INTEGRATING COMPUTATIONAL AND BIOTECHNOLOGY APPROACHES FOR ENHANCED TOCOPHEROL AND TOCOTRIENOL BIOSYNTHESIS IN PLANTS

Abstract

Tocopherols and tocotrienols collectively constitute vitamin E, a family of lipid-soluble antioxidants indispensable for human nutrition, crop quality, and plant stress tolerance. Despite their agronomic and nutraceutical importance, the molecular regulatory network governing vitamin E biosynthesis remains incompletely characterised, and conventional breeding and transgenic approaches have achieved only modest improvements in tocopherol content. The convergence of the shikimate and methylerythritol phosphate (MEP) pathway arms at homogentisate phytyldiphosphate transferase (VTE2) represents the committed step in tocopherol synthesis, highlighting it as a high-priority target for regulatory investigation and molecular intervention. In the present study, gene expression datasets (GSE59790 and GSE47518) pertaining to tocopherol biosynthesis and high-light stress in Arabidopsis thaliana were retrieved from the Gene Expression Omnibus (GEO) database and subjected to differential gene expression analysis using GEO2R. Protein-protein interaction (PPI) network construction was performed using the STRING database (confidence score > 0.900), and hub genes were identified by degree centrality analysis, with functional enrichment assessed through Gene Ontology (GO) and KEGG pathway analysis. For molecular docking, the crystal structure of VTE2 (PDB ID: 1YFP) was employed as the target, and a curated library of 75 plant-derived phytochemicals was screened using EasyDock Vina, with protein-ligand interactions characterised using LigPlot+ and PyMOL. A total of 1,148 differentially expressed genes were identified, comprising 634 upregulated and 514 downregulated genes. Hub gene analysis identified VTE1, VTE4, VTE2/HPT1, HPPD, and DXPS as the most highly connected nodes in the upregulated PPI network, confirming their central regulatory roles. Functional enrichment revealed plastidic metabolic processes, prenylquinone synthesis, and oxidative stress response as predominant biological themes. Molecular docking identified alpha mangostin as the lead compound with a binding affinity of -9.8 kcal/mol, forming critical hydrogen bonds with Ser186, Asp227, and Arg295 within the VTE2 active site, followed by beta-tocotrienol ( 9.4 kcal/mol) and quercetin-3-glucoside (-9.1 kcal/mol) as additional high-affinity candidates. These findings provide a comprehensive regulatory framework for the tocopherol biosynthetic network and nominate alpha-mangostin as a promising phytochemical modulator of VTE2 warranting further experimental validation.