In recent years, the identification of genes involved in the production and regulati specialized metabolites has led to the discovery of key molecules and new metabolic pathways that shape plant fitness, stress tolerance, and interactions with the environment. Among these, carotenoid-derived metabolic pathways mediated by Carotenoid Cleavage Dioxyge enzymes (CCDs) contribute to the biosynthesis of several apocarotenoids, a diverse class metabolites that includes phytohormones, signal molecules, chromophores and constituents; thus, enabling the characterization of novel agronomic traits in industrial crops such as Solanum lycopersicum L. In addition to the six well-characterized CCD subfamilies (NCED, CCD1, CCD2, CCD4, CCD7, CCD8), a seventh subfamily, named ZAS (zaxinone synthase), has recently been identified inOryza sativa. Functional studies showed that two rice ZAS, OsZAS and OsZAS2, cleave the apocarotenoid β-apo- 10'-zeaxanthinal (C27) yielding zaxinone (C18). Zaxinone is an important growth regulat apocarotenoid involving in plant development, arbuscular mycorrhizal (AM) symbiosis modulation of strigolactone levels in rice. Metabolic findings revealed that zaxinone is a present in tomato, prompting our interest in exploring its function and biosynthetic pathway in this species too. Through BLASTp researches againstS. lycopersicum reference proteome, we identified three putative orthologues of OsZAS in tomato genome: SlZAS, SlZAS-like 1 and SlZAS- like 2. To evaluate their functions, full-length cDNAs were expressed as thioredoxin fusion inE. coli cells and in vitro bacterial assays were performed. Among these, SlZAS displayed a cle activity in converting β-apo-10'-zeaxanthinal to zaxinone in our experimental conditions. Thi activity, together with the chloroplasts-associated localization, support its expected role carotenoid-derived metabolic pathways. To further investigate theirin vivo functions, different CRISPR/Cas9 knock-out tomato lines were generated. Specifically, phenotypic characterization of zasloss-of-function mutants (T2 generation) revealed reduced shoot growth and a less developed root system compared towild-type plants. Moreover, a strong reduction of zaxinone level was detected in mutant root system compared towild-type, highlighting its role in zaxinone biosynthesis. Furthermore, the analysis of polar and non-polar metabolic profiles in tomato zas mutants has been conducted, revealing indications of possible changes in key metabolites associated with physiological and ecological functions. Further investigations and analyses will completely elucidate the enzymatic functions of these genes and clarify the zaxinone biosynthetic pathway in tomato as well, providing new insight into apocarotenoid metabolism in solanaceous crops and new prospects for advanced breeding and the development of novel and more resilient varieties.

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