies in E. salsugineum, we optimized the EMS (Ethyl Methanesulfonate) treatment procedure for this species. We used the ver162 mutant, developed in the laboratories of Dr. Jian-Kang Zhu, which does not require vernalization in order to flower and is therefore more suitable for our studies. Different concentrations of EMS in the treatment were tested on 50 mg of seeds and the impact of the mutagenesis measured by the adverse effect on seed germination and the presence of albino sections on leaves. Based on these parameters, we have identified a method for mutagenizing E. salsugineum on a scale and efficiency comparable to A. thaliana. A final concentration of 0.6% EMS for 6 hours, which caused the desidered 40% reduction in seed germination was thus deemed optimal and used to perform bulk-mutagenesis. After 2 weeks of stratification, mutagenised seeds were germinated and transferred to soil to collect the M-2 generation in pools. As an indication of the success of the mutagenesis, we obtained 12 M-1 plants requiring vernalization, indicating a possible reverting phenotype compared to background ver162. M-2 seeds will be used to carry out large-scale screenings in order to identify genes and mechanisms important for salt, cold and drought tolerance in halophytes plants.

Osmotic stress critically limits plant growth and crop productivity. The identification of genes underlying the mechanisms of stress response is the subject of intense research in plant biology. Through microarray analyses we previously identified and isolated RGGA gene, coding for an RNA binding protein, whose expression was specifically induced in Solanumtuberosum cell cultures gradually exposed to osmotic stress. The aim of this study was to confirm the role of RGGA as a functional RNA binding protein required for a proper response to stress condition. We identified the RGGA orthologue in Arabidopsis thaliana (AtRGGA, At4g16830) and evaluated the influence of drought and salt stress on AtRGGA gene expression in cells and seedlings exposed to high concentrations of NaCl, PolyEthyleneGlycole (PEG) and abscisic acid (ABA).Interestingly, AtRGGA gene expression was up-regulated in seedlings after long-term exposure to NaCl and PEG, while short-term treatments with NaCl resulted in AtRGGA down- regulation. To investigate the protein sub-cellular localization, a YFP-RGGA fusion protein was used. Fluorescence signal indicated that RGGA is localized in the cytoplasm and the peri- nuclear region. In addition, a ?-glucuronidase (GUS) assay showed promoter activity in several tissues, including guard cells of stomata, the organs controlling transpiration. Electrophoresis Mobility Shift Assays with total RNA using recombinant His-RGGA clearly showed that RGGA is capable of binding RNA in vitro. To carry out a functional analysis, a gain- and loss-of-function approach was performed using rggaknock-out mutants and AtRGGA over-expressing plants. In addition, rggaknock-out mutant was hyper-sensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. Accordingly, the over- expressing plants showed a higher tolerance both in vitro and in soil and accumulated lower levels of proline when exposed to salt and drought stress conditions. Finally, a global analysis of gene expression using microarrays, revealed extensive alterations in the transcriptome of AtRGGA over-expressing plants and rgga mutants under osmotic stress, indicating that RGGA participates in the modulation of transcript abundance of several key genes involved in abiotic stress response. The data taken together provide compelling evidence that the RGGA gene is involved in important mechanisms of plant response to osmotic stress.

Soil salinity reduces crop yields and limits available land for agriculture. Understanding the molecular mechanisms of plant salt stress response is becoming increasingly important. RNA regulatory mechanisms such as synthesis, transport, translation, stability and degradation of transcripts participate in the modulation of abiotic stress responses. RNA binding proteins play an important role in such mechanisms, but relatively few have been characterized in plants so far. In a previous study, we have shown the involvement of AtRGGA, encoding a glycine-rich RNA-binding protein, in responses to drought and salt stress (Ambrosone et al., 2015). an Electrophoresis Mobility Shift Assays (EMSA) with total RNA using recombinant His-RGGA showed that RGGA is capable of binding RNA in vitro. Here we present protein and RNA interaction studies using AtRGGA as a bait. Results from these experiments point to a role for AtRGGA in post-transcriptional control of gene expression during osmotic stress. To identify protein partners of AtRGGA, yeast two-hybrid screening was performed using an Arabidopsis cDNA library. Most of the identified proteins are involved in salt stress response, such as BODYGUARD (BDG1, At1g64670), essential for cuticle biogenesis and regulator of the Abscisic Acid (ABA) biosynthesis and signaling. Other putative partners include RNA-binding proteins with a function in RNA export from the nucleus and involved in stress response. For the isolation of RNAs bound to AtRGGA, we performed a Ribonucleoprotein ImmunoPurification (RIP) assay. Immunopurification of FLAG-AtRGGA from stress-treated transgenic plants, followed by RNA extraction and qRT-PCR allowed the identification and quantitative comparison of RNA associated to AtRGGA under salt stress (120 mM NaCl) in vivo. Interestingly, RNA corresponding to the transcription factor DREB2A, a key regulator of drought stress responses, was significantly enriched in the AtRGGA-immunopurified samples under stress condition compared to total RNA. Taken together, our results indicate that AtRGGA could be involved in RNA export from the nucleus into the cytoplasm, and that AtRGGA is biased in the RNAs it binds, including key salt stress response transcripts. Additional experiments aimed at i) confirming putative interactions identified by yeast two- hybrid and ii) sequencing and quantification of transcripts bound to AtRGGA will address these hypotheses.

