Characterization of Rice Zaxione Synthase (ZAS) Gene Family Members and Exploration of Zaxinone Biology in Plants

Carotenoid cleavage, catalyzed by Carotenoid Cleavage Dioxygenases (CCDs), forms various apocarotenoids that act as signaling molecules and plant hormone precursors. The rice Zaxinone Synthase (ZAS), an overlooked plant CCD subfamily, catalyzes the synthesis of apocarotenoid zaxinone in vitro. Zaxinone is a growth regulator essential for normal rice growth and development. In the first project, we generated and characterized rice lines that constitutively overexpress ZAS to gain deeper insights into the role of ZAS in determining zaxinone levels and its impact on rice growth and architecture. We found that the overexpression of ZAS increased the level of endogenous zaxinone, promoted root growth, and increased the number of productive tillers. Importantly, ZAS overexpression led to about 30% higher grain yield per plant. Hormone and metabolite analysis revealed a decrease in the level of strigolactones (SLs) and an increased accumulation of monosaccharide sugars. Additionally, ZAS overexpression enhanced the uptake of nutrients, including phosphate, resulting in improved tolerance to low phosphate. Overall, ZAS plays a crucial role in regulating rice growth, yield, and various physiological and metabolic processes. The second project focused on the enzymatic and biological function of OsZAS2, which represents a separate ZAS clade. Through in vitro assay and genetic manipulation, we revealed that OsZAS2 is a further zaxinone-forming enzyme mainly localized in plastids. The loss-of-function CRISPR/Cas9-Oszas2 mutants exhibited lower levels of zaxinone in their roots, reduced root and shoot biomass, fewer tillers, and elevated levels of SLs. Furthermore, the expression of OsZAS2 is induced in arbuscule-containing cells, and in support of this, the Oszas2 mutant displayed a significant reduction in arbuscular mycorrhizal colonization. In summary, OsZAS2 is involved in zaxinone production in rice. Like OsZAS, it plays a crucial and non-redundant role in determining rice growth, architecture, and SL content and is essential for optimal mycorrhization. In the last chapter, we investigated the effect of exogenous zaxinone application on the non-mycorrhizal plant Arabidopsis that lacks ZAS orthologues but synthesizes zaxinone via an unknown mechanism. Obtained results suggest that zaxinone functions as a regulatory metabolite in Arabidopsis, stimulating the biosynthesis of the two carotenoid-derived hormones SL and abscisic acid in roots.