Biochemical and Functional Analysis of Carotenoid Biosynthetic Enzyme Complexes in Tomato Fruits
Abstract
Affiliation: Department of Biological Sciences, East Tennessee State University, Johnson City, TN, USA Abstract: Carotenoids are essential pigments in plants, playing critical roles in photosynthesis, photoprotection, and human nutrition. While the biosynthetic pathway of carotenoids is well characterized, the spatial organization and dynamic assembly of biosynthetic enzyme complexes within chromoplasts remain poorly understood. This study investigates the structural and functional organization of carotenoid biosynthetic enzymes in tomato (Solanum lycopersicum) fruits, focusing on the impact of enzyme complex compositions on metabolic flux and carotenoid composition. To explore these dynamics, we employed CRISPR-Cas9 genome editing to generate a Zeta-carotene isomerase (Z-ISO) knockout mutant, which will be analyzed alongside tomato mutants yellow flesh (r), tangerine (t), beta, and delta, which have distinct carotenoid compositions compared with wild-type (Ailsa Craig) fruits. To examine the native enzyme complexes responsible for carotenoid biosynthesis, chromoplast isolation, ultracentrifugation, and size-exclusion chromatography (SEC) were conducted. Mass spectrometry (MS) is being employed to identify and characterize protein complexes in each fraction. Protein-protein interaction assays in vivo are being used to study enzyme interaction and localization. Additionally, high-performance liquid chromatography (HPLC) is being used to analyze metabolic flux and carotenoid accumulation. By integrating genetics with biochemical and proteomic approaches, this study aims to advance our understanding of enzyme organization and metabolic channeling in chromoplasts. These findings have important implications for crop biofortification, metabolic engineering, and the development of nutritionally enhanced crops to address vitamin A deficiency. Keywords: Carotenoids, Chromoplast, CRISPR Knockout, Z-ISO, Mass Spectrometry, Tomato, Metabolic Flux, Biofortification
Start Time
16-4-2025 2:30 PM
End Time
16-4-2025 3:30 PM
Room Number
303
Presentation Type
Oral Presentation
Presentation Subtype
Grad/Comp Orals
Presentation Category
Science, Technology and Engineering
Faculty Mentor
Tianhu Sun
Biochemical and Functional Analysis of Carotenoid Biosynthetic Enzyme Complexes in Tomato Fruits
303
Affiliation: Department of Biological Sciences, East Tennessee State University, Johnson City, TN, USA Abstract: Carotenoids are essential pigments in plants, playing critical roles in photosynthesis, photoprotection, and human nutrition. While the biosynthetic pathway of carotenoids is well characterized, the spatial organization and dynamic assembly of biosynthetic enzyme complexes within chromoplasts remain poorly understood. This study investigates the structural and functional organization of carotenoid biosynthetic enzymes in tomato (Solanum lycopersicum) fruits, focusing on the impact of enzyme complex compositions on metabolic flux and carotenoid composition. To explore these dynamics, we employed CRISPR-Cas9 genome editing to generate a Zeta-carotene isomerase (Z-ISO) knockout mutant, which will be analyzed alongside tomato mutants yellow flesh (r), tangerine (t), beta, and delta, which have distinct carotenoid compositions compared with wild-type (Ailsa Craig) fruits. To examine the native enzyme complexes responsible for carotenoid biosynthesis, chromoplast isolation, ultracentrifugation, and size-exclusion chromatography (SEC) were conducted. Mass spectrometry (MS) is being employed to identify and characterize protein complexes in each fraction. Protein-protein interaction assays in vivo are being used to study enzyme interaction and localization. Additionally, high-performance liquid chromatography (HPLC) is being used to analyze metabolic flux and carotenoid accumulation. By integrating genetics with biochemical and proteomic approaches, this study aims to advance our understanding of enzyme organization and metabolic channeling in chromoplasts. These findings have important implications for crop biofortification, metabolic engineering, and the development of nutritionally enhanced crops to address vitamin A deficiency. Keywords: Carotenoids, Chromoplast, CRISPR Knockout, Z-ISO, Mass Spectrometry, Tomato, Metabolic Flux, Biofortification