Sevi, Filippo (2023) CRISPR/Cas9 for the generation of new tomato ideotypes with improved nutritional quality: a multi-omics characterization. [Tesi di dottorato]

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Abstract

Tomato (Solanum lycopersicum) is one of the most economically important vegetable crops and one of the major products of the agri-food industry. Despite tomato is a font of many nutritional compounds like carotenoids, flavonoids and vitamins, it is also a source of some antinutritional compounds including glycoalkaloids, tannins, folates and allergens. In particular glycoalkaloids are a group of compounds that provide protection against pathogens attack but also show toxic effects in humans causing gastrointestinal and neurological disorders. Another very important aspect regarding the consumption of tomatoes is their allergenicity which affects about 16% of the Italian population. The purpose of this phD thesis has been to eliminate or decrease, some of these antinutritional and allergenic molecules using the new breeding technology "genome editing" and in particular using the CRISPR/Cas9 system. In our approach, two specific gRNAs for genes of interest, GAME 4 and Sola l 4 were designed. We chose GAME 4 gene, as it is located at a crucial point in the biosynthesis of glycoalkaloids (SGAs); more in detail, it is placed at a branching point in the pathway leading, on one hand, to the production of SGAs and, on the other, to the synthesis of a class of molecules with high nutraceutical value named saponins. Thus, by intervening precisely at this enzymatic step, it is potentially possible to decrease the concentration of anti-nutritional substances, with a simultaneous increase in pro-nutritional ones. Concerning the choice of allergens, we selected Sola l 4 because it is one of the most characterized in tomatoes, as well as having a greater allergenicity in people with food allergies. The first part of this work focused on the molecular characterization of edited plant and applying a multi -omics approach (proteomics metabolomics, and volatilomics) (Chapter 2) that allow us to understand the effects derived by the knock-out of the two genes under study on plant metabolism. In fact, as a result of knock-out of Sola l 4 gene, analyzing tomato extracts through western blot analysis, no signal was observed in both edited lines, confirming the absence of the protein in its native conformation. At metabolomic level, a semi-polar and non-polar targeted characterization was carried out and as expected, a very strong reduction of total SGAs was achieved, greater than 99% in leaves and fruits. At the same time, a marked increase of some saponins, such as tigonin, was found. Moreover, a significant increase of the sugar content in edited lines was observed. Interestingly, analyzing proteomic data in leaves, we observed that among the up-regulated proteins there are numerous proteins involved in defense mechanisms against biotic stresses, such as several peroxidases, chitinases and PR-proteins. Furthermore, analysis of proteomic data in tomato fruits revealed several proteins involved in carbohydrate metabolism, according with the metabolomic data. In addition, in edited lines, an increase in branched-chain amino acids group (BCAA VOCs) was observed. In this group the most up-regulated compound was 3-methylbutanal for both MG and RR stage. Our findings suggest that as a result of genome editing engineering in glycoalkaloids and allergens content, a remodulation of secondary metabolism was observed in tomato edited plants. A second aim of this study was to compare the bacterial communities in the rhizosphere of WT and GAME 4/Sola l 4 knock-out tomatoes in order to assess if the alterations on the genotype and therefore in the content of certain molecules could be reflected in a different distribution at microbial composition and biodiversity. In the Chapter 3, the soil bacterial community has been investigated by metabarcoding of the 16S rRNA bacterial gene both in WT and in edited plants to evaluate the potential impact on microbial diversity and structure. Moreover, a metabolic analysis of roots was performed and, as expected, the same effects of gene editing observed on the secondary metabolism in fruits and leaves were also confirmed in roots, in which a reduction in total SGAs content over 98.0% was achieved in the two analyzed lines. In addition, a great accumulation of saponins in the roots was obtained. A principal coordinate analysis, PCoA, associated with PERMANOVA test, reveals a marked separation between WT and edited lines that clustered together. Consequently, our findings confirm the role of SGAs and saponins in changing the soil bacterial community. Considering that we can apply CRISPR/Casa9 technology not only to eliminate antinutritional compounds but also to increase nutritional compounds and as a consequence fruit quality, the final research of this thesis was focused on metabolic characterization of greenflesh tomato mutants (Chapter 4) generate in collaboration with the “Istituto de Biologia Molecular y Celular de Plantas” (IBMCP, laboratory of Professor Antonio Granell). Specifically, an accumulation of valuable health-promoting secondary compounds was observed, in particular an increase in carotenoid levels and a significant accumulation of vitamin E. Since genome editing technique is able to generate precise mutations within the genome, comparable to those that occur naturally or to those obtained with classical mutagenesis, the novel varieties could be efficiently generated in the future by genome-edited crops.

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