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Traditional varieties of tomato from the Mediterranean region represent a pool of biodiversity that can be mined for novel traits useful for the genetic improvement of commercial varieties. The TOMRES project aims at improving water and nutrient use efficiency in tomato through novel combinations of genotypes and management practices. Within this framework, we have tested a set of 27 accessions from Southern Italy and Spain as well as backcross inbred lines (BILs) from Israel to identify genotypes that are particularly efficient in their use of water, nitrogen, or both critical resources. Genotypes were first tested in vitro for early responses to stress. Parameters related to root architecture, such as primary root length, number and length of lateral roots were evaluated under Control conditions, Low Nutrient conditions (1/10 of control N and P dose), Osmotic stress (200 mM Mannitol), and a combined stress treatment (Low nutrients and mannitol). A subsequent evaluation involved several rounds of a large scale pot-experiment using 15 L pots filled with sand in a randomized block setup. Each genotype was grown for six weeks under four separate treatments: Control (10.2 mM NO3 -), Low Nitrate (2.88 mM NO3 -), Drought (50% water) and Combined Stress. Chlorophyll content, stomatal opening and relative water content were monitored throughout the growth cycle. At harvest above and below ground growth parameters were measured. Biometric measurements were subjected to analysis of variance (ANOVA) and Duncan post-hoc test. This showed that above ground parameters were largely in agreement and demonstrated a significantly reduced growth in stress treated compared to control plants, with two exceptions. Growth of genotype TOMRES250 was not repressed under drought or combined stress, while for genotype TOMRES271 drought stress alone did not cause reduced biomass accumulation. Interestingly, TOMRES250 had a less developed root system, stable across the different treatments, while TOMRES271 root system did not modify in response to drought stress. We thus calculated the root dry weight/shoot dry weight ratio and found that TOMRES250 and 271 did not modify biomass allocation based on the stress treatment, in contrast to the remaining tested genotypes, which had a significant alteration of this parameter due to stress. Thus, root systems that maybe preadapted to stressful environments may contribute to tolerance displayed in terms of growth of above ground tissues. Molecular analyses also showed a different transcript abundance of nitrogen uptake and transport-encoding genes in TOMRES250, indicating that a more efficient nutrient uptake may characterize this genotype. Ion content analysis is in progress to confirm this hypothesis. In conclusion, we have set up a screening system and identified two candidate genotypes that show resilience to environmental stresses. Molecular and biochemical analyses are in progress to dissect the molecular basis of this behavior. This work was supported by the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No. 727929.

Drought and salinity, which share a component of osmo-stress, impact significantly plant development and productivity, thus causing serious agricultural yield losses. The molecular mechanisms underlying the response of plants to severe osmo-stress have been studied extensively and, often, are associated to a drastic yield penalty. The current trend in improving crop resistance to osmo-stress is focused on understanding how plants adapt their growth under prolonged and progressive osmo-stress conditions, with the final aim to identify regulatory and structural gene network associated to restoration of a new cellular homeostasis and, ultimately, to resumption of plant growth under limiting environmental conditions. After 25 years of studies on plant's response to osmo-stress, we reported here our experimental evidences supporting the notion that glycophyte plants, such as potato, when acclimated gradually to osmo-stress, activate regulatory gene networks distinct from those induced upon acute stress. Potato cells gradually acclimated to PEG-induced osmo-stress sustain an active growth, due to a set of metabolic changes, including high proline content, de novo protein synthesis, changes in membrane lipid composition and ethylene accumulation, not induced in cells exposed abruptly to the same stress conditions (Leone et al., 1994, Plant Physiol. 106: 703-712); Leone et al., Plant, Cell & Environ., 1996,19: 1103-1109; Scaramagli et al., 2000, Plant Physiol. Biochem. 38, 345- 351). Extensive remodelling of gene expression depends strictly on duration and intensity of osmo- stress (Ambrosone et al., 2011, Acta Physiol. Plant. 33, 1157-1171; Ambrosone et al., 2017, Gene 15; 597:30-39). Overall, adaptation requires a major effort in terms of transcriptional regulation and distinct transcription factors promote the differential response to short- or long-term stress. Interestingly, modulation of ethylene signalling network appear to be a distinctive trait of gradual adaptation to osmo-stress. Gene expression patterns and functional analyses for adaptive mechanisms were confirmed in planta (Ambrosone et al, 2011; Ambrosone et al., 2017; Ambrosone, Batelli et al., 2015; Plant Physiol. 168:292-306) and may be part of an environmentally-driven epigenetic program, which is under investigation.

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.

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.

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.

We have studied the response of potato cell suspension culture to PEG- mediated low water potential. Gradual adaptation to high concentrations of PEG 8000 (20%) did not affect growth and endogenous ABA content of potato cells. On the contrary. direct exposure to 20% PEG (osmotic shock) drastically reduced growth in unadapted cells and also induced a six-fold increase in ABA level within 5 days from the imposition of the stress. 2D-electrophoretic pattern of in vivo labeled proteins of adapted cells was very similar to -that of control celis. Only few polypeptides were down-regulated and the synthesis of at least 31 individuaI polypeptides was increased upon adaptation. Osmotic shock as well as ABA treatment of unadapted cells determined an overall reduction of protein synthesis. Most of the induced polypeptides in PEG-shocked cells were also found to be Ïnduced in unadapted cells upon treatment with exogenous ABA. However, it was possible to identify some polypeptides specifically induced in osmotically shocked cells, a group of which was also detected in adapted celIs. The comparison of protein electrophoretic patterns demonstrated that most changes observed in PEG adapted cells were not mediated by ABA. Long-term adaptation of potato cells to PEG enhanced both the transcript and protein level of osmotin, a 26 kD protein reported to accumulate in salt-adapted tobacco celIs. In osmotic shocked ,cells a dramatic increase of osmotin transcripts was also detected, which however did not correlate with the level of the protein.