The atypical kinase ABC1K1 / PGR6 allocates geranylgeranyldiphosphate to the carotenoid biosynthesis pathway

Crop fruit species play an important role in life on Earth. Indeed, fruits provide essential nutrients and vitamins which are not synthesized by humans but are necessary for their health. The synthesis of these essential nutrients occurs during the complex process of fruit maturation and ripening, orchestrated by regulatory genes, transcription factors, and hormones. During this process, many biological, and physiological events lead to fruit changes and are indispensable for the fruit color, flavor, aroma and quality. In tomato fruit, many of these biological events occur in a colored plastid specialized in carotenoid biosynthesis and sequestration, the chromoplast. The hallmark red color of tomato fruit is due to the synthesis and accumulation of lycopene in a lipoprotein sub-organellar compartment of chloroplasts and chromoplasts called plastoglobule (PG). The hydrophobic core of chloroplast and chromoplast plastoglobules serves as a reservoir and biosynthetic compartment of phytyl esters, prenyl lipids and carotenoids. In the first chapter entitled “Plastoglobules: A hub of lipid metabolism in the chloroplast” (Shanmugabalaji et al., 2022) we reviewed plastoglobule functions and metabolism in different plastid types. In the second chapter “Chapter II: Chromoplast plastoglobules recruit the carotenoid biosynthetic pathway and contribute to carotenoid accumulation during tomato fruit maturation” (Zita et al., 2022), we used a proteomic approach to investigate plastoglobule proteome remodeling during the chloroplast to chromoplast transition in tomato fruit. This proteomic study revealed that the tomato plastoglobule contains around 30 proteins and members of different enzyme families including fibrillins (FBNs), the activity of BC1 complex kinase (ABC1Ks), tocopherol cyclase (VTE1), AD(P)Hubiquinone oxidoreductase C1 (NDC1). The study revealed the presence of the complete carotenoid biosynthesis pathway in chromoplast plastoglobules: phytoene synthase 1 (PSY1), phytoene desaturase (PDS), zeta carotene desaturase (ZDS), carotenoid isomerase (CRTISO), and lycopene beta cyclase (LYC-B). The results show that the plastoglobule becomes a platform for carotenoid biosynthesis during the chloroplast to chromoplast transition. In addition, the lipidomes of chloroplast and chromoplast plastoglobule undergo remodeling during progressive fruit ripening stages. Specifically, β-carotene and lycopene were highly enriched in chromoplast plastoglobules compared to chloroplast plastoglobules. Overall, the chapter demonstrates that the plastoglobule plays a central role in the ripening process. The third chapter “A quantitative method to measure geranylgeranyl diphosphate (GGPP) and geranylgeranyl monophosphate (GGP) in tomato (Solanum lycopersicum) fruit” (Zita et al., 2023), a method to quantify GGPP from tomato fruit tissue was developed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method has been validated by using a wild-type (WT) and mutant matrices defective in GGPP synthesis. In addition, this chapter highlight the importance of sample preparation to preserve the GGPP and limited its conversion to GGP (hydrolyzed product). This method is essential for further analysis in my thesis. Finally, in the fourth chapter of my thesis “An atypical kinase ABC1K1 allocates geranylgeranyl diphosphate to the carotenoid biosynthesis pathway in plastoglobules of chromoplasts”, I investigate the role of SlABC1K1 in tomato fruit carotenoid metabolism. The CRISPR-Cas9 mutant of SlABC1K1 (slabc1k1) showed the photosynthetic PGR6 phenotype in leaves that had previously been observed in Arabidopsis. Surprisingly, slabc1k1 fruit had an orange phenotype. The slabc1k1 phenotype was linked to reduced lycopene accumulation as well as that of its phytoene precursor. Based on our results, I propose two possible scenarios for the function of SlABC1K1 in tomato fruit plastoglobules. SlABC1K1 may acts as a key regulator for geranylgeranyl diphosphate (GGPP) allocation to the carotenoid pathway or as a co-regulator of carotenoid biosynthesis enzymes in plastoglobules. The discovery that SlABC1K1 is required for the GGPP allocation to plastoglobule is the principal result of my thesis.