Cuccurullo, Alessia (2025) Improving tomato fruit quality by editing genes of strigolactone and ascorbic acid pathway. [Tesi di dottorato]

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Abstract

Fruit quality is a general term that encompasses several aspects that, taken together, influence consumer appreciation of a food product and its market suitability. For tomato (Solanum lycopersicum L.), the most widespread and consumed vegetable in the world, the fruit nutritional profile is crucial for defining its overall quality, and in recent years its improvement has gained considerable interest in agricultural research. In this thesis, tomato nutritional quality was explored in plants employing the CRISPR/Cas9-mediated inactivation of various genes from two different pathways: the strigolactone (SL) and the ascorbic acid (AsA) pathways. In the Chapter 2 of this thesis, the characterization of a repertoire of tomato edited plants for the four core genes involved in SLs biosynthesis, namely SlD27, SlCCD7, SlCCD8 and SlMax1 is described. These edited plants did not accumulate SLs being, therefore, resistant to parasitic weeds (Phelipanche ramosa L. and P. aegyptiaca L.) colonization. It was established in various species that the partial or complete knockout (KO) of SL-biosynthesis genes has deep effects on the plant phenotype. Therefore, morphology-, flower-, and fruit-related traits were assessed in these KO-edited plants and in the non-edited plants. This comparison confirmed the effects on the phenotype of the mutations in the Slccd7, Slccd8 and Slmax1 genes. On the contrary, the Sld27 KO line was largely comparable to the non-edited wild-type (WT) line, also for fruit-related traits as fruit weight and size. Since SLs are carotenoid-derived compounds, the composition in carotenoids was analysed in fruits from the KO lines. The most significant variations were found in the production of VOCs, metabolites essential for determining aroma quality, another fundamental aspect affecting the overall quality of tomato fruit. The most affected VOCs are the derivatives of phenylpropanoids, branched-chain amino acids and phenylalanine. Also in this case, the Sld27 lines showed only minor alterations in VOC production and, conversely, significant changes emerged in mutants for genes downstream in the biosynthetic pathway, such as Slccd8 and Slmax1. In particular, pseudoionone – an apocarotenoid (ApoC)-derived metabolite positively associated to tomato flavour – was found to be moderately increased in the line Sld27. Altogether, these results displayed a gene-specific effect, never reported before for the genes involved in the SL biosynthesis, and highlighted the Sld27 as a promising line due to its WT-like phenotype. The gene D27 has been the only gene known for encoding a β-carotene isomerase so far. However, two additional D27-Likes (D27-Like 1 and D27-Like 2) genes were recently described in the rice and Arabidopsis genome. It has been hypothesised that the D27 and D27-like genes are involved in SL biosynthesis and have been proposed as key mediators of the SL-ABA interaction, which finely tunes plant physiology and development. Nonetheless, the physiological functions of these genes are far from being clear. The research line developed in Chapter 3 was focused on the functional characterization of the members of the SlD27 gene family. Herein it was described the identification of the SlD27-Like 1 and -Like 2 genes in the genome of tomato, and the generation of CRISPR/Cas9 KO lines. The effects of these mutations on the plant phenotype, physiology and secondary metabolite composition, were assessed on the complete panel of mutants, therefore including the KO lines for the already known Sld27 gene. In according to the literature, the plant phenotype of the Sld27-Likes KO lines was undistinguishable from those of the non-edited lines. The hypothesis that these genes have a role in the SL-ABA interaction was explored analysing the accumulation of ABA that resulted augmented in the leaves of all the edited lines. Consistently, the stomatal conductance (gs) was generally reduced in all the Sld27 and Sld27-Likes KO lines. Polar and non-polar secondary metabolites accumulation were further analysed in leaves. Non-polar metabolites accumulation was generally unchanged, whereas the accumulation of polar compounds exhibited relevant variations. AsA levels, in particular, increased in the Sld27 and Sld27-Like 1 KOs, and quinic acid (QA) accumulation resulted higher in all the edited lines. Other polyphenols – as naringenin chalcon (ChNar), hyperoside (quercetin-3-galactoside; Hyp) and kaempferol-3-O-glucoside (K-3-G) – were moderately decreased mostly in the Sld27 lines. Chapter 4 describes how the mutation of two ascorbate oxidase genes, which are active in the AsA recycling pathway, increases the fruit content of this essential metabolite. In particular, the role of the SlAO and the SlLAC genes encoding for two ascorbate oxidases was studied through the characterization of KO lines produced by CRISPR/Cas9-mediated editing. Minor changes were reported for the plant phenotype of the mutated lines. The soluble solids content (SSC, °Brix) was higher in the Slao KO1 and KO2 lines and in Sllac KO line. Interestingly, the KO lines were found to accumulate up to 60.5% more reduced AsA in mature red (MR) fruits compared to the non-edited lines. Moreover, this metabolite was higher in the roots of the Slao lines and in the leaves of the Sllac line. These results support the previously predicted function of SlLAC as a potential additional ascorbate oxidase. Furthermore, the accumulation pattern observed for the reduced AsA in leaves and roots suggest a differential expression these genes, with SlAO being mainly expressed in root and SlLAC in leaves. Altogether, this thesis has provided remarkable outcomes in expanding the knowledge on the effects produced by the manipulation of two different biosynthesis pathway on the tomato fruit quality, with the generation of valuable genetic materials, representing a rich resource for future studies.

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