Una delle principali attività di collaudo del progetto DERFRAM - PSR 124 della Regione Campania è stata la valutazione dell'efficacia di trattamenti biologici con microrganismi antagonistici, anche in combinazione con fungicidi di sintesi, nel controllo di malattie fungine delle piante da frutta. Poiché, inevitabilmente, parte dei prodotti antiparassitari, irrorati su chioma, cade sul suolo, nell'ambito del progetto è stata programmata e realizzata un'indagine preliminare, oggetto del presente lavoro, finalizzata alla valutazione degli effetti dei predetti prodotti sulla componente batterica della coltre pedologica più superficiale.

Drought stress in plants is among the environmental stresses causing major yield losses. Tomato has the highest acreage devoted of any vegetable crop in the world, therefore the necessity to improve the tolerance to lower water inputs is crucial and it could deliver a substantial contribution to saving of water. Several local genotypes are available within the rich Italian tomato germplasm, whose potential as source of superior adaptive alleles has not been fully exploited so far, due to the complexity of the molecular processes involved. The goal of our research was the characterization of physiological, biochemical and molecular processes occurring in tomato genotypes with different levels of tolerance to limiting water supplies, in order to dissect the complexity of the molecular events occurring in response to drought. 40-d-old potted plants of the genotype M82 and the introgression line IL.9.2.5 were grown in controlled conditions in a greenhouse and subjected to drought by withholding water for 16 days (D1), then allowed to recover by re-watering for 7 days (RW) and finally subjected to a second cycle of stress imposed for 8 days (D2). Several physiological, biochemical and molecular parameters were monitored during the experiment to gain a deeper understanding of the response of tomato to water limitations. Stomatal conductance (gs) and CO2 assimilation (A) steeply decreased in all genotypes during the two cycles of drought stress and recovered to control levels when stressed plants were rewatered. The complete recovery of the photosynthetic rate upon re-watering indicated that A was constrained by stomatal closure rather than by a permanent impairment of the photosynthetic machinery. While plant gas exchanges did not highlight a different behaviour between the tested genotypes, striking differences were noticed in leaf ABA and proline content, with M82 accumulating high levels of both metabolites during D1. Despite the induction of the nced1 (9-cisepoxycaratenoid dioxygenase) gene, plants of IL9.2.5 did not accumulate ABA during both cycles of drought stress. The physiological and metabolic adjustments were related to changes in expression of genes known as key components of the response to water deficit. At the molecular level, a dramatic upregulation of a set of genes was observed in D1. After re-watering, the expression of all target genes dropped to levels comparable to those of control plants. A second cycle of drought stress (D2), however, elicited a great up-regulation of lea (late embryogenesis abundant protein), phosph (starch phosporilase) and nced1, while the expression of aco (1-aminocyclopropane-1- carboxylic acid oxidase) and erd15 (early responsive to dehydration) was not induced, indicating that related metabolic pathways were not up-regulated. Transcriptomic changes elicited by drought and rehydration conditions were further being explored by a deep sequencing approach. Altogether, the results presented indicate that differences exist in the response of different genotypes of tomato to drought stress conditions and that genomic tools could be used to further exploit tomato genetic variability for developing drought-tolerant cultivars.

In the framework of a project for the conservation of local landraces threatened with extinction, we estimated genetic relationships and true cultivar identity of 7 and 25 accessions, respectively, of eggplant and pepper landraces from the Campania Region (Southern Italy). We used an integrated approach including SSR markers (15 for eggplant and 16 for pepper) and 9 morpho-agronomic biometric traits. Out of all the tested SSR markers, 5 and 7 markers showed polymorphism between landraces in eggplant and pepper, respectively, detecting 18 alleles in eggplant and 31 alleles in pepper. These markers generated the same genetic pattern for every accession of each investigated cultivar. Despite more markers may potentially detect polymorphism, these findings suggest an elevated genetic homogeneity between landraces. The morpho-agronomic traits investigated allowed only a partial discrimination of landraces and thus call for more detailed investigation.