Identification of oxidoreductase genes for phytoecdysteroids biosynthesis in spinach through comparative...

Phytoecdysteroids (PE) are plant-derived ecdysteroids known as insect molting hormones. Among them, 20-hydroxyecdysone (20E) is the major PE that defends against herbivorous insects. However, unlike in insects, little is known about the biosynthetic genes of 20E in plants. This study aimed to identify oxidoreductase genes involved in biosynthesis of 20E in spinach (Spinacia oleracea), a plant that contains high levels of 20E. To achieve this, spinach samples with varying 20E contents were generated, namely, 20E-positive wild-type spinach (SWT), 20E-negative callus derived from spinach roots (SCA), spinach with an increased 20E content due to pds gene knockdown via the virus-induced gene silencing (VIGS) system (SPD), and its control (STR), and then RNA sequencing-based comparative transcriptome analysis was performed. Among 8,679 differentially expressed genes, seven oxidoreductase genes were selected as candidate genes involved in 20E biosynthesis. The reliability of these genes was validated through qRT-PCR. Additionally, using the VIGS system, four oxidoreductase genes (two short-chain dehydrogenase/reductase genes and two cytochrome P450 genes) whose expression strongly correlated with the 20E content were identified. Protein-ligand docking analysis further supported these findings and showed strong interactions between the candidate proteins and 20E precursors, indicating their potential roles in 20E biosynthesis. This is the first report to identify oxidoreductase genes involved in 20E biosynthesis in spinach, providing valuable insights into the molecular mechanisms underlying 20E biosynthesis.