Identification, characterization, and expression profiling of α-tocopherol biosynthesis genes associated with anilofos metabolism in Oryza sativa

Vitamin E, particularly α-tocopherol (α-TOC), is a chloroplast-synthesized antioxidant that plays a crucial role in protecting photosynthetic membranes from oxidative damage caused by environmental stressors. Its biosynthesis involves several key enzymes: hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate phytyltransferase (HPT), tocopherol cyclase (VTE1), tocopherol methyltransferase (VTE4), and phytolkinase (VTE5). However, the expression patterns and functional roles of genes involved in α-TOC biosynthesis under pesticide stress remain poorly characterized. In this study, we identified 10 α-TOC biosynthesis genes, including 2 HPPD, 1 HPT, 2 VTE1, 1 VTE4, and 4 VTE5 genes, in rice (Oryza sativa) genome using transcriptome datasets from plants treated with anilofos and α-TOC to explore their properties under pesticide stress. Phylogenetic analysis classified the α-TOC biosynthesis gene family into five subfamilies across rice, Arabidopsis (Arabidopsis thaliana), soybean (Glycine max), wheat (Triticum aestivum), and maize (Zea mays). Chromosomal localization revealed that segmental duplications contributed to the expansion of this gene family in rice, with 10 genes distributed across 7 of its 12 chromosomes. In addition, 10 rice genes involved in α-TOC biosynthesis exhibited collinearity with corresponding genes in Arabidopsis, soybean, wheat, and maize. In silico subcellular localization prediction suggested that the proteins encoded by these 10 genes localize to multiple cellular compartments, including the nucleus, cytoplasm, mitochondria, plasma membrane, and endoplasmic reticulum. OsHPPD, OsHPT, OsVTE1, OsVTE4, and OsVTE5 possess diverse gene structures, cis-regulatory elements, motifs, and conserved domains, suggesting their diverse functions in response to biotic and abiotic stresses. RT-qPCR analysis confirmed that a subset of these genes was preferentially expressed under pesticide exposure. Notably, combined treatment with 250 mg/L α-TOC and 0.04 mg/L anilofos upregulated the expression of all 10 genes by 1.64–3.75-fold in roots and by 2.66–5.34-fold in shoots compared with anilofos-only treatment. Protein–protein interaction analysis further supported the involvement of these 10 rice α-TOC biosynthesis proteins in anilofos metabolism. These findings indicate that α-TOC biosynthesis genes respond effectively to anilofos-induced stress, likely facilitating α-TOC production and potentially regulating pesticide degradation.