INVESTIGATING THE ROLE OF AN ERF TRANSCRIPTION FACTOR IN MEDIATING STRESS RESPONSE AND TOMATO FRUIT RIPENING AND QUALITY

Tomato (Solanum lycopersicum L.) is one of the world's most consumed vegetables and its consumption has been associated with decreased risk of chronic degenerative diseases. Tomato fruit is an important source of antioxidant compounds such as carotenoids, particularly lycopene, ascorbic acid, vitamin E and phenolic compounds. Fruit ripening is regulated by ethylene. Ethylene biosynthesis and signaling are modulated during fruit development and ripening and are involved in several processes such as antioxidant accumulation and softening that affect fruit quality and shelf-life. To date, several strategies have been implemented in tomato to modulate ripening and enhance tomato fruit quality and shelf-life by regulating the expression of genes involved in ethylene biosynthesis, perception or signaling. Among others, AP2/ERF genes are transcription factors which play key roles in several processes, such as plant development, ethylene response, and pathogen resistance. In tomato fruit, they can act as positive or negative regulator of ripening and of ethylene production. Our goal is to elucidate the functional role of ERF F4 gene (Solyc07g053740) in mediating modulation of the tomato fruit ripening during the plant response to abiotic stress and investigatiing its impact on fruit redox balance and antioxidant accumulation. In particular, the ERF F4 locus was targeted in Microtom by CRISPR/Cas 9 technology to generate knockout tomato plants. One month-old T3 offspring mutant and wild-type plants underwent three level of irrigation, consisting in the complete restitution of water (FWR), restitution of 50% of lost water (HWR) and 30% of water restitution (TWR), respectively. Beside no variation was observed in the lycopene and total carotenoid levels in fruit at the red-ripe stage, edited plants showed increased levels of soluble solid content than wild type plants and responded to drought treatment with a higher increase in their antioxidant capacity. These results suggested the involvement of ERF F4 in modulating ripening associated metabolic processes and fruit redox balance in response to abiotic stresses. Ongoing experiments will further investigate the role of the tomato ERF F4 in regulating the expression of genes involved in controlling the metabolism of antioxidants, particularly glutathione and ascorbate pool

Phytochemical Constituents and Biological Activity of Wild and Cultivated <i>Rosmarinus officinalis</i> Hydroalcoholic Extracts

Rosmarinus officinalis L. is an aromatic evergreen plant from the Lamiaceae family. The purpose of this study was to compare the chemical profile and bioactivities of hydroalcoholic extracts derived from wild and cultivated R. officinalis. The chemical composition of the extracts was evaluated via LC–MS analysis, which revealed the presence of a wide range of phenolic compounds, including flavonoids, phenolic and terpenes. Both extracts showed a similar interesting antioxidant activity, probably related to their content of phenol and flavonoids. The analysis of anti-acetylcholinesterase (AChE), anti-butyrylcholinesterase (BChE), and anti-α-amylase activities showed analogous inhibition, except for AChE, in which the wild type was more active than the cultivated one. Finally, in vitro studies were performed using the J774A.1 murine macrophage cell line, to characterize the anti-inflammatory and the antioxidant effects of the extracts. As expected, pretreatment with the extracts significantly reduced the production proinflammatory cytokines and ROS through modulation of the nitric oxide pathway and the mitochondrial activity. Importantly, it is observed that the anti-inflammatory effect of the extracts was explicated through the inhibition of NF-kB and its downstream mediator COX-2. Collectively, these results demonstrated that these extracts could represent a starting point for developing novel therapeutic strategies for the treatment of inflammation-based diseases. Moreover, since no significant changes were observed in terms of composition and activity, both wild and cultivated R. officinalis extracts can be recommended for food and pharmaceutical purposes

Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer

CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies

A Snapshot of the Emerging Tomato Genome Sequence

The genome of tomato (Solanum lycopersicum L.) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy, and the United States) as part of the larger \u201cInternational Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation\u201d initiative. The tomato genome sequencing project uses an ordered bacterial artificial chromosome (BAC) approach to generate a high-quality tomato euchromatic genome sequence for use as a reference genome for the Solanaceae and euasterids. Sequence is deposited at GenBank and at the SOL Genomics Network (SGN). Currently, there are around 1000 BACs finished or in progress, representing more than a third of the projected euchromatic portion of the genome. An annotation effort is also underway by the International Tomato Annotation Group. The expected number of genes in the euchromatin is 3c40,000, based on an estimate from a preliminary annotation of 11% of finished sequence. Here, we present this first snapshot of the emerging tomato genome and its annotation, a short comparison with potato (Solanum tuberosum L.) sequence data, and the tools available for the researchers to exploit this new resource are also presented. In the future, whole-genome shotgun techniques will be combined with the BAC-by-BAC approach to cover the entire tomato genome. The high-quality reference euchromatic tomato sequence is expected to be near completion by 2010