Caratterizzazione del ruolo di Zaxinone synthase nella simbiosi micorrizica arbuscolare e nell'omeostasi dei nutrienti in Oryza sativa

In recent years, plant apocarotenoids are emerging not only as carotenoid breakdown products, but also as metabolites with active roles in regulating physiological and developmental processes and plant-(a)biotic interactions. In particular, some apocarotenoids were associated with the establishment of arbuscular mycorrhizal (AM) symbiosis. The novel discovered apocarotenoid, zaxinone, which is generated by the activity of Zaxinone Synthases (Zas1 and Zas2), was shown to control rice plant development and the extent of AM root colonization with a complex interplay with strigolactones (SLs). Rice plants express Zas1 and Zas2 genes in the roots under phosphate (Pi) deficiency conditions and during the establishment of AM symbiosis. T-DNA loss-of-function oszas1 mutant showed a decreased zaxinone content in roots, reduced shoot, and root growth, and a higher SL level compared to wild-type (WT) rice plants. My thesis work aimed at gaining a deeper understanding of the role of zaxinone in AM symbiosis and nutrient homeostasis. To address these issues, we took advantage of zaxinone biosynthesis related genotypes: a novel OsZas1 loss-of-function mutation via CRISPR-Cas9 (oszas1i) and OsZas1 over-expressing (OX-Zas) lines that were investigate by means of morphological, molecular, and physiological approaches. We firstly characterize the impact of the altered expression of OsZas1 in the content of apocarotenoids involved in AM symbiosis, such as zaxinone, SLs (4-deoxyorobanchol, orobanchol), and abscissic acid in the root exudate of WT, oszas1 and OX-Zas lines. To determine the role of OsZas1 in AM symbiosis, WT, oszas1i and OX-Zas lines were colonized by an AM fungus and the expression level of an AM marker gene was analysed during the early and late stage of AM symbiosis. Subsequently, the mycorrhization level was evaluated through a morphological analysis in order to validate the molecular data and to assess the morphology of the fungal structures in the different genotypes. In a second experimental setup, WT, oszas1i and OX-ZAS lines were grown in non-mycorrhizal condition under different Pi availability, to evaluate the nutritional profile of both leaf and root tissues of the different genotypes by using capillary electrophoresis. The results showed that, as expected, oszas1i had a lower level of zaxinone and higher level of SLs content in the root exudate. By contrast, notwithstanding the higher expression level of OsZas1, OX-Zas lines did not display a higher level of zaxinone while different levels of SLs and ABA in root exudates were detected. However, OX-Zas lines showed different apocarotenoid profiles, but the reason for this inhomogeneity is not yet understood. Concerning AM symbiosis phenotype, AM fungal colonization decreased in oszas1 while OX lines did not show any difference compared to the WT. Regarding the nutritional profile, we observed that oszas1 mutant showed, under normal Pi condition, a higher level of Pi in the roots. This data confirmed our hypothesis that OsZas1 is involved in Pi- sensing in rice roots. The nutritional profile on OX-Zas lines provided also evidence of the impact of OsZas1 on sulfate content in the roots and partially in the shoot. Overall, these findings contribute to a better understanding of the complex interactions between rice plant, AM symbiosis and zaxinone that will be instrumental to unravel other functions and potential applications of zaxinone in agriculture.